The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

PubMed

Jackson, Andrew P; Otto, Thomas D; Darby, Alistair; Ramaprasad, Abhinay; Xia, Dong; Echaide, Ignacio Eduardo; Farber, Marisa; Gahlot, Sunayna; Gamble, John; Gupta, Dinesh; Gupta, Yask; Jackson, Louise; Malandrin, Laurence; Malas, Tareq B; Moussa, Ehab; Nair, Mridul; Reid, Adam J; Sanders, Mandy; Sharma, Jyotsna; Tracey, Alan; Quail, Mike A; Weir, William; Wastling, Jonathan M; Hall, Neil; Willadsen, Peter; Lingelbach, Klaus; Shiels, Brian; Tait, Andy; Berriman, Matt; Allred, David R; Pain, Arnab

2014-06-01

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

[Application of dhfr gene negative Chinese hamster ovary cell line to express hepatitis B virus surface antigen].

PubMed

Yi, Y; Zhang, M; Liu, C

2001-06-01

To set up an efficient expressing system for recombinant hepatitis B virus surface antigen (HBsAg) in dhfr gene negative CHO cell line. HBsAg gene expressing plasmid pCI-dhfr-S was constructed by integrating HBsAg gene into plasmid pCI which carries dhfr gene. The HBsAg expressing cell line was set up by transfection of plasmid pCI-dhfr-S into dhfr gene negative CHO cell line in the way of lipofectin. Under the selective pressure of MTX, 18 of 28 clonized cell lines expressed HBsAg, 4 of them reached a high titer of 1:32 and protein content 1-3 micrograms/ml. In this study, the high level expression of HBsAg demonstrated that the dhfr negative mammalian cell line when recombined with plasmid harboring the corresponding deleted gene can efficiently express the foreign gene. The further steps toward building optimum conditions of the expressing system and the increase of expressed product are under study.

Surface antigens of Plasmodium falciparum gametocytes--a new class of transmission-blocking vaccine targets?

PubMed

Sutherland, Colin J

2009-08-01

The re-establishment of elimination and eradication on the malaria control agenda has led to calls for renewed effort in the development of parasite transmission-blocking interventions. Vaccines are ideally suited to this task, but progress towards an anti-gamete transmission-blocking vaccine, designed to act on parasites in blood-fed mosquitoes, has been slow. Recent work has confirmed that the surface of the gametocyte-infected erythrocyte presents antigens to the host immune system, and elicits specific humoral immune responses to these antigens, termed gametocyte surface antigens (GSAs). Likely candidate molecules, including antigens encoded by sub-telomeric multi-gene families, are discussed, and a hypothetical group of parasite molecules involved in spatial and temporal signal transduction in the human host is proposed, the tropins and circadins. The next steps for development of anti-gametocyte transmission-blocking vaccines for P. falciparum and the other human malaria species are considered.

Genome-Wide Prediction of the Polymorphic Ser Gene Family in Tetrahymena thermophila Based on Motif Analysis

PubMed Central

Ponsuwanna, Patrath; Kümpornsin, Krittikorn; Chookajorn, Thanat

2014-01-01

Even though antigenic variation is employed among parasitic protozoa for host immune evasion, Tetrahymena thermophila, a free-living ciliate, can also change its surface protein antigens. These cysteine-rich glycosylphosphatidylinositol (GPI)-linked surface proteins are encoded by a family of polymorphic Ser genes. Despite the availability of T. thermophila genome, a comprehensive analysis of the Ser family is limited by its high degree of polymorphism. In order to overcome this problem, a new approach was adopted by searching for Ser candidates with common motif sequences, namely length-specific repetitive cysteine pattern and GPI anchor site. The candidate genes were phylogenetically compared with the previously identified Ser genes and classified into subtypes. Ser candidates were often found to be located as tandem arrays of the same subtypes on several chromosomal scaffolds. Certain Ser candidates located in the same chromosomal arrays were transcriptionally expressed at specific T. thermophila developmental stages. These Ser candidates selected by the motif analysis approach can form the foundation for a systematic identification of the entire Ser gene family, which will contribute to the understanding of their function and the basis of T. thermophila antigenic variation. PMID:25133747

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

Longitudinal evaluation of humoral immune response and merozoite surface antigen diversity in calves naturally infected with Babesia bovis, in São Paulo, Brazil.

PubMed

Matos, Carlos António; Gonçalves, Luiz Ricardo; Alvarez, Dasiel Obregón; Freschi, Carla Roberta; Silva, Jenevaldo Barbosa da; Val-Moraes, Silvana Pompeia; Mendes, Natalia Serra; André, Marcos Rogério; Machado, Rosangela Zacarias

2017-01-01

Babesiosis is an economically important infectious disease affecting cattle worldwide. In order to longitudinally evaluate the humoral immune response against Babesia bovis and the merozoite surface antigen diversity of B. bovis among naturally infected calves in Taiaçu, Brazil, serum and DNA samples from 15 calves were obtained quarterly, from their birth to 12 months of age. Anti-B. bovis IgG antibodies were detected by means of the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The polymerase chain reaction (PCR) was used to investigate the genetic diversity of B. bovis, based on the genes that encode merozoite surface antigens (MSA-1, MSA-2b and MSA-2c). The serological results demonstrated that up to six months of age, all the calves developed active immunity against B. bovis. Among the 75 DNA samples evaluated, 2, 4 and 5 sequences of the genes msa-1, msa-2b and msa-2c were obtained. The present study demonstrated that the msa-1 and msa-2b genes sequences amplified from blood DNA of calves positive to B. bovis from Taiaçu were genetically distinct, and that msa-2c was conserved. All animals were serologically positive to ELISA and IFAT, which used full repertoire of parasite antigens in despite of the genetic diversity of MSAs.

Strains of Sarcocystis neurona exhibit differences in their surface antigens, including the absence of the major surface antigen SnSAG1.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Marsh, Antoinette E; Patil, Bhagyashree A; Saville, William J; Lindsay, David S; Dubey, J P; Granstrom, David E

2008-05-01

A gene family of surface antigens is expressed by merozoites of Sarcocystis neurona, the primary cause of equine protozoal myeloencephalitis (EPM). These surface proteins, designated SnSAGs, are immunodominant and therefore excellent candidates for development of EPM diagnostics or vaccines. Prior work had identified an EPM isolate lacking the major surface antigen SnSAG1, thus suggesting there may be some diversity in the SnSAGs expressed by different S. neurona isolates. Therefore, a bioinformatic, molecular and immunological study was conducted to assess conservation of the SnSAGs. Examination of an expressed sequence tag (EST) database revealed several notable SnSAG polymorphisms. In particular, the EST information implied that the EPM strain SN4 lacked the major surface antigen SnSAG1. The absence of this surface antigen from the SN4 strain was confirmed by both Western blot and Southern blot. To evaluate SnSAG polymorphisms in the S. neurona population, 14 strains were examined by Western blots using monospecific polyclonal antibodies against the four described SnSAGs. The results of these analyses demonstrated that SnSAG2, SnSAG3, and SnSAG4 are present in all 14 S. neurona strains tested, although some variance in SnSAG4 was observed. Importantly, SnSAG1 was not detected in seven of the strains, which included isolates from four cases of EPM and a case of fatal meningoencephalitis in a sea otter. Genetic analyses by PCR using gene-specific primers confirmed the absence of the SnSAG1 locus in six of these seven strains. Collectively, the data indicated that there is heterogeneity in the surface antigen composition of different S. neurona isolates, which is an important consideration for development of serological tests and prospective vaccines for EPM. Furthermore, the diversity reported herein likely extends to other phenotypes, such as strain virulence, and may have implications for the phylogeny of the various Sarcocystis spp. that undergo sexual stages of their life cycle in opossums.

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

PubMed

Talmadge, J E; Talmadge, C B; Zbar, B; McEwen, R; Meeker, A K; Tribble, H

1987-06-01

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

T-cell receptor revision: friend or foe?

PubMed

Hale, J Scott; Fink, Pamela J

2010-04-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

PubMed Central

Hubert, Rene S.; Vivanco, Igor; Chen, Emily; Rastegar, Shiva; Leong, Kahan; Mitchell, Steve C.; Madraswala, Rashida; Zhou, Yanhong; Kuo, James; Raitano, Arthur B.; Jakobovits, Aya; Saffran, Douglas C.; Afar, Daniel E. H.

1999-01-01

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging. PMID:10588738

Peripheral B cells latently infected with Epstein–Barr virus display molecular hallmarks of classical antigen-selected memory B cells

PubMed Central

Souza, Tatyana A.; Stollar, B. David; Sullivan, John L.; Luzuriaga, Katherine; Thorley-Lawson, David A.

2005-01-01

Epstein–Barr virus (EBV) establishes a lifelong persistent infection within peripheral blood B cells with the surface phenotype of memory cells. To date there is no proof that these cells have the genotype of true germinal-center-derived memory B cells. It is critical to understand the relative contribution of viral mimicry versus antigen signaling to the production of these cells because EBV encodes proteins that can affect the surface phenotype of infected cells and provide both T cell help and B cell receptor signals in the absence of cognate antigen. To address these questions we have developed a technique to identify single EBV-infected cells in the peripheral blood and examine their expressed Ig genes. The genes were all isotype-switched and somatically mutated. Furthermore, the mutations do not cause stop codons and display the pattern expected for antigen-selected memory cells based on their frequency, type, and location within the Ig gene. We conclude that latently infected peripheral blood B cells display the molecular hallmarks of classical antigen-selected memory B cells. Therefore, EBV does not disrupt the normal processing of latently infected cells into memory, and deviations from normal B cell biology are not tolerated in the infected cells. This article provides definitive evidence that EBV in the peripheral blood persists in true memory B cells. PMID:16330748

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

PubMed Central

Lee, John K.; Bangayan, Nathanael J.; Chai, Timothy; Smith, Bryan A.; Pariva, Tiffany E.; Yun, Sangwon; Vashisht, Ajay; Zhang, Qingfu; Park, Jung Wook; Corey, Eva; Huang, Jiaoti; Wohlschlegel, James; Witte, Owen N.

2018-01-01

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting. PMID:29686080

A rhamnose-rich O-antigen mediates adhesion, virulence, and host colonization for the xylem-limited phytopathogen Xylella fastidiosa.

PubMed

Clifford, Jennifer C; Rapicavoli, Jeannette N; Roper, M Caroline

2013-06-01

Xylella fastidiosa is a gram-negative, xylem-limited bacterium that causes a lethal disease of grapevine called Pierce's disease. Lipopolysaccharide (LPS) composes approximately 75% of the outer membrane of gram-negative bacteria and, because it is largely displayed on the cell surface, it mediates interactions between the bacterial cell and its surrounding environment. LPS is composed of a conserved lipid A-core oligosaccharide component and a variable O-antigen portion. By targeting a key O-antigen biosynthetic gene, we demonstrate the contribution of the rhamnose-rich O-antigen to surface attachment, cell-cell aggregation, and biofilm maturation: critical steps for successful infection of the host xylem tissue. Moreover, we have demonstrated that a fully formed O-antigen moiety is an important virulence factor for Pierce's disease development in grape and that depletion of the O-antigen compromises its ability to colonize the host. It has long been speculated that cell-surface polysaccharides play a role in X. fastidiosa virulence and this study confirms that LPS is a major virulence factor for this important agricultural pathogen.

Hepatitis B surface antigen gene expression is regulated by sex steroids and glucocorticoids in transgenic mice.

PubMed Central

Farza, H; Salmon, A M; Hadchouel, M; Moreau, J L; Babinet, C; Tiollais, P; Pourcel, C

1987-01-01

We have investigated the basis for liver-specific and sex-linked expression of hepatitis B surface antigen (HBsAg) gene in transgenic mice by monitoring the level of liver HBsAg mRNA and serum HBsAg at different stages of development and in response to sex-hormone regulation. Transcription of the HBsAg gene starts at day 15 of development, together with that of the albumin gene, and reaches a comparable level at birth. HBsAg mRNA level and HBsAg production are parallel in males and females during prenatal development and until the first month of life, but HBsAg gene expression increases 5-10 times in males at puberty. After castration, the level of expression decreases dramatically in both males and females and is subsequently increased by injection of testosterone or estradiol. Glucocorticoids also regulated positively expression of the HBsAg gene. Our results suggest that sex hormones play a role in hepatitis B virus gene expression during natural infection and could explain the difference in incidence of chronic carriers between men and women. Images PMID:3469661

T-cell receptor revision: friend or foe?

PubMed Central

Hale, J Scott; Fink, Pamela J

2010-01-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue. PMID:20201984

Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical, Hematompoietic Progenitor-Cell Transplant

DTIC Science & Technology

2008-05-01

adoptive therapy using CD19- specific chimeric antigen receptor re-directed T cells for recurrent/refractory follicular lymphoma. Mol Ther...T- cell therapies for B- cell malignancies we have developed a chimeric antigen receptor (CAR) which when expressed on the cell surface redirects T...that both CD4+ and CD8+ T cells expressing CD19-specific chimeric antigen receptor (CAR) can be generated usmg a novel non-viral gene

Focusing homologous recombination: pilin antigenic variation in the pathogenic Neisseria

PubMed Central

Cahoon, Laty A.; Seifert, H. Steven

2011-01-01

Summary Some pathogenic microbes utilize homologous recombination to generate antigenic variability in targets of immune surveillance. These specialized systems rely on the cellular recombination machinery to catalyze dedicated, high-frequency reactions that provide extensive diversity in the genes encoding surface antigens. A description of the specific mechanisms that allow unusually high rates of recombination without deleterious effects on the genome in the well characterized pilin antigenic variation systems of Neisseria gonorrhoeae and Neisseria meningitidis is presented. We will also draw parallels to selected bacterial and eukaryotic antigenic variation systems, and suggest the most pressing unanswered questions related to understanding these important processes. PMID:21812841

SnSAG5 is an alternative surface antigen of Sarcocystis neurona strains that is mutually exclusive to SnSAG1.

PubMed

Crowdus, Carolyn A; Marsh, Antoinette E; Saville, Willliam J; Lindsay, David S; Dubey, J P; Granstrom, David E; Howe, Daniel K

2008-11-25

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). Previous work has identified a gene family of paralogous surface antigens in S. neurona called SnSAGs. These surface proteins are immunogenic in their host animals, and are therefore candidate molecules for development of diagnostics and vaccines. However, SnSAG diversity exists in strains of S. neurona, including the absence of the major surface antigen gene SnSAG1. Instead, sequence for an alternative SnSAG has been revealed in two of the SnSAG1-deficient strains. Herein, we present data characterizing this new surface protein, which we have designated SnSAG5. The results indicated that the protein encoded by the SnSAG5 sequence is indeed a surface-associated molecule that has characteristics consistent with the other SAGs identified in S. neurona and related parasites. Importantly, Western blot analyses of a collection of S. neurona strains demonstrated that 6 of 13 parasite isolates express SnSAG5 as a dominant surface protein instead of SnSAG1. Conversely, SnSAG5 was not detected in SnSAG1-positive strains. One strain, which was isolated from the brain of a sea otter, did not express either SnSAG1 or SnSAG5. Genetic analysis with SnSAG5-specific primers confirmed the presence of the SnSAG5 gene in Western blot-positive strains, while also suggesting the presence of a novel SnSAG sequence in the SnSAG1-deficient, SnSAG5-deficient otter isolate. The findings provide further indication of S. neurona strain diversity, which has implications for diagnostic testing and development of vaccines against EPM as well as the population biology of Sarcocystis cycling in the opossum definitive host.

Inactivation of the alpha C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus.

PubMed

Li, J; Kasper, D L; Ausubel, F M; Rosner, B; Michel, J L

1997-11-25

The alpha C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The alpha C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the alpha C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the alpha C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the alpha antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of alpha-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, alpha antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of alpha-specific antibodies. In addition, antibodies to the alpha C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the alpha antigen as expressed from the bca gene. Therefore, the alpha C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

PubMed

Roszik, János; Sebestyén, Zsolt; Govers, Coen; Guri, Yakir; Szöor, Arpád; Pályi-Krekk, Zsuzsanna; Vereb, György; Nagy, Peter; Szöllosi, János; Debets, Reno

2011-05-01

T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

PubMed Central

Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

2014-01-01

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

Mouse HLA-DPA homologue H2-Pa: A pseudogene that maps between H2-Pb and H2-Oa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arimura, Y.; Koda, T.; Kishi, M.

1996-12-31

The major histocompatibility complex (MHC) class II subregion contains several subclasses of genes. The classical class II genes, HLA-DP, DQ, and DR homologues, present antigens directly to CD4{sup +} T cells. HLA-DM homologues facilitate the efficacy and transport of antigens to the cell surface by removing the CLIP peptides from the classical class II molecules. HLA-DNA/DOB homologues show unusual expression patterns and limited polymorphism, but their function is yet to be elucidated. 15 refs., 2 figs.

Antigen Loss Variants: Catching Hold of Escaping Foes.

PubMed

Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

2017-01-01

Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse. CD19 and CD20 antigen loss in acute lymphocytic leukemia and chronic lymphocytic leukemia, respectively, and lineage switching in leukemia associated with mixed lineage leukemia (MLL) gene rearrangements are well-documented evidences in this regard. Although increasing number of novel immunotherapies are being developed, majority of these do not address the control of antigen loss variants. Here, we review the occurrence of antigen loss variants in leukemia and discuss the therapeutic strategies to tackle the same. We also present an approach of dual-targeting immunoligand effectively retargeting NK cells against antigen loss variants in MLL-associated leukemia. Novel immunotherapies simultaneously targeting more than one tumor antigen certainly hold promise to completely eradicate tumor and prevent therapy-resistant relapses.

Cloning and analysis of the gene for a major surface antigen of Mycoplasma gallisepticum.

PubMed

Spencer, Denise L; Kurth, Kathy Toohey; Menon, Sreekumar A; VanDyk, Tina; Minion, F Chris

2002-01-01

Myplasma gallisepticum infects a wide variety of gallineaceous birds including chickens, turkeys, and pheasants. Infection occurs both horizontally and vertically. Thus, control of the spread of M. gallisepticum to noninfected flocks is difficult. Continual monitoring is necessary to identify infected flocks even under the most stringent infectious control practices. Monitoring, however, is usually performed by measuring hemagglutination activity (HA) in serum, an insensitive and variable test. Variability in the HA test arises differences in agglutination antigen, changes in antigenic profiles of the M. gallisepticum strain, and variability in reading the agglutination reaction. Enzyme-linked immunosorbent assays (ELISAs) are the preferred method of testing because of the ease in obtaining sera and the sensitivity and reproducibility of the assays, but the ELISA suffers from a lack of standardization in the test antigen. The ELISA test will be more easily accepted once the test antigen has been standardized. To this end, we have identified, cloned, and characterized the gene for an antigen that has potential as a species-specific antigen for M. gallisepticum The gene codes for a 75-kD protein, P75, that is recognized during natural infections. Recombinant P75 is not recognized in immunoblots by convalescent sera produced in chickens infected with Mycoplasma synoviae, Mycoplasma gallinarum, and Mycoplasma gallinaceum or in turkeys infected with Mycoplasma meleagridis.

Antigen I/II encoded by integrative and conjugative elements of Streptococcus agalactiae and role in biofilm formation.

PubMed

Chuzeville, Sarah; Dramsi, Shaynoor; Madec, Jean-Yves; Haenni, Marisa; Payot, Sophie

2015-11-01

Streptococcus agalactiae (i.e. Group B streptococcus, GBS) is a major human and animal pathogen. Genes encoding putative surface proteins and in particular an antigen I/II have been identified on Integrative and Conjugative Elements (ICEs) found in GBS. Antigens I/II are multimodal adhesins promoting colonization of the oral cavity by streptococci such as Streptococcus gordonii and Streptococcus mutans. The prevalence and diversity of antigens I/II in GBS were studied by a bioinformatic analysis. It revealed that antigens I/II, which are acquired by horizontal transfer via ICEs, exhibit diversity and are widespread in GBS, in particular in the serotype Ia/ST23 invasive strains. This study aimed at characterizing the impact on GBS biology of proteins encoded by a previously characterized ICE of S. agalactiae (ICE_515_tRNA(Lys)). The production and surface exposition of the antigen I/II encoded by this ICE was examined using RT-PCR and immunoblotting experiments. Surface proteins of ICE_515_tRNA(Lys) were found to contribute to GBS biofilm formation and to fibrinogen binding. Contribution of antigen I/II encoded by SAL_2056 to biofilm formation was also demonstrated. These results highlight the potential for ICEs to spread microbial adhesins between species. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Impact of Mutation and Gene Conversion on the Local Diversification of Antigen Genes in African Trypanosomes

PubMed Central

Gjini, Erida; Haydon, Daniel T.; Barry, J. David; Cobbold, Christina A.

2012-01-01

Patterns of genetic diversity in parasite antigen gene families hold important information about their potential to generate antigenic variation within and between hosts. The evolution of such gene families is typically driven by gene duplication, followed by point mutation and gene conversion. There is great interest in estimating the rates of these processes from molecular sequences for understanding the evolution of the pathogen and its significance for infection processes. In this study, a series of models are constructed to investigate hypotheses about the nucleotide diversity patterns between closely related gene sequences from the antigen gene archive of the African trypanosome, the protozoan parasite causative of human sleeping sickness in Equatorial Africa. We use a hidden Markov model approach to identify two scales of diversification: clustering of sequence mismatches, a putative indicator of gene conversion events with other lower-identity donor genes in the archive, and at a sparser scale, isolated mismatches, likely arising from independent point mutations. In addition to quantifying the respective probabilities of occurrence of these two processes, our approach yields estimates for the gene conversion tract length distribution and the average diversity contributed locally by conversion events. Model fitting is conducted using a Bayesian framework. We find that diversifying gene conversion events with lower-identity partners occur at least five times less frequently than point mutations on variant surface glycoprotein (VSG) pairs, and the average imported conversion tract is between 14 and 25 nucleotides long. However, because of the high diversity introduced by gene conversion, the two processes have almost equal impact on the per-nucleotide rate of sequence diversification between VSG subfamily members. We are able to disentangle the most likely locations of point mutations and conversions on each aligned gene pair. PMID:22735079

Identification of Streptococcus mitis321A vaccine antigens based on reverse vaccinology

PubMed Central

Zhang, Qiao; Lin, Kexiong; Wang, Changzheng; Xu, Zhi; Yang, Li; Ma, Qianli

2018-01-01

Streptococcus mitis (S. mitis) may transform into highly pathogenic bacteria. The aim of the present study was to identify potential antigen targets for designing an effective vaccine against the pathogenic S. mitis321A. The genome of S. mitis321A was sequenced using an Illumina Hiseq2000 instrument. Subsequently, Glimmer 3.02 and Tandem Repeat Finder (TRF) 4.04 were used to predict genes and tandem repeats, respectively, with DNA sequence function analysis using the Basic Local Alignment Search Tool (BLAST) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Groups of proteins (COG) databases. Putative gene antigen candidates were screened with BLAST ahead of phylogenetic tree analysis. The DNA sequence assembly size was 2,110,680 bp with 40.12% GC, 6 scaffolds and 9 contig. Consequently, 1,944 genes were predicted, and 119 TRF, 56 microsatellite DNA, 10 minisatellite DNA and 154 transposons were acquired. The predicted genes were associated with various pathways and functions concerning membrane transport and energy metabolism. Multiple putative genes encoding surface proteins, secreted proteins and virulence factors, as well as essential genes were determined. The majority of essential genes belonged to a phylogenetic lineage, while 321AGL000129 and 321AGL000299 were on the same branch. The current study provided useful information regarding the biological function of the S. mitis321A genome and recommends putative antigen candidates for developing a potent vaccine against S. mitis. PMID:29620181

ISOANTIGENS OF THE H-2 AND Tla LOCI OF THE MOUSE

PubMed Central

Boyse, Edward A.; Stockert, Elisabeth; Old, Lloyd J.

1968-01-01

H-2 and TL isoantigens of the mouse are specified by the closely linked genetic loci H-2 and Tla. A. study of their representation on thymocytes was performed in order to reveal any interactions between the determinant genes or their products affecting the synthesis or disposition of these components of the thymocyte surface. The method employed was quantitative absorption of cytotoxic antibody by viable thymocytes. The phenotypic expression of TL antigens was found to reduce the demonstrable amount of certain H-2 antigens to as little as 34% of the quantity demonstrable on TL- thymocytes. A reduction was observed in all three H-2 types tested, (H-2b, H-2a, and H-2k). As antigenic modulation (change of TL phenotype from TL+ to TL-, produced by TL antibody) is known to entail a compensatory increase in H-2(D) antigen, it is concluded that the TL phenotype, rather than the Tla genotype, influences the surface representation of H-2 antigens. The two known TL+ phenotypes of thymocytes (TL.2 and TL.1,2,3) depress H-2 equally. The H-2 specificities affected are those determined by the D end of the E-2 locus, which is adjacent to Tla; antigens of the K end, which is distal to Tla, are not depressed. The reduction of demonstrable H-2 antigen on the thymocytes of TL+ x TL- progeny is half that of thymocytes of TL+ x TL+ progeny and the reduction affects equally the products of both H-2 alleles (cis and trans in relation to Tla), indicating that the mechanism of H-2 reduction by TL is extrachromosomal. Whether it involves diminished synthesis of H-2 or steric masking by TL at the cell membrane is unknown, but in either case the reciprocal relation of TL and H-2(D) antigens implies that they probably occupy adjacent positions on thymocytes and that the gene order, H-2(K): H-2(D):Tla is reflected in cell surface structure. Extrachromosomal interaction, apparently involving control of synthesis, occurs also within the TL system of antigens. Thymocytes of TL.2 x TL.1,2,3 progeny express the full homozygous quantity of antigens TL.1 and TL.3 (but not of TL.2), in contrast to the half-quantity present in thymocytes of TL- x TL.1,2,3 progeny. Another example of interaction is implicit in the finding that thymocytes of TL-1,2,3 x TL.1,2,3 progeny have more TL.2 antigen than thymocytes of TL.2 x TL.2 progeny, but in this instance there is nothing to indicate whether the mechanism is chromosomal or extrachromosomal. Thus the quantitative surface representation of at least some H-2 and TL antigens is influenced by the cellular complement of H-2:Tla genes as a whole. Comparison of H-2 heterozygous thymocytes with H-2 homozygous thymocytes in quantitative absorption tests shows (a) more than the expected 50% of each parental-type H-2 antigen on heterozygous cells, and (b) a greater suppression of H-2 by TL in H-2 heterozygotes in comparison with H-2 homozygotes. Both results may be explained on the basis of differences in the density of H-2 antigenic sites and consequent differences in the efficiency of absorption of H-2 antibody. These considerations may be useful in other contexts, e.g. in estimating the representation of Rh antigens on the red cells of human subjects homozygous and heterozygous for Rh components. PMID:5662018

HD-03/ES: A Herbal Medicine Inhibits Hepatitis B Surface Antigen Secretion in Transfected Human Hepatocarcinoma PLC/PRF/5 Cells.

PubMed

Varma, Sandeep R; Sundaram, R; Gopumadhavan, S; Vidyashankar, Satyakumar; Patki, Pralhad S

2013-01-01

HD-03/ES is a herbal formulation used for the treatment of hepatitis B. However, the molecular mechanism involved in the antihepatitis B (HBV) activity of this drug has not been studied using in vitro models. The effect of HD-03/ES on hepatitis B surface antigen (HBsAg) secretion and its gene expression was studied in transfected human hepatocarcinoma PLC/PRF/5 cells. The anti-HBV activity was tested based on the inhibition of HBsAg secretion into the culture media, as detected by HBsAg-specific antibody-mediated enzyme assay (ELISA) at concentrations ranging from 125 to 1000  μ g/mL. The effect of HD-03/ES on HBsAg gene expression was analyzed using semiquantitative multiplex RT-PCR by employing specific primers. The results showed that HD-03/ES suppressed HBsAg production with an IC50 of 380  μ g/mL in PLC/PRF/5 cells for a period of 24 h. HD-03/ES downregulated HBsAg gene expression in PLC/PRF/5 cells. In conclusion, HD-03/ES exhibits strong anti-HBV properties by inhibiting the secretion of hepatitis B surface antigen in PLC/PRF/5 cells, and this action is targeted at the transcription level. Thus, HD-03/ES could be beneficial in the treatment of acute and chronic hepatitis B infections.

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells

PubMed Central

Gornalusse, Germán G.; Hirata, Roli K.; Funk, Sarah; Riolobos, Laura; Lopes, Vanda S.; Manske, Gabriel; Prunkard, Donna; Colunga, Aric G.; Hanafi, Laïla-Aïcha; Clegg, Dennis O.; Turtle, Cameron; Russell, David W.

2017-01-01

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this ‘missing self’ response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies, and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression. PMID:28504668

HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.

PubMed

Gornalusse, Germán G; Hirata, Roli K; Funk, Sarah E; Riolobos, Laura; Lopes, Vanda S; Manske, Gabriel; Prunkard, Donna; Colunga, Aric G; Hanafi, Laïla-Aïcha; Clegg, Dennis O; Turtle, Cameron; Russell, David W

2017-08-01

Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8 + T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.

Genomic analysis of Bacteroides fragilis reveals extensive DNA inversions regulating cell surface adaptation

PubMed Central

Kuwahara, Tomomi; Yamashita, Atsushi; Hirakawa, Hideki; Nakayama, Haruyuki; Toh, Hidehiro; Okada, Natsumi; Kuhara, Satoru; Hattori, Masahira; Hayashi, Tetsuya; Ohnishi, Yoshinari

2004-01-01

Bacteroides are predominant human colonic commensals, but the principal pathogenic species, Bacteroides fragilis (BF), lives closely associated with the mucosal surface, whereas a second major species, Bacteroides thetaiotaomicron (BT), concentrates within the colon. We find corresponding differences in their genomes, based on determination of the genome sequence of BF and comparative analysis with BT. Both species have acquired two mechanisms that contribute to their dominance among the colonic microbiota: an exceptional capability to use a wide range of dietary polysaccharides by gene amplification and the capacity to create variable surface antigenicities by multiple DNA inversion systems. However, the gene amplification for polysaccharide assimilation is more developed in BT, in keeping with its internal localization. In contrast, external antigenic structures can be changed more systematically in BF. Thereby, at the mucosal surface, where microbes encounter continuous attack by host defenses, BF evasion of the immune system is favored, and its colonization and infectious potential are increased. PMID:15466707

A review of mechanistic insight and application of pH-sensitive liposomes in drug delivery.

PubMed

Paliwal, Shivani Rai; Paliwal, Rishi; Vyas, Suresh P

2015-05-01

The pH-sensitive liposomes have been extensively used as an alternative to conventional liposomes in effective intracellular delivery of therapeutics/antigen/DNA/diagnostics to various compartments of the target cell. Such liposomes are destabilized under acidic conditions of the endocytotic pathway as they usually contain pH-sensitive lipid components. Therefore, the encapsulated content is delivered into the intracellular bio-environment through destabilization or its fusion with the endosomal membrane. The therapeutic efficacy of pH-sensitive liposomes enables them as biomaterial with commercial utility especially in cancer treatment. In addition, targeting ligands including antibodies can be anchored on the surface of pH-sensitive liposomes to target specific cell surface receptors/antigen present on tumor cells. These vesicles have also been widely explored for antigen delivery and serve as immunological adjuvant to enhance the immune response to antigens. The present review deals with recent research updates on application of pH-sensitive liposomes in chemotherapy/diagnostics/antigen/gene delivery etc.

High Cell Surface Expression of CD4 Allows Distinction of CD4+CD25+ Antigen-specific Effector T Cells from CD4+CD25+ Regulatory T Cells in Murine Experimental Autoimmune Encephalomyelitis

PubMed Central

Li, Jinzhu; Ridgway, William; Fathman, C. Garrison; Tse, Harley Y.; Shaw, Michael K.

2008-01-01

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up regulate cell surface expression of CD4. The CD4high sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25+ sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses. PMID:17920698

Ectopic expression of blood type antigens in inflamed mucosa with higher incidence of FUT2 secretor status in colonic Crohn's disease.

PubMed

Miyoshi, Jun; Yajima, Tomoharu; Okamoto, Susumu; Matsuoka, Katsuyoshi; Inoue, Nagamu; Hisamatsu, Tadakazu; Shimamura, Katsuyoshi; Nakazawa, Atsushi; Kanai, Takanori; Ogata, Haruhiko; Iwao, Yasushi; Mukai, Makio; Hibi, Toshifumi

2011-09-01

Host-intestinal microbial interaction plays an important role in the pathogenesis of inflammatory bowel diseases (IBDs). The surface molecules of the intestinal epithelium act as receptors for bacterial adhesion and regulate the intestinal bacteria. Some known receptors are the mucosal blood type antigens, which are regulated by the fucosyltransferase2 (FUT2) gene, and individuals who express these antigens in the gastrointestinal tract are called secretors. Recent research has revealed that the FUT2 gene is associated with Crohn's disease (CD) in western populations. To clarify the contribution of mucosal blood type antigens in IBD, we determined the incidence of five previously reported single-nucleotide polymorphisms of the FUT2 gene in Japanese patients. We also used immunohistochemistry to investigate the antigen expression in mucosal specimens from IBD patients and animal models. Genetic analysis revealed that all of the patients with colonic CD were secretors, whereas the incidence of secretors was 80, 80, 67, and 80%, respectively, for the control, ileocolonic CD, ileal CD, and ulcerative colitis groups (P = 0.036). Abnormal expression of blood type antigens was observed only in colonic CD. Interleukin-10⁻/⁻ mice, but not dextran sulfate sodium colitis mice, had enhanced colonic expression of blood type antigens, and the expression of these antigens preceded the development of colitis in the interleukin-10⁻/⁻ mice. FUT2 secretor status was associated with colonic-type CD. This finding, taken together with the immunohistochemistry data, suggests that the abnormal expression of blood type antigens in the colon may be a unique and essential factor for colonic CD.

Overview of expression of hepatitis B surface antigen in transgenic plants.

PubMed

Guan, Zheng-jun; Guo, Bin; Huo, Yan-lin; Guan, Zheng-ping; Wei, Ya-hui

2010-10-28

Hepatitis B virus (HBV), a pathogen for chronic liver infection, afflicts more than 350 million people world-wide. The effective way to control the virus is to take HBV vaccine. Hepatitis B surface antigen (HBsAg) is an effective protective antigen suitable for vaccine development. At present, "edible" vaccine based on transgenic plants is one of the most promising directions in novel types of vaccines. HBsAg production from transgenic plants has been carried out, and the transgenic plant expression systems have developed from model plants (such as tobacco, potato and tomato) to other various plant platforms. Crude or purified extracts of transformed plants have been found to conduct immunological responses and clinical trials for hepatitis B, which gave the researches of plant-based HBsAg production a big boost. The aim of this review was to summarize the recent data about plant-based HBsAg development including molecular biology of HBsAg gene, selection of expression vector, the expression of HBsAg gene in plants, as well as corresponding immunological responses in animal models or human. Copyright © 2010 Elsevier Ltd. All rights reserved.

Expression of hepatitis B surface antigen in transgenic plants.

PubMed Central

Mason, H S; Lam, D M; Arntzen, C J

1992-01-01

Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a nominally constitutive promoter. Enzyme-linked immunoassays using a monoclonal antibody directed against human serum-derived HBsAg revealed the presence of HBsAg in extracts of transformed leaves at levels that correlated with mRNA abundance. This suggests that there were no major inherent limitations of transcription or translation of this foreign gene in plants. Recombinant HBsAg was purified from transgenic plants by immunoaffinity chromatography and examined by electron microscopy. Spherical particles with an average diameter of 22 nm were observed in negatively stained preparations. Sedimentation of transgenic plant extracts in sucrose and cesium chloride density gradients showed that the recombinant HBsAg and human serum-derived HBsAg had similar physical properties. Because the HBsAg produced in transgenic plants is antigenically and physically similar to the HBsAg particles derived from human serum and recombinant yeast, which are used as vaccines, we conclude that transgenic plants hold promise as low-cost vaccine production systems. Images PMID:1465391

Molecular cloning and characterization of the spaB gene of Streptococcus sobrinus.

PubMed

Holt, R G; Perry, S E

1990-07-01

A gene of Streptococcus sobrinus 6715 (serotype g) designated spaB and encoding a surface protein antigen was isolated from a cosmid gene bank. A 5.4 kb HindIII/AvaI DNA fragment containing the gene was inserted into plasmid pBR322 to yield plasmid pXI404. Analysis of plasmid-encoded gene products showed that the 5.4 kb fragment of pXI404 encoded a 195 kDa protein. Southern blot experiments revealed that the 5.4 kb chromosomal insert DNA had sequence similarity with genomic DNA of S. sobrinus 6715, S. sobrinus B13 (serotype d) and Streptococcus cricetus HS6 (serotype a). The recombinant SpaB protein (rSpaB) was purified and monospecific antiserum was prepared. With immunological techniques and the anti-rSpaB serum, we have shown: (1) that the rSpaB protein has physico-chemical and antigenic identity with the S. sobrinus SpaB protein, (2) the presence of cross-reactive proteins in the extracellular protein of serotypes a and d of the mutans group of streptococci and (3) that the SpaB protein is expressed on the surface of mutans streptococcal serotypes a, d and g.

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

PubMed

Bartl, S; Weissman, I L

1994-01-04

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-11-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.

Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus.

PubMed Central

Knowles, D P; Cheevers, W P; McGuire, T C; Brassfield, A L; Harwood, W G; Stem, T A

1991-01-01

To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM. Images PMID:1656067

Genetic transformation of tobacco NT1 cells with Agrobacterium tumefaciens.

PubMed

Mayo, Kristin J; Gonzales, Barbara J; Mason, Hugh S

2006-01-01

This protocol is used to produce stably transformed tobacco (Nicotiana tabacum) NT1 cell lines, using Agrobacterium tumefaciens-mediated DNA delivery of a binary vector containing a gene encoding hepatitis B surface antigen and a gene encoding the kanamycin selection marker. The NT1 cultures, at the appropriate stage of growth, are inoculated with A. tumefaciens containing the binary vector. A 3-day cocultivation period follows, after which the cultures are rinsed and placed on solid selective medium. Transformed colonies ('calli') appear in approximately 4 weeks; they are subcultured until adequate material is obtained for analysis of antigen production. 'Elite' lines are selected based on antigen expression and growth characteristics. The time required for the procedure from preparation of the plant cell materials to callus development is approximately 5 weeks. Growth of selected calli to sufficient quantities for antigen screening may require 4-6 weeks beyond the initial selection. Creation of the plasmid constructs, transformation of the A. tumefaciens line, and ELISA and Bradford assays to assess protein production require additional time.

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II

PubMed Central

Quinn, Laura L.; Williams, Luke R.; White, Claire; Forrest, Calum; Rowe, Martin

2015-01-01

ABSTRACT The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8+ cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8+ cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8+ cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4+ cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8+ and CD4+ T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. IMPORTANCE Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8+ T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8+ T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8+ T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4+ T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. PMID:26468525

The Missing Link in Epstein-Barr Virus Immune Evasion: the BDLF3 Gene Induces Ubiquitination and Downregulation of Major Histocompatibility Complex Class I (MHC-I) and MHC-II.

PubMed

Quinn, Laura L; Williams, Luke R; White, Claire; Forrest, Calum; Zuo, Jianmin; Rowe, Martin

2016-01-01

The ability of Epstein-Barr virus (EBV) to spread and persist in human populations relies on a balance between host immune responses and EBV immune evasion. CD8(+) cells specific for EBV late lytic cycle antigens show poor recognition of target cells compared to immediate early and early antigen-specific CD8(+) cells. This phenomenon is due in part to the early EBV protein BILF1, whose immunosuppressive activity increases with lytic cycle progression. However, published data suggest the existence of a hitherto unidentified immune evasion protein further enhancing protection against late EBV antigen-specific CD8(+) cells. We have now identified the late lytic BDLF3 gene as the missing link accounting for efficient evasion during the late lytic cycle. Interestingly, BDLF3 also contributes to evasion of CD4(+) cell responses to EBV. We report that BDLF3 downregulates expression of surface major histocompatibility complex (MHC) class I and class II molecules in the absence of any effect upon other surface molecules screened, including CD54 (ICAM-1) and CD71 (transferrin receptor). BDLF3 both enhanced internalization of surface MHC molecules and reduced the rate of their appearance at the cell surface. The reduced expression of surface MHC molecules correlated with functional protection against CD8(+) and CD4(+) T cell recognition. The molecular mechanism was identified as BDLF3-induced ubiquitination of MHC molecules and their subsequent downregulation in a proteasome-dependent manner. Immune evasion is a necessary feature of viruses that establish lifelong persistent infections in the face of strong immune responses. EBV is an important human pathogen whose immune evasion mechanisms are only partly understood. Of the EBV immune evasion mechanisms identified to date, none could explain why CD8(+) T cell responses to late lytic cycle genes are so infrequent and, when present, recognize lytically infected target cells so poorly relative to CD8(+) T cells specific for early lytic cycle antigens. The present work identifies an additional immune evasion protein, BDLF3, that is expressed late in the lytic cycle and impairs CD8(+) T cell recognition by targeting cell surface MHC class I molecules for ubiquitination and proteasome-dependent downregulation. Interestingly, BDLF3 also targets MHC class II molecules to impair CD4(+) T cell recognition. BDLF3 is therefore a rare example of a viral protein that impairs both the MHC class I and class II antigen-presenting pathways. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides

PubMed Central

Santona, Antonella; Carta, Franco; Fraghí, Peppinetta; Turrini, Franco

2002-01-01

As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection. PMID:11748179

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires.

PubMed

DeKosky, Brandon J; Lungu, Oana I; Park, Daechan; Johnson, Erik L; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D; Ippolito, Gregory C; Gray, Jeffrey J; Georgiou, George

2016-05-10

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

Gene constellation of influenza A virus reassortants with high growth phenotype prepared as seed candidates for vaccine production.

PubMed

Fulvini, Andrew A; Ramanunninair, Manojkumar; Le, Jianhua; Pokorny, Barbara A; Arroyo, Jennifer Minieri; Silverman, Jeanmarie; Devis, Rene; Bucher, Doris

2011-01-01

Influenza A virus vaccines undergo yearly reformulations due to the antigenic variability of the virus caused by antigenic drift and shift. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. Inactivated influenza A seasonal vaccines are generated by classical reassortment. The classical method takes advantage of the ability of the influenza virus to reassort based on the segmented nature of its genome. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo. The genetic analysis was conducted using reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). The characterization was conducted to determine the parental origin of the gene segments (hy donor virus or wt virus), gene segment ratios and constellations. Fold increase in growth of reassortant viruses compared to respective parent wt viruses was determined by hemagglutination assay titers. In this study fifty-seven influenza A vaccine candidate reassortants were analyzed for the presence or absence of correlations between specific gene segment ratios, gene constellations and hy reassortant phenotype. We found two gene ratios, 6:2 and 5:3, to be the most prevalent among the hy reassortants analyzed, although other gene ratios also conferred hy in certain reassortants.

Gene Constellation of Influenza A Virus Reassortants with High Growth Phenotype Prepared as Seed Candidates for Vaccine Production

PubMed Central

Fulvini, Andrew A.; Ramanunninair, Manojkumar; Le, Jianhua; Pokorny, Barbara A.; Arroyo, Jennifer Minieri; Silverman, Jeanmarie; Devis, Rene; Bucher, Doris

2011-01-01

Background Influenza A virus vaccines undergo yearly reformulations due to the antigenic variability of the virus caused by antigenic drift and shift. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. Inactivated influenza A seasonal vaccines are generated by classical reassortment. The classical method takes advantage of the ability of the influenza virus to reassort based on the segmented nature of its genome. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo. Methodology The genetic analysis was conducted using reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). The characterization was conducted to determine the parental origin of the gene segments (hy donor virus or wt virus), gene segment ratios and constellations. Fold increase in growth of reassortant viruses compared to respective parent wt viruses was determined by hemagglutination assay titers. Significance In this study fifty-seven influenza A vaccine candidate reassortants were analyzed for the presence or absence of correlations between specific gene segment ratios, gene constellations and hy reassortant phenotype. We found two gene ratios, 6∶2 and 5∶3, to be the most prevalent among the hy reassortants analyzed, although other gene ratios also conferred hy in certain reassortants. PMID:21695145

Sarcocystis neurona Merozoites Express a Family of Immunogenic Surface Antigens That Are Orthologues of the Toxoplasma gondii Surface Antigens (SAGs) and SAG-Related Sequences†

PubMed Central

Howe, Daniel K.; Gaji, Rajshekhar Y.; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-01-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s). PMID:15664946

Sarcocystis neurona merozoites express a family of immunogenic surface antigens that are orthologues of the Toxoplasma gondii surface antigens (SAGs) and SAG-related sequences.

PubMed

Howe, Daniel K; Gaji, Rajshekhar Y; Mroz-Barrett, Meaghan; Gubbels, Marc-Jan; Striepen, Boris; Stamper, Shelby

2005-02-01

Sarcocystis neurona is a member of the Apicomplexa that causes myelitis and encephalitis in horses but normally cycles between the opossum and small mammals. Analysis of an S. neurona expressed sequence tag (EST) database revealed four paralogous proteins that exhibit clear homology to the family of surface antigens (SAGs) and SAG-related sequences of Toxoplasma gondii. The primary peptide sequences of the S. neurona proteins are consistent with the two-domain structure that has been described for the T. gondii SAGs, and each was predicted to have an amino-terminal signal peptide and a carboxyl-terminal glycolipid anchor addition site, suggesting surface localization. All four proteins were confirmed to be membrane associated and displayed on the surface of S. neurona merozoites. Due to their surface localization and homology to T. gondii surface antigens, these S. neurona proteins were designated SnSAG1, SnSAG2, SnSAG3, and SnSAG4. Consistent with their homology, the SnSAGs elicited a robust immune response in infected and immunized animals, and their conserved structure further suggests that the SnSAGs similarly serve as adhesins for attachment to host cells. Whether the S. neurona SAG family is as extensive as the T. gondii SAG family remains unresolved, but it is probable that additional SnSAGs will be revealed as more S. neurona ESTs are generated. The existence of an SnSAG family in S. neurona indicates that expression of multiple related surface antigens is not unique to the ubiquitous organism T. gondii. Instead, the SAG gene family is a common trait that presumably has an essential, conserved function(s).

Isoantigens of the H-2 and Tla loci of the mouse: interactions affecting their representation on thymocytes.

PubMed

Boyse, E A; Stockert, E; Old, L J

1968-07-01

H-2 and TL isoantigens of the mouse are specified by the closely linked genetic loci H-2 and Tla. A. study of their representation on thymocytes was performed in order to reveal any interactions between the determinant genes or their products affecting the synthesis or disposition of these components of the thymocyte surface. The method employed was quantitative absorption of cytotoxic antibody by viable thymocytes. The phenotypic expression of TL antigens was found to reduce the demonstrable amount of certain H-2 antigens to as little as 34% of the quantity demonstrable on TL- thymocytes. A reduction was observed in all three H-2 types tested, (H-2(b), H-2(a), and H-2(k)). As antigenic modulation (change of TL phenotype from TL+ to TL-, produced by TL antibody) is known to entail a compensatory increase in H-2(D) antigen, it is concluded that the TL phenotype, rather than the Tla genotype, influences the surface representation of H-2 antigens. The two known TL+ phenotypes of thymocytes (TL.2 and TL.1,2,3) depress H-2 equally. The H-2 specificities affected are those determined by the D end of the E-2 locus, which is adjacent to Tla; antigens of the K end, which is distal to Tla, are not depressed. The reduction of demonstrable H-2 antigen on the thymocytes of TL+ x TL- progeny is half that of thymocytes of TL+ x TL+ progeny and the reduction affects equally the products of both H-2 alleles (cis and trans in relation to Tla), indicating that the mechanism of H-2 reduction by TL is extrachromosomal. Whether it involves diminished synthesis of H-2 or steric masking by TL at the cell membrane is unknown, but in either case the reciprocal relation of TL and H-2(D) antigens implies that they probably occupy adjacent positions on thymocytes and that the gene order, H-2(K): H-2(D):Tla is reflected in cell surface structure. Extrachromosomal interaction, apparently involving control of synthesis, occurs also within the TL system of antigens. Thymocytes of TL.2 x TL.1,2,3 progeny express the full homozygous quantity of antigens TL.1 and TL.3 (but not of TL.2), in contrast to the half-quantity present in thymocytes of TL- x TL.1,2,3 progeny. Another example of interaction is implicit in the finding that thymocytes of TL-1,2,3 x TL.1,2,3 progeny have more TL.2 antigen than thymocytes of TL.2 x TL.2 progeny, but in this instance there is nothing to indicate whether the mechanism is chromosomal or extrachromosomal. Thus the quantitative surface representation of at least some H-2 and TL antigens is influenced by the cellular complement of H-2:Tla genes as a whole. Comparison of H-2 heterozygous thymocytes with H-2 homozygous thymocytes in quantitative absorption tests shows (a) more than the expected 50% of each parental-type H-2 antigen on heterozygous cells, and (b) a greater suppression of H-2 by TL in H-2 heterozygotes in comparison with H-2 homozygotes. Both results may be explained on the basis of differences in the density of H-2 antigenic sites and consequent differences in the efficiency of absorption of H-2 antibody. These considerations may be useful in other contexts, e.g. in estimating the representation of Rh antigens on the red cells of human subjects homozygous and heterozygous for Rh components.

[Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis].

PubMed

Wang, Jiang; Luo, Dongjiao; Sun, Aihua; Yan, Jie

2008-07-01

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engineering vaccine. In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. We used surface response analysis based on the central composite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture conditions included initial pH, induction start time, post-induction time, Isopropyl beta-D-thiogalactopyranoside (IPTG) concentration, and temperature. The maximal production of antigen protein was 37.78 mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31 degrees C. Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and enabled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.

Variability and repertoire size of T-cell receptor V alpha gene segments.

PubMed

Becker, D M; Pattern, P; Chien, Y; Yokota, T; Eshhar, Z; Giedlin, M; Gascoigne, N R; Goodnow, C; Wolf, R; Arai, K

The immune system of higher organisms is composed largely of two distinct cell types, B lymphocytes and T lymphocytes, each of which is independently capable of recognizing an enormous number of distinct entities through their antigen receptors; surface immunoglobulin in the case of the former, and the T-cell receptor (TCR) in the case of the latter. In both cell types, the genes encoding the antigen receptors consist of multiple gene segments which recombine during maturation to produce many possible peptides. One striking difference between B- and T-cell recognition that has not yet been resolved by the structural data is the fact that T cells generally require a major histocompatibility determinant together with an antigen whereas, in most cases, antibodies recognize antigen alone. Recently, we and others have found that a series of TCR V beta gene sequences show conservation of many of the same residues that are conserved between heavy- and light-chain immunoglobulin V regions, and these V beta sequences are predicted to have an immunoglobulin-like secondary structure. To extend these studies, we have isolated and sequenced eight additional alpha-chain complementary cDNA clones and compared them with published sequences. Analyses of these sequences, reported here, indicate that V alpha regions have many of the characteristics of V beta gene segments but differ in that they almost always occur as cross-hybridizing gene families. We conclude that there may be very different selective pressures operating on V alpha and V beta sequences and that the V alpha repertoire may be considerably larger than that of V beta.

Analysis of the role of tripeptidyl peptidase II in MHC class I antigen presentation in vivo1

PubMed Central

Kawahara, Masahiro; York, Ian A.; Hearn, Arron; Farfan, Diego; Rock, Kenneth L.

2015-01-01

Previous experiments using enzyme inhibitors and RNAi in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I antigen presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared to wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits antigen presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus (LCMV). The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild type and TPPII-deficient mice. These data indicate while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several antigens in vivo. PMID:19841172

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites.

PubMed

Lempereur, Laetitia; Larcombe, Stephen D; Durrani, Zeeshan; Karagenc, Tulin; Bilgic, Huseyin Bilgin; Bakirci, Serkan; Hacilarlioglu, Selin; Kinnaird, Jane; Thompson, Joanne; Weir, William; Shiels, Brian

2017-06-05

Vector-borne apicomplexan parasites are a major cause of mortality and morbidity to humans and livestock globally. The most important disease syndromes caused by these parasites are malaria, babesiosis and theileriosis. Strategies for control often target parasite stages in the mammalian host that cause disease, but this can result in reservoir infections that promote pathogen transmission and generate economic loss. Optimal control strategies should protect against clinical disease, block transmission and be applicable across related genera of parasites. We have used bioinformatics and transcriptomics to screen for transmission-blocking candidate antigens in the tick-borne apicomplexan parasite, Theileria annulata. A number of candidate antigen genes were identified which encoded amino acid domains that are conserved across vector-borne Apicomplexa (Babesia, Plasmodium and Theileria), including the Pfs48/45 6-cys domain and a novel cysteine-rich domain. Expression profiling confirmed that selected candidate genes are expressed by life cycle stages within infected ticks. Additionally, putative B cell epitopes were identified in the T. annulata gene sequences encoding the 6-cys and cysteine rich domains, in a gene encoding a putative papain-family cysteine peptidase, with similarity to the Plasmodium SERA family, and the gene encoding the T. annulata major merozoite/piroplasm surface antigen, Tams1. Candidate genes were identified that encode proteins with similarity to known transmission blocking candidates in related parasites, while one is a novel candidate conserved across vector-borne apicomplexans and has a potential role in the sexual phase of the life cycle. The results indicate that a 'One Health' approach could be utilised to develop a transmission-blocking strategy effective against vector-borne apicomplexan parasites of animals and humans.

Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells.

PubMed

Ahuja, Jaya; Kampani, Karan; Datta, Suman; Wigdahl, Brian; Flaig, Katherine E; Jain, Pooja

2006-02-01

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

Stable expression of the hepatitis B virus surface antigen containing pre-S2 protein in mouse cells using a bovine papillomavirus vector.

PubMed

Yoneyama, T; Akatsuka, T; Miyamura, T

1988-08-01

The large BglII fragment (2.8 kilobases) of hepatitis B virus DNA including the transcription unit for the hepatitis B surface antigen (HBsAg) was inserted into a bovine papillomavirus vector containing the neomycin resistance gene. The recombinant DNA was transfected into mouse C127 cells. A stable transformed cell line (MS128) secreting a large amount of 22 nm HBsAg particles containing pre-S2 protein was established. The secreted HBsAg particles had the receptor for polymerized human serum albumin. Immunoprecipitation and Western blot analyses showed that HBsAg particles consisted of two major proteins of 22K and 26K encoded by the S gene and a minor protein of 35K encoded by the pre-S2 and S genes. Southern blot analysis revealed that the transfected plasmid was integrated into the host chromosomal DNA and that most of the plasmid sequences were present. These results suggest that the stable expression of the HBsAg in MS128 cells is related to the integrated state of the recombinant DNA.

The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more?

PubMed

Akhter, Yusuf; Ehebauer, Matthias T; Mukhopadhyay, Sangita; Hasnain, Seyed E

2012-01-01

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Stable expression of hepatitis delta virus antigen in a eukaryotic cell line.

PubMed

Macnaughton, T B; Gowans, E J; Reinboth, B; Jilbert, A R; Burrell, C J

1990-06-01

The gene encoding the hepatitis delta virus structural antigen (HDAg) was linked to a neomycin resistance gene in a retrovirus expression vector, and human HepG2 cells were transfected with the recombinant plasmid. A stable cell line was cloned that expressed HDAg in the nuclei of 100% of cells, in a pattern indicating a close relationship with cell nucleoli. Analysis of partially purified recombinant HDAg by HPLC showed an Mr in the range of 7 x 10(5) to 2 x 10(6), which appeared to contain conformation-dependent epitopes, whereas the density of the antigen was 1.19 g/ml by equilibrium centrifugation in caesium chloride, and in rate zonal centrifugation it sedimented with a value of 50S, close to that of particulate hepatitis B virus surface antigen. Immunoblotting demonstrated a single polypeptide with an Mr of 24K which corresponded to the smaller of the two HDAg-specific polypeptides present in infected sera. The recombinant HDAg polypeptide was shown to be a RNA-binding protein with specificity for both genomic and antigenomic species of hepatitis delta virus RNA.

A Host-Pathogen Interaction Reduced to First Principles: Antigenic Variation in T. brucei.

PubMed

Hovel-Miner, Galadriel; Mugnier, Monica; Papavasiliou, F Nina; Pinger, Jason; Schulz, Danae

2015-01-01

Antigenic variation is a common microbial survival strategy, powered by diversity in expressed surface antigens across the pathogen population over the course of infection. Even so, among pathogens, African trypanosomes have the most comprehensive system of antigenic variation described. African trypanosomes (Trypanosoma brucei spp.) are unicellular parasites native to sub-Saharan Africa, and the causative agents of sleeping sickness in humans and of n'agana in livestock. They cycle between two habitats: a specific species of fly (Glossina spp. or, colloquially, the tsetse) and the bloodstream of their mammalian hosts, by assuming a succession of proliferative and quiescent developmental forms, which vary widely in cell architecture and function. Key to each of the developmental forms that arise during these transitions is the composition of the surface coat that covers the plasma membrane. The trypanosome surface coat is extremely dense, covered by millions of repeats of developmentally specified proteins: procyclin gene products cover the organism while it resides in the tsetse and metacyclic gene products cover it while in the fly salivary glands, ready to make the transition to the mammalian bloodstream. But by far the most interesting coat is the Variant Surface Glycoprotein (VSG) coat that covers the organism in its infectious form (during which it must survive free living in the mammalian bloodstream). This coat is highly antigenic and elicits robust VSG-specific antibodies that mediate efficient opsonization and complement mediated lysis of the parasites carrying the coat against which the response was made. Meanwhile, a small proportion of the parasite population switches coats, which stimulates a new antibody response to the prevalent (new) VSG species and this process repeats until immune system failure. The disease is fatal unless treated, and treatment at the later stages is extremely toxic. Because the organism is free living in the blood, the VSG:antibody surface represents the interface between pathogen and host, and defines the interaction of the parasite with the immune response. This interaction (cycles of VSG switching, antibody generation, and parasite deletion) results in stereotypical peaks and troughs of parasitemia that were first recognized more than 100 years ago. Essentially, the mechanism of antigenic variation in T. brucei results from a need, at the population level, to maintain an extensive repertoire, to evade the antibody response. In this chapter, we will examine what is currently known about the VSG repertoire, its depth, and the mechanisms that diversify it both at the molecular (DNA) and at the phenotypic (surface displayed) level, as well as how it could interact with antibodies raised specifically against it in the host.

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host

PubMed Central

Marques, Lucas M.; Rezende, Izadora S.; Barbosa, Maysa S.; Guimarães, Ana M. S.; Martins, Hellen B.; Campos, Guilherme B.; do Nascimento, Naíla C.; dos Santos, Andrea P.; Amorim, Aline T.; Santos, Verena M.; Farias, Sávio T.; Barrence, Fernanda Â. C.; de Souza, Lauro M.; Buzinhani, Melissa; Arana-Chavez, Victor E.; Zenteno, Maria E.; Amarante-Mendes, Gustavo P.; Messick, Joanne B.; Timenetsky, Jorge

2016-01-01

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)—Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis. PMID:27603136

Ureaplasma diversum Genome Provides New Insights about the Interaction of the Surface Molecules of This Bacterium with the Host.

PubMed

Marques, Lucas M; Rezende, Izadora S; Barbosa, Maysa S; Guimarães, Ana M S; Martins, Hellen B; Campos, Guilherme B; do Nascimento, Naíla C; Dos Santos, Andrea P; Amorim, Aline T; Santos, Verena M; Farias, Sávio T; Barrence, Fernanda  C; de Souza, Lauro M; Buzinhani, Melissa; Arana-Chavez, Victor E; Zenteno, Maria E; Amarante-Mendes, Gustavo P; Messick, Joanne B; Timenetsky, Jorge

2016-01-01

Whole genome sequencing and analyses of Ureaplasma diversum ATCC 49782 was undertaken as a step towards understanding U. diversum biology and pathogenicity. The complete genome showed 973,501 bp in a single circular chromosome, with 28.2% of G+C content. A total of 782 coding DNA sequences (CDSs), and 6 rRNA and 32 tRNA genes were predicted and annotated. The metabolic pathways are identical to other human ureaplasmas, including the production of ATP via hydrolysis of the urea. Genes related to pathogenicity, such as urease, phospholipase, hemolysin, and a Mycoplasma Ig binding protein (MIB)-Mycoplasma Ig protease (MIP) system were identified. More interestingly, a large number of genes (n = 40) encoding surface molecules were annotated in the genome (lipoproteins, multiple-banded antigen like protein, membrane nuclease lipoprotein and variable surface antigens lipoprotein). In addition, a gene encoding glycosyltransferase was also found. This enzyme has been associated with the production of capsule in mycoplasmas and ureaplasma. We then sought to detect the presence of a capsule in this organism. A polysaccharide capsule from 11 to 17 nm of U. diversum was observed trough electron microscopy and using specific dyes. This structure contained arabinose, xylose, mannose, galactose and glucose. In order to understand the inflammatory response against these surface molecules, we evaluated the response of murine macrophages J774 against viable and non-viable U. diversum. As with viable bacteria, non-viable bacteria were capable of promoting a significant inflammatory response by activation of Toll like receptor 2 (TLR2), indicating that surface molecules are important for the activation of inflammatory response. Furthermore, a cascade of genes related to the inflammasome pathway of macrophages was also up-regulated during infection with viable organisms when compared to non-infected cells. In conclusion, U. diversum has a typical ureaplasma genome and metabolism, and its surface molecules, including the identified capsular material, represent major components of the organism immunopathogenesis.

Gene Expression and Pathway Analysis of Effects of the CMAH Deactivation on Mouse Lung, Kidney and Heart

PubMed Central

Kwon, Deug-Nam; Chang, Byung-Soo; Kim, Jin-Hoi

2014-01-01

Background N-glycolylneuraminic acid (Neu5Gc) is generated by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH). However, humans lack this common mammalian cell surface molecule, Neu5Gc, due to inactivation of the CMAH gene during evolution. CMAH is one of several human-specific genes whose function has been lost by disruption or deletion of the coding frame. It has been suggested that CMAH inactivation has resulted in biochemical or physiological characteristics that have resulted in human-specific diseases. Methodology/Principal Findings To identify differential gene expression profiles associated with the loss of Neu5Gc expression, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip, using the main tissues (lung, kidney, and heart) from control mice and CMP-Neu5Ac hydroxylase (Cmah) gene knock-out mice, respectively. Out of a total of 25,697 genes, 204, 162, and 147 genes were found to be significantly modulated in the lung, kidney, and heart tissues of the Cmah null mouse, respectively. In this study, we examined the gene expression profiles, using three commercial pathway analysis software packages: Ingenuity Pathways Analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Pathway Studio. The gene ontology analysis revealed that the top 6 biological processes of these genes included protein metabolism and modification, signal transduction, lipid, fatty acid, and steroid metabolism, nucleoside, nucleotide and nucleic acid metabolism, immunity and defense, and carbohydrate metabolism. Gene interaction network analysis showed a common network that was common to the different tissues of the Cmah null mouse. However, the expression of most sialytransferase mRNAs of Hanganutziu-Deicher antigen, sialy-Tn antigen, Forssman antigen, and Tn antigen was significantly down-regulated in the liver tissue of Cmah null mice. Conclusions/Significance Mice bearing a human-like deletion of the Cmah gene serve as an important model for the study of abnormal pathogenesis and/or metabolism caused by the evolutionary loss of Neu5Gc synthesis in humans. PMID:25229777

Peptide/protein vaccine delivery system based on PLGA particles.

PubMed

Allahyari, Mojgan; Mohit, Elham

2016-03-03

Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted.

Peptide/protein vaccine delivery system based on PLGA particles

PubMed Central

Allahyari, Mojgan; Mohit, Elham

2016-01-01

abstract Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted. PMID:26513024

Production of a Recombinant Antibody Specific for i Blood Group Antigen, a Mesenchymal Stem Cell Marker

PubMed Central

Suila, Heli; Tiitinen, Sari; Natunen, Suvi; Laukkanen, Marja-Leena; Kotovuori, Annika; Reinman, Mirka; Satomaa, Tero; Alfthan, Kaija; Laitinen, Saara; Takkinen, Kristiina; Räbinä, Jarkko; Valmu, Leena

2013-01-01

Abstract Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen–positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology. PMID:24083089

Mutations in the S gene region of hepatitis B virus genotype D in Turkish patients.

PubMed

Ozaslan, Mehmet; Ozaslan, Ersan; Barsgan, Arzu; Koruk, Mehmet

2007-12-01

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the 'a'-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M -> T) and 127 (T -> P) were found on the first loop of 'a'-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.

Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli.

PubMed

Iguchi, Atsushi; Shirai, Hiroki; Seto, Kazuko; Ooka, Tadasuke; Ogura, Yoshitoshi; Hayashi, Tetsuya; Osawa, Kayo; Osawa, Ro

2011-01-01

Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci.

Molecular analysis of Toxoplasma gondii Surface Antigen 1 (SAG1) gene cloned from Toxoplasma gondii DNA isolated from Javanese acute toxoplasmosis

NASA Astrophysics Data System (ADS)

Haryati, Sri; Agung Prasetyo, Afiono; Sari, Yulia; Dharmawan, Ruben

2018-05-01

Toxoplasma gondii Surface Antigen 1 (SAG1) is often used as a diagnostic tool due to its immunodominant-specific as antigen. However, data of the Toxoplasma gondii SAG1 protein from Indonesian isolate is limited. To study the protein, genomic DNA was isolated from a Javanese acute toxoplasmosis blood samples patient. A complete coding sequence of Toxoplasma gondii SAG1 was cloned and inserted into an Escherichia coli expression plasmid and sequenced. The sequencing results were subjected to bioinformatics analysis. The Toxoplasma gondii SAG1 complete coding sequences were successfully cloned. Physicochemical analysis revealed the 336 aa of SAG1 had 34.7 kDa of weight. The isoelectric point and aliphatic index were 8.4 and 78.4, respectively. The N-terminal methionine half-life in Escherichia coli was more than 10 hours. The antigenicity, secondary structure, and identification of the HLA binding motifs also had been discussed. The results of this study would contribute information about Toxoplasma gondii SAG1 and benefits for further works willing to develop diagnostic and therapeutic strategies against the parasite.

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain

PubMed Central

Anandasabapathy, Niroshana; Victora, Gabriel D.; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L.; Nussenzweig, Michel C.; Steinman, Ralph M.

2011-01-01

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5–7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia. PMID:21788405

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain.

PubMed

Anandasabapathy, Niroshana; Victora, Gabriel D; Meredith, Matthew; Feder, Rachel; Dong, Baojun; Kluger, Courtney; Yao, Kaihui; Dustin, Michael L; Nussenzweig, Michel C; Steinman, Ralph M; Liu, Kang

2011-08-01

Antigen-presenting cells in the disease-free brain have been identified primarily by expression of antigens such as CD11b, CD11c, and MHC II, which can be shared by dendritic cells (DCs), microglia, and monocytes. In this study, starting with the criterion of Flt3 (FMS-like receptor tyrosine kinase 3)-dependent development, we characterize the features of authentic DCs within the meninges and choroid plexus in healthy mouse brains. Analyses of morphology, gene expression, and antigen-presenting function established a close relationship between meningeal and choroid plexus DCs (m/chDCs) and spleen DCs. DCs in both sites shared an intrinsic requirement for Flt3 ligand. Microarrays revealed differences in expression of transcripts encoding surface molecules, transcription factors, pattern recognition receptors, and other genes in m/chDCs compared with monocytes and microglia. Migrating pre-DC progenitors from bone marrow gave rise to m/chDCs that had a 5-7-d half-life. In contrast to microglia, DCs actively present self-antigens and stimulate T cells. Therefore, the meninges and choroid plexus of a steady-state brain contain DCs that derive from local precursors and exhibit a differentiation and antigen-presenting program similar to spleen DCs and distinct from microglia.

Infrequent and low expression of cancer-testis antigens located on the X chromosome in colorectal cancer: implications for immunotherapy in South African populations.

PubMed

Dakshinamurthy, Amirtha Ganesh; Ramesar, Rajkumar; Goldberg, Paul; Blackburn, Jonathan M

2008-11-01

Cancer-testis (CT) antigens are a group of tumor antigens that are expressed in the testis and aberrantly in cancerous tissue but not in somatic tissues. The testis is an immune-privileged site because of the presence of a blood-testis barrier; as a result, CT antigens are considered to be essentially tumor specific and are attractive targets for immunotherapy. CT antigens are classified as the CT-X and the non-X CT antigens depending on the chromosomal location to which the genes are mapped. CT-X antigens are typically highly immunogenic and hence the first step towards tailored immunotherapy is to elucidate the expression profile of CT-X antigens in the respective tumors. In this study we investigated the expression profile of 16 CT-X antigen genes in 34 colorectal cancer (CRC) patients using reverse transcription-polymerase chain reaction. We observed that 12 of the 16 CT-X antigen genes studied did not show expression in any of the CRC samples analyzed. The other 4 CT-X antigen genes showed low frequency of expression and exhibited a highly variable expression profile when compared to other populations. Thus, our study forms the first report on the expression profile of CT-X antigen genes among CRC patients in the genetically diverse South African population. The results of our study suggest that genetic and ethnic variations in population might have a role in the expression of the CT-X antigen genes. Thus our results have significant implications for anti-CT antigen-based immunotherapy trials in this population.

Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis.

PubMed

Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C

1997-06-01

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Genetic variation of the Borrelia burgdorferi gene vlsE involves cassette-specific, segmental gene conversion.

PubMed

Zhang, J R; Norris, S J

1998-08-01

The Lyme disease spirochete Borrelia burgdorferi possesses 15 silent vls cassettes and a vls expression site (vlsE) encoding a surface-exposed lipoprotein. Segments of the silent vls cassettes have been shown to recombine with the vlsE cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the vlsE cassette region, the 5' and 3' coding sequences of vlsE that flank the cassette region are not subject to sequence variation during these recombination events. The segments of the silent vls cassettes recombine in the vlsE cassette region through a unidirectional process such that the sequence and organization of the silent vls loci are not affected. As a result of recombination, the previously expressed segments are replaced by incoming segments and apparently degraded. These results provide evidence for a gene conversion mechanism in VlsE antigenic variation.

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.

PubMed

Morona, R; van den Bosch, L; Manning, P A

1995-02-01

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).

Influenza Virus PB1 and Neuraminidase Gene Segments Can Cosegregate during Vaccine Reassortment Driven by Interactions in the PB1 Coding Region

PubMed Central

Cobbin, Joanna C. A.; Ong, Chi; Verity, Erin; Gilbertson, Brad P.; Rockman, Steven P.

2014-01-01

ABSTRACT Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by “classical reassortment” of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. IMPORTANCE Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics. PMID:24872588

Influenza virus PB1 and neuraminidase gene segments can cosegregate during vaccine reassortment driven by interactions in the PB1 coding region.

PubMed

Cobbin, Joanna C A; Ong, Chi; Verity, Erin; Gilbertson, Brad P; Rockman, Steven P; Brown, Lorena E

2014-08-01

Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

PubMed Central

DeKosky, Brandon J.; Lungu, Oana I.; Park, Daechan; Johnson, Erik L.; Charab, Wissam; Chrysostomou, Constantine; Kuroda, Daisuke; Ellington, Andrew D.; Ippolito, Gregory C.; Gray, Jeffrey J.; Georgiou, George

2016-01-01

Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19+CD20+CD27− IgM-naive B cells, >120,000 antibody clusters from CD19+CD20+CD27+ antigen–experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ–Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease. PMID:27114511

Erythrocyte membrane antigen frequencies in patients with Type II congenital smell loss.

PubMed

Stateman, William A; Henkin, Robert I; Knöppel, Alexandra B; Flegel, Willy A

2015-01-01

The objective of this study was to determine whether there are genetic factors associated with Type II congenital smell loss. The expression frequencies of 16 erythrocyte antigens among patients with Type II congenital smell loss were determined and compared to those of a large control group. Blood samples were obtained from 99 patients with Type II congenital smell loss. Presence of the erythrocyte surface antigens A, B, M, N, S, s, Fy(a), Fy(b), D, C, c, E, e, K, Jk(a), and Jk(b) was analyzed by blood group serology. Comparisons of expression frequencies of these antigens were made between the patients and a large control group. Patients tested for the Duffy b antigen (Fy(b) haplotype) exhibited a statistically significant 11% decrease in expression frequency compared to the controls. There were no significant differences between patients and controls in the expression frequencies for all other erythrocyte antigens (A, B, M, N, S, s, Fy(a), D, C, c, E, e, K, Jk(a), or Jk(b)). These findings describe the presence of a previously unrevealed genetic tendency among patients with Type II congenital smell loss related to erythrocyte surface antigen expression. The deviation in expression rate of Duffy b suggests a target gene and chromosome region in which future research into this form of congenital smell loss may reveal a more specific genetic basis for Type II congenital smell loss. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression of the Major Surface Antigen of Plasmodium knowlesi Sporozoites in Yeast

NASA Astrophysics Data System (ADS)

Sharma, Shobhona; Godson, G. Nigel

1985-05-01

The circumsporozoite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor protein was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated with the 25,000g and 150,000g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.

The construction of cDNA library and the screening of related antigen of ascitic tumor cells of ovarian cancer.

PubMed

Hou, Q; Chen, K; Shan, Z

2015-01-01

To construct the cDNA library of the ascites tumor cells of ovarian cancer, which can be used to screen the related antigen for the early diagnosis of ovarian cancer and therapeutic targets of immune treatment. Four cases of ovarian serous cystadenocarcinoma, two cases of ovarian mucinous cystadenocarcinoma, and two cases of ovarian endometrial carcinoma in patients with ascitic tumor cells which were used to construct the cDNA library. To screen the ovarian cancer antigen gene, evaluate the enzyme, and analyze nucleotide sequence, serological analysis of recombinant tumor cDNA expression libraries (SEREX) and suppression subtractive hybridization technique (SSH) techniques were utilized. The detection method of recombinant expression-based serological mini-arrays (SMARTA) was used to detect the ovarian cancer antigen and the positive reaction of 105 cases of ovarian cancer patients and 105 normal women's autoantibodies correspondingly in serum. After two rounds of serologic screening and glycosides sequencing analysis, 59 candidates of ovarian cancer antigen gene fragments were finally identified, which corresponded to 50 genes. They were then divided into six categories: (1) the homologous genes which related to the known ovarian cancer genes, such as BARD 1 gene, etc; (2) the homologous genes which were associated with other tumors, such as TM4SFI gene, etc; (3) the genes which were expressed in a special organization, such as ILF3, FXR1 gene, etc; (4) the genes which were the same with some protein genes of special function, such as TIZ, ClD gene; (5) the homologous genes which possessed the same source with embryonic genes, such as PKHD1 gene, etc; (6) the remaining genes were the unknown genes without the homologous sequence in the gene pool, such as OV-189 genes. SEREX technology combined with SSH method is an effective research strategy which can filter tumor antigen with high specific character; the corresponding autoantibodies of TM4SFl, ClD, TIZ, BARDI, FXRI, and OV-189 gene's recombinant antigen in serum can be regarded as the biomarkers which are used to diagnose ovarian cancer. The combination of multiple antigen detection can improve diagnostic efficiency.

The gene coding for the B cell surface protein CD19 is localized on human chromosome 16p11.

PubMed

Stapleton, P; Kozmik, Z; Weith, A; Busslinger, M

1995-02-01

The CD19 gene codes for one of the earliest markers of the human B cell lineage and is a target for the B lymphoid-specific transcription factor BSAP (Pax-5). The transmembrane protein CD19 has been implicated in controlling proliferation of mature B lymphocytes by modulating signal transduction through the antigen receptor. In this study, we have employed Southern blot and fluorescence in situ hybridization analyses to localize the CD19 gene to human chromosome 16p11.

Immunogenicity of a novel, bivalent, plant-based oral vaccine against hepatitis B and human immunodeficiency viruses.

PubMed

Shchelkunov, Sergei N; Salyaev, Rurik K; Pozdnyakov, Sergei G; Rekoslavskaya, Natalia I; Nesterov, Andrei E; Ryzhova, Tatiana S; Sumtsova, Valentina M; Pakova, Natalia V; Mishutina, Uliana O; Kopytina, Tatiana V; Hammond, Rosemarie W

2006-07-01

A synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of human immunodeficiency virus (HIV-1) and the surface protein antigen (HBsAg) of hepatitis B virus (HBV), was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice and, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV.

Improved efficiency in amplification of Escherichia coli o-antigen gene clusters using genome-wide sequence comparison

USDA-ARS?s Scientific Manuscript database

Background: In many bacteria including E. coli, genes encoding O-antigens are clustered in the chromosome, with a 39-bp JUMPstart sequence and gnd gene located upstream and downstream of the cluster, respectively. For determining the DNA sequence of the E. coli O-antigen gene cluster, one set of P...

High-resolution profiling of linear B-cell epitopes from mucin-associated surface proteins (MASPs) of Trypanosoma cruzi during human infections

PubMed Central

Durante, Ignacio M.; La Spina, Pablo E.; Carmona, Santiago J.; Agüero, Fernán

2017-01-01

Background The Trypanosoma cruzi genome bears a huge family of genes and pseudogenes coding for Mucin-Associated Surface Proteins (MASPs). MASP molecules display a ‘mosaic’ structure, with highly conserved flanking regions and a strikingly variable central and mature domain made up of different combinations of a large repertoire of short sequence motifs. MASP molecules are highly expressed in mammal-dwelling stages of T. cruzi and may be involved in parasite-host interactions and/or in diverting the immune response. Methods/Principle findings High-density microarrays composed of fully overlapped 15mer peptides spanning the entire sequences of 232 non-redundant MASPs (~25% of the total MASP content) were screened with chronic Chagasic sera. This strategy led to the identification of 86 antigenic motifs, each one likely representing a single linear B-cell epitope, which were mapped to 69 different MASPs. These motifs could be further grouped into 31 clusters of structurally- and likely antigenically-related sequences, and fully characterized. In contrast to previous reports, we show that MASP antigenic motifs are restricted to the central and mature region of MASP polypeptides, consistent with their intracellular processing. The antigenicity of these motifs displayed significant positive correlation with their genome dosage and their relative position within the MASP polypeptide. In addition, we verified the biased genetic co-occurrence of certain antigenic motifs within MASP polypeptides, compatible with proposed intra-family recombination events underlying the evolution of their coding genes. Sequences spanning 7 MASP antigenic motifs were further evaluated using distinct synthesis/display approaches and a large panel of serum samples. Overall, the serological recognition of MASP antigenic motifs exhibited a remarkable non normal distribution among the T. cruzi seropositive population, thus reducing their applicability in conventional serodiagnosis. As previously observed in in vitro and animal infection models, immune signatures supported the concurrent expression of several MASPs during human infection. Conclusions/Significance In spite of their conspicuous expression and potential roles in parasite biology, this study constitutes the first unbiased, high-resolution profiling of linear B-cell epitopes from T. cruzi MASPs during human infection. PMID:28961244

Plasmodium vivax merozoite surface protein-3 alpha: a high-resolution marker for genetic diversity studies.

PubMed

Prajapati, Surendra Kumar; Joshi, Hema; Valecha, Neena

2010-06-01

Malaria, an ancient human infectious disease caused by five species of Plasmodium, among them Plasmodium vivax is the most widespread human malaria species and causes huge morbidity to its host. Identification of genetic marker to resolve higher genetic diversity for an ancient origin organism is a crucial task. We have analyzed genetic diversity of P. vivax field isolates using highly polymorphic antigen gene merozoite surface protein-3 alpha (msp-3 alpha) and assessed its suitability as high-resolution genetic marker for population genetic studies. 27 P. vivax field isolates collected during chloroquine therapeutic efficacy study at Chennai were analyzed for genetic diversity. PCR-RFLP was employed to assess the genetic variations using highly polymorphic antigen gene msp-3 alpha. We observed three distinct PCR alleles at msp-3 alpha, and among them allele A showed significantly high frequency (53%, chi2 = 8.22, p = 0.001). PCR-RFLP analysis revealed 14 and 17 distinct RFLP patterns for Hha1 and Alu1 enzymes respectively. Further, RFLP analysis revealed that allele A at msp-3 alpha is more diverse in the population compared with allele B and C. Combining Hha1 and Alu1 RFLP patterns revealed 21 distinct genotypes among 22 isolates reflects higher diversity resolution power of msp-3 alpha in the field isolates. P. vivax isolates from Chennai region revealed substantial amount of genetic diversity and comparison of allelic diversity with other antigen genes and microsatellites suggesting that msp-3 alpha could be a high-resolution marker for genetic diversity studies among P. vivax field isolates.

[Production, specificity and structure of immunoglobulins].

PubMed

Goujard, C; Delfraissy, J F

1991-03-21

Immunoglobulin is a key factor of the immune response resulting from B-cell activation and associated with T-cell stimulation. Because of its structure, this antibody has a dual function: it specifically recognizes the inducer antigen in the variable region and eliminates it by a constant portion which is responsible for effector properties. Surface immunoglobulin, therefore, is the B-cell antigen receptor; it differs from the T-cell receptor in that it recognizes the antigen unbound to the major istocompatibility complex; binding the antigen results in direct signal transduction first in the cytoplasm, then in the nucleus. This receptor can be secreted in the body: it is made up of circulating immunoglobulins. Human immunoglobulins are divided into 5 classes, each of them with its own response kinetics, distribution and functions. The variability of the antibody response accounts for a genetic organization involving numerous genes which may be associated with each other, or mutate, or recombine during maturation of the lymphocytes. Altogether, this system has a theoretical capacity of response to three hundred million different antigens.

Expanding and characterizing esophageal epithelial cells obtained from children with eosinophilic esophagitis.

PubMed

Sayej, Wael N; Foster, Christopher; Jensen, Todd; Chatfield, Sydney; Finck, Christine

2018-06-12

The role of epithelial cells in eosinophilic esophagitis (EoE) is not well understood. In this study, our aim was to isolate, culture, and expand esophageal epithelial cells obtained from patients with or without EoE and characterize differences observed over time in culture. Biopsies were obtained at the time of endoscopy from children with EoE or suspected to have EoE. We established patient-derived esophageal epithelial cell (PDEEC) lines utilizing conditional reprogramming methods. We determined integrin profiles, gene expression, MHC class II expression, and reactivity to antigen stimulation. The PDEECs were found to maintain their phenotype over several passages. There were differences in integrin profiles and gene expression levels in EoE-Active compared to normal controls and EoE-Remission patients. Once stimulated with antigens, PDEECs express MHC class II molecules on their surface, and when co-cultured with autologous T-cells, there is increased IL-6 and TNF-α secretion in EoE-Active patients vs. controls. We are able to isolate, culture, and expand esophageal epithelial cells from pediatric patients with and without EoE. Once stimulated with antigens, these cells express MHC class II molecules and behave as non-professional antigen-presenting cells. This method will help us in developing an ex vivo, individualized, patient-specific model for diagnostic testing for causative antigens.

A STING-activating nanovaccine for cancer immunotherapy

NASA Astrophysics Data System (ADS)

Luo, Min; Wang, Hua; Wang, Zhaohui; Cai, Haocheng; Lu, Zhigang; Li, Yang; Du, Mingjian; Huang, Gang; Wang, Chensu; Chen, Xiang; Porembka, Matthew R.; Lea, Jayanthi; Frankel, Arthur E.; Fu, Yang-Xin; Chen, Zhijian J.; Gao, Jinming

2017-07-01

The generation of tumour-specific T cells is critically important for cancer immunotherapy. A major challenge in achieving a robust T-cell response is the spatiotemporal orchestration of antigen cross-presentation in antigen-presenting cells with innate stimulation. Here, we report a minimalist nanovaccine, comprising a simple physical mixture of an antigen and a synthetic polymeric nanoparticle, PC7A NP, which generates a strong cytotoxic T-cell response with low systemic cytokine expression. Mechanistically, the PC7A NP achieves efficient cytosolic delivery of tumour antigens to antigen-presenting cells in draining lymph nodes, leading to increased surface presentation while simultaneously activating type I interferon-stimulated genes. This effect is dependent on stimulator of interferon genes (STING), but not the Toll-like receptor or the mitochondrial antiviral-signalling protein (MAVS) pathway. The nanovaccine led to potent tumour growth inhibition in melanoma, colon cancer and human papilloma virus-E6/E7 tumour models. The combination of the PC7A nanovaccine and an anti-PD-1 antibody showed great synergy, with 100% survival over 60 days in a TC-1 tumour model. Rechallenging of these tumour-free animals with TC-1 cells led to complete inhibition of tumour growth, suggesting the generation of long-term antitumour memory. The STING-activating nanovaccine offers a simple, safe and robust strategy in boosting anti-tumour immunity for cancer immunotherapy.

Differential impact of lipopolysaccharide defects caused by loss of RfaH in Yersinia pseudotuberculosis and Yersinia pestis.

PubMed

Hoffman, Jared M; Sullivan, Shea; Wu, Erin; Wilson, Eric; Erickson, David L

2017-09-07

RfaH enhances transcription of a select group of operons controlling bacterial surface features such as lipopolysaccharide (LPS). Previous studies have suggested that rfaH may be required for Yersinia pseudotuberculosis resistance to antimicrobial chemokines and survival during mouse infections. In order to further investigate the role of RfaH in LPS synthesis, resistance to host defense peptides, and virulence of Yersinia, we constructed ΔrfaH mutants of Y. pseudotuberculosis IP32953 and Y. pestis KIM6+. Loss of rfaH affected LPS synthesis in both species, resulting in a shorter core oligosaccharide. Susceptibility to polymyxin and the antimicrobial chemokine CCL28 was increased by loss of rfaH in Y. pseudotuberculosis but not in Y. pestis. Transcription of genes in the ddhD-wzz O-antigen gene cluster, but not core oligosaccharide genes, was reduced in ΔrfaH mutants. In addition, mutants with disruptions in specific ddhD-wzz O-antigen cluster genes produced LPS that was indistinguishable from the ΔrfaH mutant. This suggests that both Y. pseudotuberculosis and Y. pestis produce an oligosaccharide core with a single O-antigen unit attached in an RfaH-dependent fashion. Despite enhanced sensitivity to host defense peptides, the Y. pseudotuberculosis ΔrfaH strain was not attenuated in mice, suggesting that rfaH is not required for acute infection.

Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses.

PubMed

Yamanaka, Atsushi; Moi, Meng Ling; Takasaki, Tomohiko; Kurane, Ichiro; Matsuda, Mami; Suzuki, Ryosuke; Konishi, Eiji

2017-05-01

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections. Copyright © 2017 Elsevier B.V. All rights reserved.

Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach.

PubMed

Encinas, Paloma; Rodriguez-Milla, Miguel A; Novoa, Beatriz; Estepa, Amparo; Figueras, Antonio; Coll, Julio

2010-09-27

Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36). The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

Summaries of Research 1987

DTIC Science & Technology

1987-01-01

STRUCTURE OF THE GENE ENCODING THE CIRCUMSPOROZOITE PROTEIN OF PLASWODIUm YOELIJ. A RODENT MCDFL FOP EIAMINING ANTIMALARIAL SPOBOZOITE VACCINES. JOURNAL OF...BICLOGICAL CHEMISTRY 1987 MAR 5;262(7):2937-40 INFECTIOUS DISEASES 3M162770A870.AF.312-1 (DA301614) REPORT NO.E ANTIGENS, SURFACE PLAS!CDIU? AD A188...SURFACE CLONING, MOLECULAR PLASMCDIUM dERGHEI AD A185 330 NI!PI 87-C036 ROTd ?L CHUANG DM MULTIPLE EECHANIS!15 CF SEROTONERGIC SIGNAL TRANSDUCTION. LIFE

Downregulation of peptide transporter genes in cell lines transformed with the highly oncogenic adenovirus 12

PubMed Central

1994-01-01

The expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the high oncogenicity of this virus. In primary embryonal fibroblasts from transgenic mice that express both endogenous H-2 genes and a miniature swine class I gene (PD1), Ad12- mediated transformation results in suppression of cell surface expression of all class I antigens. Although class I mRNA levels of PD1 and H-2Db are similar to those in nonvirally transformed cells, recognition of newly synthesized class I molecules by a panel of monoclonal antibodies is impaired, presumably as a result of inefficient assembly and transport of the class I molecules. Class I expression can be partially induced by culturing cells at 26 degrees C, or by coculture of cells with class I binding peptides at 37 degrees C. Analysis of steady state mRNA levels of the TAP1 and TAP2 transporter genes for Ad12-transformed cell lines revealed that they both are significantly reduced, TAP2 by about 100-fold and TAP1 by 5-10-fold. Reconstitution of PD1 and H-2Db, but not H-2Kb, expression is achieved in an Ad12-transformed cell line by stable transfection with a TAP2, but not a TAP1, expression construct. From these data it may be concluded that suppressed expression of peptide transporter genes, especially TAP2, in Ad12-transformed cells inhibits cell surface expression of class I molecules. The failure to fully reconstitute H- 2Db and H-2Kb expression indicates that additional factors are involved in controlling class I gene expression in Ad12-transformed cells. Nevertheless, these results suggest that suppression of peptide transporter genes might be an important mechanism whereby virus- transformed cells escape immune recognition in vivo. PMID:7519239

Immune-Modulation by Epidermal Growth Factor Receptor Inhibitors: Implication on Anti-Tumor Immunity in Lung Cancer

PubMed Central

Herrmann, Amanda C.; Bernatchez, Chantale; Haymaker, Cara; Molldrem, Jeffrey J.; Hong, Waun Ki; Perez-Soler, Roman

2016-01-01

Skin toxicity is the most common toxicity caused by Epidermal Growth Factor Receptor (EGFR) inhibitors, and has been associated with clinical efficacy. As EGFR inhibitors enhance the expression of antigen presenting molecules in affected skin keratinocytes, they may concurrently facilitate neo-antigen presentation in lung cancer tumor cells contributing to anti-tumor immunity. Here, we investigated the modulatory effect of the EGFR inhibitor, erlotinib on antigen presenting molecules and PD-L1, prominent immune checkpoint protein, of skin keratinocytes and lung cancer cell lines to delineate the link between EGFR signaling pathway inhibition and potential anti-tumor immunity. Erlotinib up-regulated MHC-I and MHC-II proteins on IFNγ treated keratinocytes but abrogated IFNγ-induced expression of PD-L1, suggesting the potential role of infiltrating autoreactive T cells in the damage of keratinocytes in affected skin. Interestingly, the surface expression of MHC-I, MHC-II, and PD-L1 was up-regulated in response to IFNγ more often in lung cancer cell lines sensitive to erlotinib, but only expression of PD-L1 was inhibited by erlotinib. Further, erlotinib significantly increased T cell mediated cytotoxicity on lung cancer cells. Lastly, the analysis of gene expression dataset of 186 lung cancer cell lines from Cancer Cell Line Encyclopedia demonstrated that overexpression of PD-L1 was associated with sensitivity to erlotinib and higher expression of genes related to antigen presenting pathways and IFNγ signaling pathway. Our findings suggest that the EGFR inhibitors can facilitate anti-tumor adaptive immune responses by breaking tolerance especially in EGFR driven lung cancer that are associated with overexpression of PD-L1 and genes related to antigen presentation and inflammation. PMID:27467256

Characterization of hepatitis B virus surface antigen variability and impact on HBs antigen clearance under nucleos(t)ide analogue therapy.

PubMed

Velay, A; Jeulin, H; Eschlimann, M; Malvé, B; Goehringer, F; Bensenane, M; Frippiat, J-P; Abraham, P; Ismail, A M; Murray, J M; Combet, C; Zoulim, F; Bronowicki, J-P; Schvoerer, E

2016-05-01

For hepatitis B virus (HBV)-related chronic infection under treatment by nucleos(t)ide analogues (NUCs), HBsAg clearance is the ultimate therapeutic goal but very infrequent. We investigated how HBV envelope protein variability could lead to differential HBsAg clearance on NUCs. For 12 HBV genotype D patients receiving NUCs, six resolvers (HBsAg clearance) were compared to six matched nonresolvers (HBsAg persistence). PreS/S amino acid (aa) sequences were analysed with bioinformatics to predict HBV envelope antigenicity and aa covariance. To enrich our analyses on very rare resolvers, these were compared with other HBV genotype D strains in three characterized clinical cohorts including common chronically infected patients. The sT125M+sP127T combination was observed in four nonresolvers of six, corroborated by aa covariance analysis, associated with a lower predicted antigenicity than sT125T+sP127P. Concordant features within this HBV key functional domain, at positions 125 and 127, were reported from two of the three comparative cohorts. In our hands, a lower ELISA reactivity of HBV-vaccinated mice sera was observed against the sT125M mutant. In the S gene, 56 aa changes in minor variants were detected in non-resolvers, mainly in the major hydrophilic region, vs 28 aa changes in resolvers. Molecular features in patients showing HBsAg persistence on NUCs argue in favour of a different aa pattern in the HBV S gene compared to those showing HBsAg clearance. In nonresolvers, a decrease in HBs 'a' determinant antigenicity and more frequent mutations in the S gene suggest a role for the HBV envelope characteristics in HBsAg persistence. © 2016 John Wiley & Sons Ltd.

Molecular Cloning and Sequence of Channel Catfish (Ictalurus punctatus, Rafinesque 1818) Cathepsin S gene

USDA-ARS?s Scientific Manuscript database

Cathepsin S is a lysosomal cysteine endopeptidase of the papain family. This enzyme digests the invariant chain molecules so that antigenic peptides are able to load on the class II-associated invariant chain peptide of MHC. The complexes can subsequently be presented to the CD4 cell surface. In ...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

21 CFR 660.1 - Antibody to Hepatitis B Surface Antigen.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Antibody to Hepatitis B Surface Antigen. 660.1... Hepatitis B Surface Antigen § 660.1 Antibody to Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product shall be Antibody to Hepatitis B Surface Antigen. The product is...

Structure and genetics of the O-specific polysaccharide of Escherichia coli O27.

PubMed

Perepelov, Andrei V; Chen, Tingting; Senchenkova, Sofya N; Filatov, Andrei V; Song, Jingjie; Shashkov, Alexander S; Liu, Bin; Knirel, Yuriy A

2018-02-01

The O-specific polysaccharide (O-antigen) is a part of the lipopolysaccharide on the cell surface of Gram-negative bacteria. The O-polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Escherichia coli O27 and studied by sugar analysis and Smith degradation along with 1 H and 13 C NMR spectroscopy. The following structure of the branched hexasaccharide repeating unit was established, which is unique among known structures of bacterial polysaccharides:where GlcA is non-stoichiometrically O-acetylated at position 3 (∼22%) or 4 (∼37%). Functions of genes in the O-antigen gene cluster of E. coli O27 were tentatively assigned by comparison with sequences in the available databases and found to be consistent with the O-polysaccharide structure. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates.

PubMed

Cheng, Keding; Chui, Huixia; Domish, Larissa; Sloan, Angela; Hernandez, Drexler; McCorrister, Stuart; Robinson, Alyssia; Walker, Matthew; Peterson, Lorea A M; Majcher, Miles; Ratnam, Sam; Haldane, David J M; Bekal, Sadjia; Wylie, John; Chui, Linda; Tyler, Shaun; Xu, Bianli; Reimer, Aleisha; Nadon, Celine; Knox, J David; Wang, Gehua

2016-08-01

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing. Copyright © 2016 Cheng et al.

Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen from Plasmodium falciparum

PubMed Central

Belperron, Alexia A.; Feltquate, David; Fox, Barbara A.; Horii, Toshihiro; Bzik, David J.

1999-01-01

The liver- and blood-stage-expressed serine repeat antigen (SERA) of Plasmodium falciparum is a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein. PMID:10496891

[Designing of a candidate edible vaccine against hepatitis B and HIV on the basis of a transgenic tomato].

PubMed

Shchelkunov, S N; Saliaev, R K; Ryzhova, T S; Pozdniakov, S G; Nesterov, A E; Rekoslavskaia, N I; Sumtsova, V M; Pakova, N V; Mishutina, U O; Kopytina, T V; Hammond, R V

2004-01-01

The synthetic chimeric gene TBI-HBS encoding the synthesis of immunogenic ENV and GAC epitopes of HIV-1 (immunogenes of T- and B-lymphocytes) and of the surface protein (HBsAg) of the hepatitis B virus was introduced into tomato plants var. Ventura by agrobacterial vector pBIN35TBI-HBS; transgenic tomato plants with the integrated gene TBI-HBS were generated. The integration of the TBI-HBS target gene was confirmed by PCR. The synthesis of antigenic proteins of TBI and HBsAg in fruits of transgenic tomato plants was displayed by immunoassay. The fruits of transgenic tomato plants were fed to experimental mice with a 1-week interval. On days 14 and 28, there was discovered a sufficiently high content of antibodies to the antigenic proteins of HBV and HIV-1 in serum of experimental animals. Antibodies were found in feces of experimental mice; no antibodies were found in the control group of mice. Hence, it was established that the TBI (HIV-1) and HBsAg (HBV) antigens were synthesized in transgenic tomato fruits due to the integrated construction of pBINNp35TBI-HBS in an amount that was enough to induce the immunogenic response in mice to the oral delivery of edible vaccine.

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuhki, Naoya; O'Brien, S.J.

1990-01-01

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. The authors present here a quantitative analysis of restriction fragmentmore » length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations.« less

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history.

PubMed Central

Yuhki, N; O'Brien, S J

1990-01-01

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. We present here a quantitative analysis of restriction fragment length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations. Images PMID:1967831

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

[Effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering].

PubMed

Ding, Jun-Ying; Meng, Qing-Ling; Guo, Min-Zhuo; Yi, Yao; Su, Qiu-Dong; Lu, Xue-Xin; Qiu, Feng; Bi, Sheng-Li

2012-10-01

To study the effect of gene optimization on the expression and purification of HDV small antigen produced by genetic engineering. Based on the colon preference of E. coli, the HDV small antigen original gene from GenBank was optimized. Both the original gene and the optimized gene expressed in prokaryotic cells, SDS-PAGE was made to analyze the protein expression yield and to decide which protein expression style was more proportion than the other. Furthermore, two antigens were purified by chromatography in order to compare the purity by SDS-PAGE and Image Lab software. SDS-PAGE indicated that the molecular weight of target proteins from two groups were the same as we expected. Gene optimization resulted in the higher yield and it could make the product more soluble. After chromatography, the purity of target protein from optimized gene was up to 96.3%, obviously purer than that from original gene. Gene optimization could increase the protein expression yield and solubility of genetic engineering HDV small antigen. In addition, the product from the optimized gene group was easier to be purified for diagnosis usage.

Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

PubMed

Ranade, V V

1989-10-01

Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera for studies of chromosome structure and function, gene mapping, embryogenesis, characterization and biosynthesis of developmental and differentiation antigens. These antigens are those that are specific for various cell types and tissues, species specific antigen, antigens involved in chemotaxis, immunogenetics and clinical genetics including genetically inherited disorders, chromosome aberrations and transplantation antigens. Besides these monoclonal antibodies, their complexes have recently been investigated as exquisitely sensitive probes to be guided to target cells or organs. They have been used to deliver cytotoxic drugs to malignant cells or enzymes to specific cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge.

PubMed

Sousa, C T; Brito, T S; Lima, F J B; Siqueira, R J B; Magalhães, P J C; Lima, A A M; Santos, A A; Havt, A

2011-06-01

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Escherichia coli O-Antigen Gene Clusters of Serogroups O62, O68, O131, O140, O142, and O163: DNA Sequences and Similarity between O62 and O68, and PCR-Based Serogrouping

PubMed Central

Liu, Yanhong; Yan, Xianghe; DebRoy, Chitrita; Fratamico, Pina M.; Needleman, David S.; Li, Robert W.; Wang, Wei; Losada, Liliana; Brinkac, Lauren; Radune, Diana; Toro, Magaly; Hegde, Narasimha; Meng, Jianghong

2015-01-01

The DNA sequence of the O-antigen gene clusters of Escherichia coli serogroups O62, O68, O131, O140, O142, and O163 was determined, and primers based on the wzx (O-antigen flippase) and/or wzy (O-antigen polymerase) genes within the O-antigen gene clusters were designed and used in PCR assays to identify each serogroup. Specificity was tested with E. coli reference strains, field isolates belonging to the target serogroups, and non-E. coli bacteria. The PCR assays were highly specific for the respective serogroups; however, the PCR assay targeting the O62 wzx gene reacted positively with strains belonging to E. coli O68, which was determined by serotyping. Analysis of the O-antigen gene cluster sequences of serogroups O62 and O68 reference strains showed that they were 94% identical at the nucleotide level, although O62 contained an insertion sequence (IS) element located between the rmlA and rmlC genes within the O-antigen gene cluster. A PCR assay targeting the rmlA and rmlC genes flanking the IS element was used to differentiate O62 and O68 serogroups. The PCR assays developed in this study can be used for the detection and identification of E. coli O62/O68, O131, O140, O142, and O163 strains isolated from different sources. PMID:25664526

The SnSAG merozoite surface antigens of Sarcocystis neurona are expressed differentially during the bradyzoite and sporozoite life cycle stages.

PubMed

Gautam, A; Dubey, J P; Saville, W J; Howe, D K

2011-12-29

Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. In the present study, the expression of S. neurona merozoite surface antigens (SnSAGs) was evaluated in the sporozoite and bradyzoite stages. Western blot analysis was used to compare SnSAG expression in merozoites versus sporozoites, while immunocytochemistry was performed to examine expression of the SnSAGs in merozoites versus bradyzoites. These analyses revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed in sporozoites, while SnSAG5 was appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, it was found that SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. As shown for T. gondii, stage-specific expression of the SnSAGs may be important for the parasite to progress through its developmental stages and complete its life cycle successfully. Thus, it is possible that the SAG switching mechanism by these parasites could be exploited as a point of intervention. As well, the alterations in surface antigen expression during different life cycle stages may need to be considered when designing prospective approaches for protective vaccination. Copyright © 2011 Elsevier B.V. All rights reserved.

Three copies of a single protein II-encoding sequence in the genome of Neisseria gonorrhoeae JS3: evidence for gene conversion and gene duplication.

PubMed

van der Ley, P

1988-11-01

Gonococci express a family of related outer membrane proteins designated protein II (P.II). These surface proteins are subject to both phase variation and antigenic variation. The P.II gene repertoire of Neisseria gonorrhoeae strain JS3 was found to consist of at least ten genes, eight of which were cloned. Sequence analysis and DNA hybridization studies revealed that one particular P.II-encoding sequence is present in three distinct, but almost identical, copies in the JS3 genome. These genes encode the P.II protein that was previously identified as P.IIc. Comparison of their sequences shows that the multiple copies of this P.IIc-encoding gene might have been generated by both gene conversion and gene duplication.

Evolution of the Transmission-Blocking Vaccine Candidates Pvs28 and Pvs25 in Plasmodium vivax: Geographic Differentiation and Evidence of Positive Selection

PubMed Central

Cornejo, Omar E.; Durrego, Ester; Stanley, Craig E.; Castillo, Andreína I.; Herrera, Sócrates; Escalante, Ananias A.

2016-01-01

Transmission-blocking (TB) vaccines are considered an important tool for malaria control and elimination. Among all the antigens characterized as TB vaccines against Plasmodium vivax, the ookinete surface proteins Pvs28 and Pvs25 are leading candidates. These proteins likely originated by a gene duplication event that took place before the radiation of the known Plasmodium species to primates. We report an evolutionary genetic analysis of a worldwide sample of pvs28 and pvs25 alleles. Our results show that both genes display low levels of genetic polymorphism when compared to the merozoite surface antigens AMA-1 and MSP-1; however, both ookinete antigens can be as polymorphic as other merozoite antigens such as MSP-8 and MSP-10. We found that parasite populations in Asia and the Americas are geographically differentiated with comparable levels of genetic diversity and specific amino acid replacements found only in the Americas. Furthermore, the observed variation was mainly accumulated in the EGF2- and EGF3-like domains for P. vivax in both proteins. This pattern was shared by other closely related non-human primate parasites such as Plasmodium cynomolgi, suggesting that it could be functionally important. In addition, examination with a suite of evolutionary genetic analyses indicated that the observed patterns are consistent with positive natural selection acting on Pvs28 and Pvs25 polymorphisms. The geographic pattern of genetic differentiation and the evidence for positive selection strongly suggest that the functional consequences of the observed polymorphism should be evaluated during development of TBVs that include Pvs25 and Pvs28. PMID:27347876

Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of β Antigen Production

PubMed Central

Vasilyeva, Anastasia; Santos Sanches, Ilda; Florindo, Carlos; Dmitriev, Alexander

2015-01-01

Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for β antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed β antigen, while five strains did not express β antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of β antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of β antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of β antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S. agalactiae adaptation to the human. PMID:26047354

Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of β Antigen Production.

PubMed

Vasilyeva, Anastasia; Santos Sanches, Ilda; Florindo, Carlos; Dmitriev, Alexander

2015-01-01

Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for β antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed β antigen, while five strains did not express β antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of β antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of β antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of β antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of β antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S. agalactiae adaptation to the human.

Response of dairy calves to vaccinia viruses that express foreign genes.

PubMed Central

Gillespie, J H; Geissinger, C; Scott, F W; Higgins, W P; Holmes, D F; Perkus, M; Mercer, S; Paoletti, E

1986-01-01

Repeated intradermal inoculations of calves with wild-type vaccinia virus and recombinant vaccinia viruses expressing human hepatitis B virus surface antigen and herpes simplex virus, type 1, glycoprotein D produced characteristic pox lesions at each site of injection. In some instances, calves were inoculated as many as five times at intervals from 4 to 7 weeks. The lesions invariably were more severe after the second inoculation. Subsequent inoculations produced a less severe area of redness, swelling, necrosis, and scab formation. No other signs of illness, such as an elevation in temperature, were noted in the calves. Vaccinia virus was isolated in low titers from scabs taken at various times after inoculation. No lesions were formed at the sites injected with tissue culture fluid and cellular debris at the same time that virus inoculations were made. Calf contact controls remained normal through the 8-week exposure in isolation units with calves inoculated twice with vaccinia virus. No neutralizing antibody to vaccinia virus was detected in the contact controls. In contrast, the virus-inoculated calves developed neutralizing antibody to vaccinia virus and to herpes simplex virus glycoprotein D in serum. In all cattle, a second inoculation significantly enhanced the neutralizing antibody response within 1 week, suggesting that an anamnestic response had occurred. No antibody to hepatitis B virus surface antigen was elicited in calves after repeated inoculations with vaccinia recombinants that express hepatitis B virus surface antigen and are known to elicit in rabbits antibodies reactive with hepatitis B virus surface antigen. Images PMID:3700615

Unraveling Haplotype Diversity of the Apical Membrane Antigen-1 Gene in Plasmodium falciparum Populations in Thailand

PubMed Central

Lumkul, Lalita; Sawaswong, Vorthon; Simpalipan, Phumin; Kaewthamasorn, Morakot; Harnyuttanakorn, Pongchai; Pattaradilokrat, Sittiporn

2018-01-01

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 (AMA-1) of the human malaria parasite Plasmodium falciparum, the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA-1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA-1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA-1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation (Fst indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA-1. A total of 31 unique AMA-1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA-1 haplotypes revealed multiple clades of AMA-1, each of which contained parasites of multiple geographical origins, consistent with the Fst indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand’s borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA-1-based malaria vaccines in the GMS. PMID:29742870

Hybrid biosynthetic gene therapy vector development and dual engineering capacity.

PubMed

Jones, Charles H; Ravikrishnan, Anitha; Chen, Mingfu; Reddinger, Ryan; Kamal Ahmadi, Mahmoud; Rane, Snehal; Hakansson, Anders P; Pfeifer, Blaine A

2014-08-26

Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration.

PubMed

Ma, Bing-cun; Yang, Xin; Wang, Hong-ning; Cao, Hai-peng; Xu, Peng-wei; Ding, Meng-die; Liu, Hui

2016-01-01

To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (∆TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.

Isolation, molecular cloning and in vitro expression of rhesus monkey (Macaca mulatta) prominin-1.s1 complementary DNA encoding a potential hematopoietic stem cell antigen.

PubMed

Husain, S M; Shou, Y; Sorrentino, B P; Handgretinger, R

2006-10-01

Human prominin-1 (CD133 or AC133) is an important cell surface marker used to isolate primitive hematopoietic stem cells. The commercially available antibody to human prominin-1 does not recognize rhesus prominin-1. Therefore, we isolated, cloned and characterized the complementary DNA (cDNA) of rhesus prominin-1 gene and determined its coding potential. Following the nomenclature of prominin family of genes, we named this cDNA as rhesus prominin-1.s1. The amino acid sequence data of the putative rhesus prominin-1.s1 could be used in designing antigenic peptides to raise antibodies for use in isolation of pure populations of rhesus prominin-1(+) hematopoietic cells. To the best of our knowledge, there has been no previously published report about the isolation of a prominin-1 cDNA from rhesus monkey (Macaca mulatta).

Major surface antigen p190 of Plasmodium falciparum: detection of common epitopes present in a variety of plasmodia isolates.

PubMed Central

Gentz, R; Certa, U; Takacs, B; Matile, H; Döbeli, H; Pink, R; Mackay, M; Bone, N; Scaife, J G

1988-01-01

Plasmodium falciparum merozoites are covered with polymorphic proteins that are processed from a 190 kd (p190) precursor protein. These are candidates for an antimalarial vaccine. We cloned and expressed a number of DNA fragments, comprising almost the entire p190 gene of the K1 isolate, in Escherichia coli. Pooled human endemic-area sera and rabbit antibodies raised against p190 protein isolated from K1 parasites react with only a limited number of the recombinant proteins. From these studies we could select two antigenic polypeptides containing conserved amino acid stretches of the otherwise highly polymorphic protein. Rabbits and mice injected with the purified recombinant proteins produce antibodies reacting differentially with various isolates of P. falciparum. We obtained antibodies detecting all isolates tested and a monoclonal antibody specific for isolates containing a K1 type allele of the p190 gene. Images PMID:2452082

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis

PubMed Central

Worth, Danielle; Huang, Sherri

2018-01-01

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host. PMID:29718996

Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis.

PubMed

Radke, Joshua B; Worth, Danielle; Hong, David; Huang, Sherri; Sullivan, William J; Wilson, Emma H; White, Michael W

2018-05-01

Tachyzoite to bradyzoite development in Toxoplasma is marked by major changes in gene expression resulting in a parasite that expresses a new repertoire of surface antigens hidden inside a modified parasitophorous vacuole called the tissue cyst. The factors that control this important life cycle transition are not well understood. Here we describe an important transcriptional repressor mechanism controlling bradyzoite differentiation that operates in the tachyzoite stage. The ApiAP2 factor, AP2IV-4, is a nuclear factor dynamically expressed in late S phase through mitosis/cytokinesis of the tachyzoite cell cycle. Remarkably, deletion of the AP2IV-4 locus resulted in the expression of a subset of bradyzoite-specific proteins in replicating tachyzoites that included tissue cyst wall components BPK1, MCP4, CST1 and the surface antigen SRS9. In the murine animal model, the mis-timing of bradyzoite antigens in tachyzoites lacking AP2IV-4 caused a potent inflammatory monocyte immune response that effectively eliminated this parasite and prevented tissue cyst formation in mouse brain tissue. Altogether, these results indicate that suppression of bradyzoite antigens by AP2IV-4 during acute infection is required for Toxoplasma to successfully establish a chronic infection in the immune-competent host.

[Construction of a plant effective expression vector containing the gene of hepatitis B virus surface antigen].

PubMed

Lin, Bing-Ying; Jin, Zhi-Qiang; Li, Mei

2006-11-01

To construct a plant effective expression vector driven by a fruit specific promoter for the expression of hepatitis B virus surface antigen (HBsAg), to further improve the expression of exogenous gene in plant, and to prepare for the development of an effective anti-hepatitis vaccine. Tomato fruit-specific promoters' gene 2A12 and E8 were respectively introduced to pBPFOmega7 to form pB2A12 and pBE8. The DNA fragment containing HBsAg-s gene from plasmid YEP-HBs was inserted respectively into pB2A12 and pBE8 to form pB2A12-HBs and pBE8-HBs. The fragment containing "p35S+2A12+Omega+HBsAg-s+Tnos" of the pB2A12-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the reconstructed plant binary expression plasmid pCAM2A12-HBs, and the fragment containing "p35S+E8+Omega+HBsAg-s+Tnos" of the pBE8-HBs was sub-cloned into plasmid pCAMBIA1301 to yield the plasmid pCAME8-HBs. The inserted gene HBsAg and fruit-specific promoters in the reconstructed plant binary expression vectors were confirmed by sequencing. Then, pCAM2A12-HBs and pCAME8-HBs were directly introduced into Agrobacterium tumefaciens strain EHA105. Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments, and the sequencing results were confirmed correct. In this study, plant expression vector containing HBsAg gene driven by fruit specific promoter and CaMV35s promoter was successfully constructed.

Vaccination of domestic animals with a novel oral vaccine prevents Giardia infections, alleviates signs of giardiasis and reduces transmission to humans

PubMed Central

Serradell, Marianela C; Saura, Alicia; Rupil, Lucia L; Gargantini, Pablo R; Faya, Marcela I; Furlan, Paulina J; Lujan, Hugo D

2016-01-01

Giardia lamblia is a human intestinal parasite and one of the most frequent enteric pathogen of companion animals. Clinical manifestations of giardiasis, such as diarrhoea, anorexia, weight loss and lethargy, have been associated with Giardia infections in both domestic and farm animals. A few anti-parasitic drugs are routinely used to treat giardiasis, but re-infections are common and drug-resistant strains have already been reported. Unfortunately, efficient vaccines against Giardia are not available. Giardia undergoes antigenic variation; through this mechanism, parasites can avoid the host’s immune defenses, causing chronic infections and/or re-infections. Antigenic variation is characterised by a continuous switch in the expression of members of a homologous family of genes encoding surface antigens. In a previous report, we indicated that in Giardia, the mechanism responsible for the exchange of variant-specific surface proteins (VSPs) involves the RNA interference (RNAi) pathway. From a repertoire of ~200 VSP genes, only one is expressed on the surface of single trophozoites; however, RNAi machinery disruption generates trophozoites that express the complete VSP repertoire. We also demonstrated that gerbils orally immunised with VSPs isolated from these altered parasites showed high levels of protection. Here we tested this vaccine in cats and dogs, and found that it is highly efficient in preventing new infections and reducing chronic giardiasis in domestic animals both in experimental and natural infections. Remarkably, immunisation of dogs in a highly endemic area strongly decreased the percentage of infected children in the community, suggesting that this vaccine would block the zoonotic transmission of the disease. PMID:29263857

Antigen Presentation by Individually Transferred HLA Class I Genes in HLA-A, HLA-B, HLA-C Null Human Cell Line Generated Using the Multiplex CRISPR-Cas9 System.

PubMed

Hong, Cheol-Hwa; Sohn, Hyun-Jung; Lee, Hyun-Joo; Cho, Hyun-Il; Kim, Tai-Gyu

Human leukocyte antigens (HLAs) are essential immune molecules that affect transplantation and adoptive immunotherapy. When hematopoietic stem cells or organs are transplanted with HLA-mismatched recipients, graft-versus-host disease or graft rejection can be induced by allogeneic immune responses. The function of each HLA allele has been studied using HLA-deficient cells generated from mutant cell lines or by RNA interference, zinc finger nuclease, and the CRISPR/Cas9 system. To improve HLA gene editing, we attempted to generate an HLA class I null cell line using the multiplex CRISPR/Cas9 system by targeting exons 2 and 3 of HLA-A, HLA-B, and HLA-C genes simultaneously. Multiplex HLA editing could induce the complete elimination of HLA class I genes by bi-allelic gene disruption on target sites which was defined by flow cytometry and target-specific polymerase chain reaction. Furthermore, artificial antigen-presenting cells were generated by transfer of a single HLA class I allele and co-stimulatory molecules into this novel HLA class I null cell line. Artificial antigen-presenting cells showed HLA-restricted antigen presentation following antigen processing and were successfully used for the efficient generation of tumor antigen-specific cytotoxic T cells in vitro. The efficient editing of HLA genes may provide a basis for universal cellular therapies and transplantation.

Cytoadherence and Genotype of Plasmodium falciparum Strains from Symptomatic Children in Franceville, Southeastern Gabon

PubMed Central

Touré, Fousseyni S.; Ouwe-Missi-Oukem-Boyer, Odile; Mezui-Me-Ndong, Jérôme; Ndong-Atome, Guy Roger; Bisvigou, Ulrick; Mazier, Dominique; Bisser, Sylvie

2007-01-01

Background: Plasmodium falciparum causes severe clinical manifestations by sequestering parasitized red blood cells (PRBC) in the microvasculature of major organs such as the brain. This sequestration results from PRBC adherence to vascular endothelial cells via erythrocyte membrane protein 1, a variant parasite surface antigen. Objective: To determine whether P. falciparum multiple genotype infection (MGI) is associated with stronger PRBC cytoadherence and greater clinical severity. Methods: Nested polymerase chain reaction was used to genotype P. falciparum isolates from symptomatic children and to distinguish between single genotype infection (SGI) and MGI. PRBC cytoadhesion was studied with cultured human lung endothelial cells. Results: Analysis of two highly polymorphic regions of the merozoite surface antigen (MSP)-1 and MSP-2 genes and a dimorphic region of the erythrocyte binding antigen-175 gene showed that 21.4% and 78.6% of the 42 children had SGI and MGI, respectively. It also showed that 37 (89%) of the 42 PRBC samples expressed MSP-1 allelic family K1. Cytoadherence values ranged from 58 to 1811 PRBC/mm2 of human lung endothelial cells monolayer in SGI and from 5 to 5744 PRBC/mm2 in MGI. MGI was not associated with higher cytoadherence values or with more severe malaria. Conclusions: These results suggested that infection of the same individual by multiple clones of P. falciparum does not significantly influence PRBC cytoadherence or disease severity and confirmed the predominance of the MSP-1 K1 genotype in southeastern Gabon. PMID:17607045

The O-antigen structure of bacterium Comamonas aquatica CJG.

PubMed

Wang, Xiqian; Kondakova, Anna N; Zhu, Yutong; Knirel, Yuriy A; Han, Aidong

2017-11-01

Genus Comamonas is a group of bacteria that are able to degrade a variety of environmental waste. Comamonas aquatica CJG (C. aquatica) in this genus is able to absorb low-density lipoprotein but not high-density lipoprotein of human serum. Using 1 H and 13 C NMR spectroscopy, we found that the O-polysaccharide (O-antigen) of this bacterium is comprised of a disaccharide repeat (O-unit) of d-glucose and 2-O-acetyl-l-rhamnose, which is shared by Serratia marcescens O6. The O-antigen gene cluster of C. aquatica, which is located between coaX and tnp4 genes, contains rhamnose synthesis genes, glycosyl and acetyl transferase genes, and ATP-binding cassette transporter genes, and therefore is consistent with the O-antigen structure determined here.

Immune response elicited by two rBCG strains devoid of genes involved in c-di-GMP metabolism affect protection versus challenge with M. tuberculosis strains of different virulence.

PubMed

Segura-Cerda, Cristian Alfredo; Aceves-Sánchez, Michel de Jesús; Marquina-Castillo, Brenda; Mata-Espinoza, Dulce; Barrios-Payán, Jorge; Vega-Domínguez, Perla Jazmín; Pedroza-Roldán, César; Bravo-Madrigal, Jorge; Vallejo-Cardona, Alba Adriana; Hernández-Pando, Rogelio; Flores-Valdez, Mario Alberto

2018-04-12

Pellicles, a type of biofilm, have gathered a renewed interest in the field of tuberculosis as a structure that mimics some characteristics occurring during M. tuberculosis infection, such as antibiotic recalcitrance and chronicity of infection, and as a source of antigens for humoral response in infected guinea pigs. In other bacteria, it has been well documented that the second messenger c-di-GMP modulates the transition from planktonic cells to biofilm formation. In this work, we used the live vaccine Mycobacterium bovis BCG to determine whether deletion of genes involved in c-di-GMP metabolism would affect interaction with macrophages, capacity to induce immune response in a murine cell line and mice, and how the protein profile was modified when grown as surface pellicles. We found that deletion of the BCG1419c (Delta c-di-GMP phosphodiesterase, ΔPDE) gene, or deletion of the BCG1416c (Delta c-di-GMP diguanylate cyclase, ΔDGC) gene, altered production of TNF-α, IL-6, and IL-1β, in murine macrophages, and resulted in attenuation in intra-macrophage replication. Moreover, in addition to the improved immunogenicity of the BCGΔBCG1419c mutant already reported, deletion of the BCG1416c gene leads to increased T CD4 + and T CD8 + activation. This correlated with protection versus lethality in mice infected with the highly virulent M. tuberculosis 5186 afforded by vaccination with all the tested BCG strains, and controlled the growth of the mildly virulent M. tuberculosis H37Rv in lungs by vaccination with BCGΔBCG1419c during chronic late infection from 4 to 6 months after challenge. Furthermore, when grown as surface pellicles, a condition used to manufacture BCG vaccine, in comparison to BCG wild type, both rBCGs changed expression of antigenic proteins such as DnaK, HbhA, PstS2, 35KDa antigen, GroEL2, as well as AcpM, a protein involved in synthesis of mycolic acids, molecules relevant to modulate inflammatory responses. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response to P. falciparum Infected Erythrocytes among Asymptomatic Children in Sudan.

PubMed

Mohamed, Sara N; Hassan, Dina A; El Hussein, Abdelrahim M; Osman, Ihssan M; Ibrahim, Muntasir E; Mohamed, Hiba S; Nour, Bakri Y; Abdulhadi, Nasreldin H

2016-01-01

Background. The most prominent variant surface antigens (VSAs) of Plasmodium falciparum are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which serves as a parasite-sequestering ligand to endothelial cells. In this study we have examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes' surface antigens using flow cytometry. Methods. Ethidium-bromide-labelled erythrocytic mature forms of P. falciparum parasites obtained from symptomatic and asymptomatic children were sequentially incubated with autologous plasma and fluorescein isothiocyanate-conjugated (FITC) antihuman IgG. Plasma antibody reactivity was detected by flow cytometry. Results. Asymptomatic children had more prevalence of trophozoites in peripheral blood (66%) compared to symptomatic children (16%), p = 0.002. The mean percentage of infected RBCs reacting with autologous sera was 89.78 among symptomatic children compared to 79.62 among asymptomatic children (p = 0.09). Moreover, the mean fluorescence intensity (MFI) in the asymptomatic was significantly higher compared to symptomatic children (p value = 0.040). Conclusion. Variant surface antigens on Plasmodium falciparum infected RBCs from symptomatic malaria children tend to be better recognized by IgG antibodies. This may suggest a role of some IgG antibodies in severity of malaria.

Antigenic evaluation of a recombinant baculovirus-expressed Sarcocystis neurona SAG1 antigen.

PubMed

Gupta, G D; Lakritz, J; Saville, W J; Livingston, R S; Dubey, J P; Middleton, J R; Marsh, A E

2004-10-01

Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neospora spp., Hammondia spp., and Toxoplasma gondii. Therefore, there is a need to develop an improved method for the detection of S. neurona-specific antibodies. The sera of infected horses react strongly to surface antigen 1 (SnSAG1), an approximately 29-kDa protein, in immunoblot analysis, suggesting that it is an immunodominant antigen. The SnSAG1 gene of S. neurona was cloned, and recombinant S. neurona SAG1 protein (rSnSAG1-Bac) was expressed with the use of a baculovirus system. By immunoblot analysis, the rSnSAG1-Bac antigen detected antibodies to S. neurona from naturally infected and experimentally inoculated equids, cats, rabbit, mice, and skunk. This is the first report of a baculovirus-expressed recombinant S. neurona antigen being used to detect anti-S. neurona antibodies in a variety of host species.

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

21 CFR 660.40 - Hepatitis B Surface Antigen.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Hepatitis B Surface Antigen. 660.40 Section 660.40...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.40 Hepatitis B Surface Antigen. (a) Proper name and definition. The proper name of this product...

Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

PubMed Central

Cestari, Igor; Stuart, Ken

2015-01-01

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, L.H.; Calabi, F.; Lefebvre, F.A.

1987-12-01

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have not been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure ofmore » CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the ..beta../sub 2/-microglobulin-binding-domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.« less

Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71

PubMed Central

Varma, Nadimpalli Ravi S.; Toosa, Haryanti; Foo, Hooi Ling; Alitheen, Noorjahan Banu Mohamed; Nor Shamsudin, Mariana; Arbab, Ali S.; Yusoff, Khatijah; Abdul Rahim, Raha

2013-01-01

In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle. PMID:23476790

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus.

PubMed

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity.

A new Tla region antigen Qa-11, similar to Qa-2 and associated with B-type beta 2-microglobulin.

PubMed

van de Meugheuvel, W; van Seventer, G; Demant, P

1985-04-01

A new antigen, Qa-11, is detected as a 40,000 dalton band in the SDS-PAGE of immunoprecipitates of radiolabeled lymphocyte membrane preparations. In C57BL H-2 congenic strains, its presence is controlled by a gene in the Tla region. In strains with genetic background other than C57BL it is not expressed. Tests with recombinant inbred strains and with H-3 congenic strains show that, in addition to the Tla region, a gene linked to or identical with the beta 2-microglobulin-b-allele is required for the expression of Qa-11 as well. The mobility of the Qa-11 antigen in SDS-PAGE and in isoelectrofocusing is the same as that of Qa-2 antigen. The Cleveland peptide maps of Qa-2 and Qa-11 are identical as well. This finding, that the Tla region controlled Qa-11 antigen is structurally very similar to the Qa-2 antigen, contrasts with the fact that Tla region products do not react with anti-Qa-2 sera. This paradox could be explained by a separate Qa-11 region between Qa-2 and Tla. Alternatively, it is possible that the Qa-11 antigen is the result of the action of a modifying gene in the Tla region upon a Qa-2 gene product, or that the structural gene for Qa-11 is located in the Qa-2 region and a Tla region gene controls its expression.

Embryonal carcinoma antigen and the T/t locus of the mouse.

PubMed Central

Kemler, R; Babinet, C; Condamine, H; Gachelin, G; Guenet, J L; Jacob, F

1976-01-01

The presence of the F9 antigen and of four other antigens related to the T/t locus of the mouse was investigated by immunofluorescence on preimplantation embryos. In morulae heterozygous for any of these t haplotypes, both the appropriate t antigen and the F9 antigen are expressed. The F9 antigen segregates among the progeny of crosses producing embryos homozygous for some (tw32 and tw5) but not for other haplotypes. It is concluded that (i) whatever the time of action of a t haplotype, its corresponding antigen is expressed during cleavage and (ii) the F9 antigen is specified by a gene(s) in the region of the T/t locus. Images PMID:1069295

A critical examination of the numerology of antigen-binding cells: evidence for multiple receptor specificities on single cells.

PubMed

Miller, A

1977-01-01

The data available from other laboratories as well as our own on the frequency of cells recognizing major histocompatibility antigens or conventional protein and hapten antigens is critically evaluated. The frequency of specific binding for a large number of antigens is sufficiently high to support the idea that at least part of the antigen-binding cell population must have multiple specificities. Our results suggest that these multiple specific cells result from single cells synthesizing and displaying as many as 50-100 species of receptor, each at a frequency of 10(4) per cell. A model involving gene expansion of constant-region genes is suggested and some auxilliary evidence consistent with such C-gene expansion is presented.

Statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza A viruses.

PubMed Central

Ina, Y; Gojobori, T

1994-01-01

To examine whether positive selection operates on the hemagglutinin 1 (HA1) gene of human influenza A viruses (H1 subtype), 21 nucleotide sequences of the HA1 gene were statistically analyzed. The nucleotide sequences were divided into antigenic and nonantigenic sites. The nucleotide diversities for antigenic and nonantigenic sites of the HA1 gene were computed at synonymous and nonsynonymous sites separately. For nonantigenic sites, the nucleotide diversities were larger at synonymous sites than at nonsynonymous sites. This is consistent with the neutral theory of molecular evolution. For antigenic sites, however, the nucleotide diversities at nonsynonymous sites were larger than those at synonymous sites. These results suggest that positive selection operates on antigenic sites of the HA1 gene of human influenza A viruses (H1 subtype). PMID:8078892

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

PubMed Central

Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

2012-01-01

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

PubMed

Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

2017-02-16

Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.

PubMed

Bryant, Jessica M; Regnault, Clément; Scheidig-Benatar, Christine; Baumgarten, Sebastian; Guizetti, Julien; Scherf, Artur

2017-07-11

Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression. Copyright © 2017 Bryant et al.

Rhesus Cytomegalovirus Contains Functional Homologues of US2, US3, US6, and US11

PubMed Central

Pande, Nupur T.; Powers, Colin; Ahn, Kwangseog; Früh, Klaus

2005-01-01

Human cytomegalovirus (HCMV) is a paradigm for mechanisms subverting antigen presentation by major histocompatibility complex (MHC) molecules. Due to its limited host range, HCMV cannot be studied in animals. Thus, the in vivo importance of inhibiting antigen presentation for the establishment and maintenance of infection with HCMV is unknown. Rhesus cytomegalovirus (RhCMV) is an emerging animal model that shares many of the features of HCMV infection. The recent completion of the genomic sequence of RhCMV revealed a significant degree of homology to HCMV. Strikingly, RhCMV contains several genes with low homology to the HCMV US6 gene family of inhibitors of the MHC I antigen presentation pathway. Here, we examine whether the RhCMV US6 homologues (open reading frames Rh182, -184, -185, -186, -187, and -189) interfere with the MHC I antigen-processing pathway. We demonstrate that Rh182 and Rh189 function similarly to HCMV US2 and US11, respectively, mediating the proteasomal degradation of newly synthesized MHC I. The US3 homologue, Rh184, delayed MHC I maturation. Unlike US3, MHC I molecules eventually escaped retention by Rh184, so that steady-state surface levels of MHC I remained unchanged. Rh185 acted similarly to US6 and inhibited peptide transport by TAP and, consequently, peptide loading of MHC I molecules. Thus, despite relatively low sequence conservation, US6 family-related genes in RhCMV are functionally closely related to the conserved structural features of HCMV immunomodulators. The conservation of these mechanisms implies their importance for immune evasion in vivo, a question that can now be addressed experimentally. PMID:15827193

Interleukin-1 and cutaneous inflammation: a crucial link between innate and acquired immunity.

PubMed

Murphy, J E; Robert, C; Kupper, T S

2000-03-01

As our primary interface with the environment, the skin is constantly subjected to injury and invasion by pathogens. The fundamental force driving the evolution of the immune system has been the need to protect the host against overwhelming infection. The ability of T and B cells to recombine antigen receptor genes during development provides an efficient, flexible, and powerful immune system with nearly unlimited specificity for antigen. The capacity to expand subsets of antigen-specific lymphocytes that become activated by environmental antigens (memory response) is termed "acquired" immunity. Immunologic memory, although a fundamental aspect of mammalian biology, is a relatively recent evolutionary event that permits organisms to live for years to decades. "Innate" immunity, mediated by genes that remain in germ line conformation and encode for proteins that recognize conserved structural patterns on microorganisms, is a much more ancient system of host defense. Defensins and other antimicrobial peptides, complement and opsonins, and endocytic receptors are all considered components of the innate immune system. None of these, however, are signal-transducing receptors. Most recently, a large family of cell surface receptors that mediate signaling through the NF-kappaB transcription factor has been identified. This family of proteins shares striking homology with plant and Drosophila genes that mediate innate immunity. In mammals, this family includes the type I interleukin-1 receptor, the interleukin-18 receptor, and a growing family of Toll-like receptors, two of which were recently identified as signal-transducing receptors for bacterial endotoxin. In this review, we discuss how interleukin-1 links the innate and acquired immune systems to provide synergistic host defense activities in skin.

Effects of Trx2p and Sec23p expression on stable production of hepatitis B surface antigen S domain in recombinant Saccharomyces cerevisiae.

PubMed

Park, Young-Kyoung; Jung, Sang-Min; Lim, Hyung-Kwon; Son, Young-Jin; Park, Yong-Cheol; Seo, Jin-Ho

2012-08-31

The S domain of hepatitis B virus surface antigen (sHBsAg) is the primary component for vaccine development against virus infection. For stable expression of sHBsAg in recombinant Saccharomyces cerevisiae, new accessory genes necessary for foreign protein expression were screened by DNA microarray. Among 600 genes of interest, genes coding for an activating protein of ATPase in Hsp90 (Aha1p), S. cerevisiae DnaJ (Scj1p), thioredoxin 2 (Trx2p) and a GTPase-activator specific for Sar1 (Sec23p) as well as Pdi1p were selected in transcriptome analysis, which are known to facilitate disulfide bond formation or induce protein transport in the secretion pathway. Individual and combinatorial expression of SEC23, TRX2 and PDI1 increased total sHBsAg concentration by 1.9-6.5-fold, relative to the control strain expressing sHBsAg only. Additionally, moderate expression of Kex2p protease able to cut off the signal peptide enhanced the portion of the authentic sHBsAg to total sHBsAg. Fed-batch fermentation of the S. cerevisiae 2805 strain coexpressing the sHBsAg, SEC23, PDI1 and KEX2 genes resulted in 70.6mg/L final sHBsAg concentration which was 5.6 times higher than that of the control. Transmission electron microscopic analysis of the yeast cells elucidated the effects of the accessory gene coexpression on the intracellular localization of sHBsAg. Like PDI1, coexpression of both SEC23 and/or TRX2 newly isolated in this study is expected to improve the target protein expression in S. cerevisiae. Copyright © 2012 Elsevier B.V. All rights reserved.

Protective Immunity Against a Lethal Respiratory Yersinia pestis Challenge Induced by V Antigen or the F1 Capsular Antigen Incorporated into Adenovirus Capsid

PubMed Central

Boyer, Julie L.; Sofer-Podesta, Carolina; Ang, John; Hackett, Neil R.; Chiuchiolo, Maria J.; Senina, Svetlana; Perlin, David

2010-01-01

Abstract The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1–, E3– serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime–boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective immune responses than equivalent recombinant protein-based subunit vaccines administered with conventional adjuvant, suggesting that F1-and/or V-modified capsid Ad-based recombinant vaccines should be considered for development as anti-plague vaccines. PMID:20180652

Identification, expression and phylogenetic analysis of EgG1Y162 from Echinococcus granulosus

PubMed Central

Zhang, Fengbo; Ma, Xiumin; Zhu, Yuejie; Wang, Hongying; Liu, Xianfei; Zhu, Min; Ma, Haimei; Wen, Hao; Fan, Haining; Ding, Jianbing

2014-01-01

Objective: This study was to clone, identify and analyze the characteristics of egG1Y162 gene from Echinococcus granulosus. Methods: Genomic DNA and total RNAs were extracted from four different developmental stages of protoscolex, germinal layer, adult and egg of Echinococcus granulosus, respectively. Fluorescent quantitative PCR was used for analyzing the expression of egG1Y162 gene. Prokaryotic expression plasmid of pET41a-EgG1Y162 was constructed to express recombinant His-EgG1Y162 antigen. Western blot analysis was performed to detect antigenicity of EgG1Y162 antigen. Gene sequence, amino acid alignment and phylogenetic tree of EgG1Y162 were analyzed by BLAST, online Spidey and MEGA4 software, respectively. Results: EgG1Y162 gene was expressed in four developmental stages of Echinococcus granulosus. And, egG1Y162 gene expression was the highest in the adult stage, with the relative value of 19.526, significantly higher than other three stages. Additionally, Western blot analysis revealed that EgG1Y162 recombinant protein had good reaction with serum samples from Echinococcus granulosus infected human and dog. Moreover, EgG1Y162 antigen was phylogenetically closest to EmY162 antigen, with the similarity over 90%. Conclusion: Our study identified EgG1Y162 antigen in Echinococcus granulosus for the first time. EgG1Y162 antigen had a high similarity with EmY162 antigen, with the genetic differences mainly existing in the intron region. And, EgG1Y162 recombinant protein showed good antigenicity. PMID:25337206

[Changes in Cell Surface Properties and Biofilm Formation Efficiency in Azospirillum brasilense Sp245 Mutants in the Putative Genes of Lipid Metabolism mmsB1 and fabG1].

PubMed

Shumilova, E; Shelud'ko, A V; Filip'echeva, Yu A; Evstigneeva, S S; Ponomareva, E G; Petrova, L P; Katsy, E I

2016-01-01

The previously obtained insertion mutants ofAzospirillum brasilense Sp245 in the genes mmsBl and fabG1 (strains SK039 and Sp245.1610, respectively) were characterized by impaired flagellation and motility. The putative products of expression of these genes are 3-hydroxyisobutyrate dehydrogenase and 3-oxoacyl-[acyl-carrier protein] reductase, respectively. In the present work, A. brasilense- Sp245 strains SK039 and Sp245.1610 were found to have differences in the content of 3-hydroxyhexadecanoic, hexadecanoic, 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, and nonadecanoic acids in their lipopolysaccharide prepa- rations, as well as in cell hydrophobicity and hemagglutination activity and dynamics of cell aggregation, in biomass amount, and in the relative content of lipopolysaccharide antigens in mature biofilms formed on hydrophilic or hydrophobic surfaces.

Analysis of peroxidase-negative acute unclassifiable leukemias by monoclonal antibodies. 1. Acute myelogenous leukemia and acute myelomonocytic leukemia.

PubMed

Imamura, N; Tanaka, R; Kajihara, H; Kuramoto, A

1988-11-01

In this study, pretreatment peripheral and/or bone marrow blasts from 12 patients with acute unclassifiable leukemia (AUL) expressing the myeloid-related cell-surface antigen (CD 11) were isolated for further analysis. Despite a lack of myeloperoxidase (MPO) activity, 1 patient's blasts contained cytoplasmic Auer rods. The circulating blasts from another patient expressed MPO while maintaining the same surface phenotype during 20 months of clinical follow-up. In addition, the blasts from 3 cases demonstrated both myelomonocytic and monocyte-specific surface antigens, whereas the remaining 9 cases completely lacked any monocyte-specific antigen detectable by monoclonal antibodies, Mo2, My4 and Leu M3 (CD 14). The first case eventually was diagnosed as acute myelomonocytic leukemia and the second as acute myelogenous leukemia by means of immunophenotypic analysis using flow cytometry (FACS IV). In addition, the presence of MPO protein was identified in the cytoplasm of blast cells from 5 patients with AUL by means of a cytoplasmic immunofluorescence test using a monoclonal antibody (MA1). Our study indicates that non-T, non-B AUL expressing OKM1 (CD 11) antigens include acute leukemias which are unequivocally identifiable as being of either myeloid or myelomonocytic origin. However, further investigations, including immunophenotypic and cytoplasmic analysis, ultrastructural cytochemistry and gene analysis with molecular probes (tests applicable to normal myeloid cells), are necessary in order to determine the actual origin of blasts and to recognize the differentiation stages of the various types of leukemic cells from patients with undifferentiated forms of leukemia.

Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer1

PubMed Central

Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

2007-01-01

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informativemodel for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction. PMID:18030364

Enhanced expression of interferon-gamma-induced antigen-processing machinery components in a spontaneously occurring cancer.

PubMed

Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

2007-11-01

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informative model for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction.

In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: A strategy for efficient display of large full-length proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shivachandra, Sathish B.; Rao, Mangala; Janosi, Laszlo

2006-02-05

An in vitro binding system is described to display large full-length proteins on bacteriophage T4 capsid surface at high density. The phage T4 icosahedral capsid features 155 copies of a nonessential highly antigenic outer capsid protein, Hoc, at the center of each major capsid protein hexon. Gene fusions were engineered to express the 83-kDa protective antigen (PA) from Bacillus anthracis fused to the N-terminus of Hoc and the 130-kDa PA-Hoc protein was expressed in Escherichia coli and purified. The purified PA-Hoc was assembled in vitro on hoc {sup -} phage particles. Binding was specific, stable, and of high affinity. Thismore » defined in vitro system allowed manipulation of the copy number of displayed PA and imposed no significant limitation on the size of the displayed antigen. In contrast to in vivo display systems, the in vitro approach allows all the capsid binding sites to be occupied by the 130-kDa PA-Hoc fusion protein. The PA-T4 particles were immunogenic in mice in the absence of an adjuvant, eliciting strong PA-specific antibodies and anthrax lethal toxin neutralizing antibodies. The in vitro display on phage T4 offers a novel platform for potential construction of customized vaccines against anthrax and other infectious diseases.« less

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

NASA Astrophysics Data System (ADS)

Yang, Lili; Baltimore, David

2005-03-01

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

Conservation of myeloid surface antigens on primate granulocytes.

PubMed

Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

1983-02-01

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

Genetics and evolution of Yersinia pseudotuberculosis O-specific polysaccharides: a novel pattern of O-antigen diversity

PubMed Central

Kenyon, Johanna J.; Cunneen, Monica M.

2017-01-01

Abstract O-antigen polysaccharide is a major immunogenic feature of the lipopolysaccharide of Gram-negative bacteria, and most species produce a large variety of forms that differ substantially from one another. There are 18 known O-antigen forms in the Yersinia pseudotuberculosis complex, which are typical in being composed of multiple copies of a short oligosaccharide called an O unit. The O-antigen gene clusters are located between the hemH and gsk genes, and are atypical as 15 of them are closely related, each having one of five downstream gene modules for alternative main-chain synthesis, and one of seven upstream modules for alternative side-branch sugar synthesis. As a result, many of the genes are in more than one gene cluster. The gene order in each module is such that, in general, the earlier a gene product functions in O-unit synthesis, the closer the gene is to the 5΄ end for side-branch modules or the 3΄ end for main-chain modules. We propose a model whereby natural selection could generate the observed pattern in gene order, a pattern that has also been observed in other species. PMID:28364730

Cloning and Expression of Genes for Dengue Virus Type-2 Encoded-Antigens for Rapid Diagnosis and Vaccine Development

DTIC Science & Technology

1988-10-31

00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...11. TITLE (include Security Classification) Cloning and Expression of Genes f or Dengue Virus Type 2 Fncoded Antigens for Rapid Diagnosis and Vaccine ...epidemics in Central and South Americas and the Caribbean is a cause of major concern. An effective vaccine is not available to protect individuals

Characterization of two in vivo-expressed methyltransferases of the Mycobacterium tuberculosis complex: antigenicity and genetic regulation

PubMed Central

Golby, Paul; Nunez, Javier; Cockle, Paul J.; Ewer, Katie; Logan, Karen; Hogarth, Philip; Vordermeier, H. Martin; Hinds, Jason; Hewinson, R. Glyn; Gordon, Stephen V.

2011-01-01

Genome sequencing of Mycobacterium tuberculosis complex members has accelerated the search for new disease-control tools. Antigen mining is one area that has benefited enormously from access to genome data. As part of an ongoing antigen mining programme, we screened genes that were previously identified by transcriptome analysis as upregulated in response to an in vitro acid shock for their in vivo expression profile and antigenicity. We show that the genes encoding two methyltransferases, Mb1438c/Rv1403c and Mb1440c/Rv1404c, were highly upregulated in a mouse model of infection, and were antigenic in M. bovis-infected cattle. As the genes encoding these antigens were highly upregulated in vivo, we sought to define their genetic regulation. A mutant was constructed that was deleted for their putative regulator, Mb1439/Rv1404; loss of the regulator led to increased expression of the flanking methyltransferases and a defined set of distal genes. This work has therefore generated both applied and fundamental outputs, with the description of novel mycobacterial antigens that can now be moved into field trials, but also with the description of a regulatory network that is responsive to both in vivo and in vitro stimuli. PMID:18375799

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

PubMed

Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul

2012-09-01

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes.

PubMed

Feder-Mengus, C; Ghosh, S; Weber, W P; Wyler, S; Zajac, P; Terracciano, L; Oertli, D; Heberer, M; Martin, I; Spagnoli, G C; Reschner, A

2007-04-10

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A(*)0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A(*)0201+, TAA+) and NA8 (HLA-A(*)0201+, TAA-) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-gamma) production by HLA-A(*)0201-restricted Melan-A/MART-1(27-35) or gp 100(280-288)-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-gamma production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

PubMed

Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

2017-09-02

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

A Novel Protective Vaccine Antigen from the Core Escherichia coli Genome

PubMed Central

Moriel, Danilo G.; Tan, Lendl; Goh, Kelvin G. K.; Ipe, Deepak S.; Lo, Alvin W.; Peters, Kate M.

2016-01-01

ABSTRACT Escherichia coli is a versatile pathogen capable of causing intestinal and extraintestinal infections that result in a huge burden of global human disease. The diversity of E. coli is reflected by its multiple different pathotypes and mosaic genome composition. E. coli strains are also a major driver of antibiotic resistance, emphasizing the urgent need for new treatment and prevention measures. Here, we used a large data set comprising 1,700 draft and complete genomes to define the core and accessory genome of E. coli and demonstrated the overlapping relationship between strains from different pathotypes. In combination with proteomic investigation, this analysis revealed core genes that encode surface-exposed or secreted proteins that represent potential broad-coverage vaccine antigens. One of these antigens, YncE, was characterized as a conserved immunogenic antigen able to protect against acute systemic infection in mice after vaccination. Overall, this work provides a genomic blueprint for future analyses of conserved and accessory E. coli genes. The work also identified YncE as a novel antigen that could be exploited in the development of a vaccine against all pathogenic E. coli strains—an important direction given the high global incidence of infections caused by multidrug-resistant strains for which there are few effective antibiotics. IMPORTANCE E. coli is a multifaceted pathogen of major significance to global human health and an important contributor to increasing antibiotic resistance. Given the paucity of therapies still effective against multidrug-resistant pathogenic E. coli strains, novel treatment and prevention strategies are urgently required. In this study, we defined the core and accessory components of the E. coli genome by examining a large collection of draft and completely sequenced strains available from public databases. This data set was mined by employing a reverse-vaccinology approach in combination with proteomics to identify putative broadly protective vaccine antigens. One such antigen was identified that was highly immunogenic and induced protection in a mouse model of bacteremia. Overall, our study provides a genomic and proteomic framework for the selection of novel vaccine antigens that could mediate broad protection against pathogenic E. coli. PMID:27904885

Expression of mouse Tla region class I genes in tissues enriched for gamma delta cells.

PubMed

Eghtesady, P; Brorson, K A; Cheroutre, H; Tigelaar, R E; Hood, L; Kronenberg, M

1992-01-01

The Tla region of the BALB/c mouse major histocompatibility complex contains at least 20 class I genes. The function of the products of these genes is unknown, but recent evidence demonstrates that some Tla region gene products could be involved in presentation of antigens to gamma delta T cells. We have generated a set of polymerase chain reaction (PCR) oligonucleotide primers and hybridization probes that permit us to specifically amplify and detect expression of 11 of the 20 BALB/c Tla region genes. cDNA prepared from 12 adult and fetal tissues and from seven cell lines was analyzed. In some cases, northern blot analysis or staining with monoclonal antibodies specific for the Tla-encoded thymus leukemia (TL) antigen were used to confirm the expression pattern of several of the genes as determined by PCR. Some Tla region genes, such as T24d and the members of the T10d/T22d gene pair, are expressed in a wide variety of tissues in a manner similar to the class I transplantation antigens. The members of the TL antigen encoding gene pair, T3d/T18d, are expressed in only a limited number of organs, including several sites enriched for gamma delta T cells. Other Tla region genes, including T1d, T2d, T16d, and T17d, are transcriptionally silent and transcripts from the T8d/T20d gene pair do not undergo proper splicing. In general, sites that contain gamma delta T lymphocytes have Tla region transcripts. The newly identified pattern of expression of the genes analyzed in sites containing gamma delta T cells further extends the list of potential candidates for antigen presentation to gamma delta T cells.

Mapping epitopes and antigenicity by site-directed masking

NASA Astrophysics Data System (ADS)

Paus, Didrik; Winter, Greg

2006-06-01

Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets*

PubMed Central

Rhoden, John J.; Dyas, Gregory L.

2016-01-01

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. PMID:27022022

Enhancement of Poly(orthoester) Microspheres for DNA Vaccine Delivery by Blending with Poly(ethylenimine)

PubMed Central

Nguyen, David N.; Raghavan, Shyam S.; Tashima, Lauren M.; Lin, Elizabeth C.; Fredette, Stephen J.; Langer, Robert S.; Wang, Chun

2008-01-01

Poly(ortho ester) (POE) microspheres have been previously shown to possess certain advantages for the in vivo delivery of DNA vaccines. In particular, timing of DNA release from POE microspheres in response to acidic phagosomal pH was shown to be an important factor in determining immunogenicity, which was hypothesized to be linked to the natural progression of antigen presenting cell uptake, transfection, maturation, and antigen presentation. Here we report in vitro characterization of the enhanced the efficacy of POE microspheres by blending poly(ethylenimine) (PEI), a well-characterized cationic transfection agent, into the POE matrix. Blending of a tiny amount of PEI (approximately 0.04 wt%) with POE caused large alterations in POE microsphere properties. PEI provided greater control over the rate of pH-triggered DNA release by doubling the total release time of plasmid DNA and enhanced gene transfection efficiency of the microspheres up to 50-fold without any significant cytotoxicity. Confocal microscopy with labeled PEI and DNA plasmids revealed that PEI caused a surface-localizing distribution of DNA and PEI within the POE microsphere as well as focal co-localization of PEI with DNA. We provide evidence that upon degradation, the microspheres of POE-PEI blends released electrostatic complexes of DNA and PEI, which are responsible for the enhanced gene transfection. Furthermore, blending PEI into the POE microsphere induced 50% to 60% greater phenotypic maturation and activation of bone marrow-derived dendritic cells in vitro, judged by up-regulation of co-stimulatory markers on the cell surface. Physically blending PEI with POE is a simple approach for modulating the properties of biodegradable microspheres in terms of gene transfection efficiency and DNA release kinetics. Combined with the ability to induce maturation of antigen-presenting cells, POE-PEI blended microspheres may be excellent carriers for DNA vaccines. PMID:18400294

An exonic missense mutation c.28G>A is associated with weak B blood group by affecting RNA splicing of the ABO gene.

PubMed

Cai, Xiaohong; Qian, Chengrui; Wu, Wenman; Lei, Hang; Ding, Qiulan; Zou, Wei; Xiang, Dong; Wang, Xuefeng

2017-09-01

The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change. Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing. While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells. ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene. © 2017 AABB.

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-02-01

The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation

PubMed Central

Dobson, Rachel; Stockdale, Christopher; Lapsley, Craig; Wilkes, Jonathan; McCulloch, Richard

2011-01-01

Homologous recombination in Trypanosoma brucei is used for moving variant surface glycoprotein (VSG) genes into expression sites during immune evasion by antigenic variation. A major route for such VSG switching is gene conversion reactions in which RAD51, a universally conserved recombinase, catalyses homology-directed strand exchange. In any eukaryote, RAD51-directed strand exchange in vivo is mediated by further factors, including RAD51-related proteins termed Rad51 paralogues. These appear to be ubiquitously conserved, although their detailed roles in recombination remain unclear. In T. brucei, four putative RAD51 paralogue genes have been identified by sequence homology. Here we show that all four RAD51 paralogues act in DNA repair, recombination and RAD51 subnuclear dynamics, though not equivalently, while mutation of only one RAD51 paralogue gene significantly impedes VSG switching. We also show that the T. brucei RAD51 paralogues interact, and that the complexes they form may explain the distinct phenotypes of the mutants as well as observed expression interdependency. Finally, we document the Rad51 paralogues that are encoded by a wide range of protists, demonstrating that the Rad51 paralogue repertoire in T. brucei is unusually large among microbial eukaryotes and that one member of the protein family corresponds with a key, conserved eukaryotic Rad51 paralogue. PMID:21615552

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

Characterization of occult hepatitis B virus infection among HIV positive patients in Cameroon.

PubMed

Gachara, George; Magoro, Tshifhiwa; Mavhandu, Lufuno; Lum, Emmaculate; Kimbi, Helen K; Ndip, Roland N; Bessong, Pascal O

2017-03-08

Occult hepatitis B infection (OBI) among HIV positive patients varies widely in different geographic regions. We undertook a study to determine the prevalence of occult hepatitis B infection among HIV infected individuals visiting a health facility in South West Cameroon and characterized occult HBV strains based on sequence analyses. Plasma samples (n = 337), which previously tested negative for hepatitis B surface antigen (HBsAg), were screened for antibodies against hepatitis B core (anti-HBc) and surface (anti-HBs) antigens followed by DNA extraction. A 366 bp region covering the overlapping surface/polymerase gene of HBV was then amplified in a nested PCR and the amplicons sequenced using Sanger sequencing. The resulting sequences were then analyzed for genotypes and for escape and drug resistance mutations. Twenty samples were HBV DNA positive and were classified as OBI giving a prevalence of 5.9%. Out of these, 9 (45%) were anti-HBs positive, while 10 (52.6%) were anti-HBc positive. Additionally, 2 had dual anti-HBs and anti-HBc reactivity, while 6 had no detectable HBV antibodies. Out of the ten samples that were successfully sequenced, nine were classified as genotype E and one as genotype A. Three sequences possessed mutations associated with lamivudine resistance. We detected a number of mutations within the major hydrophilic region of the surface gene where most immune escape mutations occur. Findings from this study show the presence of hepatitis B in patients without any of the HBV serological markers. Further prospective studies are required to determine the risk factors and markers of OBI.

The molecular relationship between antigenic domains and epitopes on hCG.

PubMed

Berger, Peter; Lapthorn, Adrian J

2016-08-01

Antigenic domains are defined to contain a limited number of neighboring epitopes recognized by antibodies (Abs) but their molecular relationship remains rather elusive. We thoroughly analyzed the antigenic surface of the important pregnancy and tumor marker human chorionic gonadotropin (hCG), a cystine knot (ck) growth factor, and set antigenic domains and epitopes in molecular relationships to each other. Antigenic domains on hCG, its free hCGα and hCGβ subunits are dependent on appropriate inherent molecular features such as molecular accessibility and protrusion indices that determine bulging structures accessible to Abs. The banana-shaped intact hCG comprises ∼7500Å(2) of antigenic surface with minimally five antigenic domains that encompass a continuum of overlapping non-linear composite epitopes, not taking into account the C-terminal peptide extension of hCGβ (hCGβCTP). Epitopes within an antigenic domain are defined by specific Abs, that bury nearly 1000Å(2) of surface accessible area on the antigen and recognize a few up to 15 amino acid (aa) residues, whereby between 2 and 5 of these provide the essential binding energy. Variability in Ab binding modes to the contact aa residues are responsible for the variation in affinity and intra- and inter-species specificity, e.g. cross-reactions with luteinizing hormone (LH). Each genetically distinct fragment antigen binding (Fab) defines its own epitope. Consequently, recognition of the same epitope by different Abs is only possible in cases of genetically identical sequences of its binding sites. Due to combinatorial V(D)J gene segment variability of heavy and light chains, Abs defining numerous epitopes within an antigenic domain can be generated by different individuals and species. Far more than hundred Abs against the immuno-dominant antigenic domains of either subunit at both ends of the hCG-molecule, the tips of peptide loops one and three (Ł1+3) protruding from the central ck, encompassing hCGβŁ1+3 (aa 20-25+64+68-81) and hCGαŁ1 (aa 13-22; Pro16, Phe17, Phe18) plus hCGαŁ3 (Met71, Phe74), respectively, have been identified in the two "ISOBM Tissue Differentiation-7 Workshops on hCG and Related Molecules" and in other studies. These Abs recognize distinct but overlapping epitopes with slightly different specificity profiles and affinities. Heterodimeric-specific epitopes involve neighboring αŁ1 plus βŁ2 (hCGβ44/45 and 47/48). Diagnostically important Abs recognize the middle of the molecule, the ck (aa Arg10, Arg60 and possibly Gln89) and the linear hCGβCTP "tail" (aa 135-145; Asp139, Pro144, Gln145), respectively. Identification of antigenic domains and of specific epitopes is essential for harmonization of Abs in methods that are used for reliable and robust hCG measurements for the management of pregnancy, pregnancy-related disease and tumors. Copyright © 2016. Published by Elsevier Ltd.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii.

PubMed

Kim, Seon-Hee; Bae, Young-An; Seoh, Ju-Young; Yang, Hyun-Jong

2017-06-01

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium . Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii -infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

PubMed

Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

Cryptosporidium hominis gene catalog: a resource for the selection of novel Cryptosporidium vaccine candidates

PubMed Central

Ifeonu, Olukemi O.; Simon, Raphael; Tennant, Sharon M.; Sheoran, Abhineet S.; Daly, Maria C.; Felix, Victor; Kissinger, Jessica C.; Widmer, Giovanni; Levine, Myron M.; Tzipori, Saul; Silva, Joana C.

2016-01-01

Human cryptosporidiosis, caused primarily by Cryptosporidium hominis and a subset of Cryptosporidium parvum, is a major cause of moderate-to-severe diarrhea in children under 5 years of age in developing countries and can lead to nutritional stunting and death. Cryptosporidiosis is particularly severe and potentially lethal in immunocompromised hosts. Biological and technical challenges have impeded traditional vaccinology approaches to identify novel targets for the development of vaccines against C. hominis, the predominant species associated with human disease. We deemed that the existence of genomic resources for multiple species in the genus, including a much-improved genome assembly and annotation for C. hominis, makes a reverse vaccinology approach feasible. To this end, we sought to generate a searchable online resource, termed C. hominis gene catalog, which registers all C. hominis genes and their properties relevant for the identification and prioritization of candidate vaccine antigens, including physical attributes, properties related to antigenic potential and expression data. Using bioinformatic approaches, we identified ∼400 C. hominis genes containing properties typical of surface-exposed antigens, such as predicted glycosylphosphatidylinositol (GPI)-anchor motifs, multiple transmembrane motifs and/or signal peptides targeting the encoded protein to the secretory pathway. This set can be narrowed further, e.g. by focusing on potential GPI-anchored proteins lacking homologs in the human genome, but with homologs in the other Cryptosporidium species for which genomic data are available, and with low amino acid polymorphism. Additional selection criteria related to recombinant expression and purification include minimizing predicted post-translation modifications and potential disulfide bonds. Forty proteins satisfying these criteria were selected from 3745 proteins in the updated C. hominis annotation. The immunogenic potential of a few of these is currently being tested. Database URL: http://cryptogc.igs.umaryland.edu PMID:28095366

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

PubMed

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

PubMed Central

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

2010-01-01

Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes.

PubMed

Yokoyama, Naoaki; Sivakumar, Thillaiampalam; Tuvshintulga, Bumduuren; Hayashida, Kyoko; Igarashi, Ikuo; Inoue, Noboru; Long, Phung Thang; Lan, Dinh Thi Bich

2015-03-01

The genes that encode merozoite surface antigens (MSAs) in Babesia bovis are genetically diverse. In this study, we analyzed the genetic diversity of B. bovis MSA-1, MSA-2b, and MSA-2c genes in Vietnamese cattle and water buffaloes. Blood DNA samples from 258 cattle and 49 water buffaloes reared in the Thua Thien Hue province of Vietnam were screened with a B. bovis-specific diagnostic PCR assay. The B. bovis-positive DNA samples (23 cattle and 16 water buffaloes) were then subjected to PCR assays to amplify the MSA-1, MSA-2b, and MSA-2c genes. Sequencing analyses showed that the Vietnamese MSA-1 and MSA-2b sequences are genetically diverse, whereas MSA-2c is relatively conserved. The nucleotide identity values for these MSA gene sequences were similar in the cattle and water buffaloes. Consistent with the sequencing data, the Vietnamese MSA-1 and MSA-2b sequences were dispersed across several clades in the corresponding phylogenetic trees, whereas the MSA-2c sequences occurred in a single clade. Cattle- and water-buffalo-derived sequences also often clustered together on the phylogenetic trees. The Vietnamese MSA-1, MSA-2b, and MSA-2c sequences were then screened for recombination with automated methods. Of the seven recombination events detected, five and two were associated with the MSA-2b and MSA-2c recombinant sequences, respectively, whereas no MSA-1 recombinants were detected among the sequences analyzed. Recombination between the sequences derived from cattle and water buffaloes was very common, and the resultant recombinant sequences were found in both host animals. These data indicate that the genetic diversity of the MSA sequences does not differ between cattle and water buffaloes in Vietnam. They also suggest that recombination between the B. bovis MSA sequences in both cattle and water buffaloes might contribute to the genetic variation in these genes in Vietnam. Copyright © 2015 Elsevier B.V. All rights reserved.

[Virulence markers of Escherichia coli O1 strains].

PubMed

Makarova, M A; Kaftyreva, L A; Grigor'eva, N S; Kicha, E V; Lipatova, L A

2011-01-01

To detect virulence genes in clinical isolates of Escherichia coli O1 using polymerase chain reaction (PCR). One hundred and twenty strains of E.coli O1 strains isolated from faeces of patients with acute diarrhea (n = 45) and healthy persons (n = 75) were studied. PCR with primers for rfb and fliC genes, which control synthesis of O- and H- antigens respectively, was used. Fourteen virulence genes (pap, aaf, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, st, and aer) were detected by PCR primers. K1-antigen was determined by Pastorex Meningo B/E. coli O1 kit (Bio-Rad). rfb gene controlling O-antigen synthesis in serogroup O1 as well as fliC gene controlling synthesis of H7 and K1 antigens were detected in all strains. Thus all E. coli strains had antigenic structure O1:K1 :H-:F7. Virulence genes aafl, sfa, afa, eaeA, bfpA, ial, hly, cnf, stx1, stx2, lt, and st were not detected. All strains owned pap and aer genes regardless of the presence of acute diarrhea symptoms. It was shown that E. coli O1:KI:H-:F7 strains do not have virulence genes which are characteristic for diarrhea-causing Escherichia. In accordance with the presence of pap and aer genes they could be attributed to uropathogenic Escherichia (UPEC) or avian-pathogenic Escherichia (APEC). It is necessary to detect virulence factors in order to determine E. coli as a cause of intestinal infection.

Comprehensive red blood cell and platelet antigen prediction from whole genome sequencing: proof of principle

PubMed Central

Westhoff, Connie M.; Uy, Jon Michael; Aguad, Maria; Smeland‐Wagman, Robin; Kaufman, Richard M.; Rehm, Heidi L.; Green, Robert C.; Silberstein, Leslie E.

2015-01-01

BACKGROUND There are 346 serologically defined red blood cell (RBC) antigens and 33 serologically defined platelet (PLT) antigens, most of which have known genetic changes in 45 RBC or six PLT genes that correlate with antigen expression. Polymorphic sites associated with antigen expression in the primary literature and reference databases are annotated according to nucleotide positions in cDNA. This makes antigen prediction from next‐generation sequencing data challenging, since it uses genomic coordinates. STUDY DESIGN AND METHODS The conventional cDNA reference sequences for all known RBC and PLT genes that correlate with antigen expression were aligned to the human reference genome. The alignments allowed conversion of conventional cDNA nucleotide positions to the corresponding genomic coordinates. RBC and PLT antigen prediction was then performed using the human reference genome and whole genome sequencing (WGS) data with serologic confirmation. RESULTS Some major differences and alignment issues were found when attempting to convert the conventional cDNA to human reference genome sequences for the following genes: ABO, A4GALT, RHD, RHCE, FUT3, ACKR1 (previously DARC), ACHE, FUT2, CR1, GCNT2, and RHAG. However, it was possible to create usable alignments, which facilitated the prediction of all RBC and PLT antigens with a known molecular basis from WGS data. Traditional serologic typing for 18 RBC antigens were in agreement with the WGS‐based antigen predictions, providing proof of principle for this approach. CONCLUSION Detailed mapping of conventional cDNA annotated RBC and PLT alleles can enable accurate prediction of RBC and PLT antigens from whole genomic sequencing data. PMID:26634332

Inactivation of DNA-Binding Response Regulator Sak189 Abrogates β-Antigen Expression and Affects Virulence of Streptococcus agalactiae

PubMed Central

Rozhdestvenskaya, Anastasia S.; Totolian, Artem A.; Dmitriev, Alexander V.

2010-01-01

Background Streptococcus agalactiae is able to colonize numerous tissues employing different mechanisms of gene regulation, particularly via two-component regulatory systems. These systems sense the environmental stimuli and regulate expression of the genes including virulence genes. Recently, the novel two-component regulatory system Sak188/Sak189 was identified. In S. agalactiae genome, it was adjacent to the bac gene encoding for β-antigen, an important virulence factor. Methodology/Principal Findings In this study, the sak188 and sak189 genes were inactivated, and the functional role of Sak188/Sak189 two-component system in regulation of the β-antigen expression was investigated. It was demonstrated that both transcription of bac gene and expression of encoded β-antigen were controlled by Sak189 response regulator, but not Sak188 histidine kinase. It was also found that the regulation occurred at transcriptional level. Finally, insertional inactivation of sak189 gene, but not sak188 gene, significantly affected virulent properties of S. agalactiae. Conclusions/Significance Sak189 response regulator is necessary for activation of bac gene transcription. It also controls the virulent properties of S. agalactiae. Given that the primary functional role of Sak188/Sak189 two-component systems is a control of bac gene transcription, this system can be annotated as BgrR/S (bac gene regulatory system). PMID:20419089

Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein

PubMed Central

Sahu, Tejram; Malkov, Vlad; Morrison, Robert; Pei, Ying; Juompan, Laure; Milman, Neta; Zarling, Stasya; Anderson, Charles; Wong-Madden, Sharon; Wendler, Jason; Ishizuka, Andrew; MacMillen, Zachary W.; Garcia, Valentino; Kappe, Stefan H. I.; Krzych, Urszula; Duffy, Patrick E.

2016-01-01

Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP. PMID:27434123

Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

PubMed

Speake, Cate; Pichugin, Alexander; Sahu, Tejram; Malkov, Vlad; Morrison, Robert; Pei, Ying; Juompan, Laure; Milman, Neta; Zarling, Stasya; Anderson, Charles; Wong-Madden, Sharon; Wendler, Jason; Ishizuka, Andrew; MacMillen, Zachary W; Garcia, Valentino; Kappe, Stefan H I; Krzych, Urszula; Duffy, Patrick E

2016-01-01

Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P. falciparum genes transcribed at higher levels during liver stage versus sporozoite or blood stages of development. We prepared DNA vaccines for 21 genes using the predicted orthologues in P. yoelii and P. berghei and tested their efficacy using different delivery methods against pre-erythrocytic malaria in rodent models. In our primary screen using P. yoelii in BALB/c mice, we found that 16 antigens significantly reduced liver stage parasite burden. In our confirmatory screen using P. berghei in C57Bl/6 mice, we confirmed 6 antigens that were protective in both models. Two antigens, when combined with CSP, provided significantly greater protection than CSP alone in both models. Based on the observations reported here, transcriptional patterns of Plasmodium genes can be useful in identifying novel pre-erythrocytic antigens that induce protective immunity alone or in combination with CSP.

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets.

PubMed

Rhoden, John J; Dyas, Gregory L; Wroblewski, Victor J

2016-05-20

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

PubMed

Zhang, Qing; Yuan, Xiang-Fei; Lu, Yang; Li, Zhen-Zhen; Bao, Shi-Qi; Zhang, Xiao-Long; Yang, Yuan-Yuan; Fan, Dong-Mei; Zhang, Yi-Zhi; Wu, Chen-Xuan; Guo, Hong-Xing; Zhang, Yan-Jun; Ye, Zhou; Xiong, Dong-Sheng

2017-10-01

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy. © 2017 UICC.

FOXP3, CBLB and ITCH gene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

PubMed Central

Sellebjerg, F; Krakauer, M; Khademi, M; Olsson, T; Sørensen, P S

2012-01-01

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4+CD25high T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4+CD25high T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4+CD25high T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4+CD25high T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4+CD25high T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function. PMID:23039885

Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum

PubMed Central

Artzy-Randrup, Yael; Rorick, Mary M; Day, Karen; Chen, Donald; Dobson, Andrew P; Pascual, Mercedes

2012-01-01

The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity. DOI: http://dx.doi.org/10.7554/eLife.00093.001 PMID:23251784

Analysis of density and epitopes of D antigen on the surface of erythrocytes from DEL phenotypic individuals carrying the RHD1227A allele.

PubMed

Gu, Juan; Sun, An-Yuan; Wang, Xue-Dong; Shao, Chao-Peng; Li, Zheng; Huang, Li-Hua; Pan, Zhao-Lin; Wang, Qing-Ping; Sun, Guang-Ming

2014-04-01

The characteristics of the D antigen are important as they influence the immunogenicity of D variant cells. Several studies on antigenic sites have been reported in normal D positive, weak D and partial D cases, including a comprehensive analysis of DEL types in Caucasians. The aim of this study was to assess D antigen density and epitopes on the erythrocyte surface of Asian type DEL phenotypic individuals carrying the RHD1227A allele in the Chinese population. A total of 154 DEL phenotypic individuals carrying the RHD1227A allele were identified through adsorption and elution tests and polymerase chain reaction analysis with sequence-specific primers in the Chinese population. D antigen density on the erythrocyte surface of these individuals was detected using a flow cytometric method. An erythrocyte sample with known D antigen density was used as a standard. Blood samples from D-negative and D-positive individuals were used as controls. In addition, D antigen epitopes on the erythrocyte surface of DEL individuals carrying the RHD1227A allele were investigated with 18 monoclonal anti-D antibodies specific for different D antigen epitopes. The means of the median fluorescence intensity of D antigen on the erythrocyte membrane surface of D-negative, D-positive and DEL individuals were 2.14±0.25, 193.61±11.43 and 2.45±0.82, respectively. The DEL samples were estimated to have approximately 22 D antigens per cell. The samples from all 154 DEL individuals reacted positively with 18 monoclonal anti-D antibodies specific for different D antigen epitopes. In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was extremely low, there being only very few antigenic molecules per cell, but the D antigen epitopes were grossly complete.

Induction of the c-myc protooncogene following antigen binding to hapten-specific B cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Snow, E.C.; Fetherston, J.; Zimmer, S.

1986-03-01

Considerable controversy has centered on the role that the surface immunoglobulin (sIg) receptor for antigen plays during the induction of B cell activation. Stimulation by anti-Ig reagents has been shown to activate G/sub 0/ B cells to enter the cell cycle. The binding of thymus-dependent antigens to hapten-specific B cell populations apparently does not result in the movement of the antigen-binding cells (ABC) into the G/sub 1/ stage of the cell cycle. However, the authors have recently demonstrated that antigen binding to such hapten-specific B cells does result in the initiation of the membrane phosphatidylinositol cycle. In the present experiments,more » hapten-specific B cells (80-90% ABC, 99% in G/sub 0/) were incubated with either the correct hapten-carrier conjugate, with the carrier protein, or only media for 2 hours at 37/sup 0/C. At that time, total cellular RNA was isolated and subsequently analyzed by either dot blots or Northern gel techniques. The blots were probed with a (/sup 32/P)-c-myc SstI-Xhol fragment. The results indicate that hapten carrier stimulation of the hapten-specific B cells induces enhanced transcription of the c-myc gene. These observations lend further support to the premise that antigen binding to the sIg receptor results in the transduction to the cell of important signals and implicates the active participation of sIg during the process of antigen-mediated B cell activation.« less

The Klebsiella pneumoniae O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia

PubMed Central

Shankar-Sinha, Sunita; Valencia, Gabriel A.; Janes, Brian K.; Rosenberg, Jessica K.; Whitfield, Chris; Bender, Robert A.; Standiford, Ted J.; Younger, John G.

2004-01-01

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type K. pneumoniae and the O-antigen-deficient mutant in regard to avid binding by murine complement C3 or resistance to serum- or whole-blood-mediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 × 103 versus 6 × 104 CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by K. pneumoniae, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection. PMID:14977947

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki

2014-02-01

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite. © 2013.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

HLA Immune Function Genes in Autism

PubMed Central

Torres, Anthony R.; Westover, Jonna B.; Rosenspire, Allen J.

2012-01-01

The human leukocyte antigen (HLA) genes on chromosome 6 are instrumental in many innate and adaptive immune responses. The HLA genes/haplotypes can also be involved in immune dysfunction and autoimmune diseases. It is now becoming apparent that many of the non-antigen-presenting HLA genes make significant contributions to autoimmune diseases. Interestingly, it has been reported that autism subjects often have associations with HLA genes/haplotypes, suggesting an underlying dysregulation of the immune system mediated by HLA genes. Genetic studies have only succeeded in identifying autism-causing genes in a small number of subjects suggesting that the genome has not been adequately interrogated. Close examination of the HLA region in autism has been relatively ignored, largely due to extraordinary genetic complexity. It is our proposition that genetic polymorphisms in the HLA region, especially in the non-antigen-presenting regions, may be important in the etiology of autism in certain subjects. PMID:22928105

Sequence Analysis of Different Domains of Plasmodium vivax Apical Membrane Antigen (PvAMA-1 gene) Locus in Iran.

PubMed

Motevalli Haghi, A; Nateghpour, M; Edrissian, Ghh; Sepehrizadeh, Z; Mohebali, M; Khoramizade, Mr; Shahrbabak, S Sabouri; Moghimi, H

2012-01-01

Plasmodium vivax is responsible for approximately 80 million malaria cases in the world. Apical membrane antigen1 (AMA-1) is a type I integral membrane protein present in all Plasmodium species. AMA-1 interferes in critical steps of invasion of human hepatocytes by sporozoites and red blood cells by merozoites and is one of the most immunodominant antigens for eliciting a protective immune response in human. It is considered as a promising antigen for inclusion in a vaccine against P. vivax. Since more knowledge is needed to lighten the scope of such antigen we compared genetic variation in P. vivax AMA-1from an Iranian isolate with those reported from some of the other malarious countries so far. P. vivax genomic DNA was extracted from the whole blood of an Iranian patient with patent P. vivax infection. The nucleotide sequence for 446 amino acid (AA) residues (42-488 of PvAMA-1) was amplified by PCR and cloned in pUC19 vector for sequencing. Sequence analysis of the antigen showed a high degree of identity (99%) with strong homology to the PvAMA-1 gene of P. vivax S3 and SKO814 isolates from India and Korea (Asian isolates) respectively, and 96% similarity with P. vivax Sal-1 AMA-1 gene from El Salvador. We cloned and characterized three domains of PvAMA-1 gene from an Iranian patient. Predicted protein sequence of this gene showed some discrepancies in corresponding protein in comparing with similar genes reported from other malarious countries.

Capsule null locus meningococci: typing of antigens used in an investigational multicomponent meningococcus serogroup B vaccine.

PubMed

Claus, Heike; Jördens, Markus S; Kriz, Pavla; Musilek, Martin; Jarva, Hanna; Pawlik, Marie-Christin; Meri, Seppo; Vogel, Ulrich

2012-01-05

The investigational multicomponent meningococcus serogroup B vaccine (4CMenB) targets the antigenetically variable population of serogroup B meningococci. Forty-one strains of capsule null locus (cnl) meningococci, which are frequent among healthy carriers, were selected from nine sequence types (ST), which belong to four clonal complexes (cc), and three countries. They were antigen sequence typed and analyzed for antigen expression to predict whether these strains harbor the genes and express the four vaccine antigens of 4CMenB as measured by the meningococcal antigen typing system (MATS). The PorA variant used in the vaccine was not found. The nadA gene was absent in all but one strain, which did not express the antigen in vitro. Only strains of clonal complex ST-198 harbored a factor H binding protein (FHBP) allele of the cross-reactive variant 1 family which is included in the vaccine. All these strains expressed the antigen. Five variants of the Neisserial heparin binding antigen (NHBA) gene were identified. Expression of NHBA was observed in all strains with highest levels in ST-198 cc and ST-845. The data suggest a potential impact of 4CMenB immunization at least on cnl meningococci of the ST-198 cc and ST-845. Copyright © 2011 Elsevier Ltd. All rights reserved.

Peptide-Conjugated Nanoparticles Reduce Positive Co-stimulatory Expression and T Cell Activity to Induce Tolerance.

PubMed

Kuo, Robert; Saito, Eiji; Miller, Stephen D; Shea, Lonnie D

2017-07-05

Targeted approaches to treat autoimmune diseases would improve upon current therapies that broadly suppress the immune system and lead to detrimental side effects. Antigen-specific tolerance was induced using poly(lactide-co-glycolide) nanoparticles conjugated with disease-relevant antigen to treat a model of multiple sclerosis. Increasing the nanoparticle dose and amount of conjugated antigen both resulted in more durable immune tolerance. To identify active tolerance mechanisms, we investigated downstream cellular and molecular events following nanoparticle internalization by antigen-presenting cells. The initial cell response to nanoparticles indicated suppression of inflammatory signaling pathways. Direct and functional measurement of surface MHC-restricted antigen showed positive correlation with both increasing particle dose from 1 to 100 μg/mL and increasing peptide conjugation by 2-fold. Co-stimulatory analysis of cells expressing MHC-restricted antigen revealed most significant decreases in positive co-stimulatory molecules (CD86, CD80, and CD40) following high doses of nanoparticles with higher peptide conjugation, whereas expression of a negative co-stimulatory molecule (PD-L1) remained high. T cells isolated from mice immunized against myelin proteolipid protein (PLP 139-151 ) were co-cultured with antigen-presenting cells administered PLP 139-151 -conjugated nanoparticles, which resulted in reduced T cell proliferation, increased T cell apoptosis, and a stronger anti-inflammatory response. These findings indicate several potential mechanisms used by peptide-conjugated nanoparticles to induce antigen-specific tolerance. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Evaluation of single-round infectious, chimeric dengue type 1 virus as an antigen for dengue functional antibody assays.

PubMed

Yamanaka, Atsushi; Suzuki, Ryosuke; Konishi, Eiji

2014-07-23

Dengue fever and dengue hemorrhagic fever are endemic throughout tropical and subtropical countries. Four serotypes of dengue viruses (DENV-1 to DENV-4), each with several genotypes including various subclades, are co-distributed in most endemic areas. Infection-neutralizing and -enhancing antibodies are believed to play protective and pathogenic roles, respectively. Measurement of these functional antibodies against a variety of viral strains is thus important for evaluating coverage and safety of dengue vaccine candidates. Although transportation of live virus materials beyond national borders is increasingly limited, this difficulty may be overcome using biotechnology that enables generation of an antibody-assay antigen equivalent to authentic virus based on viral sequence information. A rapid system to produce flavivirus single-round infectious particles (SRIPs) was recently developed using a Japanese encephalitis virus (JEV) subgenomic replicon plasmid. This system allows production of chimeric SRIPs that have surface proteins of other flaviviruses. In the present study, SRIPs of DENV-1 (D1-SRIPs) were evaluated as an antigen for functional antibody assays. Inclusion of the whole mature capsid gene of JEV into the replicon plasmid provided higher D1-SRIP yields than did its exclusion in cases where a DENV-1 surface-protein-expressing plasmid was used for co-transfection of 293T cells with the replicon plasmid. In an assay to measure the balance between neutralizing and enhancing activities, dose (antibody dilution)-dependent activity curves in dengue-immune human sera or mouse monoclonal antibodies obtained using D1-SRIP antigen were equivalent to those obtained using DENV-1 antigen. Similar results were obtained using additional DENV-2 and DENV-3 systems. In a conventional Vero-cell neutralization test, a significant correlation was shown between antibody titers obtained using D1-SRIP and DENV-1 antigens. These results demonstrate the utility of D1-SRIPs as an alternative antigen to authentic DENV-1 in functional antibody assays. SRIP antigens may contribute to dengue vaccine candidate evaluation, understanding of dengue pathogenesis, and development of serodiagnostic systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prostate Stem Cell Antigen DNA Vaccination Breaks Tolerance to Self-antigen and Inhibits Prostate Cancer Growth

PubMed Central

Ahmad, Sarfraz; Casey, Garrett; Sweeney, Paul; Tangney, Mark; O'Sullivan, Gerald C

2009-01-01

Prostate stem cell antigen (PSCA) is a cell surface antigen expressed in normal human prostate and over expressed in prostate cancer. Elevated levels of PSCA protein in prostate cancer correlate with increased tumor stage/grade, with androgen independence and have higher expression in bone metastases. In this study, the PSCA gene was isolated from the transgenic adenocarcinoma mouse prostate cell line (TRAMPC1), and a vaccine plasmid construct was generated. This plasmid PSCA (pmPSCA) was delivered by intramuscular electroporation (EP) and induced effective antitumor immune responses against subcutaneous TRAMPC1 tumors in male C57 BL/6 mice. The pmPSCA vaccination inhibited tumor growth, resulting in cure or prolongation in survival. Similarly, the vaccine inhibited metastases in PSCA expressing B16 F10 tumors. There was activation of Th-1 type immunity against PSCA, indicating the breaking of tolerance to a self-antigen. This immunity was tumor specific and was transferable by adoptive transfer of splenocytes. The mice remained healthy and there was no evidence of collateral autoimmune responses in normal tissues. EP-assisted delivery of the pmPSCA evoked strong specific responses and could, in neoadjuvant or adjuvant settings, provide a safe and effective immune control of prostate cancer, given that there is significant homology between human and mouse PSCA. PMID:19337234

B-cell acquisition of antigen: Sensing the surface.

PubMed

Knight, Andrew M

2015-06-01

B-cell antigen receptor (BCR) recognition and acquisition of antigen by B cells is the essential first step in the generation of effective antibody responses. As B-cell-mediated antigen presentation is also believed to play a significant role in the activation of CD4(+) Th-cell responses, considerable effort has focused on clarifying the nature of antigen/BCR interactions. Following earlier descriptions of interactions of soluble antigens with the BCR, it is now clear that B cells also recognize, physically extract and present antigens that are tethered to, or integral components of, the surfaces or extracellular matrix of other cells. In this issue of the European Journal of Immunology, Zeng et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] examine how the physical property or "stiffness" of the surface displaying antigens to B cells influences the B-cell response. This commentary reports that antigen tethered on "less stiff" surfaces induces increased B-cell activation and antibody responses. I then infer how "sensing the surface" by B cells may represent a new component of the immune system's ability to detect "damage," and how this understanding may influence approaches to clinical therapies where immune activity is either unwanted or desired. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antibody profiling using a recombinant protein-based multiplex ELISA array accelerates recombinant vaccine development: Case study on red sea bream iridovirus as a reverse vaccinology model.

PubMed

Matsuyama, Tomomasa; Sano, Natsumi; Takano, Tomokazu; Sakai, Takamitsu; Yasuike, Motoshige; Fujiwara, Atushi; Kawato, Yasuhiko; Kurita, Jun; Yoshida, Kazunori; Shimada, Yukinori; Nakayasu, Chihaya

2018-05-03

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center for... used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen. [40 FR...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

21 CFR 660.4 - Potency test.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.4 Potency test. To be satisfactory for release, each filling of Antibody to Hepatitis B Surface Antigen shall be tested against the Reference Hepatitis B Surface Antigen Panel and shall be sufficiently potent...

21 CFR 660.3 - Reference panel.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.3 Reference panel. A Reference Hepatitis B Surface Antigen Panel shall be obtained from the Center... shall be used for determining the potency and specificity of Antibody to Hepatitis B Surface Antigen...

Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes.

PubMed

Borhani Dizaji, Nahid; Basseri, Hamid Reza; Naddaf, Saied Reza; Heidari, Mansour

2014-10-25

Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could be utilized as a potential target for future studies in TBV area for malaria control. Copyright © 2014 Elsevier B.V. All rights reserved.

[A study on mutations of the overlapping hepatitis B virus surface and polymerase gene in patients with HBV reinfection after liver transplantations].

PubMed

Song, Hong-li; Shen, Zhong-yang; Wang, Jian; Zheng, Wei-ping; Wang, Zheng-lu

2008-04-01

To investigate the influence of combined hepatitis B immune globulin (HBIG) and lamivudine (LMV) treatment on hepatitis B virus (HBV) surface antigen and polymerase overlapping gene mutations in HBV reinfected liver transplant recipients. From June 2002 to December 2003, 320 patients who underwent liver transplantations due to HBV-related end-stage liver diseases were followed-up for 1.5 to 3 years postoperatively. Fourteen patients developed HBV reinfection. They had LMV before their liver transplantations and had LMV and HBIG after the transplantations to prevent HBV infections. Their serum levels of HBV DNA were measured by polymerase chain reaction. Gene sequencing method was used to analyze HBV genotype and mutations of the S gene. Micro-particle enzyme immunoassay was used to measure the serum concentration of HBIG. (1) There was no obvious difference in the number of amino acid mutation sites in S and P regions before and after the transplantations. (2) The HBV genotypes were B-type (n=2) and C-type (n=12) in the reinfected group before the transplantations, and genotypes after the transplantations remained the same. (3) HBIG concentrations were 0 U/L in 7 patients, less than 100 U/L in 5 patients, and more than 100 U/L in 2 patients. Mutations were detected as I126S, T131N, S143T and G145R in 'a' determinant and L110F, I113S, T160K in up- or down-stream of 'a' determinant. (4) Mutations in S gene caused missense mutation in the surface antigen region. These mutations also caused corresponding missense mutations in the polymerase region. The missense mutation in the polymerase region involved lamivudine mutation sites and other mutation sites. Immunosuppressant therapy has no obvious influence on the numbers of mutations, but it can influence the sites of the mutations. Besides 'a' determinant mutations, there exist mutations in up- or down-streams of 'a' determinant and they may cause HBV reinfection.

A Molecular Simulation Study of Antibody-Antigen Interactions on Surfaces for the Rational Design of Next-Generation Antibody Microarrays

NASA Astrophysics Data System (ADS)

Bush, Derek B.

Antibody microarrays constitute a next-generation sensing platform that has the potential to revolutionize the way that molecular detection is conducted in many scientific fields. Unfortunately, current technologies have not found mainstream use because of reliability problems that undermine trust in their results. Although several factors are involved, it is believed that undesirable protein interactions with the array surface are a fundamental source of problems where little detail about the molecular-level biophysics are known. A better understanding of antibody stability and antibody-antigen binding on the array surface is needed to improve microarray technology. Despite the availability of many laboratory methods for studying protein stability and binding, these methods either do not work when the protein is attached to a surface or they do not provide the atomistic structural information that is needed to better understand protein behavior on the surface. As a result, molecular simulation has emerged as the primary method for studying proteins on surfaces because it can provide metrics and views of atomistic structures and molecular motion. Using an advanced, coarse-grain, protein-surface model this study investigated how antibodies react to and function on different types of surfaces. Three topics were addressed: (1) the stability of individual antibodies on surfaces, (2) antibody binding to small antigens while on a surface, and (3) antibody binding to large antigens while on a surface. The results indicate that immobilizing antibodies or antibody fragments in an upright orientation on a hydrophilic surface can provide the molecules with thermal stability similar to their native aqueous stability, enhance antigen binding strength, and minimize the entropic cost of binding. Furthermore, the results indicate that it is more difficult for large antigens to approach the surface than small antigens, that multiple binding sites can aid antigen binding, and that antigen flexiblity simultaneously helps and hinders the binding process as it approaches the surface. The results provide hope that next-generation microarrays and other devices decorated with proteins can be improved through rational design.

Diversity of vir Genes in Plasmodium vivax from Endemic Regions in the Republic of Korea: an Initial Evaluation.

PubMed

Son, Ui-Han; Dinzouna-Boutamba, Sylvatrie-Danne; Lee, Sanghyun; Yun, Hae Soo; Kim, Jung-Yeon; Joo, So-Young; Jeong, Sookwan; Rhee, Man Hee; Hong, Yeonchul; Chung, Dong-Il; Kwak, Dongmi; Goo, Youn-Kyoung

2017-04-01

Variant surface antigens (VSAs) encoded by pir families are considered to be the key proteins used by many Plasmodium spp. to escape the host immune system by antigenic variation. This attribute of VSAs is a critical issue in the development of a novel vaccine. In this regard, a population genetic study of vir genes from Plasmodium vivax was performed in the Republic of Korea (ROK). Eighty-five venous blood samples and 4 of the vir genes, namely vir 27, vir 21, vir 12, and vir 4, were selected for study. The number of segregating sites (S), number of haplotypes (H), haplotype diversity (Hd), DNA diversity (π and Θ w ), and Tajima's D test value were conducted. Phylogenetic trees of each gene were constructed. The vir 21 (S=143, H=22, Hd=0.827) was the most genetically diverse gene, and the vir 4 (S=6, H=4, Hd=0.556) was the opposite one. Tajima's D values for vir 27 (1.08530, P >0.1), vir 12 (2.89007, P <0.01), and vir 21 (0.40782, P >0.1) were positive, and that of vir 4 (-1.32162, P >0.1) was negative. All phylogenetic trees showed 2 clades with no particular branching according to the geographical differences and cluster. This study is the first survey on the vir genes in ROK, providing information on the genetic level. The sample sequences from vir 4 showed a clear difference to the Sal-1 reference gene sequence, whereas they were very similar to those from Indian isolates.

Novel reassortant H9N2 viruses in pigeons and evidence for antigenic diversity of H9N2 viruses isolated from quails in Egypt.

PubMed

Kandeil, Ahmed; El-Shesheny, Rabeh; Maatouq, Asmaa; Moatasim, Yassmin; Cai, Zhipeng; McKenzie, Pamela; Webby, Richard; Kayali, Ghazi; Ali, Mohamed A

2017-04-01

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011-2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk.

Biochemical and genetic characterization of serologically untypable Streptococcus mutans strains isolated from patients with bacteremia.

PubMed

Fujiwara, T; Nakano, K; Kawaguchi, M; Ooshima, T; Sobue, S; Kawabata, S; Nakagawa, I; Hamada, S

2001-10-01

Four out of 522 streptococcal isolates from the peripheral blood of patients with bacteremia exhibited typical properties of Streptococcus mutans in terms of sucrose-dependent adhesion, expression of glucosyltransferases, fermentation profiles of sugars, the presence of surface protein antigen, and DNA-DNA hybridization. Two strains were determined as serotype f and e by immunodiffusion, whereas the other two isolates did not react with the specific antiserum to S. mutans serotype c. e. or f of the eight different serotypes of mutans streptococci. The latter two untypable isolates, however, expressed a new antigenic determinant that was different from serotype c/e/f specificity as revealed by immunodiffusion. Analysis of the cell wall polysaccharides revealed very low contents of glucose in the untypable isolates. Furthermore, Southern blot analysis demonstrated that the untypable strains lacked at least one gene corresponding to a glucose-adding enzyme. These results indicate that the serologically untypable nature is due to the loss of glucosidic residue from the serotype-specific polysaccharide antigens of S. mutans.

ABH and Lewis histo-blood group antigens, a model for the meaning of oligosaccharide diversity in the face of a changing world.

PubMed

Marionneau, S; Cailleau-Thomas, A; Rocher, J; Le Moullac-Vaidye, B; Ruvoën, N; Clément, M; Le Pendu, J

2001-07-01

Antigens of the ABH and Lewis histo-blood group family have been known for a long time. Yet their biological meaning is still largely obscure. Based on the available knowledge about the genes involved in their biosynthesis and about their tissue distribution in humans and other mammals, we discuss here the selective forces that may maintain or propagate these oligosaccharide antigens. The ABO, alpha 1,2fucosyltransferase and alpha 1,3fucosyltransferase enzyme families have been generated by gene duplications. Members of these families contribute to biosynthesis of the antigens through epistatic interactions. We suggest that the highly polymorphic genes of each family provide intraspecies diversity that allows coping with diverse and rapidly evolving pathogens. In contrast, the genes of low frequency polymorphism are expected to play roles at the cellular level, although they may be dispensable at the individual level. In addition, some members of these three gene families are expected to be functionally redundant and may either provide a reservoir for additional diversity in the future or become inactivated. We also discuss the role of the ABH and Lewis histo-blood group antigens in pathologies such as cancer and cardiovascular diseases, but argue that it is merely incidental and devoid of evolutionary impact.

Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-12-15

Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI) and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL) for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as "cellular drugs". As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs), transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR) gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next-generation sequencing.

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected. © 2017 AABB.

Multiple mechanisms underlie defective recognition of melanoma cells cultured in three-dimensional architectures by antigen-specific cytotoxic T lymphocytes

PubMed Central

Feder-Mengus, C; Ghosh, S; Weber, W P; Wyler, S; Zajac, P; Terracciano, L; Oertli, D; Heberer, M; Martin, I; Spagnoli, G C; Reschner, A

2007-01-01

Cancer cells' growth in three-dimensional (3D) architectures promotes resistance to drugs, cytokines, or irradiation. We investigated effects of 3D culture as compared to monolayers (2D) on melanoma cells' recognition by tumour-associated antigen (TAA)-specific HLA-A*0201-restricted cytotoxic T-lymphocytes (CTL). Culture of HBL, D10 (both HLA-A*0201+, TAA+) and NA8 (HLA-A*0201+, TAA−) melanoma cells on polyHEMA-coated plates, resulted in generation of 3D multicellular tumour spheroids (MCTS). Interferon-gamma (IFN-γ) production by HLA-A*0201-restricted Melan-A/MART-127–35 or gp100280–288-specific CTL clones served as immunorecognition marker. Co-culture with melanoma MCTS, resulted in defective TAA recognition by CTL as compared to 2D as witnessed by decreased IFN-γ production and decreased Fas Ligand, perforin and granzyme B gene expression. A multiplicity of mechanisms were potentially involved. First, MCTS per se limit CTL capacity of recognising HLA class I restricted antigens by reducing exposed cell surfaces. Second, expression of melanoma differentiation antigens is downregulated in MCTS. Third, expression of HLA class I molecules can be downregulated in melanoma MCTS, possibly due to decreased interferon-regulating factor-1 gene expression. Fourth, lactic acid production is increased in MCTS, as compared to 2D. These data suggest that melanoma cells growing in 3D, even in the absence of immune selection, feature characteristics capable of dramatically inhibiting TAA recognition by specific CTL. PMID:17342088

Pro-Inflammatory Activated Kupffer Cells by Lipids Induce Hepatic NKT Cells Deficiency through Activation-Induced Cell Death

PubMed Central

Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Background Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. Aims The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Methods Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. Results High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. Conclusion High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD. PMID:24312613

The use of phage display in neurobiology.

PubMed

Bradbury, Andrew R M

2010-04-01

Phage display has been extensively used to study protein-protein interactions, receptor- and antibody-binding sites, and immune responses, to modify protein properties, and to select antibodies against a wide range of different antigens. In the format most often used, a polypeptide is displayed on the surface of a filamentous phage by genetic fusion to one of the coat proteins, creating a chimeric coat protein, and coupling phenotype (the protein) to genotype (the gene within). As the gene encoding the chimeric coat protein is packaged within the phage, selection of the phage on the basis of the binding properties of the polypeptide displayed on the surface simultaneously results in the isolation of the gene encoding the polypeptide. This unit describes the background to the technique, and illustrates how it has been applied to a number of different problems, each of which has its neurobiological counterparts. Although this overview concentrates on the use of filamentous phage, which is the most popular platform, other systems are also described. (c) 2010 by John Wiley & Sons, Inc.

Molecular Cloning and Sequence Analysis of the Sta58 Major Antigen Gene of Rickettsia tsutsugamushi: Sequence homology and Antigenic Comparison of Sta58 to the 60-Kilodalton Family of Stress Proteins

DTIC Science & Technology

1990-05-01

Sta58 antigen and the Sta56 strain- GroES, C. burnetii HtpA, Mycobacterium tuberculosis 12- specific major antigen of R. tsutsugamushi (strain Karp...kb HindlIl fragment carrying the gene for the Sta58 tuberculosis, and Mycobacterium smegmatis (65-kDa anti- protein was subjected to DNA sequence...the Hsp6O and HsplO proteins. R. tsu., R. isutsugamushi; M. lep., Mvtcobacteriutn leprae : C. bur., C. burneiii; Synech.. Synechococcus strain 6301; T

Identification of two bvg-repressed surface proteins of Bordetella pertussis.

PubMed Central

Stenson, T H; Peppler, M S

1995-01-01

Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen. PMID:7558280

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis.

PubMed

Lysakowska, Monika Eliza; Denys, Andrzej; Sienkiewicz, Monika

2012-12-01

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren't. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ(2) test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ(2) test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

PubMed Central

Raymond, Frédéric; Boisvert, Sébastien; Roy, Gaétan; Ritt, Jean-François; Légaré, Danielle; Isnard, Amandine; Stanke, Mario; Olivier, Martin; Tremblay, Michel J.; Papadopoulou, Barbara; Ouellette, Marc; Corbeil, Jacques

2012-01-01

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage. PMID:21998295

Evidence of the Red-Queen Hypothesis from Accelerated Rates of Evolution of Genes Involved in Biotic Interactions in Pneumocystis.

PubMed

Delaye, Luis; Ruiz-Ruiz, Susana; Calderon, Enrique; Tarazona, Sonia; Conesa, Ana; Moya, Andrés

2018-06-01

Pneumocystis species are ascomycete fungi adapted to live inside the lungs of mammals. These ascomycetes show extensive stenoxenism, meaning that each species of Pneumocystis infects a single species of host. Here, we study the effect exerted by natural selection on gene evolution in the genomes of three Pneumocystis species. We show that genes involved in host interaction evolve under positive selection. In the first place, we found strong evidence of episodic diversifying selection in Major surface glycoproteins (Msg). These proteins are located on the surface of Pneumocystis and are used for host attachment and probably for immune system evasion. Consistent with their function as antigens, most sites under diversifying selection in Msg code for residues with large relative surface accessibility areas. We also found evidence of positive selection in part of the cell machinery used to export Msg to the cell surface. Specifically, we found that genes participating in glycosylphosphatidylinositol (GPI) biosynthesis show an increased rate of nonsynonymous substitutions (dN) versus synonymous substitutions (dS). GPI is a molecule synthesized in the endoplasmic reticulum that is used to anchor proteins to membranes. We interpret the aforementioned findings as evidence of selective pressure exerted by the host immune system on Pneumocystis species, shaping the evolution of Msg and several proteins involved in GPI biosynthesis. We suggest that genome evolution in Pneumocystis is well described by the Red-Queen hypothesis whereby genes relevant for biotic interactions show accelerated rates of evolution.

Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tabuchi, Yoshiaki; Kondo, Takashi; Suzuki, Yoshihisa

2005-04-15

Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 deg C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 deg C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21{sup waf1}, milk fat globule membrane protein E8, heat-responsive protein 12, and selenoproteinmore » P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.« less

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

PubMed Central

den Reijer, Paul Martijn; Lemmens-den Toom, Nicole; Kant, Samantha; Snijders, Susan V.; Boelens, Hélène; Tavakol, Mehri; Verkaik, Nelianne J.; van Belkum, Alex; Verbrugh, Henri A.; van Wamel, Willem J. B.

2013-01-01

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BµG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688. PMID:23308212

Rapid screening and identification of dominant B cell epitopes of HBV surface antigen by quantum dot-based fluorescence polarization assay

NASA Astrophysics Data System (ADS)

Meng, Zhongji; Song, Ruihua; Chen, Yue; Zhu, Yang; Tian, Yanhui; Li, Ding; Cui, Daxiang

2013-03-01

A method for quickly screening and identifying dominant B cell epitopes was developed using hepatitis B virus (HBV) surface antigen as a target. Eleven amino acid fragments from HBV surface antigen were synthesized by 9-fluorenylmethoxy carbonyl solid-phase peptide synthesis strategy, and then CdTe quantum dots were used to label the N-terminals of all peptides. After optimizing the factors for fluorescence polarization (FP) immunoassay, the antigenicities of synthetic peptides were determined by analyzing the recognition and combination of peptides and standard antibody samples. The results of FP assays confirmed that 10 of 11 synthetic peptides have distinct antigenicities. In order to screen dominant antigenic peptides, the FP assays were carried out to investigate the antibodies against the 10 synthetic peptides of HBV surface antigen respectively in 159 samples of anti-HBV surface antigen-positive antiserum. The results showed that 3 of the 10 antigenic peptides may be immunodominant because the antibodies against them existed more widely among the samples and their antibody titers were higher than those of other peptides. Using three dominant antigenic peptides, 293 serum samples were detected for HBV infection by FP assays; the results showed that the antibody-positive ratio was 51.9% and the sensitivity and specificity were 84.3% and 98.2%, respectively. In conclusion, a quantum dot-based FP assay is a very simple, rapid, and convenient method for determining immunodominant antigenic peptides and has great potential in applications such as epitope mapping, vaccine designing, or clinical disease diagnosis in the future.

Ultrastructural localization of human HL-A membrane antigens by use of hybrid antibodies

PubMed Central

Neauport-Sautes, Catherine; Silvestre, Daniele; Niccolai, Marie-Gabrielle; Kourilsky, F. M.; Levy, J. P.

1972-01-01

The localization of HL-A histocompatibility antigens at the surface of human lymphocytes in electron microscopy has been studied using hybrid antibodies to bind electron-dense particles (ferritin and plant viruses) to anti-HL-A antibody. A discontinuous distribution of the markers is observed at the cell surface, which is identical with that described for H-2 antigens on mouse lymphocytes with the same technique. Double labelling experiments suggest that the areas of the cell surface where HL-A antigens are detected contain also the heterologous lymphocyte antigens detected by an anti-thymocyte serum and that HL-A antigens are not renewed at a detectable level during the period of the labelling procedure in the areas of the cell surface which are not labelled primarily with ferritin-anti-IgG-anti-HL-A complexes. The interpretation of the discontinuous labelling of HL-A antigens with direct immunoferritin techniques is discussed. ImagesFIG. 2FIG. 3FIG. 4FIG. 5 PMID:5063188

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum.

PubMed

Lyon, J A; Haynes, J D; Diggs, C L; Chulay, J D; Pratt-Rossiter, J M

1986-03-15

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.

Advantages and applications of CAR-expressing natural killer cells

PubMed Central

Glienke, Wolfgang; Esser, Ruth; Priesner, Christoph; Suerth, Julia D.; Schambach, Axel; Wels, Winfried S.; Grez, Manuel; Kloess, Stephan; Arseniev, Lubomir; Koehl, Ulrike

2015-01-01

In contrast to donor T cells, natural killer (NK) cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD). In order to improve cytotoxicity against resistant cancer cells, auspicious efforts have been made with chimeric antigen receptor (CAR) expressing T- and NK cells. These CAR-modified cells express antigen receptors against tumor-associated surface antigens, thus redirecting the effector cells and enhancing tumor-specific immunosurveillance. However, many cancer antigens are also expressed on healthy tissues, potentially leading to off tumor/on target toxicity by CAR-engineered cells. In order to control such potentially severe side effects, the insertion of suicide genes into CAR-modified effectors can provide a means for efficient depletion of these cells. While CAR-expressing T cells have entered successfully clinical trials, experience with CAR-engineered NK cells is mainly restricted to pre-clinical investigations and predominantly to NK cell lines. In this review we summarize the data on CAR expressing NK cells focusing on the possible advantage using these short-lived effector cells and discuss the necessity of suicide switches. Furthermore, we address the compliance of such modified NK cells with regulatory requirements as a new field in cellular immunotherapy. PMID:25729364

Diversity of Pneumolysin and Pneumococcal Histidine Triad Protein D of Streptococcus pneumoniae Isolated from Invasive Diseases in Korean Children.

PubMed

Yun, Ki Wook; Lee, Hyunju; Choi, Eun Hwa; Lee, Hoan Jong

2015-01-01

Pneumolysin (Ply) and pneumococcal histidine triad protein D (PhtD) are candidate proteins for a next-generation pneumococcal vaccine. We aimed to analyze the genetic diversity and antigenic heterogeneity of Ply and PhtD for 173 pneumococci isolated from invasive diseases in Korean children. Allele was designated based on the variation of amino acid sequence. Antigenicity was predicted by the amino acid hydrophobicity of the region. There were seven and 39 allele types for the ply and phtD genes, respectively. The nucleotide sequence identity was 97.2%-99.9% for ply and 91.4%-98.0% for phtD gene. Only minor variations in hydrophobicity were noted among the antigenicity plots of Ply and PhtD. Overall, the allele types of the ply and phtD genes were remarkably homogeneous, and the antigenic diversity of the corresponding proteins was very limited. The Ply and PhtD could be useful antigens for universal pneumococcal vaccines.

Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi.

PubMed Central

Ramamoorthy, R; Povinelli, L; Philipp M, T

1996-01-01

An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete. PMID:8606088

Pathogenesis and spectrum of autoimmunity.

PubMed

Perl, Andras

2012-01-01

The immune system specifically recognizes and eliminates foreign antigens and, thus, protects integrity of the host. During maturation of the immune system, tolerance mechanisms develop that prevent or inhibit potentially harmful reactivities to self-antigens. Autoreactive B and T cells that are generated during immune responses are eliminated by apoptosis in the thymus, lymph nodes, or peripheral circulation or actively suppressed by regulatory T cells. However, autoreactive cells may survive due to failure of apoptosis or molecular mimicry, i.e., presentation and recognition of cryptic epitopes of self-antigens, or aberrant lymphokine production. Preservation of the host requires the development of immune responses to foreign antigen and tolerance to self-antigens. Autoimmunity results from a breakdown of tolerance to self-antigens through an interplay of genetic and environmental factors.One of the basic functions of the immune system is to specifically recognize and eliminate foreign antigens and, thus, protect integrity of the host. Through rearrangements and somatic mutations of various gene segments encoding T and B cell receptors and antibody molecules, the immune system acquires tremendous diversity. During maturation of the immune system, recognition of self-antigens plays an important role in shaping the repertoires of immune receptors. Tolerance mechanisms develop that prevent or inhibit potentially harmful reactivities to self-antigens. These self-defense mechanisms are mediated on the levels of central and peripheral tolerance, i.e., autoreactive T cells are either eliminated by apoptosis in the thymus, lymph nodes, or peripheral circulation or actively suppressed by regulatory T cells. Likewise, autoreactive B cells are eliminated in the bone marrow or peripheral lymphoid organs. However, immune responses triggered by foreign antigens may be sustained by molecular mimicry, i.e., presentation and recognition of cryptic epitopes of self-antigens. Further downstream, execution of immune responses depends on the functioning of intracellular signaling networks and the cooperation of many cell types communicating via surface receptors, cytokines, chemokines, and antibody molecules. Therefore, autoimmunity represents the end result of the breakdown of one or multiple basic mechanisms of immune tolerance (Table 1).

Identified OAS3 gene variants associated with coexistence of HBsAg and anti-HBs in chronic HBV infection.

PubMed

Wang, S; Wang, J; Fan, M-J; Li, T-Y; Pan, H; Wang, X; Liu, H-K; Lin, Q-F; Zhang, J-G; Guan, L-P; Zhernakova, D V; O'Brien, S J; Feng, Z-R; Chang, L; Dai, E-H; Lu, J-H; Xi, H-L; Zeng, Z; Yu, Y-Y; Wang, B-B

2018-03-27

The underlying mechanism of coexistence of hepatitis B surface antigen (HBsAg) and hepatitis B surface antigen antibody (anti-HBs) is still controversial. To identify the host genetic factors related to this unusual clinical phenomenon, a two-stage study was conducted in the Chinese Han population. In the first stage, we performed a case-control (1:1) age- and gender-matched study of 101 cases with concurrent HBsAg and anti-HBs and 102 controls with negative HBsAg and positive anti-HBs using whole exome sequencing. In the second validation stage, we directly sequence the 16 exons on the OAS3 gene in two dependent cohorts of 48 cases and 200 controls. Although, in the first stage, a genome-wide association study of 58,563 polymorphism variants in 101 cases and 102 controls found no significant loci (P-value ≤ .05/58563), and neither locus achieved a conservative genome-wide significance threshold (P-value ≤ 5e-08), gene-based burden analysis showed that OAS3 gene rare variants were associated with the coexistence of HBsAg and anti-HBs. (P-value = 4.127e-06 ≤ 0.05/6994). A total of 16 rare variants were screened out from 21 cases and 3 controls. In the second validation stage, one case with a stop-gained rare variant was identified. Fisher's exact test of all 149 cases and 302 controls showed that the rare coding sequence mutations were more frequent in cases vs controls (P-value = 7.299e-09, OR = 17.27, 95% CI [5.01-58.72]). Protein-coding rare variations on the OAS3 gene are associated with the coexistence of HBsAg and anti-HBs in patients with chronic HBV infection in Chinese Han population. © 2018 John Wiley & Sons Ltd.

Genetic transformation of novel isolates of chicken Lactobacillus bearing probiotic features for expression of heterologous proteins: a tool to develop live oral vaccines

PubMed Central

Mota, Rodrigo M; Moreira, João Luiz S; Souza, Marcelo R; Fátima Horta, M; Teixeira, Santuza MR; Neumann, Elisabeth; Nicoli, Jacques R; Nunes, Álvaro C

2006-01-01

Background The use of lactic acid bacteria as vehicles to delivery antigens to immunize animals is a promising issue. When genetically modified, these bacteria can induce a specific local and systemic immune response against selected pathogens. Gastric acid and bile salts tolerance, production of antagonistic substances against pathogenic microorganisms, and adhesive ability to gut epithelium are other important characteristics that make these bacteria useful for oral immunization. Results Bacteria isolated on de Man, Rogosa and Sharpe medium (MRS) from different gastrointestinal portions of broiler chicks were evaluated for their resistance to artificial gastric acid and bile salts, production of hydrogen peroxide, and cell surface hydrophobicity. Thirty-eight isolates were first typed at species level by PCR amplification of 16S-23S rRNA intergenic spacers using universal primers that anneal within 16S and 23S genes, followed by restriction digestion analyses of PCR amplicons (PCR-ARDRA). An expression cassette was assembled onto the pCR2.1-Topo vector by cloning the promoter, leader peptide, cell wall anchor and terminator sequences derived from the laminin binding S-layer protein gene of L. crispatus strain F5.7 (lbs gene). A sequence encoding the green fluorescent protein (GFP) was inserted as reporter gene, and an erythromycin resistance gene was added as selective marker. All constructs were able to express GFP in the cloning host E. coli XL1-Blue and different Lactobacillus strains as verified by FACS and laser scanning confocal microscopy. Conclusion Lactobacillus isolated from gastrointestinal tract of broiler chickens and selected for probiotic characteristics can be genetically modified by introducing an expression cassette into the lbs locus. The transformed bacteria expressed on its cell wall surface different fluorescent proteins used as reporters of promoter function. It is possible then that similar bacterial model expressing pathogen antigens can be used as live oral vaccines to immunize broilers against infectious diseases. PMID:16396687

Differentiation of EL4 lymphoma cells by tumoral environment is associated with inappropriate expression of the large chondroitin sulfate proteoglycan PG-M and the tumor-associated antigen HTgp-175.

PubMed

Rottiers, P; Verfaillie, T; Contreras, R; Revets, H; Desmedt, M; Dooms, H; Fiers, W; Grooten, J

1998-11-09

Progression to malignancy of transformed cells involves complex genetic alterations and aberrant gene expression patterns. While aberrant gene expression is often caused by alterations in individual genes, the contribution of the tumoral environment to the triggering of this gene expression is less well established. The stable but heterogeneous expression in cultured EL4/13 cells of a novel tumor-associated antigen, designated as HTgp-175, was chosen for the investigation of gene expression during tumor formation. Homogeneously HTgp-175-negative EL4/13 cells, isolated by cell sorting or obtained by subcloning, acquired HTgp-175 expression as a result of tumor formation. The tumorigenicity of HTgp-175-negative vs. HTgp-175-positive EL4 variants was identical, indicating that induction but not selection accounted for the phenotypic switch from HTgp-175-negative to HTgp-175-positive. Although mutagenesis experiments showed that the protein was not essential for tumor establishment, tumor-derived cells showed increased malignancy, linking HTgp-175 expression with genetic changes accompanying tumor progression. This novel gene expression was not an isolated event, since it was accompanied by ectopic expression of the large chondroitin sulfate proteoglycan PG-M and of normal differentiation antigens. We conclude that signals derived from the tumoral microenvironment contribute significantly to the aberrant gene expression pattern of malignant cells, apparently by fortuitous activation of differentiation processes and cause expression of novel differentiation antigens as well as of inappropriate tumor-associated and ectopic antigens.

Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome

PubMed Central

Haddad, Diana; Bilcikova, Erika; Witney, Adam A.; Carlton, Jane M.; White, Charles E.; Blair, Peter L.; Chattopadhyay, Rana; Russell, Joshua; Abot, Esteban; Charoenvit, Yupin; Aguiar, Joao C.; Carucci, Daniel J.; Weiss, Walter R.

2004-01-01

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens. PMID:14977966

Evaluation of Recombinant Attenuated Salmonella Vaccine Strains for Broad Protection against Extraintestinal Pathogenic Escherichia coli.

PubMed

Maddux, Jacob T; Stromberg, Zachary R; Curtiss Iii, Roy; Mellata, Melha

2017-01-01

Antibiotic-resistant bacterial infections are difficult to treat, producing a burden on healthcare and the economy. Extraintestinal pathogenic Escherichia coli (ExPEC) strains frequently carry antibiotic resistance genes, cause infections outside of the intestine, and are causative agents of hospital-acquired infections. Developing a prevention strategy against this pathogen is challenging due to its antibiotic resistance and antigenic diversity. E. coli common pilus (ECP) is frequently found in ExPEC strains and may serve as a common antigen to induce protection against several ExPEC serotypes. In addition, live recombinant attenuated Salmonella vaccine (RASV) strains have been used to prevent Salmonella infection and can also be modified to deliver foreign antigens. Thus, the objective of this study was to design a RASV to produce ECP on its surface and assess its ability to provide protection against ExPEC infections. To constitutively display ECP in a RASV strain, we genetically engineered a vector (pYA4428) containing aspartate-β-semialdehyde dehydrogenase and E. coli ecp genes and introduced it into RASV χ9558. RASV χ9558 containing an empty vector (pYA3337) was used as a control to assess protection conferred by the RASV strain without ECP. We assessed vaccine efficacy in in vitro bacterial inhibition assays and mouse models of ExPEC-associated human infections. We found that RASV χ9558(pYA4428) synthesized the major pilin (EcpA) and tip pilus adhesin (EcpD) on the bacterial surface. Mice orally vaccinated with RASV χ9558(pYA3337) without ECP or χ9558(pYA4428) with ECP, produced anti- Salmonella LPS and anti- E. coli EcpA and EcpD IgG and IgA antibodies. RASV strains showed protective potential against some E. coli and Salmonella strains as assessed using in vitro assays. In mouse sepsis and urinary tract infection challenge models, both vaccines had significant protection in some internal organs. Overall, this work showed that RASVs can elicit an immune response to E . coli and Salmonella antigens in some mice, provide significant protection in some internal organs during ExPEC challenge, and thus this study is a promising initial step toward developing a vaccine for prevention of ExPEC infections. Future studies should optimize the ExPEC antigens displayed by the RASV strain for a more robust immune response and enhanced protection against ExPEC infection.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

PubMed Central

McConnell, Sean C.; Hernandez, Kyle M.; Wcisel, Dustin J.; Kettleborough, Ross N.; Stemple, Derek L.; Andrade, Jorge; de Jong, Jill L. O.

2016-01-01

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates. PMID:27493218

Targeting gene therapy to cancer: a review.

PubMed

Dachs, G U; Dougherty, G J; Stratford, I J; Chaplin, D J

1997-01-01

In recent years the idea of using gene therapy as a modality in the treatment of diseases other than genetically inherited, monogenic disorders has taken root. This is particularly obvious in the field of oncology where currently more than 100 clinical trials have been approved worldwide. This report will summarize some of the exciting progress that has recently been made with respect to both targeting the delivery of potentially therapeutic genes to tumor sites and regulating their expression within the tumor microenvironment. In order to specifically target malignant cells while at the same time sparing normal tissue, cancer gene therapy will need to combine highly selective gene delivery with highly specific gene expression, specific gene product activity, and, possibly, specific drug activation. Although the efficient delivery of DNA to tumor sites remains a formidable task, progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods. In this report emphasis will be placed on targeted rather than high-efficiency delivery, although those would need to be combined in the future for effective therapy. To date delivery has been targeted to tumor-specific and tissue-specific antigens, such as epithelial growth factor receptor, c-kit receptor, and folate receptor, and these will be described in some detail. To increase specificity and safety of gene therapy further, the expression of the therapeutic gene needs to be tightly controlled within the target tissue. Targeted gene expression has been analyzed using tissue-specific promoters (breast-, prostate-, and melanoma-specific promoters) and disease-specific promoters (carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC). Alternatively, expression could be regulated externally with the use of radiation-induced promoters or tetracycline-responsive elements. Another novel possibility that will be discussed is the regulation of therapeutic gene products by tumor-specific gene splicing. Gene expression could also be targeted at conditions specific to the tumor microenvironment, such as glucose deprivation and hypoxia. We have concentrated on hypoxia-targeted gene expression and this report will discuss our progress in detail. Chronic hypoxia occurs in tissue that is more than 100-200 microns away from a functional blood supply. In solid tumors hypoxia is widespread both because cancer cells are more prolific than the invading endothelial cells that make up the blood vessels and because the newly formed blood supply is disorganized. Measurements of oxygen partial pressure in patients' tumors showed a high percentage of severe hypoxia readings (less than 2.5 mmHg), readings not seen in normal tissue. This is a major problem in the treatment of cancer, because hypoxic cells are resistant to radiotherapy and often to chemotherapy. However, severe hypoxia is also a physiological condition specific to tumors, which makes it a potentially exploitable target. We have utilized hypoxia response elements (HRE) derived from the oxygen-regulated phosphoglycerate kinase gene to control gene expression in human tumor cells in vitro and in experimental tumors. The list of genes that have been considered for use in the treatment of cancer is extensive. It includes cytokines and costimulatory cell surface molecules intended to induce an effective systemic immune response against tumor antigens that would not otherwise develop. Other inventive strategies include the use of internally expressed antibodies to target oncogenic proteins (intrabodies) and the use of antisense technology (antisense oligonucleotides, antigenes, and ribozymes). This report will concentrate more on novel genes encoding prodrug activating enzymes, so-called suicide genes (Herpes simplex virus thymidine kinase, Escherichia coli nitroreductase, E. (ABSTRACT TRUNCATED)

Deletion of a 77-base-pair inverted repeat element alters the synthesis of surface polysaccharides in Porphyromonas gingivalis.

PubMed

Bainbridge, Brian W; Hirano, Takanori; Grieshaber, Nicole; Davey, Mary E

2015-04-01

Bacterial cell surface glycans, such as capsular polysaccharides and lipopolysaccharides (LPS), influence host recognition and are considered key virulence determinants. The periodontal pathogen Porphyromonas gingivalis is known to display at least three different types of surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown that PG0121 (in strain W83) encodes a DNABII histone-like protein and that this gene is transcriptionally linked to the K-antigen capsule synthesis genes, generating a large ∼19.4-kb transcript (PG0104-PG0121). Furthermore, production of capsule is deficient in a PG0121 mutant strain. In this study, we report on the identification of an antisense RNA (asRNA) molecule located within a 77-bp inverted repeat (77bpIR) element located near the 5' end of the locus. We show that overexpression of this asRNA decreases the amount of capsule produced, indicating that this asRNA can impact capsule synthesis in trans. We also demonstrate that deletion of the 77bpIR element and thereby synthesis of the large 19.4-kb transcript also diminishes, but does not eliminate, capsule synthesis. Surprisingly, LPS structures were also altered by deletion of the 77bpIR element, and reactivity to monoclonal antibodies specific to both O-LPS and A-LPS was eliminated. Additionally, reduced reactivity to these antibodies was also observed in a PG0106 mutant, indicating that this putative glycosyltransferase, which is required for capsule synthesis, is also involved in LPS synthesis in strain W83. We discuss our finding in the context of how DNABII proteins, an antisense RNA molecule, and the 77bpIR element may modulate expression of surface polysaccharides in P. gingivalis. The periodontal pathogen Porphyromonas gingivalis displays at least three different types of cell surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown using Northern analysis that the K-antigen capsule locus encodes a large transcript (∼19.4 kb), encompassing a 77-bp inverted repeat (77bpIR) element near the 5' end. Here, we report on the identification of an antisense RNA (asRNA) encoded within the 77bpIR. We show that overexpression of this asRNA or deletion of the element decreases the amount of capsule. LPS structures were also altered by deletion of the 77bpIR, and reactivity to monoclonal antibodies to both O-LPS and A-LPS was eliminated. Our data indicate that the 77bpIR element is involved in modulating both LPS and capsule synthesis in P. gingivalis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Deletion of a 77-Base-Pair Inverted Repeat Element Alters the Synthesis of Surface Polysaccharides in Porphyromonas gingivalis

PubMed Central

Bainbridge, Brian W.; Hirano, Takanori; Grieshaber, Nicole

2015-01-01

ABSTRACT Bacterial cell surface glycans, such as capsular polysaccharides and lipopolysaccharides (LPS), influence host recognition and are considered key virulence determinants. The periodontal pathogen Porphyromonas gingivalis is known to display at least three different types of surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown that PG0121 (in strain W83) encodes a DNABII histone-like protein and that this gene is transcriptionally linked to the K-antigen capsule synthesis genes, generating a large ∼19.4-kb transcript (PG0104-PG0121). Furthermore, production of capsule is deficient in a PG0121 mutant strain. In this study, we report on the identification of an antisense RNA (asRNA) molecule located within a 77-bp inverted repeat (77bpIR) element located near the 5′ end of the locus. We show that overexpression of this asRNA decreases the amount of capsule produced, indicating that this asRNA can impact capsule synthesis in trans. We also demonstrate that deletion of the 77bpIR element and thereby synthesis of the large 19.4-kb transcript also diminishes, but does not eliminate, capsule synthesis. Surprisingly, LPS structures were also altered by deletion of the 77bpIR element, and reactivity to monoclonal antibodies specific to both O-LPS and A-LPS was eliminated. Additionally, reduced reactivity to these antibodies was also observed in a PG0106 mutant, indicating that this putative glycosyltransferase, which is required for capsule synthesis, is also involved in LPS synthesis in strain W83. We discuss our finding in the context of how DNABII proteins, an antisense RNA molecule, and the 77bpIR element may modulate expression of surface polysaccharides in P. gingivalis. IMPORTANCE The periodontal pathogen Porphyromonas gingivalis displays at least three different types of cell surface glycans: O-LPS, A-LPS, and K-antigen capsule. We have shown using Northern analysis that the K-antigen capsule locus encodes a large transcript (∼19.4 kb), encompassing a 77-bp inverted repeat (77bpIR) element near the 5′ end. Here, we report on the identification of an antisense RNA (asRNA) encoded within the 77bpIR. We show that overexpression of this asRNA or deletion of the element decreases the amount of capsule. LPS structures were also altered by deletion of the 77bpIR, and reactivity to monoclonal antibodies to both O-LPS and A-LPS was eliminated. Our data indicate that the 77bpIR element is involved in modulating both LPS and capsule synthesis in P. gingivalis. PMID:25622614

What helminth genomes have taught us about parasite evolution.

PubMed

Zarowiecki, Magdalena; Berriman, Matt

2015-02-01

The genomes of more than 20 helminths have now been sequenced. Here we perform a meta-analysis of all sequenced genomes of nematodes and Platyhelminthes, and attempt to address the question of what are the defining characteristics of helminth genomes. We find that parasitic worms lack systems for surface antigenic variation, instead maintaining infections using their surfaces as the first line of defence against the host immune system, with several expanded gene families of genes associated with the surface and tegument. Parasite excretory/secretory products evolve rapidly, and proteases even more so, with each parasite exhibiting unique modifications of its protease repertoire. Endoparasitic flatworms show striking losses of metabolic capabilities, not matched by nematodes. All helminths do however exhibit an overall reduction in auxiliary metabolism (biogenesis of co-factors and vitamins). Overall, the prevailing pattern is that there are few commonalities between the genomes of independently evolved parasitic worms, with each parasite having undergone specific adaptations for their particular niche.

Sequence, distribution and chromosomal context of class I and class II pilin genes of Neisseria meningitidis identified in whole genome sequences

PubMed Central

2014-01-01

Background Neisseria meningitidis expresses type four pili (Tfp) which are important for colonisation and virulence. Tfp have been considered as one of the most variable structures on the bacterial surface due to high frequency gene conversion, resulting in amino acid sequence variation of the major pilin subunit (PilE). Meningococci express either a class I or a class II pilE gene and recent work has indicated that class II pilins do not undergo antigenic variation, as class II pilE genes encode conserved pilin subunits. The purpose of this work was to use whole genome sequences to further investigate the frequency and variability of the class II pilE genes in meningococcal isolate collections. Results We analysed over 600 publically available whole genome sequences of N. meningitidis isolates to determine the sequence and genomic organization of pilE. We confirmed that meningococcal strains belonging to a limited number of clonal complexes (ccs, namely cc1, cc5, cc8, cc11 and cc174) harbour a class II pilE gene which is conserved in terms of sequence and chromosomal context. We also identified pilS cassettes in all isolates with class II pilE, however, our analysis indicates that these do not serve as donor sequences for pilE/pilS recombination. Furthermore, our work reveals that the class II pilE locus lacks the DNA sequence motifs that enable (G4) or enhance (Sma/Cla repeat) pilin antigenic variation. Finally, through analysis of pilin genes in commensal Neisseria species we found that meningococcal class II pilE genes are closely related to pilE from Neisseria lactamica and Neisseria polysaccharea, suggesting horizontal transfer among these species. Conclusions Class II pilins can be defined by their amino acid sequence and genomic context and are present in meningococcal isolates which have persisted and spread globally. The absence of G4 and Sma/Cla sequences adjacent to the class II pilE genes is consistent with the lack of pilin subunit variation in these isolates, although horizontal transfer may generate class II pilin diversity. This study supports the suggestion that high frequency antigenic variation of pilin is not universal in pathogenic Neisseria. PMID:24690385

[Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product].

PubMed

Luo, Dong-jiao; Hu, Ye; Dennin, R H; Yan, Jie

2007-09-01

To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a favorable condition to use its product for further developing a novel universal vaccine as well as detection kit of leptospirosis.

Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBsAg and antiHBc negative blood donor.

PubMed

Datta, Sibnarayan; Banerjee, Arup; Chandra, Partha K; Chakraborty, Subhasis; Basu, Subir Kumar; Chakravarty, Runu

2007-11-01

In blood donors, HBV infection is detected by the presence of serum hepatitis B surface antigen (HBsAg). However, some mutations in the surface gene region may result in altered or truncated HBsAg that can escape from immunoassay-based diagnosis. Such diagnostic escape mutants pose a potential risk for blood transfusion services. In the present study, we report a blood donor seronegative for HBsAg and antiHBc, but positive for antiHBs who was HBV DNA positive by PCR. Sequencing of the HBsAg gene revealed presence of a point mutation (T-A) at 207th nucleotide of the HBsAg ORF, which resulted in a premature stop codon at position 69. This results in a truncated HBsAg gene lacking the entire 'a' determinant region. However, follow-up of the donor after 2 years revealed clearance of HBV DNA from the serum. The case illustrates an unusual mutation, which causes HBsAg negativity. The finding emphasizes the importance of molecular assays in reducing the possibility of HBV transmission through blood transfusion. However, developing more sensitive serological assays, capable of detecting HBV mutants, is an alternative to expensive and complex amplification-based assays for developing countries.

Genetic engineering with T cell receptors.

PubMed

Zhang, Ling; Morgan, Richard A

2012-06-01

In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers. Published by Elsevier B.V.

Expression of Immunoglobulin Receptors with Distinctive Features Indicating Antigen Selection by Marginal Zone B Cells from Human Spleen

PubMed Central

Colombo, Monica; Cutrona, Giovanna; Reverberi, Daniele; Bruno, Silvia; Ghiotto, Fabio; Tenca, Claudya; Stamatopoulos, Kostas; Hadzidimitriou, Anastasia; Ceccarelli, Jenny; Salvi, Sandra; Boccardo, Simona; Calevo, Maria Grazia; De Santanna, Amleto; Truini, Mauro; Fais, Franco; Ferrarini, Manlio

2013-01-01

Marginal zone (MZ) B cells, identified as surface (s)IgMhighsIgDlowCD23low/−CD21+CD38− B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27+ and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared (“stereotyped”) sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli. PMID:23877718

AgdbNet – antigen sequence database software for bacterial typing

PubMed Central

Jolley, Keith A; Maiden, Martin CJ

2006-01-01

Background Bacterial typing schemes based on the sequences of genes encoding surface antigens require databases that provide a uniform, curated, and widely accepted nomenclature of the variants identified. Due to the differences in typing schemes, imposed by the diversity of genes targeted, creating these databases has typically required the writing of one-off code to link the database to a web interface. Here we describe agdbNet, widely applicable web database software that facilitates simultaneous BLAST querying of multiple loci using either nucleotide or peptide sequences. Results Databases are described by XML files that are parsed by a Perl CGI script. Each database can have any number of loci, which may be defined by nucleotide and/or peptide sequences. The software is currently in use on at least five public databases for the typing of Neisseria meningitidis, Campylobacter jejuni and Streptococcus equi and can be set up to query internal isolate tables or suitably-configured external isolate databases, such as those used for multilocus sequence typing. The style of the resulting website can be fully configured by modifying stylesheets and through the use of customised header and footer files that surround the output of the script. Conclusion The software provides a rapid means of setting up customised Internet antigen sequence databases. The flexible configuration options enable typing schemes with differing requirements to be accommodated. PMID:16790057

Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production.

PubMed

Boulnois, G J; Roberts, I S; Hodge, R; Hardy, K R; Jann, K B; Timmis, K N

1987-06-01

Transposon and deletion analysis of the cloned K1 capsule biosynthesis genes of Escherichia coli revealed that approximately 17 kb of DNA, split into three functional regions, is required for capsule production. One block (region 1) is required for translocation of polysaccharide to the cell surface and mutations in this region result in the intracellular appearance of polymer indistinguishable on immunoelectrophoresis to that found on the surface of K1 encapsulated bacteria. This material was released from the cell by osmotic shock indicating that the polysaccharide was probably present in the periplasmic space. Insertions in a second block (region 2) completely abolished polymer production and this second region is believed to encode the enzymes for the biosynthesis and polymerisation of the K1 antigen. Addition of exogenous N-acetylneuraminic acid to one insertion mutant in this region restored its ability to express surface polymer as judged by K1 phage sensitivity. This insertion probably defines genes involved in biosynthesis of N-acetylneuraminic acid. Insertions in a third block (region 3) result in the intracellular appearance of polysaccharide with a very low electrophoretic mobility. The presence of the cloned K1 capsule biosynthesis genes on a multicopy plasmid in an E. coli K-12 strain did not increase the yields of capsular polysaccharide produced compared to the K1+ isolate from which the genes were cloned.

Imaging of blood antigen distribution on blood cells by thermal lens microscopy

NASA Astrophysics Data System (ADS)

Kimura, Hiroko; Sekiguchi, Kazuya; Nagao, Fumiko; Mukaida, Masahiro; Kitamori, Takehiko; Sawada, Tsuguo

2000-05-01

Blood group antigens on a cell were measured by a new microscopic method, i.e. thermal lens microscopy which involves spectrometry using a laser-induced thermal-lens effect. The blood group antigen was immunologically stained using antibody labeled with colloidal gold. Human leukocyte antigens (HLA) on lymphocytes and mononuclear leukocytes were observed by the thermal lens microscope, and Lewis blood group antigens on erythrocytes and polymorphonuclear leukocytes were also observed. The antigen distribution on each cell-surface was imaged using this technique. In spite of convex surface of living cells, colloidal gold was correctly quantified by adjusting the deviation of the focal point of the probe laser by the phase of the signal. In the measurement of leukocyte antigens, antigens of HLA-A, -B, -C loci on the lymphocytes were identified and quantitated by using a single cell. The image of HLA-A, -B, -C antigen distribution on a mononuclear leukocyte was obtained. In the measurement of erythrocyte antigens, a small quantity of Lewis antigens was detected on the cord erythrocytes. Localized small quantities of membrane antigens are better quantitated without extraction or cytolysis. Our thermal lens microscope is a powerful and highly sensitive analytical tool for detecting and quantitating localized antigens in single cells and/or cell-surface-associated molecules.

JPRS Report, Science & Technology, Japan, Government and Private Sector Joint R&D Projects

DTIC Science & Technology

1988-08-16

is that if this system uses exogenous genes represented by the DNA that codes for hepatitis B surface antigen, polyvaccines can be produced easily...that can express in large quantities molecular clones of genetic products is awaited. Vectors using cells of higher mammals including man have been...functions of IVM and MMD using cultured spinal nerve cells with a great advantage that the same sample can be used for both electrophysiological and

Genotypic Characterization of Egypt Enterotoxigenic Escherichia coli Isolates Expressing Coli Surface Antigen 6

DTIC Science & Technology

2013-02-01

47%) of these isolates were resistant to ampicillin, a third (37%) of the isolates were resistant to trimethoprim -sulfamethoxazole, and 24% of the...isolates were tetracycline-resistant. A blaTEM gene was detected in 24 (83%) ampicillin-resistant isolates. Trimethoprim -sulfamethoxazole-resistant...ciprofloxacin (CIP) 5 μg, amikacin (AN) 30 μg, gentamicin (GEN) 10 μg, tetracycline (TET) 30 μg, trimethoprim 1.25 μg / sulfamethoxazole 23.75 μg (SXT

Pim-1: A Molecular Target to Modulate Cellular Resistance to Therapy in Prostate Cancer

DTIC Science & Technology

2005-10-01

Reiter RE, Lilly MB: Gene expression profiling in R- flurbiprofen -treated prostate cancer: Identification of prostate stem cell antigen as a... flurbiprofen -regulated gene. (submitted, 2006). 51. Holder SL, Zemskova M, Bremner R, Neidigh J, Lilly MB: Identification of specific, cell-permeable...profiling in R- flurbiprofen - treated prostate cancer: Identification of prostate stem cell antigen as a flurbiprofen - regulated gene. (poster

Expressed var gene repertoire and variant surface antigen diversity in a shrinking Plasmodium falciparum population.

PubMed

Carlos, Bianca C; Fotoran, Wesley L; Menezes, Maria J; Cabral, Fernanda J; Bastos, Marcele F; Costa, Fabio T M; Sousa-Neto, Jayme A; Ribolla, Paulo E M; Wunderlich, Gerhard; Ferreira, Marcelo U

2016-11-01

The var gene-encoded erythrocyte membrane protein-1 of Plasmodium falciparum (PfEMP-1) is the main variant surface antigen (VSA) expressed on infected erythrocytes. The rate at which antibody responses to VSA expressed by circulating parasites are acquired depends on the size of the local VSA repertoire and the frequency of exposure to new VSA. Because parasites from areas with declining malaria endemicity, such as the Amazon, typically express a restricted PfEMP-1 repertoire, we hypothesized that Amazonians would rapidly acquire antibodies to most locally circulating VSA. Consistent with our expectations, the analysis of 5878 sequence tags expressed by 10 local P. falciparum samples revealed little PfEMP-1 DBL1α domain diversity. Among the most commonly expressed DBL1α types, 45% were shared by two or more independent parasite lines. Nevertheless, Amazonians displayed major gaps in their repertoire of anti-VSA antibodies, although the breadth of anti-VSA antibody responses correlated positively with their cumulative exposure to malaria. We found little antibody cross-reactivity even when testing VSA from related parasites expressing the same dominant DBL1α types. We conclude that variant-specific immunity to P. falciparum VSAs develops slowly despite the relatively restricted PfEMP-1 repertoire found in low-endemicity settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Cloning and expression of colonization factor antigen I (CFA/I) epitopes of enterotoxigenic Escherichia coli (ETEC) in Salmonella flagellin.

PubMed

Luna, M G; Martins, M M; Newton, S M; Costa, S O; Almeida, D F; Ferreira, L C

1997-01-01

Oligonucleotides coding for linear epitopes of the fimbrial colonization factor antigen I (CFA/I) of enterotoxigenic Escherichia coli (ETEC) were cloned and expressed in a deleted form of the Salmonella muenchen flagellin fliC (H1-d) gene. Four synthetic oligonucleotide pairs coding for regions corresponding to amino acids 1 to 15 (region I), amino acids 11 to 25 (region II), amino acids 32 to 45 (region III) and amino acids 88 to 102 (region IV) were synthesized and cloned in the Salmonella flagellin-coding gene. All four hybrid flagellins were exported to the bacterial surface where they produced flagella, but only three constructs were fully motile. Sera recovered from mice immunized with intraperitoneal injections of purified flagella containing region II (FlaII) or region IV (FlaIV) showed high titres against dissociated solid-phase-bound CFA/I subunits. Hybrid flagellins containing region I (FlaI) or region III (FlaIII) elicited a weak immune response as measured in enzyme-linked immunosorbent assay (ELISA) with dissociated CFA/I subunits. None of the sera prepared with purified hybrid flagella were able to agglutinate or inhibit haemagglutination promoted by CFA/I-positive strains. Moreover, inhibition ELISA tests indicated that antisera directed against region I, II, III or IV cloned in flagellin were not able to recognize surface-exposed regions on the intact CFA/I fimbriae.

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

PubMed

Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2015-04-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. © 2015 The Authors.

A newly identified protein of Leptospira interrogans mediates binding to laminin.

PubMed

Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2009-10-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

Scavenger receptor WC1 contributes to the γδ T cell response to Leptospira.

PubMed

Wang, Fei; Herzig, Carolyn T A; Chen, Chuang; Hsu, Haoting; Baldwin, Cynthia L; Telfer, Janice C

2011-03-01

WC1 molecules are exclusively expressed on the surface of γδ T cells. They belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. WC1 molecules have been grouped on the basis of antibody reactivity. The expression of WC1 molecules from these serologically defined groups is correlated with differences in γδ T cell responses. The expression of receptors within the WC1.1 group correlates with the capacity of γδ T cells to respond to Leptospira antigen. In this study, we used RNA interference to directly investigate the role of WC1 expression in the response to Leptospira borgpetersenii. We found that when three out of thirteen WC1 gene products were downregulated by RNA interference, γδ T cell proliferation and IFN-γ production in response to Leptospira antigen was significantly reduced. Our data demonstrate that specific receptors in the WC1 family directly participate in Leptospira recognition and/or activation of γδ T cells. Copyright © 2010 Elsevier Ltd. All rights reserved.

Antigenic Determinants of Alpha-Like Proteins of Streptococcus agalactiae

PubMed Central

Maeland, Johan A.; Bevanger, Lars; Lyng, Randi Valsoe

2004-01-01

The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Cα) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Cα. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments. PMID:15539502

Detection of lamivudine-resistant variants and mutations related to reduced antigenicity of HBsAg in individuals from the cities of Santos and São Paulo, Brazil

PubMed Central

2013-01-01

Background Continuous long-term treatment is recommended to reduce the hepatitis B virus (HBV) viral load. However, as a consequence, resistance mutations can emerge and be transmitted to other individuals. The polymerase (POL) gene overlaps the surface (S) gene. Thus, during treatment, mutations in the POL gene may lead to changes in hepatitis B surface antigen (HBsAg). The purpose of this study was to evaluate the frequency of lamivudine and vaccine escape mutations in HBsAg-positive blood donors from the city of Santos and in untreated HBV mono-infected patients from the city of São Paulo, Brazil. Methods HBV DNA was extracted from 80 serum samples, of which 61 were from volunteer blood donors and 19 were from untreated HBV patients. A fragment of the POL/S genes containing 593 base pairs was amplified using nested PCR. Thirty four were PCR-positive and sequencing was performed using an ABI Prism 3130 Genetic Analyzer. Alignments and mutation mapping were performed using BioEdit software. Results HBV DNA from 21 blood donors and 13 untreated patient samples were characterized using nucleotide sequencing PCR products from the POL/S genes. We were able to detect one sample with the resistance mutation to lamivudine rtM204V + rtL180M (2.94%), which was found in a volunteer blood donor that has never used antiviral drugs. The other samples showed only compensatory mutations, such as rtL80F (5.88%), rtL80V (2.94%), rtL82V + rtV207L (2.94%), rtT128P (5.88%), rtT128N/S (2.94%) and rtS219A (5.88%). We found modifications in the S gene in 14 of the 34 samples (41.16%). The mutations detected were as follows: sM133L + sI195T (2.94%), sI195M (2.94%), sP120T (2.94%), sY100S/F (2.94%), sY100C (17.64%), sI/T126P + sQ129P (2.94%), sM198I + sF183C (2.94%) and sS210R (5.88%). Conclusions Our results suggest the transmission of lamivudine-resistant forms. Thus, the evaluation of HBV-infected subjects for lamivudine resistance would improve treatment regime. Moreover, the mutations in the S gene may impair HBsAg antigenicity and contribute to HBsAg failure detection and vaccine escape. PMID:24165277

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

PubMed

Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

2016-11-01

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

First detection of Candidatus Rickettsia barbariae in the flea Vermipsylla alakurt from north-western China.

PubMed

Zhao, Shan-Shan; Li, Hong-Yu; Yin, Xiao-Ping; Liu, Zhi-Qiang; Chen, Chuang-Fu; Wang, Yuan-Zhi

2016-06-07

Vermipsylla is a genus of the family Vermipsyllidae within the order Siphonaptera of fleas. Vermipsylla alakurt is mainly distributed in alpine pastoral areas of Kazakhstan, Mongolia, China and Nepal, and infests sheep, yaks and horses, causing irritation, poor condition, anaemia and even death. However, to date, no rickettsial agents have been reported in V. alakurt. A total of 133 fleas were collected directly from the tails of three sheep flocks (n = 335) in Minfeng County, Xinjiang Uygur Autonomous Region, north-western China. Of these, 55 fleas were identified by morphological examination and molecular analysis of four loci (the ribosomal 18S and 28S rDNA genes and the mitochondrial genes cytochrome  c oxidase subunit II and elongation factor 1-alpha). Eight Rickettsia-specific gene fragments originated from seven genes: the 17-kilodalton antigen gene (17-kDa), citrate synthase gene (gltA), 16S rRNA gene (rrs), outer membrane protein A gene (ompA), surface cell antigen 1 gene (sca1), PS120 protein gene (gene D), and outer membrane protein B gene (ompB, two fragments), were used to identify the species of Rickettsia in 53 fleas. The amplified products were sequenced and included in a phylogenetic analysis to verify the taxonomic identification of the rickettsial agents. Based on morphological and molecular evidence, the flea was identified as Vermipsylla alakurt. Nine samples were positive (16.98 %, 9/53) for Rickettsia spp. The phylogenetic tree revealed that the rickettsial agents found in V. alakurt cluster with Candidatus Rickettsia barbariae. Our study suggests that: (i) V. alakurt may serve as a carrier for Candidatus R. barbariae; and (ii) Candidatus R. barbariae, previously reported in Israel, is the eighth newly discovered validated Rickettsia species in China. This finding extends our knowledge of the distribution of Candidatus R. barbariae and the profile of carriers, which not only comprise ticks but also fleas.

Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation.

PubMed

Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin; Wang, Lei

2017-11-01

Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy.

Progesterone impairs antigen-non-specific immune protection by CD8 T memory cells via interferon-γ gene hypermethylation

PubMed Central

Yao, Yushi; Li, Hui; Ding, Jie; Xia, Yixin

2017-01-01

Pregnant women and animals have increased susceptibility to a variety of intracellular pathogens including Listeria monocytogenes (LM), which has been associated with significantly increased level of sex hormones such as progesterone. CD8 T memory(Tm) cell-mediated antigen-non-specific IFN-γ responses are critically required in the host defense against LM. However, whether and how increased progesterone during pregnancy modulates CD8 Tm cell-mediated antigen-non-specific IFN-γ production and immune protection against LM remain poorly understood. Here we show in pregnant women that increased serum progesterone levels are associated with DNA hypermethylation of IFN-γ gene promoter region and decreased IFN-γ production in CD8 Tm cells upon antigen-non-specific stimulation ex vivo. Moreover, IFN-γ gene hypermethylation and significantly reduced IFN-γ production post LM infection in antigen-non-specific CD8 Tm cells are also observed in pregnant mice or progesterone treated non-pregnant female mice, which is a reversible phenotype following demethylation treatment. Importantly, antigen-non-specific CD8 Tm cells from progesterone treated mice have impaired anti-LM protection when adoptive transferred in either pregnant wild type mice or IFN-γ-deficient mice, and demethylation treatment rescues the adoptive protection of such CD8 Tm cells. These data demonstrate that increased progesterone impairs immune protective functions of antigen-non-specific CD8 Tm cells via inducing IFN-γ gene hypermethylation. Our findings thus provide insights into a new mechanism through which increased female sex hormone regulate CD8 Tm cell functions during pregnancy. PMID:29155896

Immunoelectrophoretic study of cell surface antigens from different Streptococcus mutans serotypes and Streptococcus sanguis.

PubMed

Ogier, J A; Klein, J P; Niddam, R; Frank, R M

1985-06-01

Antigens prepared from culture supernatants or whole cells of several cariogenic strains were examined by immunoelectrophoresis for their crossed antigenicity, with reference to Streptococcus mutans OMZ175, serotype f. Crossed immunoelectrophoresis revealed a crossreactivity between soluble extracellular and wall associated antigens of six strains of Streptococcus mutans and one strain of Streptococcus sanguis. Protease destroyed the immunoreactivity of crossreactive antigens. One of them was shown to be localized on the bacterial surface.

[Novel therapy for malignant lymphoma: adoptive immuno-gene therapy using chimeric antigen receptor(CAR)-expressing T lymphocytes].

PubMed

Ozawa, Keiya

2014-03-01

Adoptive T-cell therapy using chimeric antigen receptor (CAR) technology is a novel approach to cancer immuno-gene therapy. CARs are hybrid proteins consisting of target-antigen-specific single-chain antibody fragment fused to intracellular T-cell activation domains (CD28 or CD137/CD3 zeta receptor). CAR-expressing engineered T lymphocytes can directly recognize and kill tumor cells in an HLA independent manner. In the United States, promising results have been obtained in the clinical trials of adoptive immuno-gene therapy using CD19-CAR-T lymphocytes for the treatment of refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL). In this review article, CD19-CAR-T gene therapy for refractory B-cell non-Hodgkin lymphoma is discussed.

[Gene deletion and functional analysis of the heptyl glycosyltransferase (waaF) gene in Vibrio parahemolyticus O-antigen cluster].

PubMed

Zhao, Feng; Meng, Songsong; Zhou, Deqing

2016-02-04

To construct heptyl glycosyltransferase gene II (waaF) gene deletion mutant of Vibrio parahaemolyticus, and explore the function of the waaF gene in Vibrio parahaemolyticus. The waaF gene deletion mutant was constructed by chitin-based transformation technology using clinical isolates, and then the growth rate, morphology and serotypes were identified. The different sources (O3, O5 and O10) waaF gene complementations were constructed through E. coli S17λpir strains conjugative transferring with Vibrio parahaemolyticus, and the function of the waaF gene was further verified by serotypes. The waaF gene deletion mutant strain was successfully constructed and it grew normally. The growth rate and morphology of mutant were similar with the wild type strains (WT), but the mutant could not occurred agglutination reaction with O antisera. The O3 and O5 sources waaF gene complementations occurred agglutination reaction with O antisera, but the O10 sources waaF gene complementations was not. The waaF gene was related with O-antigen synthesis and it was the key gene of O-antigen synthesis pathway in Vibrio parahaemolyticus. The function of different sources waaF gene were not the same.

Structure of a gene encoding a murine thymus leukemia antigen, and organization of Tla genes in the BALB/c mouse

PubMed Central

1985-01-01

We have determined the DNA sequence of a gene encoding a thymus leukemia (TL) antigen in the BALB/c mouse, and have more definitively mapped the cloned BALB/c Tla-region class I gene clusters. Analysis of the sequence shows that the Tla gene is less closely related to the H-2 genes than H-2 genes are to one another or to a Qa-2,3-region genes. The Tla gene, 17.3A, contains an apparent gene conversion. Comparison of the BALB/c Tla genes with those from C57BL shows that BALB/c has more Tla-region class I genes, and that one of the genes absent in C57BL is gene 17.3A. PMID:3894562

SYNTHESIS, INTRACELLULAR DISTRIBUTION, AND SECRETION OF IMMUNOGLOBULIN AND H-2 ANTIGEN IN MURINE SPLENOCYTES

PubMed Central

Wernet, Dorothee; Vitetta, Ellen S.; Uhr, Jonathan W.; Boyse, Edward A.

1973-01-01

A/J spleen cells were labeled with [3H]leucine and at intervals thereafter were homogenized and separated into microsomes and cell sap. Ig and H-2 antigens were assayed in the cell fractions and cell supernatants using immunoprecipitation. In addition, cells labeled by enzymatic radioiodination were incubated to determine the rates of release of Ig and H-2 antigens from the surface. The results indicate that the majority of Ig and H-2 antigens remain membrane bound throughout their intracellular life. In contrast to Ig, H-2 antigens are neither secreted nor shed from the cell surface. It is suggested that Ig is a peripheral protein of the cell membrane, whereas H-2 antigens are integral ones. The release of Ig on a fragment of plasma membrane could occur at fixed cell surface areas that contain no H-2 antigens or from which they have migrated before release. PMID:4200648

Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.

PubMed Central

van Endert, P M; Lopez, M T; Patel, S D; Monaco, J J; McDevitt, H O

1992-01-01

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes. Images PMID:1360671

Noninvasive ocular drug delivery: potential transcorneal and other alternative delivery routes for therapeutic molecules in glaucoma.

PubMed

Foldvari, Marianna

2014-01-01

Drug delivery to the eye is made difficult by multiple barriers (such as the tear film, cornea, and vitreous) between the surface of the eye and the treatment site. These barriers are difficult to surmount for the purposes of drug delivery without causing toxicity. Using nanotechnology tools to control, manipulate, and study delivery systems, new approaches to delivering drugs, genes, and antigens that are effective and safe can be developed. Topical administration to the ocular surface would be the safest method for delivery, as it is noninvasive and painless compared with other delivery methods. However, there is only limited success using topical delivery methods, especially for gene therapy. Current thinking on treatments of the future enabled by nanodelivery systems and the identification of target specificity parameters that require deeper understanding to develop successful topical delivery systems for glaucoma is highlighted.

Correlation of the cell surface antigens with stage and grade in cancer of the bladder.

PubMed

Emmott, R C; Javadpour, N; Bergman, S M; Soares, T

1979-01-01

We examined 76 bladder tumors of various stages and grades for the presence of the ABO (H) cell surface antigen, using the specific red cell adherence technique. Of the grade I lesions studied 70 per cent were positive for the cell surface antigen and none of the 26 grade III tumors retained the antigens. When correlated with clinical stage the tumors showed no antigens for those of stages B1 to D, while 12 of 16 stage A lesions were positive for the antigen. When stage A lesions were studied and the findings were correlated with recurrence and metastasis/invasion rates the cell surface antigen was present on the initial tumor in only 1 lesion that recurred at an invasive stage. The findings of this study show that the specific red cell adherence technique may be valuable for predicting malignant potential in low grade, low stage cancer of the bladder. If supported by further investigation this technique may offer the capability of selecting low grade, low stage bladder tumors that are destined to invade or metastasize while they are at curable stages.

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

PubMed

Tapia, Lorena I; Shaw, Chad A; Aideyan, Letisha O; Jewell, Alan M; Dawson, Brian C; Haq, Taha R; Piedra, Pedro A

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community.

Gene Sequence Variability of the Three Surface Proteins of Human Respiratory Syncytial Virus (HRSV) in Texas

PubMed Central

Tapia, Lorena I.; Shaw, Chad A.; Aideyan, Letisha O.; Jewell, Alan M.; Dawson, Brian C.; Haq, Taha R.; Piedra, Pedro A.

2014-01-01

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004–2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community. PMID:24625544

Roles for SH2 and SH3 domains in Lyn kinase association with activated FcepsilonRI in RBL mast cells revealed by patterned surface analysis.

PubMed

Hammond, Stephanie; Wagenknecht-Wiesner, Alice; Veatch, Sarah L; Holowka, David; Baird, Barbara

2009-10-01

In mast cells, antigen-mediated cross-linking of IgE bound to its high-affinity surface receptor, FcepsilonRI, initiates a signaling cascade that culminates in degranulation and release of allergic mediators. Antigen-patterned surfaces, in which the antigen is deposited in micron-sized features on a silicon substrate, were used to examine the spatial relationship between clustered IgE-FcepsilonRI complexes and Lyn, the signal-initiating tyrosine kinase. RBL mast cells expressing wild-type Lyn-EGFP showed co-redistribution of this protein with clustered IgE receptors on antigen-patterned surfaces, whereas Lyn-EGFP containing an inhibitory point mutation in its SH2 domain did not significantly accumulate with the patterned antigen, and Lyn-EGFP with an inhibitory point mutation in its SH3 domain exhibited reduced interactions. Our results using antigen-patterned surfaces and quantitative cross-correlation image analysis reveal that both the SH2 and SH3 domains contribute to interactions between Lyn kinase and cross-linked IgE receptors in stimulated mast cells.

Genome-Scale Protein Microarray Comparison of Human Antibody Responses in Plasmodium vivax Relapse and Reinfection

PubMed Central

Chuquiyauri, Raul; Molina, Douglas M.; Moss, Eli L.; Wang, Ruobing; Gardner, Malcolm J.; Brouwer, Kimberly C.; Torres, Sonia; Gilman, Robert H.; Llanos-Cuentas, Alejandro; Neafsey, Daniel E.; Felgner, Philip; Liang, Xiaowu; Vinetz, Joseph M.

2015-01-01

Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions. PMID:26149860

Transcriptional changes induced by candidate malaria vaccines and correlation with protection against malaria in a human challenge model

PubMed Central

Dunachie, Susanna; Berthoud, Tamara; Hill, Adrian V.S.; Fletcher, Helen A.

2015-01-01

Introduction The complexity of immunity to malaria is well known, and clear correlates of protection against malaria have not been established. A better understanding of immune markers induced by candidate malaria vaccines would greatly enhance vaccine development, immunogenicity monitoring and estimation of vaccine efficacy in the field. We have previously reported complete or partial efficacy against experimental sporozoite challenge by several vaccine regimens in healthy malaria-naïve subjects in Oxford. These include a prime-boost regimen with RTS,S/AS02A and modified vaccinia virus Ankara (MVA) expressing the CSP antigen, and a DNA-prime, MVA-boost regimen expressing the ME TRAP antigens. Using samples from these trials we performed transcriptional profiling, allowing a global assessment of responses to vaccination. Methods We used Human RefSeq8 Bead Chips from Illumina to examine gene expression using PBMC (peripheral blood mononuclear cells) from 16 human volunteers. To focus on antigen-specific changes, comparisons were made between PBMC stimulated with CSP or TRAP peptide pools and unstimulated PBMC post vaccination. We then correlated gene expression with protection against malaria in a human Plasmodium falciparum malaria challenge model. Results Differentially expressed genes induced by both vaccine regimens were predominantly in the IFN-γ pathway. Gene set enrichment analysis revealed antigen-specific effects on genes associated with IFN induction and proteasome modules after vaccination. Genes associated with IFN induction and antigen presentation modules were positively enriched in subjects with complete protection from malaria challenge, while genes associated with haemopoietic stem cells, regulatory monocytes and the myeloid lineage modules were negatively enriched in protected subjects. Conclusions These results represent novel insights into the immune repertoires involved in malaria vaccination. PMID:26256523

[Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes activated by T-cells with different differentiation status in vitro].

PubMed

Yurova, K A; Sokhonevich, N A; Khaziakhmatova, O G; Litvinova, L S

2016-01-01

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common g-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vitro using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+-cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common g-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal "surrogate" cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important for construction of a general model of self-maintenance and differentiation of immune cells.

Transcriptional changes induced by candidate malaria vaccines and correlation with protection against malaria in a human challenge model.

PubMed

Dunachie, Susanna; Berthoud, Tamara; Hill, Adrian V S; Fletcher, Helen A

2015-09-29

The complexity of immunity to malaria is well known, and clear correlates of protection against malaria have not been established. A better understanding of immune markers induced by candidate malaria vaccines would greatly enhance vaccine development, immunogenicity monitoring and estimation of vaccine efficacy in the field. We have previously reported complete or partial efficacy against experimental sporozoite challenge by several vaccine regimens in healthy malaria-naïve subjects in Oxford. These include a prime-boost regimen with RTS,S/AS02A and modified vaccinia virus Ankara (MVA) expressing the CSP antigen, and a DNA-prime, MVA-boost regimen expressing the ME TRAP antigens. Using samples from these trials we performed transcriptional profiling, allowing a global assessment of responses to vaccination. We used Human RefSeq8 Bead Chips from Illumina to examine gene expression using PBMC (peripheral blood mononuclear cells) from 16 human volunteers. To focus on antigen-specific changes, comparisons were made between PBMC stimulated with CSP or TRAP peptide pools and unstimulated PBMC post vaccination. We then correlated gene expression with protection against malaria in a human Plasmodium falciparum malaria challenge model. Differentially expressed genes induced by both vaccine regimens were predominantly in the IFN-γ pathway. Gene set enrichment analysis revealed antigen-specific effects on genes associated with IFN induction and proteasome modules after vaccination. Genes associated with IFN induction and antigen presentation modules were positively enriched in subjects with complete protection from malaria challenge, while genes associated with haemopoietic stem cells, regulatory monocytes and the myeloid lineage modules were negatively enriched in protected subjects. These results represent novel insights into the immune repertoires involved in malaria vaccination. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Primary cutaneous B-cell lymphoma is associated with somatically hypermutated immunoglobulin variable genes and frequent use of VH1-69 and VH4-59 segments.

PubMed

Perez, M; Pacchiarotti, A; Frontani, M; Pescarmona, E; Caprini, E; Lombardo, G A; Russo, G; Faraggiana, T

2010-03-01

Accurate assessment of the somatic mutational status of clonal immunoglobulin variable region (IgV) genes is relevant in elucidating tumour cell origin in B-cell lymphoma; virgin B cells bear unmutated IgV genes, while germinal centre and postfollicular B cells carry mutated IgV genes. Furthermore, biases in the IgV repertoire and distribution pattern of somatic mutations indicate a possible antigen role in the pathogenesis of B-cell malignancies. This work investigates the cellular origin and antigenic selection in primary cutaneous B-cell lymphoma (PCBCL). We analysed the nucleotide sequence of clonal IgV heavy-chain gene (IgVH) rearrangements in 51 cases of PCBCL (25 follicle centre, 19 marginal zone and seven diffuse large B-cell lymphoma, leg-type) and compared IgVH sequences with their closest germline segment in the GenBank database. Molecular data were then correlated with histopathological features. We showed that all but one of the 51 IgVH sequences analysed exhibited extensive somatic hypermutations. The detected mutation rate ranged from 1.6% to 21%, with a median rate of 9.8% and was independent of PCBCL histotype. Calculation of antigen-selection pressure showed that 39% of the mutated IgVH genes displayed a number of replacement mutations and silent mutations in a pattern consistent with antigenic selection. Furthermore, two segments, VH1-69 (12%) and VH4-59 (14%), were preferentially used in our case series. Data indicate that neoplastic B cells of PBCBL have experienced germinal centre reaction and also suggest that the involvement of IgVH genes is not entirely random in PCBCL and that common antigen epitopes could be pathologically relevant in cutaneous lymphomagenesis.

Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomavo, S.; Schwarz, R.T.; Dubremetz, J.F.

1989-10-01

The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with {sup 3}H-fatty acids, ({sup 3}H)ethanolamine, and ({sup 3}H)carbohydrates. Treatment of {sup 3}H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

Induction of Interferon-Stimulated Genes by Simian Virus 40 T Antigens

PubMed Central

Rathi, Abhilasha V.; Cantalupo, Paul G.; Sarkar, Saumendra N.; Pipas, James M.

2010-01-01

Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAgwt) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAgwt and a truncated TAg (TAgN136), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAgwt including many interferon stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAgwt. Our genetic studies using several TAg mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs. PMID:20692676

Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection.

PubMed

Moeller, Maria; Haynes, Nicole M; Kershaw, Michael H; Jackson, Jacob T; Teng, Michele W L; Street, Shayna E; Cerutti, Loretta; Jane, Stephen M; Trapani, Joseph A; Smyth, Mark J; Darcy, Phillip K

2005-11-01

Because CD4+ T cells play a key role in aiding cellular immune responses, we wanted to assess whether increasing numbers of gene-engineered antigen-restricted CD4+ T cells could enhance an antitumor response mediated by similarly gene-engineered CD8+ T cells. In this study, we have used retroviral transduction to generate erbB2-reactive mouse T-cell populations composed of various proportions of CD4+ and CD8+ cells and then determined the antitumor reactivity of these mixtures. Gene-modified CD4+ and CD8+ T cells were shown to specifically secrete Tc1 (T cytotoxic-1) or Tc2 cytokines, proliferate, and lyse erbB2+ tumor targets following antigen ligation in vitro. In adoptive transfer experiments using severe combined immunodeficient (scid) mice, we demonstrated that injection of equivalent numbers of antigen-specific engineered CD8+ and CD4+ T cells led to significant improvement in survival of mice bearing established lung metastases compared with transfer of unfractionated (largely CD8+) engineered T cells. Transferred CD4+ T cells had to be antigen-specific (not just activated) and secrete interferon gamma (IFN-gamma) to potentiate the antitumor effect. Importantly, antitumor responses in these mice correlated with localization and persistence of gene-engineered T cells at the tumor site. Strikingly, mice that survived primary tumor challenge could reject a subsequent rechallenge. Overall, this study has highlighted the therapeutic potential of using combined transfer of antigen-specific gene-modified CD8+ and CD4+ T cells to significantly enhance T-cell adoptive transfer strategies for cancer therapy.

Count of splenic stromal precursor cells in mice and expression of cytokine genes in these cells in primary cultures during different periods after immunization of animals with S. typhimurium antigens.

PubMed

Gorskaya, Yu F; Danilova, T A; Mezentseva, M V; Shapoval, I M; Narovlyanskii, A N; Nesterenko, V G

2011-06-01

Injection of S. typhimurium antigens significantly (9-fold) increased cloning efficiency and, hence, the content of stromal precursor cells in the spleen as soon as after 24 h. These parameters returned to normal by days 6-15 after immunization. Cultured splenocytes collected from immune (but not intact) animals expressed the genes of proinflammatory cytokines IL-1β (on days 1, 6, 15) and IL-6 (on days 1 and 6), TNF-α (on days 6 and 15), and of IFN-α and IL-18 (on days 6 and 15). The expression of IL-4 gene was suppressed on day 6 after immunization, of IL-10 gene on days 1 and 6, of IL-6 gene on day 15. Hence, no signs of immune response suppression by stromal cells were found in this system. The spectrum and dynamics of the expression of pro- and anti-inflammatory cytokine genes in stromal cell cultures from the spleen of immunized mice seemed to correspond to those needed for support of the immune response to S. typhimurium antigens, observed in immunized animals. The results indicate possible involvement of stromal cells in the realization of immune response in vivo. The increase of stromal precursor cells cloning efficiency in response to antigen injection could not be reproduced in vitro: the presence of S. typhimurium antigens in primary cultures of intact mouse bone marrow and spleen throughout the entire period of culturing ≈ 20-fold reduced cloning efficiency in cultures.

Structural and genetic relationships of closely related O-antigens of Cronobacter spp. and Escherichia coli: C. sakazakii G2594 (serotype O4)/E. coli O103 and C. malonaticus G3864 (serotype O1)/E. coli O29.

PubMed

Shashkov, Alexander S; Wang, Min; Turdymuratov, Eldar M; Hu, Shaohui; Arbatsky, Nikolay P; Guo, Xi; Wang, Lei; Knirel, Yuriy A

2015-03-02

O-Antigen (O-polysaccharide) variation is the basis for bacterial serotyping and is important in bacterial virulence and niche adaptation. In this work, we present structural and genetic evidences for close relationships between the O-antigens of the Cronobacter spp. and Escherichia coli. Cronobacter sakazakii G2594 (serotype O4) and Cronobacter malonaticus G3864 (serotype O1) are structurally related to those of E. coli O103 and O29, respectively, and some other members of the Enterobacteriaceae family differing in the patterns of lateral glucosylation (C. sakazakii G2594) or O-acetylation (C. malonaticus G3864). The O-antigen gene clusters of the corresponding Cronobacter and E. coli strains contain the same genes with high-level similarity, and the structural differences within both O-antigen pairs were suggested to be due to modification genes carried by prophages. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lack of gender-specific antibody recognition of products from domains of a var gene implicated in pregnancy-associated Plasmodium falciparum malaria.

PubMed

Jensen, Anja T R; Zornig, Hanne D; Buhmann, Caecilie; Salanti, Ali; Koram, Kwadwo A; Riley, Eleanor M; Theander, Thor G; Hviid, Lars; Staalsoe, Trine

2003-07-01

Gender-specific and parity-dependent acquired antibody recognition is characteristic of variant surface antigens (VSA) expressed by chondroitin sulfate A (CSA)-adherent Plasmodium falciparum involved in pregnancy-associated malaria (PAM). However, antibody recognition of recombinant products of a specific VSA gene (2O2var1) implicated in PAM and transcribed by a CSA-adhering parasite line did not have these characteristics. Furthermore, we could not demonstrate preferential transcription of 2O2var1 in the CSA-adhering line versus the unselected, parental isolate. Our data call for circumspection regarding the molecular identity of the parasite ligand mediating adhesion to CSA in PAM.

Structure and function of three novel MHC class I antigens derived from a C3H ultraviolet-induced fibrosarcoma

PubMed Central

1986-01-01

The UV-induced, C3H fibrosarcoma, 1591, expresses at least three unique MHC class I antigens not found on normal C3H tissue. Here we report the complete DNA sequence of the three novel class I genes encoding these molecules, and describe in detail the recognition of the individual products by tumor-reactive and allospecific CTL. Remarkably, although C3H does not appear to express H-2L locus information, this C3H tumor expresses two distinct antigens, termed A149 and A166, which are extremely homologous to each other and to the H-2Ld antigen from BALB/c. The gene encoding the third novel class I antigen from 1591, A216, is quite homologous to H-2Kk) throughout its 3' end. Since all three of these genes account for polymorphic restriction fragments not found in C3H, it is likely that they were derived by recombination from the endogenous class I genes of C3H. The DNA sequence homology of A149, A166, and H-2Ld is especially significant given the functional conservation observed between the products of these genes. Limited sequence substitutions appear to correlate with some of the discrete serological differences observed between these molecules. In addition, both A149 and A166 crossreact, but to differing extents, with H-2Ld at the level of T cell recognition. Our results are consistent with the view that CTL recognize complex conformational determinants on class I molecules, but extend previous observations by comparing a set of antigens with discrete and overlapping structural and functional differences. PMID:3489061

Phase Variable O Antigen Biosynthetic Genes Control Expression of the Major Protective Antigen and Bacteriophage Receptor in Vibrio cholerae O1

PubMed Central

Seed, Kimberley D.; Faruque, Shah M.; Mekalanos, John J.; Calderwood, Stephen B.; Qadri, Firdausi; Camilli, Andrew

2012-01-01

The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage. PMID:23028317

Genetic diversity of merozoite surface antigens in Babesia bovis detected from Sri Lankan cattle.

PubMed

Sivakumar, Thillaiampalam; Okubo, Kazuhiro; Igarashi, Ikuo; de Silva, Weligodage Kumarawansa; Kothalawala, Hemal; Silva, Seekkuge Susil Priyantha; Vimalakumar, Singarayar Caniciyas; Meewewa, Asela Sanjeewa; Yokoyama, Naoaki

2013-10-01

Babesia bovis, the causative agent of severe bovine babesiosis, is endemic in Sri Lanka. The live attenuated vaccine (K-strain), which was introduced in the early 1990s, has been used to immunize cattle populations in endemic areas of the country. The present study was undertaken to determine the genetic diversity of merozoite surface antigens (MSAs) in B. bovis isolates from Sri Lankan cattle, and to compare the gene sequences obtained from such isolates against those of the K-strain. Forty-four bovine blood samples isolated from different geographical regions of Sri Lanka and judged to be B. bovis-positive by PCR screening were used to amplify MSAs (MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b), AMA-1, and 12D3 genes from parasite DNA. Although the AMA-1 and 12D3 gene sequences were highly conserved among the Sri Lankan isolates, the MSA gene sequences from the same isolates were highly diverse. Sri Lankan MSA-1, MSA-2c, MSA-2a1, MSA-2a2, and MSA-2b sequences clustered within 5, 2, 4, 1, and 9 different clades in the gene phylograms, respectively, while the minimum similarity values among the deduced amino acid sequences of these genes were 36.8%, 68.7%, 80.3%, 100%, and 68.3%, respectively. In the phylograms, none of the Sri Lankan sequences fell within clades containing the respective K-strain sequences. Additionally, the similarity values for MSA-1 and MSA-2c were 40-61.8% and 90.9-93.2% between the Sri Lankan isolates and the K-strain, respectively, while the K-strain MSA-2a/b sequence shared 64.5-69.8%, 69.3%, and 70.5-80.3% similarities with the Sri Lankan MSA-2a1, MSA-2a2, and MSA-2b sequences, respectively. The present study has shown that genetic diversity among MSAs of Sri Lankan B. bovis isolates is very high, and that the sequences of field isolates diverged genetically from the K-strain. Copyright © 2013 Elsevier B.V. All rights reserved.

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

PubMed

Oyebola, Kolapo M; Idowu, Emmanuel T; Olukosi, Yetunde A; Awolola, Taiwo S; Amambua-Ngwa, Alfred

2017-06-29

The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known genomic signatures of selection from drug pressure and host immunity. This is evidence that P. falciparum populations explore common adaptive strategies that can be targeted for the development of new interventions.

Structure of Adeno-Associated Virus Type 4

PubMed Central

Padron, Eric; Bowman, Valorie; Kaludov, Nikola; Govindasamy, Lakshmanan; Levy, Hazel; Nick, Phillip; McKenna, Robert; Muzyczka, Nicholas; Chiorini, John A.; Baker, Timothy S.; Agbandje-McKenna, Mavis

2005-01-01

Adeno-associated virus (AAV) is a member of the Parvoviridae, belonging to the Dependovirus genus. Currently, several distinct isolates of AAV are in development for use in human gene therapy applications due to their ability to transduce different target cells. The need to manipulate AAV capsids for specific tissue delivery has generated interest in understanding their capsid structures. The structure of AAV type 4 (AAV4), one of the most antigenically distinct serotypes, was determined to 13-Å resolution by cryo-electron microscopy and image reconstruction. A pseudoatomic model was built for the AAV4 capsid by use of a structure-based sequence alignment of its major capsid protein, VP3, with that of AAV2, to which AAV4 is 58% identical and constrained by its reconstructed density envelope. The model showed variations in the surface loops that may account for the differences in receptor binding and antigenicity between AAV2 and AAV4. The AAV4 capsid surface topology also shows an unpredicted structural similarity to that of Aleutian mink disease virus and human parvovirus B19, autonomous members of the genus, despite limited sequence homology. PMID:15795290

Gene Transfer Efficiency in Gonococcal Biofilms: Role of Biofilm Age, Architecture, and Pilin Antigenic Variation.

PubMed

Kouzel, Nadzeya; Oldewurtel, Enno R; Maier, Berenike

2015-07-01

Extracellular DNA is an important structural component of many bacterial biofilms. It is unknown, however, to which extent external DNA is used to transfer genes by means of transformation. Here, we quantified the acquisition of multidrug resistance and visualized its spread under selective and nonselective conditions in biofilms formed by Neisseria gonorrhoeae. The density and architecture of the biofilms were controlled by microstructuring the substratum for bacterial adhesion. Horizontal transfer of antibiotic resistance genes between cocultured strains, each carrying a single resistance, occurred efficiently in early biofilms. The efficiency of gene transfer was higher in early biofilms than between planktonic cells. It was strongly reduced after 24 h and independent of biofilm density. Pilin antigenic variation caused a high fraction of nonpiliated bacteria but was not responsible for the reduced gene transfer at later stages. When selective pressure was applied to dense biofilms using antibiotics at their MIC, the double-resistant bacteria did not show a significant growth advantage. In loosely connected biofilms, the spreading of double-resistant clones was prominent. We conclude that multidrug resistance readily develops in early gonococcal biofilms through horizontal gene transfer. However, selection and spreading of the multiresistant clones are heavily suppressed in dense biofilms. Biofilms are considered ideal reaction chambers for horizontal gene transfer and development of multidrug resistances. The rate at which genes are exchanged within biofilms is unknown. Here, we quantified the acquisition of double-drug resistance by gene transfer between gonococci with single resistances. At early biofilm stages, the transfer efficiency was higher than for planktonic cells but then decreased with biofilm age. The surface topography affected the architecture of the biofilm. While the efficiency of gene transfer was independent of the architecture, spreading of double-resistant bacteria under selective conditions was strongly enhanced in loose biofilms. We propose that while biofilms help generating multiresistant strains, selection takes place mostly after dispersal from the biofilm. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of Actinobacillus pleuropneumoniae Genes Preferentially Expressed During Infection Using In Vivo-Induced Antigen Technology (IVIAT).

PubMed

Zhang, Fei; Zhang, Yangyi; Wen, Xintian; Huang, Xiaobo; Wen, Yiping; Wu, Rui; Yan, Qigui; Huang, Yong; Ma, Xiaoping; Zhao, Qin; Cao, Sanjie

2015-10-01

Porcine pleuropneumonia is an infectious disease caused by Actinobacillus pleuropneumoniae. The identification of A. pleuropneumoniae genes, specially expressed in vivo, is a useful tool to reveal the mechanism of infection. IVIAT was used in this work to identify antigens expressed in vivo during A. pleuropneumoniae infection, using sera from individuals with chronic porcine pleuropneumonia. Sequencing of DNA inserts from positive clones showed 11 open reading frames with high homology to A. pleuropneumoniae genes. Based on sequence analysis, proteins encoded by these genes were involved in metabolism, replication, transcription regulation, and signal transduction. Moreover, three function-unknown proteins were also indentified in this work. Expression analysis using quantitative real-time PCR showed that most of the genes tested were up-regulated in vivo relative to their expression levels in vitro. IVI (in vivoinduced) genes that were amplified by PCR in different A. pleuropneumoniae strains showed that these genes could be detected in almost all of the strains. It is demonstrated that the identified IVI antigen may have important roles in the infection of A. pleuropneumoniae.

Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.

PubMed

Bien, Christian G; Vincent, Angela; Barnett, Michael H; Becker, Albert J; Blümcke, Ingmar; Graus, Francesc; Jellinger, Kurt A; Reuss, David E; Ribalta, Teresa; Schlegel, Jürgen; Sutton, Ian; Lassmann, Hans; Bauer, Jan

2012-05-01

Classical paraneoplastic encephalitis syndromes with 'onconeural' antibodies directed to intracellular antigens, and the recently described paraneoplastic or non-paraneoplastic encephalitides and antibodies against both neural surface antigens (voltage-gated potassium channel-complexes, N-methyl-d-aspartate receptors) and intracellular antigens (glutamic acid decarboxylase-65), constitute an increasingly recognized group of immune-mediated brain diseases. Evidence for specific immune mechanisms, however, is scarce. Here, we report qualitative and quantitative immunopathology in brain tissue (biopsy or autopsy material) of 17 cases with encephalitis and antibodies to either intracellular (Hu, Ma2, glutamic acid decarboxylase) or surface antigenic targets (voltage-gated potassium channel-complex or N-methyl-d-aspartate receptors). We hypothesized that the encephalitides with antibodies against intracellular antigens (intracellular antigen-onconeural and intracellular antigen-glutamic acid decarboxylase groups) would show neurodegeneration mediated by T cell cytotoxicity and the encephalitides with antibodies against surface antigens would be antibody-mediated and would show less T cell involvement. We found a higher CD8/CD3 ratio and more frequent appositions of granzyme-B(+) cytotoxic T cells to neurons, with associated neuronal loss, in the intracellular antigen-onconeural group (anti-Hu and anti-Ma2 cases) compared to the patients with surface antigens (anti-N-methyl-d-aspartate receptors and anti-voltage-gated potassium channel complex cases). One of the glutamic acid decarboxylase antibody encephalitis cases (intracellular antigen-glutamic acid decarboxylase group) showed multiple appositions of GrB-positive T cells to neurons. Generally, however, the glutamic acid decarboxylase antibody cases showed less intense inflammation and also had relatively low CD8/CD3 ratios compared with the intracellular antigen-onconeural cases. Conversely, we found complement C9neo deposition on neurons associated with acute neuronal cell death in the surface antigen group only, specifically in the voltage-gated potassium channel-complex antibody patients. N-methyl-d-aspartate receptors-antibody cases showed no evidence of antibody and complement-mediated tissue injury and were distinguished from all other encephalitides by the absence of clear neuronal pathology and a low density of inflammatory cells. Although tissue samples varied in location and in the stage of disease, our findings strongly support a central role for T cell-mediated neuronal cytotoxicity in encephalitides with antibodies against intracellular antigens. In voltage-gated potassium channel-complex encephalitis, a subset of the surface antigen antibody encephalitides, an antibody- and complement-mediated immune response appears to be responsible for neuronal loss and cerebral atrophy; the apparent absence of these mechanisms in N-methyl-d-aspartate receptors antibody encephalitis is intriguing and requires further study.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

PubMed

Lapirattanakul, Jinthana; Nomura, Ryota; Matsumoto-Nakano, Michiyo; Srisatjaluk, Ratchapin; Ooshima, Takashi; Nakano, Kazuhiko

2015-05-01

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates. Copyright © 2015 Elsevier GmbH. All rights reserved.

Characterisation of monoclonal antibodies to common protein epitopes on the cell surface of Streptococcus mutans and Streptococcus sobrinus.

PubMed

Smith, R; Lehner, T

1989-09-01

Three monoclonal antibodies (MAb) were prepared against a cell surface antigen which cross-react between Streptococcus mutans (serotypes c, e and f) and Streptococcus sobrinus (serotypes d and g). Two of the MAb also recognise a determinant on the surface of Streptococcus cricetus (serotype a). The common antigen shared between S. mutans and S. sobrinus was demonstrated by Western blotting to be about 200 kD in size. This antigen is shared not only by the cell surfaces of serotypes a, c, d, e, f and g, but also by the major cell surface antigen of S. mutans of 185 kD and another of 150 kD. These MAb identify all but one mutans type of streptococci and can be utilised as analytical reagents.

Defining the Genetic Features of O-Antigen Biosynthesis Gene Cluster and Performance of an O-Antigen Serotyping Scheme for Escherichia albertii.

PubMed

Wang, Hong; Zheng, Han; Li, Qun; Xu, Yanmei; Wang, Jianping; Du, Pengcheng; Li, Xinqiong; Liu, Xiang; Zhang, Ling; Zou, Nianli; Yan, Guodong; Zhang, Zhengdong; Jing, Huaiqi; Xu, Jianguo; Xiong, Yanwen

2017-01-01

Escherichia albertii is a newly described and emerging diarrheagenic pathogen responsible for outbreaks of gastroenteritis. Serotyping plays an important role in diagnosis and epidemiological studies for pathogens of public health importance. The diversity of O-antigen biosynthesis gene clusters (O-AGCs) provides the primary basis for serotyping. However, little is known about the distribution and diversity of O-AGCs of E. albertii strains. Here, we presented a complete sequence set for the O-AGCs from 52 E. albertii strains and identified seven distinct O-AGCs. Six of these were also found in 15 genomes of E. albertii strains deposited in the public database. Possession of wzy / wzx genes in each O-AGC strongly suggest that O-antigens of E. albertii were synthesized by the Wzx/Wzy-dependent pathway. Furthermore, we performed an O-antigen serotyping scheme for E. albertii based on specific antisera against seven O-antigens and a high throughput xTAG Luminex assay to simultaneously detect seven O-AGCs. Both methods accurately identified serotypes of 64 tested E. albertii strains. Our data revealed the high-level diversity of O-AGCs in E. albertii . We also provide valuable methods to reliably identify and serotype this bacterium.

Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii.

PubMed

Zheng, Li; Pang, Wei; Qi, Zanmei; Luo, Enjie; Cui, Liwang; Cao, Yaming

2016-08-08

Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Membrane Asymmetry and Expression of Cell Surface Antigens of Micrococcus lysodeikticus Established by Crossed Immunoelectrophoresis

PubMed Central

Owen, Peter; Salton, Milton R. J.

1977-01-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions. Images PMID:144722

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

PubMed

Owen, P; Salton, M R

1977-12-01

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56▿

PubMed Central

Krücken, Jürgen; Hosse, Ralf J.; Mouafo, Aimdip N.; Entzeroth, Rolf; Bierbaum, Stefan; Marinovski, Predrag; Hain, Karolina; Greif, Gisela; Wunderlich, Frank

2008-01-01

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins. PMID:18083827

Excystation of Eimeria tenella sporozoites impaired by antibody recognizing gametocyte/oocyst antigens GAM22 and GAM56.

PubMed

Krücken, Jürgen; Hosse, Ralf J; Mouafo, Aimdip N; Entzeroth, Rolf; Bierbaum, Stefan; Marinovski, Predrag; Hain, Karolina; Greif, Gisela; Wunderlich, Frank

2008-02-01

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine- and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.

Modulation of alloimmune response by commensal gut microbiota and potential new avenues to influence the outcome of allogeneic transplantation by modification of the 'gut culture'.

PubMed

Kanangat, S

2017-02-01

Host defence response against microbial infections was the foundation for the Science of Immunology. Now, we know the mechanisms of such host defence which include innate immune responses that is generally nonspecific but effective in many cases and lead to more specific responses called adaptive immune response. The gene loci of class I, II and III of the major histocompatibility complex (MHC) play a major role in directing the adaptive immune responses by presenting processed antigens to T and B cells to induce appropriate antigen-specific cellular and or humoral immune responses. In humans, these are commonly referred to as human leucocyte antigens class I/II-HLA I/II). The class III region, the gamma region in the MHC complex, is mostly associated with regulation of immune responses along with genes associated with complement activation. The adaptive immune responses are orchestrated by T and B cells that are tuned to respond to antigens that are normally foreign to the body, because these cells are educated to avoid self-antigens by a process of thymic education and selection of the T cells that are mostly non-self-reactive which also helps the B cells in eliciting specific immune responses to non-self-antigens. A by-product of this is the ability of the T and B cells to elicit strong immune responses to foreign HLA/MHC (alloimmune response), which developed into the field of histocompatibility testing for allogeneic transplantation of stem cells and organs. Now, we are beginning to learn that such alloimmune responses can be influenced by the microbiota that symbiotically live in our body especially on the mucosal surfaces and on the skin. This review deals with new and emerging data on how the commensal mucosal and skin microbiota influence the immune homeostasis, and how manipulating the commensal microbiota of the mucosa and skin could influence the survival and long-term functions of the allografts. Also, alterations of the microbiota by the inevitable immunosuppression prior to and following allogeneic transplantation could contribute towards the outcome of the allografts by alloimmune responses generated due to microbial antigen vs HLA cross-reactivity. © 2017 John Wiley & Sons Ltd.

Antigen processing and presentation: evolution from a bird's eye view.

PubMed

Kaufman, Jim

2013-09-01

Most detailed knowledge of the MHC outside of mammals has come from studies of chickens, originally due to the economic importance of the poultry industry. We have used our discoveries about the chicken MHC to develop a framework for understanding the evolution of the MHC, based on the importance of genomic organisation for gene co-evolution. In humans, MHC class I molecules are polymorphic and determine the specificity of peptide presentation, while the molecules involved in antigen processing are functionally monomorphic. The genes for tapasin, transporters associated with antigen presentation (TAPs) and inducible proteasome components (LMPs) are located in and beyond the class II region, far away from the class I genes in the class I region. In contrast, chickens express only one class I locus at high levels, which can result in strong MHC associations with resistance to particular infectious pathogens. The chicken TAP and tapasin genes are located very close to the class I genes, and have high levels of allelic polymorphism and moderate sequence diversity, co-evolving their specificities to work optimally with the dominantly expressed class I molecule. The salient features of the chicken MHC are found in many if not most non-mammalian species examined, and are likely to represent the ancestral organisation of the MHC. Comparison with the MHC organisation of humans and typical mammals suggests that a large inversion brought the class III region into the middle of the MHC, separating the antigen processing genes from the class I gene, breaking the co-evolutionary relationships and allowing a multigene family of well-expressed class I genes. Such co-evolution in the primordial MHC was likely responsible for the appearance of the antigen presentation pathways and receptor-ligand interactions at the birth of the adaptive immune system. Of course, much further work is required to understand this evolutionary framework in more detail. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification and Characterization of Genes Required for Early Myxococcus xanthus Developmental Gene Expression

PubMed Central

Guo, Dongchuan; Wu, Yun; Kaplan, Heidi B.

2000-01-01

Starvation and cell density regulate the developmental expression of Myxococcus xanthus gene 4521. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn5 lac Ω4521 fusion are Lac+. One class of these mutants inactivates SasN, a negative regulator of 4521 expression; another class activates SasS, a sensor kinase-positive regulator of 4521 expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of 4521 expression, 11 Lac− TnV.AS transposon insertion mutants were isolated from a screen of 18,000 Lac+ LPS O-antigen mutants containing Tn5 lac Ω4521 (Tcr). Ten mutations identified genes that could encode positive regulators of 4521 developmental expression based on their ability to abolish 4521 expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the sasB locus, which encodes the known 4521 regulators SasS and SasN. One mapped to sasS, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated sasR, and four others mapped to a gene designated sasP. One mutation, designated ssp10, specifically suppressed the LPS O-antigen defect; the ssp10 mutation had no effect on 4521 expression in an otherwise wild-type background but reduced 4521 developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in sasP conferred defects during growth and development. These data indicate that a number of elements are required for 4521 developmental expression and that most of these are necessary for normal growth and fruiting body development. PMID:10913090

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

VH gene family expression in mice with the xid defect

PubMed Central

1991-01-01

Preferential use of particular VH gene families in the response to specific antigens has been demonstrated in several systems. The lack of responses to certain types of antigens, therefore, could be the result of deletion of or failure to express some VH genes. Because CBA/N mice, which carry the X-linked immunodeficiency (xid) gene defect, have been shown to be unresponsive to thymus-independent polysaccharide antigens, it was of interest to examine if this unresponsiveness could be accounted for by abnormal expression of particular VH gene families. Using in situ hybridization on B cell colonies, we determined the expression of nine VH gene families in CBA/CaHN females (genotypically normal), CBA/N males (xid) and females (xid), and (CBA/N x CBA/CaHN)F1 males (xid) and females (phenotypically normal). Our results indicate that VH gene family expression, including the S107 family, in CBA/N males and F1 males, is similar to that of CBA/CaHN and F1 females with predominant expression of J558, the largest gene family, in all individuals. Interestingly, CBA/N female mice, which carry two defective X chromosomes, as a group expressed significantly reduced levels of the J558 gene family, and as individuals showed variation in which family was predominantly expressed. We conclude that the unresponsiveness of mice with the xid defect to polysaccharide antigens can not attributed to a failure to express the nine VH gene families that we examined. Our findings do not support previous studies (Primi, D., and P.-A. Cazenave 1986. J. Exp. Med. 165:357), which found an absence of expression of the S107 family in xid mice. PMID:1711566

Domain-swapped T cell receptors improve the safety of TCR gene therapy

PubMed Central

Bethune, Michael T; Gee, Marvin H; Bunse, Mario; Lee, Mark S; Gschweng, Eric H; Pagadala, Meghana S; Zhou, Jing; Cheng, Donghui; Heath, James R; Kohn, Donald B; Kuhns, Michael S; Uckert, Wolfgang; Baltimore, David

2016-01-01

T cells engineered to express a tumor-specific αβ T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αβ chains with endogenous αβ chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and β chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy. DOI: http://dx.doi.org/10.7554/eLife.19095.001 PMID:27823582

Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B.

PubMed

Savardashtaki, Amir; Sarkari, Bahador; Arianfar, Farzane; Mostafavi-Pour, Zohreh

2017-06-01

Cystic echinococcosis (CE), as a chronic parasitic disease, is a major health problem in many countries. The performance of the currently available serodiagnostic tests for the diagnosis of CE is unsatisfactory. The current study aimed at sub-cloning a gene, encoding the B8/1 subunit of antigen B (AgB) from Echinococcus granulosus, using gene optimization for the immunodiagnosis of human CE. The coding sequence for AgB8/1 subunit of Echinococcus granulosus was selected from GenBank and was gene-optimized. The sequence was synthesized and inserted into pGEX-4T-1 vector. Purification was performed with GST tag affinity column. Diagnostic performance of the produced recombinant antigen, native antigen B and a commercial ELISA kit were further evaluated in an ELISA system, using a panel of sera from CE patients and controls. SDS-PAGE demonstrated that the protein of interest had a high expression level and purity after GST tag affinity purification. Western blotting verified the immunoreactivity of the produced recombinant antigen with the sera of CE patients. In an ELISA system, the sensitivity and specificity (for human CE diagnosis) of the recombinant antigen, native antigen B and commercial kit were respectively 93% and 92%, 87% and 90% and 97% and 95%. The produced recombinant antigen showed a high diagnostic value which can be recommended for serodiagnosis of CE in Iran and other CE-endemic areas. Utilizing the combination of other subunits of AgB8 would improve the performance value of the introduced ELISA system.

CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response

PubMed Central

Peixoto, António; Evaristo, César; Munitic, Ivana; Monteiro, Marta; Charbit, Alain; Rocha, Benedita; Veiga-Fernandes, Henrique

2007-01-01

To study in vivo CD8 T cell differentiation, we quantified the coexpression of multiple genes in single cells throughout immune responses. After in vitro activation, CD8 T cells rapidly express effector molecules and cease their expression when the antigen is removed. Gene behavior after in vivo activation, in contrast, was quite heterogeneous. Different mRNAs were induced at very different time points of the response, were transcribed during different time periods, and could decline or persist independently of the antigen load. Consequently, distinct gene coexpression patterns/different cell types were generated at the various phases of the immune responses. During primary stimulation, inflammatory molecules were induced and down-regulated shortly after activation, generating early cells that only mediated inflammation. Cytotoxic T cells were generated at the peak of the primary response, when individual cells simultaneously expressed multiple killer molecules, whereas memory cells lost killer capacity because they no longer coexpressed killer genes. Surprisingly, during secondary responses gene transcription became permanent. Secondary cells recovered after antigen elimination were more efficient killers than cytotoxic T cells present at the peak of the primary response. Thus, primary responses produced two transient effector types. However, after boosting, CD8 T cells differentiate into long-lived killer cells that persist in vivo in the absence of antigen. PMID:17485515

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation

PubMed Central

Almeida, Freya M Freyre; Blanco, Aracelys; Trujillo, Heidy; Hernández, Dunia; García, Daymir; Alba, José S; Abad, Matilde López; Merino, Nelson; Lobaina, Yadira

2016-01-01

ABSTRACT The development of therapeutic vaccines against chronic hepatitis B requires the capacity of the formulation to subvert a tolerated immune response as well as the evaluation of histopathological damage resulting from the treatment. In the present study, the dynamicity of induced immune response to hepatitis B surface antigen (HBsAg) was evaluated in transgenic mice that constitutively express the HBsAg gene (HBsAg-tg mice). After immunization with a vaccine candidate containing both surface (HBsAg) and core (HBcAg) antigens of hepatitis B virus (HBV), the effect of vaccination on clearance of circulating HBsAg and the potential histological alterations were examined. Transgenic (tg) and non-transgenic (Ntg) mice were immunized by intranasal (IN) and subcutaneous (SC) routes simultaneously. A control group received phosphate-buffered saline (PBS) by IN route and aluminum by SC route. Positive responses, at both humoral and cellular levels, were obtained after five immunizations in HBsAg-tg mice. Such responses were delayed and of lower intensity in tg mice, compared to vaccinated Ntg mice. Serum IgG response was characterized by a similar IgG subclass pattern. Even when HBsAg-specific CD8+ T cell responses were clearly detectable by gamma-interferon ELISPOT assay, histopathological alterations were not detected in any organ, including the liver and kidneys. Our study demonstrated, that it is possible to subvert the immune tolerance against HBsAg in tg mice, opening a window for new studies to optimize the schedule, dose, and formulation to improve the immune response to the therapeutic vaccine candidate. These results can be considered a safety proof to support clinical developments for the formulation under study. How to cite this article Freyre FM, Blanco A, Trujillo H, Hernández D, García D, Alba JS, Lopez M, Merino N, Lobaina Y, Aguilar JC. Dynamic of Immune Response induced in Hepatitis B Surface Antigen-transgenic Mice Immunized with a Novel Therapeutic Formulation. Euroasian J Hepato-Gastroenterol 2016;6(1):25-30. PMID:29201720

[Recombinant OspC identification and antigenicity detection from Borrelia burgdorferi PD91 in China].

PubMed

Chen, Jian; Wan, Kang-Lin

2003-10-01

To recombine OspC gene from Borrelia burgdorferi PD91 of China and expressed it in E. coli for early diagnosis of Lyme disease. The OspC gene was amplified from the genome of Borrelia burgdorferi PD91 strain by polymerase chain reaction and recombined with plasmid PET-11D. The recombinant plasmid PET-11D-OspC was identified with PCR, restriction endonuclease analysis and sequencing. The antigenicity was verified with Western Blot. OspC gene was cloned correctly into vector PET-11D. The resultant sequence was definitely different from the published sequence. The recombinant OspC seemed to have had strong antigenicity. The findings laid basis for the studies on early diagnosis of Lyme disease.

Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus

PubMed Central

Graham, Deborah S Cunninghame; Pinder, Christopher L; Tombleson, Philip; Behrens, Timothy W; Martín, Javier; Fairfax, Benjamin P; Knight, Julian C; Chen, Lingyan; Replogle, Joseph; Syvänen, Ann-Christine; Rönnblom, Lars; Graham, Robert R; Wither, Joan E; Rioux, John D; Alarcón-Riquelme, Marta E; Vyse, Timothy J

2015-01-01

Systemic lupus erythematosus (SLE; OMIM 152700) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry: a new GWAS, meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including 10 novel associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n=16) of transcription factors among SLE susceptibility genes. This supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE. PMID:26502338

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

PubMed Central

Balish, Amanda; Hoang, Anh Nguyen; Gustin, Kortney M.; Nhung, Pham Thi; Jones, Joyce; Thu, Ngoc Nguyen; Davis, William; Ngoc, Thao Nguyen Thi; Jang, Yunho; Sleeman, Katrina; Villanueva, Julie; Kile, James; Gubareva, Larisa V.; Lindstrom, Stephen; Tumpey, Terrence M.; Davis, C. Todd; Long, Nguyen Thanh

2015-01-01

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses. PMID:26244768

AbMiner: a bioinformatic resource on available monoclonal antibodies and corresponding gene identifiers for genomic, proteomic, and immunologic studies.

PubMed

Major, Sylvia M; Nishizuka, Satoshi; Morita, Daisaku; Rowland, Rick; Sunshine, Margot; Shankavaram, Uma; Washburn, Frank; Asin, Daniel; Kouros-Mehr, Hosein; Kane, David; Weinstein, John N

2006-04-06

Monoclonal antibodies are used extensively throughout the biomedical sciences for detection of antigens, either in vitro or in vivo. We, for example, have used them for quantitation of proteins on "reverse-phase" protein lysate arrays. For those studies, we quality-controlled > 600 available monoclonal antibodies and also needed to develop precise information on the genes that encode their antigens. Translation among the various protein and gene identifier types proved non-trivial because of one-to-many and many-to-one relationships. To organize the antibody, protein, and gene information, we initially developed a relational database in Filemaker for our own use. When it became apparent that the information would be useful to many other researchers faced with the need to choose or characterize antibodies, we developed it further as AbMiner, a fully relational web-based database under MySQL, programmed in Java. AbMiner is a user-friendly, web-based relational database of information on > 600 commercially available antibodies that we validated by Western blot for protein microarray studies. It includes many types of information on the antibody, the immunogen, the vendor, the antigen, and the antigen's gene. Multiple gene and protein identifier types provide links to corresponding entries in a variety of other public databases, including resources for phosphorylation-specific antibodies. AbMiner also includes our quality-control data against a pool of 60 diverse cancer cell types (the NCI-60) and also protein expression levels for the NCI-60 cells measured using our high-density "reverse-phase" protein lysate microarrays for a selection of the listed antibodies. Some other available database resources give information on antibody specificity for one or a couple of cell types. In contrast, the data in AbMiner indicate specificity with respect to the antigens in a pool of 60 diverse cell types from nine different tissues of origin. AbMiner is a relational database that provides extensive information from our own laboratory and other sources on more than 600 available antibodies and the genes that encode the antibodies' antigens. The data will be made freely available at http://discover.nci.nih.gov/abminer.

Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis.

PubMed

Ogrodzki, P; Forsythe, S

2015-10-08

Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell(+)] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes.

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors.

PubMed

Schröder, R; Maassen, A; Lippoldt, A; Börner, T; von Baehr, R; Dobrowolski, P

1991-08-01

Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids.

A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach

PubMed Central

Vicente, Sara; Diaz-Freitas, Belen; Peleteiro, Mercedes; Sanchez, Alejandro; Pascual, David W.; Gonzalez-Fernandez, Africa; Alonso, Maria J.

2013-01-01

The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarrier's surface (using the recombinant hepatitis B surface antigen –rHBsAg– as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC−), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg. PMID:23614052

Novel reassortant H9N2 viruses in pigeons and evidence for antigenic diversity of H9N2 viruses isolated from quails in Egypt

PubMed Central

Kandeil, Ahmed; El-Shesheny, Rabeh; Maatouq, Asmaa; Moatasim, Yassmin; Cai, Zhipeng; McKenzie, Pamela; Webby, Richard

2017-01-01

The endemicity of avian influenza viruses (AIVs) among Egyptian poultry represents a public health risk. Co-circulation of low pathogenic AIV H9N2 subtype with highly pathogenic AIV H5N1 subtype in Egyptian farms provides a possibility to generate novel reassortant viruses. Here, the genetic characteristics of surface glycoproteins of 59 Egyptian H9N2 viruses, isolated between 2013 and 2015, were analysed. To elucidate the potential of genetic reassortment, 10 H9N2 isolates were selected based on different avian hosts (chickens, ducks, pigeons and quails) and phylogenetic analyses of their full genome sequences were conducted. Additionally, we performed antigenic analysis to further investigate the antigenic evolution of H9N2 viruses isolated during 2011–2015. Different viral characteristics including receptor-binding affinity and drug resistance of representative Egyptian H9N2 viruses were further investigated. The surface glycoproteins of current Egyptian H9N2 viruses were closely related to viruses of the G1-like lineage isolated from Egypt. Several genetic markers that enhance virulence in poultry and transmission to humans were detected. Analysis of the full genome of 10 H9N2 isolates indicated that two pigeon isolates inherited five internal genes from Eurasian AIVs circulating in wild birds. Antigenic conservation of different Egyptian H9N2 isolates from chickens, pigeons and ducks was observed, whereas quail isolates showed antigenic drift. The Egyptian H9N2 viruses preferentially bound to the human-like receptor rather than to the avian-like receptor. Our results suggest that the endemic H9N2 viruses in Egypt contain elements that may favour avian-to-human transmission and thus represent a public health risk. PMID:27902350

Telomere interactions may condition the programming of antigen expression in Trypanosoma brucei.

PubMed Central

Van der Werf, A; Van Assel, S; Aerts, D; Steinert, M; Pays, E

1990-01-01

The AnTat 1.1 antigen type typically occurs late in a chronic infection by the EATRO 1125 stock of Trypanosoma brucei. The AnTat 1.1 gene, which is located 24 kb from a chromosome end, seems exclusively expressed by acting as a donor in gene conversion events targeted to the telomeric expression site. We report that this gene is sufficiently provided with the homology blocks required for recombination with the expression site, and is not interrupted by stop codons up to the 3' block of homology. A possible reason for its low probability of activation is an inverse orientation with respect to the proximal chromosome end, since, if correctly positioned, it is readily expressed at an early stage of infection, following gene conversion. This suggests that interactions between chromosome ends may precede and favour the rearrangements leading to antigenic variation. Images Fig. 1. Fig. 3. Fig. 4. Fig. 5. PMID:2323332

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles.

PubMed

Somiya, Masaharu; Yoshimoto, Nobuo; Iijima, Masumi; Niimi, Tomoaki; Dewa, Takehisa; Jung, Joohee; Kuroda, Shun'ichi

2012-06-15

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy. Copyright © 2012 Elsevier Ltd. All rights reserved.

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

DOE PAGES

Menachery, Vineet D.; Schafer, Alexandra; Burnum-Johnson, Kristin E.; ...

2018-01-16

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigenmore » presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Altogether, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.« less

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Menachery, Vineet D.; Schäfer, Alexandra; Burnum-Johnson, Kristin E.

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigenmore » presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.« less

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion

PubMed Central

2010-01-01

Background Occult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India. Methods A total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing. Results A total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples. Conclusion Thus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India. PMID:20799931

Anti-hepatitis B core antigen testing with detection and characterization of occult hepatitis B virus by an in-house nucleic acid testing among blood donors in Behrampur, Ganjam, Orissa in southeastern India: implications for transfusion.

PubMed

Panigrahi, Rajesh; Biswas, Avik; Datta, Sibnarayan; Banerjee, Arup; Chandra, Partha K; Mahapatra, Pradip K; Patnaik, Bharat; Chakrabarti, Sekhar; Chakravarty, Runu

2010-08-27

Occult hepatitis B virus (HBV) infection might transmit viremic units into the public blood supply if only hepatitis B surface antigen (HBsAg) testing is used for donor screening. Our aim was to evaluate the prevalence of occult HBV infection among the HBsAg negative/antiHBc positive donations from a highly HIV prevalent region of India. A total of 729 HBsAg negative donor units were included in this study. Surface gene and precore region were amplified by in house nucleic acid test (NAT) for detection of occult HBV infection and surface gene was analyzed after direct sequencing. A total of 220 (30.1%) HBsAg negative donors were antiHBc positive, of them 66 (30%) were HBV DNA positive by NAT. HBV DNA positivity among 164 antiHBc only group, was 27.1% and among 40 antiHBs positive group was 30.0%. HBV/D (93.3%) was predominant and prevalence of both HBV/C and HBV/A was 3.3%. Single or multiple amino acids substitutions were found in 95% samples. Thus, a considerable number of HBV infected donors remain undiagnosed, if only HBsAg is used for screening. Addition of antiHBc testing for donor screening, although will lead to rejection of a large number of donor units, will definitely eliminate HBV infected donations and help in reducing HBV transmission with its potential consequences, especially among the immunocompromised population. The HBV genetic diversity found in this donor population are in accordance with other parts of India.

Characterization of a gene coding for a type IIo bacterial IgG-binding protein.

PubMed

Boyle, M D; Weber-Heynemann, J; Raeder, R; Podbielski, A

1995-06-01

Two antigenic classes of non-immune IgG-binding proteins can be expressed by group A streptococci. One antigenic group of proteins is recognized by an antibody prepared against the product of a cloned fcrA gene (anti-FcRA). In this study, the immunogen used to prepare the antibody that defines the second antigenic class was shown to be the product of the emm-like (emmL) gene of M serotype 55 group A isolate, A928. The emmL55 gene expressed in E. coli produced an M(r) approximately 58,000 molecule which bound human IgG1, IgG2, IgG3 and IgG4, as well as horse, rabbit and pig IgG in a non-immune fashion. These properties are characteristic of the previously described type IIo IgG-binding protein isolated from this strain. In addition, the recombinant protein was reactive with human serum albumin and fibrinogen. The emmL 55 gene sequence was analysed and found to have the organization and sequence characteristics of a typical class I emm-like gene.

Evolution of Alternative Adaptive Immune Systems in Vertebrates.

PubMed

Boehm, Thomas; Hirano, Masayuki; Holland, Stephen J; Das, Sabyasachi; Schorpp, Michael; Cooper, Max D

2018-04-26

Adaptive immunity in jawless fishes is based on antigen recognition by three types of variable lymphocyte receptors (VLRs) composed of variable leucine-rich repeats, which are differentially expressed by two T-like lymphocyte lineages and one B-like lymphocyte lineage. The T-like cells express either VLRAs or VLRCs of yet undefined antigen specificity, whereas the VLRB antibodies secreted by B-like cells bind proteinaceous and carbohydrate antigens. The incomplete VLR germline genes are assembled into functional units by a gene conversion-like mechanism that employs flanking variable leucine-rich repeat sequences as templates in association with lineage-specific expression of cytidine deaminases. B-like cells develop in the hematopoietic typhlosole and kidneys, whereas T-like cells develop in the thymoid, a thymus-equivalent region at the gill fold tips. Thus, the dichotomy between T-like and B-like cells and the presence of dedicated lymphopoietic tissues emerge as ancestral vertebrate features, whereas the somatic diversification of structurally distinct antigen receptor genes evolved independently in jawless and jawed vertebrates.

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

PubMed Central

MacKay, P; Pasek, M; Magazin, M; Kovacic, R T; Allet, B; Stahl, S; Gilbert, W; Schaller, H; Bruce, S A; Murray, K

1981-01-01

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals. PMID:6170067

Evolution of HBV S-gene in the backdrop of HDV co-infection.

PubMed

Baig, Samina; Abidi, Syed H; Azam, Zahid; Majid, Shahid; Khan, Saeed; Khanani, Muhammad R; Ali, Syed

2018-04-16

HBV-HDV co-infected people have a higher chance of developing cirrhosis, fulminant hepatitis, and hepatocellular carcinoma (HCC) compared to those infected only with HBV. The present study was conducted to investigate HBV genotypes and phylogeny among HBV mono-infected and HBV-HDV co-infected patients, as well as analyze mutations in the surface gene of HBV in mono-infected and co-infected patients. A total of 100 blood samples (50 co-infected with HBV and HDV, and 50 mono-infected with HBV only) were collected for this study. HBV DNA was extracted from patient sera and partial surface antigen gene was amplified from HBV genome using polymerase chain reaction. HBV S gene was sequenced from 49 mono-infected and 36 co-infected patients and analyzed to identify HBV genotypes and phylogenetic patterns. Subsequently, HBV S amino acid sequences were analyzed for mutational differences between sequences from mono- and co-infected patients. HBV genotype D was predominantly found in both mono-infected as well as co-infected patients. Phylogenetic analysis showed the divergence of HBV sequences, between mono- and co-infected patients, into two distinct clusters. HBV S gene mutation analysis revealed certain mutations in HBV-HDV co-infected subjects to be distinct from those found in mono-infected patients. This might indicate the evolution of HBV S gene under selection pressures generated from HDV coinfection. © 2018 Wiley Periodicals, Inc.

Antibodies to dendritic neuronal surface antigens in opsoclonus myoclonus ataxia syndrome

PubMed Central

Panzer, Jessica A.; Anand, Ronan; Dalmau, Josep; Lynch, David R.

2015-01-01

Opsoclonus myoclonus ataxia syndrome (OMAS) is an autoimmune disorder characterized by rapid, random, conjugate eye movements (opsoclonus), myoclonus, and ataxia. Given these symptoms, autoantibodies targeting the cerebellum or brainstem could mediate the disease or be markers of autoimmunity. In a subset of patients with OMAS, we identified such autoantibodies, which bind to non-synaptic puncta on the surface of live cultured cerebellar and brainstem neuronal dendrites. These findings implicate autoimmunity to a neuronal surface antigen in the pathophysiology of OMAS. Identification of the targeted antigen(s) could elucidate the mechanisms underlying OMAS and provide a biomarker for diagnosis and response to therapy. PMID:26298330

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation

PubMed Central

Seydoux, Emilie; Rothen-Rutishauser, Barbara; Nita, Izabela M; Balog, Sandor; Gazdhar, Amiq; Stumbles, Philip A; Petri-Fink, Alke; Blank, Fabian; von Garnier, Christophe

2014-01-01

Introduction Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results The frequency of PS particle–positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4+ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4+ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles. PMID:25152619

Integrating Molecular Imaging Approaches to Monitor Prostate Targeted Suicide and Anti-angiogenic Gene Therapy

DTIC Science & Technology

2005-02-01

tissue-specific expression of prostate-specific antigen. Cancer Res. 57: 495–499. 11. Schuur, E. R ., Henderson, G . A., Kmetec, L. A., Miller, J. D...Lamparski, H. G ., and Henderson, D. R . (1996). Prostate-specific antigen expression is regulated by an up- stream enhancer. J. Biol. Chem. 271: 7043...5: 223–232. 29. Blasberg, R . G ., and Tjuvajev, J. G . (1999). Herpes simplex virus thymidine kinase as a marker/reporter gene for PET imaging of gene

ACE/DD genotype is associated with hemostasis balance disturbances reflecting hypercoagulability and endothelial dysfunction in patients with untreated hypertension.

PubMed

Makris, T K; Stavroulakis, G A; Dafni, U G; Gialeraki, A E; Krespi, P G; Hatzizacharias, A N; Tsoukala, C G; Vythoulkas, J S; Kyriakidis, M K

2000-11-01

Angiotensin-converting enzyme (ACE) gene polymorphism has been associated with an increased incidence of myocardial infarction. Recent studies have investigated a potential influence of ACE gene polymorphism on fibrinolysis or endothelial function. It has been previously established that essential hypertension is accompanied by endothelial dysfunction and fibrinolytic balance disorders. The aim of our study was to study the relation between ACE gene polymorphism and fibrinolytic/hemostatic factors as well as endothelial cell damage markers in patients with hypertension. The following parameters were evaluated in 104 patients with previously untreated hypertension: plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) antigen, fibrinogen, D-dimer, and von Willebrand factor (vWF). The genotype of the ACE gene was also determined (by the polymerase chain reaction method), and patients were characterized according to the observed alleles as deletion/deletion (DD), insertion/insertion (II), or insertion/deletion (ID). Those with DD genotype (n = 42) had significantly higher plasma levels of PAI-1 antigen (P =. 012), tPA antigen (P =.0001), fibrinogen (P =.0002), D-dimer (P =. 0001) and vWF (P =.0004) compared with ID (n = 30) or II (n = 32) genotypes. The ACE gene genotypes appeared to be significant predictors for plasma PAI-1 antigen, tPA antigen, fibrinogen, D -dimer, and vWF even after adjustment for age, sex, body mass index, triglyceride and cholesterol levels, and blood pressure. Our findings suggest that the ACE/DD genotype is associated with hemostasis balance disturbances reflecting hypercoagulability and endothelial damage in patients with untreated hypertension.

Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses.

PubMed

Yeh, Kuo-Ming; Chiu, Sheng-Kung; Lin, Chii-Lan; Huang, Li-Yueh; Tsai, Yu-Kuo; Chang, Jen-Chang; Lin, Jung-Chung; Chang, Feng-Yee; Siu, Leung-Kei

2016-01-01

The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

Genome-Scale Protein Microarray Comparison of Human Antibody Responses in Plasmodium vivax Relapse and Reinfection.

PubMed

Chuquiyauri, Raul; Molina, Douglas M; Moss, Eli L; Wang, Ruobing; Gardner, Malcolm J; Brouwer, Kimberly C; Torres, Sonia; Gilman, Robert H; Llanos-Cuentas, Alejandro; Neafsey, Daniel E; Felgner, Philip; Liang, Xiaowu; Vinetz, Joseph M

2015-10-01

Large scale antibody responses in Plasmodium vivax malaria remains unexplored in the endemic setting. Protein microarray analysis of asexual-stage P. vivax was used to identify antigens recognized in sera from residents of hypoendemic Peruvian Amazon. Over 24 months, of 106 participants, 91 had two symptomatic P. vivax malaria episodes, 11 had three episodes, 3 had four episodes, and 1 had five episodes. Plasmodium vivax relapse was distinguished from reinfection by a merozoite surface protein-3α restriction fragment length polymorphism polymerase chain reaction (MSP3α PCR-RFLP) assay. Notably, P. vivax reinfection subjects did not have higher reactivity to the entire set of recognized P. vivax blood-stage antigens than relapse subjects, regardless of the number of malaria episodes. The most highly recognized P. vivax proteins were MSP 4, 7, 8, and 10 (PVX_003775, PVX_082650, PVX_097625, and PVX_114145); sexual-stage antigen s16 (PVX_000930); early transcribed membrane protein (PVX_090230); tryptophan-rich antigen (Pv-fam-a) (PVX_092995); apical merozoite antigen 1 (PVX_092275); and proteins of unknown function (PVX_081830, PVX_117680, PVX_118705, PVX_121935, PVX_097730, PVX_110935, PVX_115450, and PVX_082475). Genes encoding reactive proteins exhibited a significant enrichment of non-synonymous nucleotide variation, an observation suggesting immune selection. These data identify candidates for seroepidemiological tools to support malaria elimination efforts in P. vivax-endemic regions. © The American Society of Tropical Medicine and Hygiene.

An antibody that confers plant disease resistance targets a membrane-bound glyoxal oxidase in Fusarium.

PubMed

Song, Xiu-Shi; Xing, Shu; Li, He-Ping; Zhang, Jing-Bo; Qu, Bo; Jiang, Jin-He; Fan, Chao; Yang, Peng; Liu, Jin-Long; Hu, Zu-Quan; Xue, Sheng; Liao, Yu-Cai

2016-05-01

Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Agglutination Assays of the Plasmodium falciparum-Infected Erythrocyte.

PubMed

Tan, Joshua; Bull, Peter C

2015-01-01

The agglutination assay is used to determine the ability of antibodies to recognize parasite variant antigens on the surface of Plasmodium falciparum-infected erythrocytes. In this technique, infected erythrocytes are selectively labelled with a DNA-binding fluorescent dye and mixed with antibodies of interest to allow antibody-surface antigen binding. Recognition of surface antigens by the antibodies can result in the formation of agglutinates containing multiple parasite-infected erythrocytes. These can be viewed and quantified using a fluorescence microscope.

Patterns of protective associations differ for antibodies to P. falciparum-infected erythrocytes and merozoites in immunity against malaria in children.

PubMed

Chan, Jo-Anne; Stanisic, Danielle I; Duffy, Michael F; Robinson, Leanne J; Lin, Enmoore; Kazura, James W; King, Christopher L; Siba, Peter M; Fowkes, Freya Ji; Mueller, Ivo; Beeson, James G

2017-12-01

Acquired antibodies play an important role in immunity to P. falciparum malaria and are typically directed towards surface antigens expressed by merozoites and infected erythrocytes (IEs). The importance of specific IE surface antigens as immune targets remains unclear. We evaluated antibodies and protective associations in two cohorts of children in Papua New Guinea. We used genetically-modified P. falciparum to evaluate the importance of PfEMP1 and a P. falciparum isolate with a virulent phenotype. Our findings suggested that PfEMP1 was the dominant target of antibodies to the IE surface, including functional antibodies that promoted opsonic phagocytosis by monocytes. Antibodies were associated with increasing age and concurrent parasitemia, and were higher among children exposed to a higher force-of-infection as determined using molecular detection. Antibodies to IE surface antigens were consistently associated with reduced risk of malaria in both younger and older children. However, protective associations for antibodies to merozoite surface antigens were only observed in older children. This suggests that antibodies to IE surface antigens, particularly PfEMP1, play an earlier role in acquired immunity to malaria, whereas greater exposure is required for protective antibodies to merozoite antigens. These findings have implications for vaccine design and serosurveillance of malaria transmission and immunity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional Genomics of the Aeromonas salmonicida Lipopolysaccharide O-Antigen and A-Layer from Typical and Atypical Strains

PubMed Central

Merino, Susana; de Mendoza, Elena; Canals, Rocío; Tomás, Juan M.

2015-01-01

The A. salmonicida A450 LPS O-antigen, encoded by the wbsalmo gene cluster, is exported through an ABC-2 transporter-dependent pathway. It represents the first example of an O-antigen LPS polysaccharide with three different monosaccharides in their repeating unit assembled by this pathway. Until now, only repeating units with one or two different monosaccharides have been described. Functional genomic analysis of this wbsalmo region is mostly in agreement with the LPS O-antigen structure of acetylated l-rhamnose (Rha), d-glucose (Glc), and 2-amino-2-deoxy-d-mannose (ManN). Between genes of the wbsalmo we found the genes responsible for the biosynthesis and assembly of the S-layer (named A-layer in these strains). Through comparative genomic analysis and in-frame deletions of some of the genes, we concluded that all the A. salmonicida typical and atypical strains, other than A. salmonicida subsp. pectinolytica strains, shared the same wbsalmo and presence of A-layer. A. salmonicida subsp. pectinolytica strains lack wbsalmo and A-layer, two major virulence factors, and this could be the reason they are the only ones not found as fish pathogens. PMID:26082990

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

PubMed

Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

2012-01-01

TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

Optimization of a gene electrotransfer procedure for efficient intradermal immunization with an hTERT-based DNA vaccine in mice

PubMed Central

Calvet, Christophe Y; Thalmensi, Jessie; Liard, Christelle; Pliquet, Elodie; Bestetti, Thomas; Huet, Thierry; Langlade-Demoyen, Pierre; Mir, Lluis M

2014-01-01

DNA vaccination consists in administering an antigen-encoding plasmid in order to trigger a specific immune response. This specific vaccine strategy is of particular interest to fight against various infectious diseases and cancer. Gene electrotransfer is the most efficient and safest non-viral gene transfer procedure and specific electrical parameters have been developed for several target tissues. Here, a gene electrotransfer protocol into the skin has been optimized in mice for efficient intradermal immunization against the well-known telomerase tumor antigen. First, the luciferase reporter gene was used to evaluate gene electrotransfer efficiency into the skin as a function of the electrical parameters and electrodes, either non-invasive or invasive. In a second time, these parameters were tested for their potency to generate specific cellular CD8 immune responses against telomerase epitopes. These CD8 T-cells were fully functional as they secreted IFNγ and were endowed with specific cytotoxic activity towards target cells. This simple and optimized procedure for efficient gene electrotransfer into the skin using the telomerase antigen is to be used in cancer patients for the phase 1 clinical evaluation of a therapeutic cancer DNA vaccine called INVAC-1. PMID:26015983

Surface grafted antibodies: controlled architecture permits enhanced antigen detection.

PubMed

Sebra, Robert P; Masters, Kristyn S; Bowman, Christopher N; Anseth, Kristi S

2005-11-22

The attachment of antibodies to substrate surfaces is useful for achieving specific detection of antigens and toxins associated with clinical and field diagnostics. Here, acrylated whole antibodies were produced through conjugation chemistry, with the goal of covalently photografting these proteins from surfaces in a controlled fashion, to facilitate rapid and sensitive antigenic detection. A living radical photopolymerization chemistry was used to graft the acrylated whole antibodies on polymer surfaces at controlled densities and spatial locations by controlling the exposure time and area, respectively. Copolymer grafts containing these antibodies were synthesized to demonstrate two principles. First, PEG functionalities were introduced to prevent nonspecific protein interactions and improve the reaction kinetics by increasing solvation and mobility of the antibody-containing chains. Both of these properties lead to sensitive (pM) and rapid (<20 min) detection of antigens with this surface modification technique. Second, graft composition was tailored to include multiple antibodies on the same grafted chains, establishing a means for simultaneously detecting multiple antigens on one grafted surface area. Finally, the addition of PEG spacers between the acrylate functionality and the pendant detection antibodies was tuned to enhance the detection of a short-half-life molecule, glucagon, in a complex biological environment, plasma.

Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

PubMed

Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

2015-01-01

In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. © 2015 by the Association of Clinical Scientists, Inc.

Molecular characterization of the pL40 protein in Leptospira interrogans.

PubMed

Zhao, Wei; Chen, Chun-Yan; Zhang, Xiang-Yan; Lai, Wei-Qiang; Hu, Bao-Yu; Zhao, Guo-Ping; Qin, Jin-Hong; Guo, Xiao-Kui

2009-06-01

Leptospirosis is a widespread zoonotic disease caused by pathogenic leptospires. The identification of outer membrane proteins (OMPs) conserved among pathogenic leptospires, which are exposed on the leptospiral surface and expressed during mammalian infection, has become a major focus of leptospirosis research. pL40, a 40 kDa protein coded by the LA3744 gene in Leptospira interrogans, was found to be unique to Leptospira. Triton X-114 fractionation and flow cytometry analyses indicate that pL40 is a component of the leptospiral outer membrane. The conservation of pL40 among Leptospira strains prevalent in China was confirmed by both Western blotting and PCR screening. Furthermore, the pL40 antigen could be recognized by sera from guinea pigs and mice infected with low-passage L. interrogans. These findings indicate that pL40 may serve as a useful serodiagnostic antigen and vaccine candidate for L. interrogans.

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

21 CFR 660.44 - Specificity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.44 Specificity. Each filling of the product shall be specific for Hepatitis B Surface Antigen as determined by...

First report of Rickettsia raoultii and R. slovaca in Melophagus ovinus, the sheep ked.

PubMed

Liu, Dan; Wang, Yuan-Zhi; Zhang, Huan; Liu, Zhi-Qiang; Wureli, Ha-Zi; Wang, Shi-Wei; Tu, Chang-Chun; Chen, Chuang-Fu

2016-11-25

Melophagus ovinus (Diptera: Hippoboscidae), a hematophagous ectoparasite, is mainly found in Europe, Northwestern Africa, and Asia. This wingless fly infests sheep, rabbits, and red foxes, and causes inflammation, wool loss and skin damage. Furthermore, this parasite has been shown to transmit diseases, and plays a role as a vector. Herein, we investigated the presence of various Rickettsia species in M. ovinus. In this study, a total of 95 sheep keds were collected in Kuqa County and Alaer City southern region of Xinjiang Uygur Autonomous Region, northwestern China. First, collected sheep keds were identified on the species level using morphological keys and molecular methods based on a fragment of the 18S ribosomal DNA gene (18S rDNA). Thereafter, to assess the presence of rickettsial DNA in sheep keds, the DNA of individual samples was screened by PCR based on six Rickettsia-specific gene fragments originating from six genes: the 17-kilodalton antigen gene (17-kDa), 16S rRNA gene (rrs), surface cell antigen 4 gene (sca4), citrate synthase gene (gltA), and outer membrane protein A and B genes (ompA and ompB). The amplified products were confirmed by sequencing and BLAST analysis ( https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome ). According to its morphology and results of molecular analysis, the species was identified as Melophagus ovinus, with 100% identity to M. ovinus from St. Kilda, Australia (FN666411). DNA of Rickettsia spp. were found in 12 M. ovinus samples (12.63%, 12/95). Rickettsia raoultii and R. slovaca were confirmed based on phylogenetic analysis, although the genetic markers of these two rickettsial agents amplified in this study showed molecular diversity. This is the first report of R. raoultii and R. slovaca DNA in M. ovinus. Rickettsia slovaca was found for the first time around the Taklimakan Desert located in China. This finding extends the geographical range of spotted fever group rickettsiae.

Simian Virus 40 Large T Antigen Interacts with Human TFIIB-Related Factor and Small Nuclear RNA-Activating Protein Complex for Transcriptional Activation of TATA-Containing Polymerase III Promoters

PubMed Central

Damania, Blossom; Mital, Renu; Alwine, James C.

1998-01-01

The TATA-binding protein (TBP) is common to the basal transcription factors of all three RNA polymerases, being associated with polymerase-specific TBP-associated factors (TAFs). Simian virus 40 large T antigen has previously been shown to interact with the TBP-TAFII complexes, TFIID (B. Damania and J. C. Alwine, Genes Dev. 10:1369–1381, 1996), and the TBP-TAFI complex, SL1 (W. Zhai, J. Tuan, and L. Comai, Genes Dev. 11:1605–1617, 1997), and in both cases these interactions are critical for transcriptional activation. We show a similar mechanism for activation of the class 3 polymerase III (pol III) promoter for the U6 RNA gene. Large T antigen can activate this promoter, which contains a TATA box and an upstream proximal sequence element but cannot activate the TATA-less, intragenic VAI promoter (a class 2, pol III promoter). Mutants of large T antigen that cannot activate pol II promoters also fail to activate the U6 promoter. We provide evidence that large T antigen can interact with the TBP-containing pol III transcription factor human TFIIB-related factor (hBRF), as well as with at least two of the three TAFs in the pol III-specific small nuclear RNA-activating protein complex (SNAPc). In addition, we demonstrate that large T antigen can cofractionate and coimmunoprecipitate with the hBRF-containing complex TFIIIB derived from HeLa cells infected with a recombinant adenovirus which expresses large T antigen. Hence, similar to its function with pol I and pol II promoters, large T antigen interacts with TBP-containing, basal pol III transcription factors and appears to perform a TAF-like function. PMID:9488448

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

PubMed Central

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Sialylated multivalent antigens engage CD22 in trans and inhibit B cell activation.

PubMed

Courtney, Adam H; Puffer, Erik B; Pontrello, Jason K; Yang, Zhi-Qiang; Kiessling, Laura L

2009-02-24

CD22 is an inhibitory coreceptor on the surface of B cells that attenuates B cell antigen receptor (BCR) signaling and, therefore, B cell activation. Elucidating the molecular mechanisms underlying the inhibitory activity of CD22 is complicated by the ubiquity of CD22 ligands. Although antigens can display CD22 ligands, the receptor is known to bind to sialylated glycoproteins on the cell surface. The propinquity of CD22 and cell-surface glycoprotein ligands has led to the conclusion that the inhibitory properties of the receptor are due to cis interactions. Here, we examine the functional consequences of trans interactions by employing sialylated multivalent antigens that can engage both CD22 and the BCR. Exposure of B cells to sialylated antigens results in the inhibition of key steps in BCR signaling. These results reveal that antigens bearing CD22 ligands are powerful suppressors of B cell activation. The ability of sialylated antigens to inhibit BCR signaling through trans CD22 interactions reveals a previously unrecognized role for the Siglec-family of receptors as modulators of immune signaling.

Use of JH4 joining segment gene by an anti-arsonate antibody that bears the major A-strain cross-reactive idiotype but displays diminished antigen binding.

PubMed

Slaughter, C A; Jeske, D J; Kuziel, W A; Milner, E C; Capra, J D

1984-06-01

One of the antibody families utilized by the A/J mouse in its response to p-azophenylarsonate (Ars) is characterized by the expression of the major anti-arsonate cross-reactive idiotype (CRI) of the A strain. This family has been termed the Ars-A family. A hybridoma antibody (HP 101F11 ) obtained after immunization of an A/J mouse with Ars was identified initially as displaying the CRI, but was subsequently found to bind antigen at a level much lower than most members of the Ars-A family. The results of binding studies suggested that HP 101F11 possesses reduced avidity for antigen. When isolated light and heavy chains were allowed to recombine with the heavy and light chains of a strongly antigen-binding, strongly CRI-positive antibody of the Ars-A family (HP 93G7 ), the low level of antigen binding by HP 101F11 was found to be due to a structurally variant heavy chain. Whereas antibodies of the Ars-A family with normal avidity for antigen had been shown to use the JH2 joining segment gene, amino acid sequence analysis of HP 101F11 revealed that this antibody has a JH segment with a sequence identical to that encoded by a portion of a different JH gene, JH4 . The implication that 101F11 uses the JH4 gene instead of JH2 was supported by the observation that the productively rearranged gene is associated with an Eco R1 restriction fragment 0.95 Kb smaller than the corresponding fragments of Ars-A hybridomas with normal avidity for antigen. The size difference of 0.95 Kb corresponds exactly to the known distance between the JH2 and JH4 genes in BALB/c germline DNA. In addition to the structural differences immediately attributable to the use of JH4 , HP 101F11 has shown an amino acid interchange in the DH segment, and a single amino acid deletion at the DH-JH boundary. These results show that variation among members of the Ars-A family in the DH and/or JH segments provides alternative structural forms of Ars-A antibodies upon which selective processes can operate during the course of an immune response.

The heptanucleotide motif GAGACGC is a key component of a cis-acting promoter element that is critical for SnSAG1 expression in Sarcocystis neurona.

PubMed

Gaji, Rajshekhar Y; Howe, Daniel K

2009-07-01

The apicomplexan parasite Sarcocystis neurona undergoes a complex process of intracellular development, during which many genes are temporally regulated. The described study was undertaken to begin identifying the basic promoter elements that control gene expression in S. neurona. Sequence analysis of the 5'-flanking region of five S. neurona genes revealed a conserved heptanucleotide motif GAGACGC that is similar to the WGAGACG motif described upstream of multiple genes in Toxoplasma gondii. The promoter region for the major surface antigen gene SnSAG1, which contains three heptanucleotide motifs within 135 bases of the transcription start site, was dissected by functional analysis using a dual luciferase reporter assay. These analyses revealed that a minimal promoter fragment containing all three motifs was sufficient to drive reporter molecule expression, with the presence and orientation of the 5'-most heptanucleotide motif being absolutely critical for promoter function. Further studies should help to identify additional sequence elements important for promoter function and for controlling gene expression during intracellular development by this apicomplexan pathogen.

Mouse models expressing human carcinoembryonic antigen (CEA) as a transgene: Evaluation of CEA-based cancer vaccines

PubMed Central

Hance, Kenneth W.; Zeytin, Hasan E.; Greiner, John W.

2010-01-01

In recent years, investigators have carried out several studies designed to evaluate whether human tumor-associated antigens might be exploited as targets for active specific immunotherapy, specifically human cancer vaccines. Not too long ago such an approach would have been met with considerable skepticism because the immune system was believed to be a rigid discriminator between self and non-self which, in turn, protected the host from a variety of pathogens. That viewpoint has been challenged in recent years by a series of studies indicating that antigenic determinants of self have not induced absolute host immune tolerance. Moreover, under specific conditions that evoke danger signals, peptides from self-antigen can be processed by the antigen-presenting cellular machinery, loaded onto the major histocompatibility antigen groove to serve as targets for immune intervention. Those findings provide the rationale to investigate a wide range of tumor-associated antigens, including differentiation antigens, oncogenes, and tumor suppressor genes as possible immune-based targets. One of those tumor-associated antigens is the carcinoembryonic antigen (CEA). Described almost 40 years ago, CEA is a Mr 180–200,000 oncofetal antigen that is one of the more widely studied human tumor-associated antigens. This review will provide: (i) a brief overview of the CEA gene family, (ii) a summary of early preclinical findings on overcoming immune tolerance to CEA, and (iii) the rationale to develop mouse models which spontaneously develop gastrointestinal tumors and express the CEA transgene. Those models have been used extensively in the study of overcoming host immune tolerance to CEA, a self, tumor-associated antigen, and the experimental findings have served as the rationale for the design of early clinical trials to evaluate CEA-based cancer vaccines. PMID:15888344

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

21 CFR 660.5 - Specificity.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Antibody to Hepatitis B Surface Antigen § 660.5 Specificity. Each filling of the product shall be specific for antibody to hepatitis B surface antigen, as...

Critical biological parameters modulate affinity as a determinant of function in T-cell receptor gene-modified T-cells.

PubMed

Spear, Timothy T; Wang, Yuan; Foley, Kendra C; Murray, David C; Scurti, Gina M; Simms, Patricia E; Garrett-Mayer, Elizabeth; Hellman, Lance M; Baker, Brian M; Nishimura, Michael I

2017-11-01

T-cell receptor (TCR)-pMHC affinity has been generally accepted to be the most important factor dictating antigen recognition in gene-modified T-cells. As such, there is great interest in optimizing TCR-based immunotherapies by enhancing TCR affinity to augment the therapeutic benefit of TCR gene-modified T-cells in cancer patients. However, recent clinical trials using affinity-enhanced TCRs in adoptive cell transfer (ACT) have observed unintended and serious adverse events, including death, attributed to unpredicted off-tumor or off-target cross-reactivity. It is critical to re-evaluate the importance of other biophysical, structural, or cellular factors that drive the reactivity of TCR gene-modified T-cells. Using a model for altered antigen recognition, we determined how TCR-pMHC affinity influenced the reactivity of hepatitis C virus (HCV) TCR gene-modified T-cells against a panel of naturally occurring HCV peptides and HCV-expressing tumor targets. The impact of other factors, such as TCR-pMHC stabilization and signaling contributions by the CD8 co-receptor, as well as antigen and TCR density were also evaluated. We found that changes in TCR-pMHC affinity did not always predict or dictate IFNγ release or degranulation by TCR gene-modified T-cells, suggesting that less emphasis might need to be placed on TCR-pMHC affinity as a means of predicting or augmenting the therapeutic potential of TCR gene-modified T-cells used in ACT. A more complete understanding of antigen recognition by gene-modified T-cells and a more rational approach to improve the design and implementation of novel TCR-based immunotherapies is necessary to enhance efficacy and maximize safety in patients.

Investigation of SnSPR1, a novel and abundant surface protein of Sarcocystis neurona merozoites.

PubMed

Zhang, Deqing; Howe, Daniel K

2008-04-15

An expressed sequence tag (EST) sequencing project has produced over 15,000 partial cDNA sequences from the equine pathogen Sarcocystis neurona. While many of the sequences are clear homologues of previously characterized genes, a significant number of the S. neurona ESTs do not exhibit similarity to anything in the extensive sequence databases that have been generated. In an effort to characterize parasite proteins that are novel to S. neurona, a seemingly unique gene was selected for further investigation based on its abundant representation in the collection of ESTs and the predicted presence of a signal peptide and glycolipid anchor addition on the encoded protein. The gene was expressed in E. coli, and monospecific polyclonal antiserum against the recombinant protein was produced by immunization of a rabbit. Characterization of the native protein in S. neurona merozoites and schizonts revealed that it is a low molecular weight surface protein that is expressed throughout intracellular development of the parasite. The protein was designated Surface Protein 1 (SPR1) to reflect its display on the outer surface of merozoites and to distinguish it from the ubiquitous SAG/SRS surface antigens of the heteroxenous Coccidia. Interestingly, infection assays in the presence of the polyclonal antiserum suggested that SnSPR1 plays some role in attachment and/or invasion of host cells by S. neurona merozoites. The work described herein represents a general template for selecting and characterizing the various unidentified gene sequences that are plentiful in the EST databases for S. neurona and other apicomplexans. Furthermore, this study illustrates the value of investigating these novel sequences since it can offer new candidates for diagnostic or vaccine development while also providing greater insight into the biology of these parasites.

An Alternative Strategy for Trypanosome Survival in the Mammalian Bloodstream Revealed through Genome and Transcriptome Analysis of the Ubiquitous Bovine Parasite Trypanosoma (Megatrypanum) theileri

PubMed Central

Kelly, Steven; Ivens, Alasdair; Mott, G. Adam; O’Neill, Ellis; Emms, David; Macleod, Olivia; Voorheis, Paul; Tyler, Kevin; Clark, Matthew; Matthews, Jacqueline

2017-01-01

Abstract There are hundreds of Trypanosoma species that live in the blood and tissue spaces of their vertebrate hosts. The vast majority of these do not have the ornate system of antigenic variation that has evolved in the small number of African trypanosome species, but can still maintain long-term infections in the face of the vertebrate adaptive immune system. Trypanosoma theileri is a typical example, has a restricted host range of cattle and other Bovinae, and is only occasionally reported to cause patent disease although no systematic survey of the effect of infection on agricultural productivity has been performed. Here, a detailed genome sequence and a transcriptome analysis of gene expression in bloodstream form T. theileri have been performed. Analysis of the genome sequence and expression showed that T. theileri has a typical kinetoplastid genome structure and allowed a prediction that it is capable of meiotic exchange, gene silencing via RNA interference and, potentially, density-dependent growth control. In particular, the transcriptome analysis has allowed a comparison of two distinct trypanosome cell surfaces, T. brucei and T. theileri, that have each evolved to enable the maintenance of a long-term extracellular infection in cattle. The T. theileri cell surface can be modeled to contain a mixture of proteins encoded by four novel large and divergent gene families and by members of a major surface protease gene family. This surface composition is distinct from the uniform variant surface glycoprotein coat on African trypanosomes providing an insight into a second mechanism used by trypanosome species that proliferate in an extracellular milieu in vertebrate hosts to avoid the adaptive immune response. PMID:28903536

Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

PubMed Central

Boulassel, Mohamed-Rachid; Galal, Ahmed

2012-01-01

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

Comparative Genomics of Early-Diverging Brucella Strains Reveals a Novel Lipopolysaccharide Biosynthesis Pathway

PubMed Central

Wattam, Alice R.; Inzana, Thomas J.; Williams, Kelly P.; Mane, Shrinivasrao P.; Shukla, Maulik; Almeida, Nalvo F.; Dickerman, Allan W.; Mason, Steven; Moriyón, Ignacio; O’Callaghan, David; Whatmore, Adrian M.; Sobral, Bruno W.; Tiller, Rebekah V.; Hoffmaster, Alex R.; Frace, Michael A.; De Castro, Cristina; Molinaro, Antonio; Boyle, Stephen M.; De, Barun K.; Setubal, João C.

2012-01-01

ABSTRACT Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. PMID:22930339

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

PubMed Central

Deressa, Tekalign; Strandt, Helen; Florindo Pinheiro, Douglas; Mittermair, Roberta; Pizarro Pesado, Jennifer; Thalhamer, Josef; Hammerl, Peter; Stoecklinger, Angelika

2015-01-01

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin+ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin+ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation. PMID:26030383

Natural anti-carbohydrate antibodies contributing to evolutionary survival of primates in viral epidemics?

PubMed

Galili, Uri

2016-11-01

Humans produce multiple natural antibodies against carbohydrate antigens on gastrointestinal bacteria. Two such antibodies appeared in primates in recent geological times. Anti-Gal, abundant in humans, apes and Old-World monkeys, appeared 20-30 million years ago (mya) following inactivation of the α1,3GT gene (GGTA1). This gene encodes in other mammals the enzyme α1,3galactosyltransferase (α1,3GT) that synthesizes α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R) which bind anti-Gal. Anti-Neu5Gc, found only in humans, appeared in hominins <6 mya, following elimination of N-glycolylneuraminic-acid (Neu5Gc) because of inactivation of CMAH, the gene encoding hydroxylase that converts N-acetylneuraminic-acid (Neu5Ac) into Neu5Gc. These antibodies, were initially produced in few individuals that acquired random mutations inactivating the corresponding genes and eliminating α-gal epitopes or Neu5Gc, which became nonself antigens. It is suggested that these evolutionary selection events were induced by epidemics of enveloped viruses, lethal to ancestral Old World primates or hominins. Such viruses presented α-gal epitopes or Neu5Gc, synthesized in primates that conserved active GGTA1 or CMAH, respectively, and were lethal to their hosts. The natural anti-Gal or anti-Neu5Gc antibodies, produced in offspring lacking the corresponding carbohydrate antigens, neutralized and destroyed viruses presenting α-gal epitopes or Neu5Gc. These antibodies further induced rapid, effective immune responses against virus antigens, thus preventing infections from reaching lethal stages. These epidemics ultimately resulted in extinction of primate populations synthesizing these carbohydrate antigens and their replacement with offspring populations lacking the antigens and producing protective antibodies against them. Similar events could mediate the elimination of various carbohydrate antigens, thus preventing the complete extinction of other vertebrate species. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mapping antigenic motifs in the trypomastigote small surface antigen from Trypanosoma cruzi.

PubMed

Balouz, Virginia; Cámara, María de Los Milagros; Cánepa, Gaspar E; Carmona, Santiago J; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán; Buscaglia, Carlos A

2015-03-01

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mapping Antigenic Motifs in the Trypomastigote Small Surface Antigen from Trypanosoma cruzi

PubMed Central

Balouz, Virginia; Cámara, María de los Milagros; Cánepa, Gaspar E.; Carmona, Santiago J.; Volcovich, Romina; Gonzalez, Nicolás; Altcheh, Jaime; Agüero, Fernán

2015-01-01

The trypomastigote small surface antigen (TSSA) is a mucin-like molecule from Trypanosoma cruzi, the etiological agent of Chagas disease, which displays amino acid polymorphisms in parasite isolates. TSSA expression is restricted to the surface of infective cell-derived trypomastigotes, where it functions as an adhesin and engages surface receptors on the host cell as a prerequisite for parasite internalization. Previous results have established TSSA-CL, the isoform encoded by the CL Brener clone, as an appealing candidate for use in serology-based diagnostics for Chagas disease. Here, we used a combination of peptide- and recombinant protein-based tools to map the antigenic structure of TSSA-CL at maximal resolution. Our results indicate the presence of different partially overlapping B-cell epitopes clustering in the central portion of TSSA-CL, which contains most of the polymorphisms found in parasite isolates. Based on these results, we assessed the serodiagnostic performance of a 21-amino-acid-long peptide that spans TSSA-CL major antigenic determinants, which was similar to the performance of the previously validated glutathione S-transferase (GST)-TSSA-CL fusion molecule. Furthermore, the tools developed for the antigenic characterization of the TSSA antigen were also used to explore other potential diagnostic applications of the anti-TSSA humoral response in Chagasic patients. Overall, our present results provide additional insights into the antigenic structure of TSSA-CL and support this molecule as an excellent target for molecular intervention in Chagas disease. PMID:25589551

The Trophic Life Cycle Stage of the Opportunistic Fungal Pathogen Pneumocystis murina Hinders the Ability of Dendritic Cells To Stimulate CD4+ T Cell Responses

PubMed Central

Evans, Heather M.; Simpson, Andrew; Shen, Shu; Stromberg, Arnold J.; Pickett, Carol L.

2017-01-01

ABSTRACT The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including β-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with β-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells. PMID:28694293

Dendritic cells from CML patients have altered actin organization, reduced antigen processing, and impaired migration.

PubMed

Dong, Rong; Cwynarski, Kate; Entwistle, Alan; Marelli-Berg, Federica; Dazzi, Francesco; Simpson, Elizabeth; Goldman, John M; Melo, Junia V; Lechler, Robert I; Bellantuono, Ilaria; Ridley, Anne; Lombardi, Giovanna

2003-05-01

Chronic myeloid leukemia (CML) is characterized by expression of the BCR-ABL fusion gene that encodes a 210-kDa protein, which is a constitutively active tyrosine kinase. At least 70% of the oncoprotein is localized to the cytoskeleton, and several of the most prominent tyrosine kinase substrates for p210(BCR-ABL) are cytoskeletal proteins. Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells responsible for the initiation of immune responses. In CML patients, up to 98% of myeloid DCs generated from peripheral blood mononuclear cells are BCR-ABL positive. In this study we have compared the morphology and behavior of myeloid DCs derived from CML patients with control DCs from healthy individuals. We show that the actin cytoskeleton and shape of CML-DCs of myeloid origin adherent to fibronectin differ significantly from those of normal DCs. CML-DCs are also defective in processing and presentation of exogenous antigens such as tetanus toxoid. The antigen-processing defect may be a consequence of the reduced capacity of CML-DCs to capture antigen via macropinocytosis or via mannose receptors when compared with DCs generated from healthy individuals. Furthermore, chemokine-induced migration of CML-DCs in vitro was significantly reduced. These observations cannot be explained by a difference in the maturation status of CML and normal DCs, because phenotypic analysis by flow cytometry showed a similar surface expression of maturation makers. Taken together, these results suggest that the defects in antigen processing and migration we have observed in CML-DCs may be related to underlying cytoskeletal changes induced by the p210(BCR-ABL) fusion protein.

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System

PubMed Central

Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya

2018-01-01

ABSTRACT We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica, which is required for O antigen biosynthesis, into the chromosome of B. pertussis, which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti-B. bronchiseptica antibody but not the anti-B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica. O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis, which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant mutant strain of B. pertussis. The present results demonstrate that this system successfully accomplished the above-described purpose. We consider this system to be applicable to a number of bacteria other than Bordetella. PMID:29404410

Ectopic Expression of O Antigen in Bordetella pertussis by a Novel Genomic Integration System.

PubMed

Ishigaki, Keisuke; Shinzawa, Naoaki; Nishikawa, Sayaka; Suzuki, Koichiro; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko

2018-01-01

We describe a novel genome integration system that enables the introduction of DNA fragments as large as 50 kbp into the chromosomes of recipient bacteria. This system, named BPI, comprises a bacterial artificial chromosome vector and phage-derived gene integration machinery. We introduced the wbm locus of Bordetella bronchiseptica , which is required for O antigen biosynthesis, into the chromosome of B. pertussis , which intrinsically lacks O antigen, using the BPI system. After the introduction of the wbm locus, B. pertussis presented an additional substance in the lipooligosaccharide fraction that was specifically recognized by the anti- B. bronchiseptica antibody but not the anti- B. pertussis antibody, indicating that B. pertussis expressed O antigen corresponding to that of B. bronchiseptica . O antigen-expressing B. pertussis was less sensitive to the bactericidal effects of serum and polymyxin B than the isogenic parental strain. In addition, an in vivo competitive infection assay showed that O antigen-expressing B. pertussis dominantly colonized the mouse respiratory tract over the parental strain. These results indicate that the BPI system provides a means to alter the phenotypes of bacteria by introducing large exogenous DNA fragments. IMPORTANCE Some bacterial phenotypes emerge through the cooperative functions of a number of genes residing within a large genetic locus. To transfer the phenotype of one bacterium to another, a means to introduce the large genetic locus into the recipient bacterium is needed. Therefore, we developed a novel system by combining the advantages of a bacterial artificial chromosome vector and phage-derived gene integration machinery. In this study, we succeeded for the first time in introducing a gene locus involved in O antigen biosynthesis of Bordetella bronchiseptica into the chromosome of B. pertussis , which intrinsically lacks O antigen, and using this system we analyzed phenotypic alterations in the resultant mutant strain of B. pertussis . The present results demonstrate that this system successfully accomplished the above-described purpose. We consider this system to be applicable to a number of bacteria other than Bordetella .

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

PubMed

Wieland, Eberhard; Shipkova, Maria

2016-04-01

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules. Surface antigen expression can be followed by flow cytometry using monoclonal antibodies in either cell function assays using donor-specific or nonspecific stimulation of isolated cells or whole blood and without stimulation on circulating lymphocytes. Molecules such as CD30 can be proteolytically cleaved off the surface of activated cells in vivo, and the determination of the soluble protein (sCD30) in serum or plasma is performed by immunoassays. As promising biomarkers for rejection and long-term transplant outcome, CD28 (costimulatory receptor for CD80 and CD86), CD154 (CD40 ligand), and sCD30 (tumor necrosis factor receptor superfamily, member 8) have been identified. Whereas cell function assays are time-consuming laboratory-developed tests which are difficult to standardize, commercial assays are frequently available for soluble proteins. Therefore, more data from clinical trials have been published for sCD30 compared with the surface antigens on activated T cells. This short review summarizes the association between selected surface antigens and immunosuppression, and rejection in solid organ transplantation.

In Azospirillum brasilense, mutations in flmA or flmB genes affect polar flagellum assembly, surface polysaccharides, and attachment to maize roots.

PubMed

Rossi, Fernando Ariel; Medeot, Daniela Beatriz; Liaudat, Juan Pablo; Pistorio, Mariano; Jofré, Edgardo

2016-09-01

Azospirillum brasilense is a soil bacterium capable of promoting plant growth. Several surface components were previously reported to be involved in the attachment of A. brasilense to root plants. Among these components are the exopolysaccharide (EPS), lipopolysaccharide (LPS) and the polar flagellum. Flagellin from polar flagellum is glycosylated and it was suggested that genes involved in such a posttranslational modification are the same ones involved in the biosynthesis of sugars present in the O-antigen of the LPS. In this work, we report on the characterization of two homologs present in A. brasilense Cd, to the well characterized flagellin modification genes, flmA and flmB, from Aeromonas caviae. We show that mutations in either flmA or flmB genes of A. brasilense resulted in non-motile cells due to alterations in the polar flagellum assembly. Moreover, these mutations also affected the capability of A. brasilense cells to adsorb to maize roots and to produce LPS and EPS. By generating a mutant containing the polar flagellum affected in their rotation, we show the importance of the bacterial motility for the early colonization of maize roots. Copyright © 2016 Elsevier GmbH. All rights reserved.

Characterization of swine leucocyte antigen alleles in a crossbred pig to be used in xenotransplant studies.

PubMed

Reyes, L M; Blosser, R J; Smith, R F; Miner, A C; Paris, L L; Blankenship, R L; Tector, M F; Tector, A J

2014-11-01

We have characterized swine leucocyte antigen (SLA) classes I and II molecules of a domestic pig as a model for use in our xenotransplant program. Molecular characterization of the SLA classes I and II genes is critical to understanding the adaptive immune responses between swine and humans in the event of xenotransplantation. Seven swine leucocyte antigen genes (SLA-1, SLA-2, SLA-3, DQB1, DRB1, DQA and DRA) were analyzed and 15 alleles were identified. A novel DRA*w04re01 is reported for this limited polymorphic class II gene. The heterozygous haplotypes, Hp-32.0/35.0 and Hp-0.13/0.23 were deduced for our IU-pig model, for SLA classes I and II regions, respectively. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Characterisation of surface antigens of Strongylus vulgaris of potential immunodiagnostic importance.

PubMed

Nichol, C; Masterson, W J

1987-08-01

When antigens prepared by detergent washes of Strongylus vulgaris and Parascaris equorum were probed in an enzyme-linked immunosorbent assay test with horse sera from single species infections of S. vulgaris and P. equorum, a high degree of cross-reaction between the species was demonstrated. Western blot analysis of four common horse nematode species showed a large number of common antigens when probed with horse infection sera. Antisera raised in rabbits against the four species, including S. vulgaris, were also found to cross-react considerably. Rabbit anti-S. vulgaris sera were affinity adsorbed over a series of affinity chromatography columns, bound with cross-reactive surface antigens, to obtain S. vulgaris-specific antisera and thereby identify S. vulgaris-specific antigens by Western blotting. These studies revealed potentially specific antigens of apparent molecular weights of 100,000, 52,000, and 36,000. Of these bands, only the 52 kDa and 36 kDa appeared to be found on the surface as judged by 125I-labelling of intact worms by the Iodogen method, although neither protein was immunoprecipitated by horse infection sera. Finally, immunoprecipitation of in vitro translated proteins derived from larval S. vulgaris RNA suggests that two proteins may be parasite-derived. These findings are discussed both with respect to the surface of S. vulgaris and to the use of these species-specific antigens in immunodiagnosis.

Prospects for personalized combination immunotherapy for solid tumors based on adoptive cell therapies and immune checkpoint blockade therapies.

PubMed

Kato, Daiki; Yaguchi, Tomonori; Iwata, Takashi; Morii, Kenji; Nakagawa, Takayuki; Nishimura, Ryohei; Kawakami, Yutaka

2017-01-01

  Immune checkpoint blockade (ICB) and adoptive cell therapies (ACT) with antigen-receptor gene-engineered T cells have been shown to be successful for a limited number of patients with solid tumors. Responders to ICB therapy typically have T cell-inflamed tumors. Thus, it is important to develop strategies that convert non-T cell-inflamed tumors to T cell-inflamed tumors. Although chimeric antigen receptor transduced T (CAR-T) cell therapy targeting hematological malignancies demonstrated durable clinical responses, the success of gene-engineered T cell therapies in solid tumors is hampered by a lack of unique antigens, antigen loss in cancer cells, and the immune-suppressive tumor microenvironment (TME) of solid tumors. However, gene-engineered T cells possess strong killing activity and cytokine production capacity, which can induce antigen spreading and modulate the TME of non-T cell-inflamed tumors seen in non-responders to ICB therapy. Immune responses against cancer are highly heterogeneous, not only between tumor types, but also within a patient or between different patients with the same type of cancer, indicating that personalized immunotherapy should be employed, based on the immune status of the individual patient. Here, we offer our perspective for personalized combination immunotherapy for solid tumors based on ACT and ICB therapies.

Characterization of the Aspergillus nidulans aspnd1 gene demonstrates that the ASPND1 antigen, which it encodes, and several Aspergillus fumigatus immunodominant antigens belong to the same family.

PubMed Central

Calera, J A; Ovejero, M C; López-Medrano, R; Segurado, M; Puente, P; Leal, F

1997-01-01

For the first time, an immunodominant Aspergillus nidulans antigen (ASPND1) consistently reactive with serum samples from aspergilloma patients has been purified and characterized, and its coding gene (aspnd1) has been cloned and sequenced. ASPND1 is a glycoprotein with four N-glycosidically-bound sugar chains (around 2.1 kDa each) which are not necessary for reactivity with immune human sera. The polypeptide part is synthesized as a 277-amino-acid precursor of 30.6 kDa that after cleavage of a putative signal peptide of 16 amino acids, affords a mature protein of 261 amino acids with a molecular mass of 29 kDa and a pI of 4.24 (as deduced from the sequence). The ASPND1 protein is 53.1% identical to the AspfII allergen from Aspergillus fumigatus and 48% identical to an unpublished Candida albicans antigen. All of the cysteine residues and most of the glycosylation sites are perfectly conserved in the three proteins, suggesting a similar but yet unknown function. Analysis of the primary structure of the ASPND1 coding gene (aspnd1) has allowed the establishment of a clear relationship between several previously reported A. fumigatus and A. nidulans immunodominant antigens. PMID:9119471

Strong positive selection and recombination drive the antigenic variation of the PilE protein of the human pathogen Neisseria meningitidis.

PubMed

Andrews, T Daniel; Gojobori, Takashi

2004-01-01

The PilE protein is the major component of the Neisseria meningitidis pilus, which is encoded by the pilE/pilS locus that includes an expressed gene and eight homologous silent fragments. The silent gene fragments have been shown to recombine through gene conversion with the expressed gene and thereby provide a means by which novel antigenic variants of the PilE protein can be generated. We have analyzed the evolutionary rate of the pilE gene using the nucleotide sequence of two complete pilE/pilS loci. The very high rate of evolution displayed by the PilE protein appears driven by both recombination and positive selection. Within the semivariable region of the pilE and pilS genes, recombination appears to occur within multiple small sequence blocks that lie between conserved sequence elements. Within the hypervariable region, positive selection was identified from comparison of the silent and expressed genes. The unusual gene conversion mechanism that operates at the pilE/pilS locus is a strategy employed by N. meningitidis to enhance mutation of certain regions of the PilE protein. The silent copies of the gene effectively allow "parallelized" evolution of pilE, thus enabling the encoded protein to rapidly explore a large area of sequence space in an effort to find novel antigenic variants.

A new technique to detect antibody-antigen reaction (biological interactions) on a localized surface plasmon resonance (LSPR) based nano ripple gold chip

NASA Astrophysics Data System (ADS)

Saleem, Iram; Widger, William; Chu, Wei-Kan

2017-07-01

We demonstrate that the gold nano-ripple localized surface plasmon resonance (LSPR) chip is a low cost and a label-free method for detecting the presence of an antigen. A uniform stable layer of an antibody was coated on the surface of a nano-ripple gold pattern chip followed by the addition of different concentrations of the antigen. A red shift was observed in the LSPR spectral peak caused by the change in the local refractive index in the vicinity of the nanostructure. The LSPR chip was fabricated using oblique gas cluster ion beam (GCIB) irradiation. The plasmon-resonance intensity of the scattered light was measured by a simple optical spectroscope. The gold nano ripple chip shows monolayer scale sensitivity and high selectivity. The LSPR substrate was used to detect antibody-antigen reaction of rabbit X-DENTT antibody and DENTT blocking peptide (antigen).

Mature parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum binds to the 30-kDa domain of protein 4.1 in malaria-infected red blood cells.

PubMed

Waller, Karena L; Nunomura, Wataru; An, Xiuli; Cooke, Brian M; Mohandas, Narla; Coppel, Ross L

2003-09-01

The Plasmodium falciparum mature parasite-infected erythrocyte surface antigen (MESA) is exported from the parasite to the infected red blood cell (IRBC) membrane skeleton, where it binds to protein 4.1 (4.1R) via a 19-residue MESA sequence. Using purified RBC 4.1R and recombinant 4.1R fragments, we show MESA binds the 30-kDa region of RBC 4.1R, specifically to a 51-residue region encoded by exon 10 of the 4.1R gene. The 3D structure of this region reveals that the MESA binding site overlaps the region of 4.1R involved in the p55, glycophorin C, and 4.1R ternary complex. Further binding studies using p55, 4.1R, and MESA showed competition between p55 and MESA for 4.1R, implying that MESA bound at the IRBC membrane skeleton may modulate normal 4.1R and p55 interactions in vivo. Definition of minimal binding domains involved in critical protein interactions in IRBCs may aid the development of novel therapies for falciparum malaria.

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

PubMed Central

Stahl, S; MacKay, P; Magazin, M; Bruce, S A; Murray, K

1982-01-01

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples. Images PMID:7041126

Affinity Maturation of an Anti-V Antigen IgG Expressed In Situ Via Adenovirus Gene Delivery Confers Enhanced Protection Against Yersinia pestis Challenge

PubMed Central

Van Blarcom, Thomas J.; Sofer-Podesta, Carolina; Ang, John; Boyer, Julie L.; Crystal, Ronald G.; Georgiou, George

2013-01-01

Genetic transfer of neutralizing antibodies has been shown to confer strong and persistent protection against bacterial and viral infectious agents. While it is well established that for many exogenous neutralizing antibodies increased antigen affinity correlates with protection, the effect of antigen affinity on antibodies produced in situ following adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal antibody 2C12.4 recognizes the Yersinia pestis Type III secretion apparatus protein LcrV (V antigen) and confers protection in mice when administered as an IgG intraperitoneally or, following genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad) 1. 2C12.4 was expressed as a scFv fragment in E. coli and was shown to display a KD=3.5 nM by surface plasmon resonance (SPR) analysis. The 2C12.4 scFv was subjected to random mutagenesis and variants with increased affinity were isolated by flow cytometry using the Anchored Periplasmic Expression (APEx) bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower KD values (H8, KD=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdαV giving rise to AdαV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen antibodies 3 days post-immunization with 109, 1010 or 1011 particle units. Following intranasal challenge with 363 LD50Y. pestis CO92, 54% of the mice immunized with 1010 pu of AdαV.H8 survived at the 14 day end point compared to only 15% survivors for the group immunized with AdαV expressing the lower affinity 2C12.4 (P<0.04, AdαV versus AdαV.H8). These results indicate that affinity maturation of a neutralizing antibody delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but possibly for other pathogens. PMID:20393511

Isolation of Separate Ureaplasma Species From Endotracheal Secretions of Twin Patients.

PubMed

Beeton, Michael L; Maxwell, Nicola C; Chalker, Victoria J; Brown, Rebecca J; Aboklaish, Ali F; Spiller, O Brad

2016-08-01

Isolation of Ureaplasma spp. from preterm neonates and the association with development of bronchopulmonary dysplasia has been previously investigated. However, few studies have contrasted the nature of infection in twins. In this article, we report that dizygotic twins (1 girl, 1 boy) born at 24 weeks gestation both yielded culturable Ureaplasma from endotracheal secretions. The samples were part of a serial blind collection cohort of ventilated premature neonates, and analysis of repeat cultures showed stable, separate infections over a period of 17 and 21 days, respectively. Immunoblot and probe-specific quantitative polymerase chain reaction analysis determined that Twin 1 was solely infected with Ureaplasma parvum (specifically, serovar 6 by gene sequencing), whereas Twin 2 was solely infected with Ureaplasma urealyticum (specifically, genotype A- serovars 2, 5, and 8 by gene sequencing). Immunoblot analysis found that the major surface antigen (multiple-banded antigen) altered relative mass for both strains during the course of infection. Quantitative polymerase chain reaction analysis of extracted endotracheal aspirates confirmed no evidence of mixed infection for either twin. Failure of sentinel ventilated preterm infants on the same ward to acquire Ureaplasma infection after the first week of birth suggests no cot-to-cot transfer of Ureaplasma infection occurred. This study demonstrated not only a contrasting clinical outcome for a set of twins infected with 2 separate species of Ureaplasma, but also the first real-time demonstration of multiple-banded antigen alteration and evolution of Ureaplasma over the course of a clinical infection. Copyright © 2016 by the American Academy of Pediatrics.

Protamine-based nanoparticles as new antigen delivery systems.

PubMed

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Dry eye syndrome: developments and lifitegrast in perspective

PubMed Central

Lollett, Ivonne V; Galor, Anat

2018-01-01

Dry eye (DE) is a chronic ocular condition with high prevalence and morbidity. It has a complex pathophysiology and is multifactorial in nature. Chronic ocular surface inflammation has emerged as a key component of DE that is capable of perpetuating ocular surface damage and leading to symptoms of ocular pain, discomfort, and visual phenomena. It begins with stress to the ocular surface leading to the production of proinflammatory mediators that induce maturation of resident antigen-presenting cells which then migrate to the lymph nodes to activate CD4 T cells. The specific antigen(s) targeted by these pathogenic CD4+ T cells remains unknown. Two emerging theories include self-antigens by autoreactive CD4 T cells or harmless exogenous antigens in the setting of mucosal immunotolerance loss. These CD4 T cells migrate to the ocular surface causing additional inflammation and damage. Lifitegrast is the second topical anti-inflammatory agent to be approved by the US Food and Drug Administration for the treatment of DE and the first to show improvement in DE symptoms. Lifitegrast works by blocking the interaction between intercellular adhesion molecule-1 and lymphocyte functional associated antigen-1, which has been shown to be critical for the migration of antigen-presenting cells to the lymph nodes as well as CD4+ T cell activation and migration to the ocular surface. In four large multicenter, randomized controlled trials, lifitegrast has proven to be effective in controlling both the signs and symptoms of DE with minimal side effects. Further research should include comparative and combination studies with other anti-inflammatory therapies used for DE. PMID:29391773

Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation.

PubMed

Dagenais, P; Desprez, B; Albert, J; Escher, E

1994-10-01

Direct adsorption of small peptide antigens to unaltered, commercially available polystyrene surfaces may be too weak to permit suitable assay by ELISA. We therefore developed a simple method for the covalent attachment of small, potentially single epitope antigens to polystyrene surfaces. Chemical activation of polystyrene plates with carbodiimide considerably improves the total and covalent attachment of radioactive octapeptides. The covalent attachment was demonstrated by washing with hot detergent. A 3.5 Mrad gamma-irradiation of plates also increases total binding, particularly in combination with chemical activation. The covalent attachment presumably occurs through formation and chemical activation of carboxylate functions on the polystyrene surface which form amide bonds with peptides. ELISA test was performed with CGRP and successive smaller CGRP fragments. Covalent attachment of C-terminal peptide fragments as detection antigens allows optimal recognition and sensitivity even for hexapeptides, while decapeptide antigens were already poorly recognized using a conventional antigen plating technique. Repetitive detergent washes and/or prolonged storage of plates with covalently bound antigens did not reduce their ELISA sensitivity. The method with storage and reutilization capacities that we present here will be useful for the development of preplated antibody screening test.

Cross-reactive antigens and lectin as determinants of symbiotic specificity in the Rhizobium-clover association.

PubMed Central

Dazzo, F B; Hubbell, D H

1975-01-01

Cross-reactive antigens of clover roots and Rhizobium trifolii were detected on their cell surfaces by tube agglutination, immunofluorescent, and radioimmunoassay techniques. Anti-clover root antiserum had a higher agglutinating titer with infective strains of R. trifolii than with noninfective strains. The root antiserum previously adsorbed with noninfective R. trifolii cells remained reactive only with infective cells, including infective revertants. When adsorbed with infective cells, the root antiserum was reactive with neither infective nor noninfective cells. Other Rhizobium species incapable of infecting clover did not demonstrate surface antigens cross-reactive with clover. Radioimmunoassay indicated twice as much antigenic cross-reactivity of clover roots and R. trifolii 403 (infective) than R. trifolii Bart A (noninfective). Immunofluorescence with anti-R. trifolii (infective) antiserum was detected on the exposed surface of the root epidermal cells and diminished at the root meristem. The immunofluorescent crossreaction on clover roots was totally removed by adsorption of anti-R. trifolii (infective) antiserum with encapsulated infective cells but not with noninfective cells. The cross-reactive capsular antigens from R. trifolii strains were extracted and purified. The ability of these antigens to induce clover root hair deformation was much greater when they were obtained from the infective than noninfective strains. The cross-reactive capsular antigen of R. trifolii 403 was characterized as a high-molecular-weight (greater than 4.6 times 10(6) daltons), beta-linked, acidic heteropolysaccharide containing 2-deoxyglucose, galactose, glucose, and glucuronic acid. A soluble, nondialyzable, substance (clover lectin) capable of binding to the cross-reactive antigen and agglutinating only infective cells of R. trifolii was extracted from white clover seeds. This lectin was sensitive to heat, Pronase, and trypsin. inhibition studies indicated that 2-deoxyglucose was the most probable haptenic determinant of the cross-reactive capsular antigen capable of binding to the root antiserum and the clover lectin. A model is proposed suggesting the preferential adsorption of infective versus noninfective cells of R. trifolii on the surface of clover roots by a cross-bridging of their common surface antigens with a multivalent clover lectin. Images PMID:55100

Genetic susceptibility to Grave's disease.

PubMed

Li, Hong; Chen, Qiuying

2013-06-01

The variety of clinical presentations of eye changes in patients with Graves' disease (GD) suggests that complex interactions between genetic, environmental, endogenous and local factors influence the severity of Graves' ophthalmopathy (GO). It is thought that the development of GO might be influenced by genetic factors and environmental factors, such as cigarette smoking. At present, however, the role of genetic factors in the development of GO is not known. On the basis of studies with candidate genes and other genetic approaches, several susceptibility loci in GO have been proposed, including immunological genes, human leukocyte antigen (HLA), cytotoxic T-lymphocyte antigen-4 (CTLA-4), regulatory T-cell genes and thyroid-specific genes. This review gives a brief overview of the current range of major susceptibility genes found for GD.

Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery.

PubMed

Degan, Massimo; Bomben, Riccardo; Bo, Michele Dal; Zucchetto, Antonella; Nanni, Paola; Rupolo, Maurizio; Steffan, Agostino; Attadia, Vincenza; Ballerini, Pier Ferruccio; Damiani, Daniela; Pucillo, Carlo; Poeta, Giovanni Del; Colombatti, Alfonso; Gattei, Valter

2004-07-01

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

PubMed Central

Siddle, Hannah V.; Kreiss, Alexandre; Tovar, Cesar; Yuen, Chun Kit; Cheng, Yuanyuan; Belov, Katherine; Swift, Kate; Pearse, Anne-Maree; Hamede, Rodrigo; Jones, Menna E.; Skjødt, Karsten; Woods, Gregory M.; Kaufman, Jim

2013-01-01

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host–immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD. PMID:23479617

DISTINCT ANTIBODY SPECIES: STRUCTURAL DIFFERENCES CREATING THERAPEUTIC OPPORTUNITIES

PubMed Central

Muyldermans, Serge; Smider, Vaughn V.

2016-01-01

Antibodies have been a remarkably successful class of molecules for binding a large number of antigens in therapeutic, diagnostic, and research applications. Typical antibodies derived from mouse or human sources use the surface formed by complementarity determining regions (CDRs) on the variable regions of the heavy chain/light chain heterodimer, which typically forms a relatively flat binding surface. Alternative species, particularly camelids and bovines, provide a unique paradigm for antigen recognition through novel domains which form the antigen binding paratope. For camelids, heavy chain antibodies bind antigen with only a single heavy chain variable region, in the absence of light chains. In bovines, ultralong CDR-H3 regions form an independently folding minidomain, which protrudes from the surface of the antibody and is diverse in both its sequence and disulfide patterns. The atypical paratopes of camelids and bovines potentially provide the ability to interact with different epitopes, particularly recessed or concave surfaces, compared to traditional antibodies. PMID:26922135

Merozoite surface protein-1 genetic diversity in Plasmodium malariae and Plasmodium brasilianum from Brazil.

PubMed

Guimarães, Lilian O; Wunderlich, Gerhard; Alves, João M P; Bueno, Marina G; Röhe, Fabio; Catão-Dias, José L; Neves, Amanda; Malafronte, Rosely S; Curado, Izilda; Domingues, Wilson; Kirchgatter, Karin

2015-11-16

The merozoite surface protein 1 (MSP1) gene encodes the major surface antigen of invasive forms of the Plasmodium erythrocytic stages and is considered a candidate vaccine antigen against malaria. Due to its polymorphisms, MSP1 is also useful for strain discrimination and consists of a good genetic marker. Sequence diversity in MSP1 has been analyzed in field isolates of three human parasites: P. falciparum, P. vivax, and P. ovale. However, the extent of variation in another human parasite, P. malariae, remains unknown. This parasite shows widespread, uneven distribution in tropical and subtropical regions throughout South America, Asia, and Africa. Interestingly, it is genetically indistinguishable from P. brasilianum, a parasite known to infect New World monkeys in Central and South America. Specific fragments (1 to 5) covering 60 % of the MSP1 gene (mainly the putatively polymorphic regions), were amplified by PCR in isolates of P. malariae and P. brasilianum from different geographic origin and hosts. Sequencing of the PCR-amplified products or cloned PCR fragments was performed and the sequences were used to construct a phylogenetic tree by the maximum likelihood method. Data were computed to give insights into the evolutionary and phylogenetic relationships of these parasites. Except for fragment 4, sequences from all other fragments consisted of unpublished sequences. The most polymorphic gene region was fragment 2, and in samples where this region lacks polymorphism, all other regions are also identical. The low variability of the P. malariae msp1 sequences of these isolates and the identification of the same haplotype in those collected many years apart at different locations is compatible with a low transmission rate. We also found greater diversity among P. brasilianum isolates compared with P. malariae ones. Lastly, the sequences were segregated according to their geographic origins and hosts, showing a strong genetic and geographic structure. Our data show that there is a low level of sequence diversity and a possible absence of allelic dimorphism of MSP1 in these parasites as opposed to other Plasmodium species. P. brasilianum strains apparently show greater divergence in comparison to P. malariae, thus P. malariae could derive from P. brasilianum, as it has been proposed.

[Experimental study on TCRbeta idiotypic antigenic determinants DNA vaccine to induce anti-lymphoma antibodies].

PubMed

Zhang, Yeping; Zhu, Ping; Shi, Yongjin; Liu, Jihua; Pu, Dingfang; Cao, Xianghong; Zhu, Qiang; Wang, Yijia; Ma, Mingxin; Yu, Jiren

2002-02-01

To investigate the anti-human CEM lymphoma cell activities induced by TCR idiotypic DNA vaccine containing different antigen determinants in BALB/c mice. The specific rearranged gene fragment encoding TCRVbeta region of CEM cell line was obtained by RT-PCR technique. The PCR product was cloned into eukaryocytic expression vector pcDNA3, which was used as DNA vaccine and template for PCR amplifying different antigen determinant. Gene fragments encoding different antigen determinant were amplified and cloned into pcDNA3, separately. The experimental mice were immunized by intramuscular injection of the DNA vaccines. The specific anti-idiotype antibodies were detected by indirect immunofluorescence assay. TCRbetaV of CEM cell line contains five antigen determinants. Specific anti-idiotype antibody was detected in all of the six mice immunized with DNA vaccine containing all the five determinants (the highest titer was 1:480). Although the antibody could also be detected in four of the six mice immunized with DNA vaccine containing four of the five antigen determinants, the antibody titer was lower (the highest titer was 1:80). DNA vaccine containing two of the five determinants could not induce the specific antibody. The idiotypic DNA vaccine containing the whole TCRbetaV five antigen determinants could induce the specific anti-lymphoma idiotypic antibody in BALB/c mice.

Simplification of genotyping techniques of the ABO blood type experiment and exploration of population genetics.

PubMed

Hu, Jian; Zhou, Yi-ren; Ding, Jia-lin; Wang, Zhi-yuan; Liu, Ling; Wang, Ye-kai; Lou, Hui-ling; Qiao, Shou-yi; Wu, Yan-hua

2017-05-20

The ABO blood type is one of the most common and widely used genetic traits in humans. Three glycosyltransferase-encoding gene alleles, I A , I B and i, produce three red blood cell surface antigens, by which the ABO blood type is classified. By using the ABO blood type experiment as an ideal case for genetics teaching, we can easily introduce to the students several genetic concepts, including multiple alleles, gene interaction, single nucleotide polymorphism (SNP) and gene evolution. Herein we have innovated and integrated our ABO blood type genetics experiments. First, in the section of Molecular Genetics, a new method of ABO blood genotyping was established: specific primers based on SNP sites were designed to distinguish three alleles through quantitative real-time PCR. Next, the experimental teaching method of Gene Evolution was innovated in the Population Genetics section: a gene-evolution software was developed to simulate the evolutionary tendency of the ABO genotype encoding alleles under diverse conditions. Our reform aims to extend the contents of genetics experiments, to provide additional teaching approaches, and to improve the learning efficiency of our students eventually.

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

USDA-ARS?s Scientific Manuscript database

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

PubMed Central

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

PubMed

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.

Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

PubMed

Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

2016-03-01

T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes

PubMed Central

Bishop, R.P.; Hemmink, J.D.; Morrison, W.I.; Weir, W.; Toye, P.G.; Sitt, T.; Spooner, P.R.; Musoke, A.J.; Skilton, R.A.; Odongo, D.O.

2015-01-01

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. ‘Deep 454 pyrosequencing’ of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva. PMID:26543804

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

PubMed

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

Codon optimisation to improve expression of a Mycobacterium avium ssp. paratuberculosis-specific membrane-associated antigen by Lactobacillus salivarius.

PubMed

Johnston, Christopher; Douarre, Pierre E; Soulimane, Tewfik; Pletzer, Daniel; Weingart, Helge; MacSharry, John; Coffey, Aidan; Sleator, Roy D; O'Mahony, Jim

2013-06-01

Subunit and DNA-based vaccines against Mycobacterium avium ssp. paratuberculosis (MAP) attempt to overcome inherent issues associated with whole-cell formulations. However, these vaccines can be hampered by poor expression of recombinant antigens from a number of disparate hosts. The high G+C content of MAP invariably leads to a codon bias throughout gene expression. To investigate if the codon bias affects recombinant MAP antigen expression, the open reading frame of a MAP-specific antigen MptD (MAP3733c) was codon optimised for expression against a Lactobacillus salivarius host. Of the total 209 codons which constitute MAP3733c, 172 were modified resulting in a reduced G+C content from 61% for the native gene to 32.7% for the modified form. Both genes were placed under the transcriptional control of the PnisA promoter; allowing controlled heterologous expression in L. salivarius. Expression was monitored using fluorescence microscopy and microplate fluorometry via GFP tags translationally fused to the C-termini of the two MptD genes. A > 37-fold increase in expression was observed for the codon-optimised MAP3733synth variant over the native gene. Due to the low cost and improved expression achieved, codon optimisation significantly improves the potential of L. salivarius as an oral vaccine stratagem against Johne's disease. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Roles of oral bacteria in cardiovascular diseases--from molecular mechanisms to clinical cases: Cell-surface structures of novel serotype k Streptococcus mutans strains and their correlation to virulence.

PubMed

Nakano, Kazuhiko; Nomura, Ryota; Matsumoto, Michiyo; Ooshima, Takashi

2010-01-01

Streptococcus mutans is generally known as a pathogen of dental caries, and it is also considered to cause bacteremia and infective endocarditis (IE). S. mutans was previously classified into 3 serotypes, c, e, and f, due to the different chemical compositions of the serotype-specific polysaccharides, which are composed of a rhamnose backbone and glucose side chains. We recently designated non-c/e/f serotype S. mutans strains as novel serotype k, which is characterized by a drastic reduction in the amount of the glucose side chain. A common biological feature of novel serotype-k strains is a lower level of cariogenicity due to alterations of several major cell surface protein antigens. As for virulence in blood, these strains survive in blood for a longer duration due to lower antigenicity, while the detection rate of all strains carrying the gene encoding collagen-binding adhesin has been shown to be high. Furthermore, molecular biological analyses of infected heart valve specimens obtained from IE patients revealed a high detection rate of serotype-k S. mutans. Together, these findings suggest that serotype-k S. mutans strains show low cariogenicity but high virulence in blood as compared to the other serotypes, due to alterations of several cell surface structures.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujiwara, Daisuke; Kato, Kazunori, E-mail: kzkatou@juntendo.ac.jp; Department of Atopy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421

2013-05-17

Highlights: •Spheroids were created from esophageal carcinoma cells using NanoCulture® Plates. •The proportion of strongly ALDH-positive cells increased in 3-D culture. •Expression of cancer stem cell-related genes was enhanced in 3-D culture. •CA-9 expression was enhanced, suggesting hypoxia had been induced in 3-D culture. •Drug resistance was increased. 3-D culture is useful for inducing cancer stem cells. -- Abstract: In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present inmore » esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.« less

O-Antigens of Escherichia coli Strains O81 and HS3-104 Are Structurally and Genetically Related, Except O-Antigen Glucosylation in E. coli HS3-104.

PubMed

Zdorovenko, E L; Wang, Y; Shashkov, A S; Chen, T; Ovchinnikova, O G; Liu, B; Golomidova, A K; Babenko, V V; Letarov, A V; Knirel, Y A

2018-05-01

Glycerophosphate-containing O-specific polysaccharides (OPSs) were obtained by mild acidic degradation of lipopolysaccharides isolated from Escherichia coli type strain O81 and E. coli strain HS3-104 from horse feces. The structures of both OPSs and of the oligosaccharide derived from the strain O81 OPS by treatment with 48% HF were studied by monosaccharide analysis and one- and two-dimensional 1H- and 13C-NMR spectroscopy. Both OPSs had similar structures and differed only in the presence of a side-chain glucose residue in the strain HS3-104 OPS. The genes and the organization of the O-antigen biosynthesis gene cluster in both strains are almost identical with the exception of the gtr gene cluster responsible for glucosylations in the strain HS3-104, which is located elsewhere in the genome.

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

PubMed

Criscitiello, Michael F; Ohta, Yuko; Graham, Matthew D; Eubanks, Jeannine O; Chen, Patricia L; Flajnik, Martin F

2012-03-01

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates. Copyright © 2011 Elsevier Ltd. All rights reserved.

Antigenic differences in the surfaces of hyphae and rhizoids in allomyces.

PubMed

Fultz, S A; Sussman, A S

1966-05-06

Immunofluorescent techniques have demonstrated a difference in surface components of hyphae and rhizoids of Allomyces macrogynus. An antigenic component detected on the rhizoidal surface may be present, but masked, in the hyphal-wall matrix material. The system also allows visualization of the hyphal wall during aging, when changes from a smooth to a fissured surface are noted, and differences in adsorptive properties occur.

Comparison of O-Antigen gene clusters of all O-Serogroups of Escherichia coli and proposal for adopting a new nomenclature for O-Typing

USDA-ARS?s Scientific Manuscript database

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interaction. Because they are hi...

Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test.

PubMed

Kumar, Navin; Pande, Veena; Bhatt, R M; Shah, Naman K; Mishra, Neelima; Srivastava, Bina; Valecha, Neena; Anvikar, Anupkumar R

2013-01-01

Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India. Copyright © 2012 Elsevier B.V. All rights reserved.

Novel Antigen Identification Method for Discovery of Protective Malaria Antigens by Rapid Testing of DNA Vaccines Encoding Exons from the Parasite Genome

DTIC Science & Technology

2004-03-01

EAA21673 1,443 — — Xeroderma pigmentosum G N&I region, helix-hairpin-helix class P.f., P.k., P.b., P.v. PY02286 EAA21722 696 — — Hypothetical protein...ND PY01828 Gene gun 0.1 2,560 640 Pos IM 0.1 Neg Neg ND CSP Gene gun 0.1 2,560 Neg Neg IM 2.7* 2,560 Neg ND a Parasite burden in liver is in...negative; Pos , positive; ND, not done. c Sera tested at a single dilution (1:80). VOL. 72, 2004 DISCOVERY OF PROTECTIVE MALARIA PARASITE ANTIGENS 1599

Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgaria.

PubMed

Strateva, Tanya; Atanasova, Daniela; Savov, Encho; Petrova, Guergana; Mitov, Ivan

2016-01-01

To evaluate the prevalence of some virulence genes among 510 clinical Enterococcus spp. isolates and to assess the association of those genes with the species, infection site, and patient group (inpatients/outpatients). Adhesins genes (aggregation substances agg and asa1 of Enterococcus faecalis and Enterococcus faecium, respectively), enterococcal surface protein (esp), endocarditis-specific antigen A (efaA), collagen-binding proteins (ace/acm)); invasins (hyaluronidase (hyl) and gelatinase (gelE)); cytotoxines (activation of cytolysin (cylA) in E. faecalis); and modulators of the host immunity and inflammation (enhanced expression pheromone (eep) in E. faecalis) were detected by polymerase chain reaction. The overall prevalence was: esp - 44.3%, agg/asa1 - 38.4%, ace/acm - 64.3%, efaA - 85.9%, eep - 69.4%, gelE - 64.3%, hyl - 25.1%, and cylA - 47.1%. E. faecalis isolates had significantly higher frequency of adhesin genes (esp and agg/asa1) and gelatinase in comparison to E. faecium. Multiple virulence genes in E. faecalis were significantly more prevalent than in E. faecium isolates. Domination of E. faecium with or without only one gene compared to the isolates of E. faecalis were found. Enterococcus spp. isolates obtained from outpatients compared to inpatients isolates had significantly higher frequency of agg/asa1, eep, gelE and cylA. Some adhesins genes (esp, agg/asa1 and efaA) had higher prevalence among the non-invasive Enterococcus spp. isolates compared to those causing invasive bacteremia, while ace/acm revealed higher dissemination in isolates causing invasive infections compared to non-invasive isolates. Most E. faecalis attaches to abiotic surfaces in hospital environment, which correlates with higher prevalence of gene encoding for virulence factors involved in biofilm formation, such as enterococcal surface protein, aggregation substance, and gelatinase. The intestinal tract is an important reservoir for opportunistic enterococcal pathogens and allows them to access infectious sites through different virulence factors, demonstrated in outpatient isolates in this study. Copyright © 2016 Elsevier Editora Ltda. All rights reserved.

Antigenic topology of chlamydial PorB protein and identification of targets for immune neutralization of infectivity.

PubMed

Kawa, Diane E; Stephens, Richard S

2002-05-15

The outer membrane protein PorB is a conserved chlamydial protein that functions as a porin and is capable of eliciting neutralizing Abs. A topological antigenic map was developed using overlapping synthetic peptides representing the Chlamydia trachomatis PorB sequence and polyclonal immune sera. To identify which antigenic determinants were surface accessible, monospecific antisera were raised to the PorB peptides and were used in dot-blot and ELISA-based absorption studies with viable chlamydial elementary bodies (EBs). The ability of the surface-accessible antigenic determinants to direct neutralizing Ab responses was investigated using standardized in vitro neutralization assays. Four major antigenic clusters corresponding to Phe(34)-Leu(59) (B1-2 and B1-3), Asp(112) -Glu(145) (B2-3 and B2-4), Gly(179)-Ala(225) (B3-2 to B3-4), and Val(261)-Asn(305) (B4-4 to B5-2) were identified. Collectively, the EB absorption and dot-blot assays established that the immunoreactive PorB Ags were exposed on the surface of chlamydial EBs. Peptide-specific antisera raised to the surface-accessible Ags neutralized chlamydial infectivity and demonstrated cross-reactivity to synthetic peptides representing analogous C. pneumoniae PorB sequences. Furthermore, neutralization of chlamydial infectivity by C. trachomatis PorB antisera was inhibited by synthetic peptides representing the surface-exposed PorB antigenic determinants. These findings demonstrate that PorB Ags may be useful for development of chlamydial vaccines.

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt, as an inactivator of hepatitis B surface antigen.

PubMed Central

Sugimoto, Y; Toyoshima, S

1979-01-01

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt (CAE), exhibited a strong inactivating effect on hepatitis B surface antigen. Concentrations of CAE required for 50 and 100% inactivation of the antigen were 0.01 to 0.025% and 0.025 to 0.05% respectively. CAE completely inactivated hepatitis B surface antigen at the lowest concentration compared with various compounds including about 500 amino acid derivatives, sodium hypochlorite, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, and some detergents. Furthermore, CAE inactivated vaccinia virus, herpes simplex virus, and influenza virus, whereas poliovirus was not inactivated at all. The results suggest that the inactivating effects of CAE are related to interaction with lipid-containing viral envelopes. PMID:228595

The Plasmodium falciparum transcriptome in severe malaria reveals altered expression of genes involved in important processes including surface antigen–encoding var genes

PubMed Central

Tonkin-Hill, Gerry Q.; Trianty, Leily; Noviyanti, Rintis; Nguyen, Hanh H. T.; Sebayang, Boni F.; Lampah, Daniel A.; Marfurt, Jutta; Cobbold, Simon A.; Rambhatla, Janavi S.; McConville, Malcolm J.; Rogerson, Stephen J.; Brown, Graham V.; Day, Karen P.; Price, Ric N.; Anstey, Nicholas M.

2018-01-01

Within the human host, the malaria parasite Plasmodium falciparum is exposed to multiple selection pressures. The host environment changes dramatically in severe malaria, but the extent to which the parasite responds to—or is selected by—this environment remains unclear. From previous studies, the parasites that cause severe malaria appear to increase expression of a restricted but poorly defined subset of the PfEMP1 variant, surface antigens. PfEMP1s are major targets of protective immunity. Here, we used RNA sequencing (RNAseq) to analyse gene expression in 44 parasite isolates that caused severe and uncomplicated malaria in Papuan patients. The transcriptomes of 19 parasite isolates associated with severe malaria indicated that these parasites had decreased glycolysis without activation of compensatory pathways; altered chromatin structure and probably transcriptional regulation through decreased histone methylation; reduced surface expression of PfEMP1; and down-regulated expression of multiple chaperone proteins. Our RNAseq also identified novel associations between disease severity and PfEMP1 transcripts, domains, and smaller sequence segments and also confirmed all previously reported associations between expressed PfEMP1 sequences and severe disease. These findings will inform efforts to identify vaccine targets for severe malaria and also indicate how parasites adapt to—or are selected by—the host environment in severe malaria. PMID:29529020

Designing two-in-one antibodies.

PubMed

Valladares, Ignacio Garcia; Espinoza, Luis R

2009-09-01

Evaluation of: Bostrom J, Shang-Fan Y, Kan D et al.: Variants of the antibody Herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610-1614 (2009). The longstanding held notion that one antibody equals one antigen and, hence, one function has been challenged in recent years. Improved technology in antibody production, especially the accumulation of sequence data of immunoglobulin genes and the advent of PCR have made it possible to clone antibody gene repertoires. The current paper provides further challenge to the notion of one antibody = one antigen by developing 'two-in-one' antibodies with an antigen-binding site that binds two distinct proteins with high affinity. A therapeutic variant antibody of Herceptin (Genentech, CA, USA) was isolated that binds the human EGF receptor (HER)2 and also to VEGF. This development may represent a breakthrough discovery and may have significant implications in the therapy of malignant, infectious, allergic and autoimmune disorders.

Autodisplay: Development of an Efficacious System for Surface Display of Antigenic Determinants in Salmonella Vaccine Strains

PubMed Central

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B.; Lattemann, Claus T.

2003-01-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp6074-86), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp6074-86 was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp6074-86-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-γ secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains. PMID:12654812

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains.

PubMed

Kramer, Uwe; Rizos, Konstantin; Apfel, Heiko; Autenrieth, Ingo B; Lattemann, Claus T

2003-04-01

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp60(74-86)), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp60(74-86) was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp60(74-86)-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-gamma secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains.

Myelin-oligodendrocyte glycoprotein is a member of a subset of the immunoglobulin superfamily encoded within the major histocompatibility complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pham-Dinh, D.; Dautigny, A.; Mattei, M.G.

1993-09-01

Myelin/oligodendrocyte glycoprotein (MOG) is found on the surface of myelinating oligodendrocytes and external lamellae of myelin sheaths in the central nervous system, and it is target antigen in experimental autoimmune encephalomyelitis and multiple sclerosis. The authors have isolated bovine, mouse, and rat MOG cDNA clones and shown that the developmental pattern of MOG expression in the rat central nervous system coincides with the late stages of myelination. The amino-terminal, extracellular domain of MOG has characteristics of an immunoglobulin variable domain and is 46% and 41% identical with the amino terminus of bovine butyrophilin (expressed in the lactating mammary gland) andmore » B-G antigens of the chicken major histocompatibility complex (MHC), respectively; these proteins thus form a subset of the immunoglobulin superfamily. The homology between MOG and B-G extends beyond their structure and genetic mapping to their ability to induce strong antibody responses and has implications for the role of MOG in pathological, autoimmune conditions. The authors colocalized the MOG and BT genes to the human MHC on chromosome 6p21.3-p22. The mouse MOG gene was mapped to the homologous band C of chromosome 17, within the M region of the mouse MHC. 38 refs., 6 figs.« less

Limited genetic diversity among Sarcocystis neurona strains infecting southern sea otters precludes distinction between marine and terrestrial isolates

PubMed Central

Wendte, J.M.; Miller, M.A.; Nandra, A.K.; Peat, S.M.; Crosbie, P.R.; Conrad, P.A.; Grigg, M.E.

2010-01-01

Sarcocystis neurona is an apicomplexan parasite identified as a cause of fatal neurological disease in the threatened southern sea otter (Enhydra lutris nereis). In an effort to characterize virulent S. neurona strains circulating in the marine ecosystem, this study developed a range of markers relevant for molecular genotyping. Highly conserved sequences within the 18S ribosomal gene array, the plastid-encoded RNA polymerase (RPOb) and the cytochrome c oxidase subunit 1 mitochondrial gene (CO1) were assessed for their ability to distinguish isolates at the genus and species level. For within-species comparisons, five surface antigens (SnSAG1-SnSAG5) and one high resolution microsatellite marker (Sn9) were developed as genotyping markers to evaluate intra-strain diversity. Molecular analysis at multiple loci revealed insufficient genetic diversity to distinguish terrestrial isolates from strains infecting marine mammals. Furthermore, SnSAG specific primers applied against DNA from the closely related species, Sarcocystis falcatula, lead to the discovery of highly similar orthologs to SnSAG2, 3, and 4, calling into question the specificity of diagnostic tests based on these antigens. The results of this study suggest a population genetic structure for S. neurona similar to that reported for the related parasite, Toxoplasma gondii, dominated by a limited number of successful genotypes. PMID:20071081

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins

PubMed Central

2018-01-01

ABSTRACT African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates. PMID:29386289

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

PubMed

Jancovich, James K; Chapman, Dave; Hansen, Debra T; Robida, Mark D; Loskutov, Andrey; Craciunescu, Felicia; Borovkov, Alex; Kibler, Karen; Goatley, Lynnette; King, Katherine; Netherton, Christopher L; Taylor, Geraldine; Jacobs, Bertram; Sykes, Kathryn; Dixon, Linda K

2018-04-15

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates. Copyright © 2018 Jancovich et al.

Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.

PubMed

Tsaltas, G; Ford, C H

1993-02-01

Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.

Specific immune response genes of new inbred strains of guinea pigs.

PubMed

Chiba, J; Egashira, Y

1978-01-01

Distribution of specific immune-response (Ir) genes controlling responsiveness to synthetic polypeptide antigens, homopolymer of poly-L-lysine (PLL), copolymer of L-glutamic acid and L-alanine (GA) and copolymer of L-glutamic acid and L-tyrosine (GT), and limiting doses of 2,4-dinitrophenyl guinea pig serum albumin (DNP-GPA) was surveyed in new inbred strains of guinea pigs, JY 1, JY 2, JY 9 and JY 10, established in this Institute. The PLL gene was not found in any of the guinea pigs. The GA gene was found in JY 1 and JY 2 guinea pigs and the GT gene in all the guinea pigs. The gene controlling responsiveness to low doses (1 microgram) of DNP-GPA was found in JY 1, JY 9 and JY 10 guinea pigs. The associated (Ia) antigens was discussed.

A virus vector based on Canine Herpesvirus for vaccine applications in canids.

PubMed

Strive, T; Hardy, C M; Wright, J; Reubel, G H

2007-01-31

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.

MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

PubMed

Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A M

2013-01-01

Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.

PubMed

Li, Y; Spellerberg, M B; Stevenson, F K; Capra, J D; Potter, K N

1996-03-01

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Identification and Characterization of an Immunodominant 28-Kilodalton Coxiella burnetii Outer Membrane Protein Specific to Isolates Associated with Acute Disease

PubMed Central

Zhang, Guoquan; To, Ho; Russell, Kasi E.; Hendrix, Laura R.; Yamaguchi, Tsuyoshi; Fukushi, Hideto; Hirai, Katsuya; Samuel, James E.

2005-01-01

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an ∼28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a ∼28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever. PMID:15731054

Diversity of Sarcocystis spp shed by opossums in Brazil inferred with phylogenetic analysis of DNA coding ITS1, cytochrome B, and surface antigens.

PubMed

Valadas, Samantha Y O B; da Silva, Juliana I G; Lopes, Estela Gallucci; Keid, Lara B; Zwarg, Ticiana; de Oliveira, Alice S; Sanches, Thaís C; Joppert, Adriana M; Pena, Hilda F J; Oliveira, Tricia M F S; Ferreira, Helena L; Soares, Rodrigo M

2016-05-01

Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits. Copyright © 2016 Elsevier Inc. All rights reserved.

An epidemiological survey of bovine Babesia and Theileria parasites in cattle, buffaloes, and sheep in Egypt.

PubMed

Elsify, Ahmed; Sivakumar, Thillaiampalam; Nayel, Mohammed; Salama, Akram; Elkhtam, Ahmed; Rizk, Mohamed; Mosaab, Omar; Sultan, Khaled; Elsayed, Shimaa; Igarashi, Ikuo; Yokoyama, Naoaki

2015-02-01

Cattle, buffaloes, and sheep are the main sources of meat and milk in Egypt, but their productivity is thought to be greatly reduced by hemoprotozoan parasitic diseases. In this study, we analyzed the infection rates of Babesia bovis, Babesia bigemina, Theileria annulata, and Theileria orientalis, using parasite-specific PCR assays in blood-DNA samples sourced from cattle (n=439), buffaloes (n=50), and sheep (n=105) reared in Menoufia, Behera, Giza, and Sohag provinces of Egypt. In cattle, the positive rates of B. bovis, B. bigemina, T. annulata, and T. orientalis were 3.18%, 7.97%, 9.56%, and 0.68%, respectively. On the other hand, B. bovis and T. orientalis were the only parasites detected in buffaloes and each of these parasites was only found in two individual DNA samples (both 2%), while one (0.95%) and two (1.90%) of the sheep samples were positive for B. bovis and B. bigemina, respectively. Sequence analysis showed that the B. bovis Rhoptry Associated Protein-1 and the B. bigemina Apical Membrane Antigen-1 genes were highly conserved among the samples, with 99.3-100% and 95.3-100% sequence identity values, respectively. In contrast, the Egyptian T. annulata merozoite surface antigen-1 gene sequences were relatively diverse (87.8-100% identity values), dispersing themselves across several clades in the phylogenetic tree containing sequences from other countries. Additionally, the T. orientalis Major Piroplasm Surface Protein (MPSP) gene sequences were classified as types 1 and 2. This is the first report of T. orientalis in Egypt, and of type 2 MPSP in buffaloes. Detection of MPSP type 2, which is considered a relatively virulent genotype, suggests that T. orientalis infection may have veterinary and economic significance in Egypt. In conclusion, the present study, which analyzed multiple species of Babesia and Theileria parasites in different livestock animals, may shed an additional light on the epidemiology of hemoprotozoan parasites in Egypt. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Phenotypically heterogeneous deletion of the ABH antigen from the transformed bladder urothelium. A scanning electron microscope study.

PubMed

De Harven, E; He, S; Hanna, W; Bootsma, G; Connolly, J G

1987-10-01

The deletion of ABH blood group antigens from the luminal surface of the bladder mucosa in cases of well differentiated transitional cell carcinomata, and the formation of pleomorphic microvilli have both been associated with aggressive biological behaviour and invasiveness of the tumors. We have studied cold cup biopsies from 8 normal mucosae and 17 papillary transitional cell carcinomata of the urinary bladder. The aim of our study was to correlate the formation of uniform or pleomorphic microvilli with the extent of deletion of the ABH blood group antigens on the surface of normal and transformed bladder urothelium. Immunogold scanning electron microscopy (SEM) in the backscattered electron (BE) imaging mode was used for this purpose. In the normal urothelium, uniform labeling of the luminal cells was demonstrated. In well differentiated tumors, the superficial cells exhibited uniform microvilli and a heterogeneous expression of the ABH antigens, giving characteristic 'mosaic' patterns of the antigenic labeling across the mucosal surface. These patterns were sharply delimitated at cell junctions when viewed by SEM; these observations were confirmed by transmission electron microscopy. In higher grade tumors, decreased ABH antigen expression, pleomorphic microvilli and/or featureless luminal cells were observed. In the transformed urothelium, the formation of uniform microvilli appeared to precede the loss of ABH antigen in most cases.

Genetic diversity of pneumococcal surface protein A in invasive pneumococcal isolates from Korean children, 1991-2016.

PubMed

Yun, Ki Wook; Choi, Eun Hwa; Lee, Hoan Jong

2017-01-01

Pneumococcal surface protein A (PspA) is an important virulence factor of pneumococci and has been investigated as a primary component of a capsular serotype-independent pneumococcal vaccine. Thus, we sought to determine the genetic diversity of PspA to explore its potential as a vaccine candidate. Among the 190 invasive pneumococcal isolates collected from Korean children between 1991 and 2016, two (1.1%) isolates were found to have no pspA by multiple polymerase chain reactions. The full length pspA genes from 185 pneumococcal isolates were sequenced. The length of pspA varied, ranging from 1,719 to 2,301 base pairs with 55.7-100% nucleotide identity. Based on the sequences of the clade-defining regions, 68.7% and 49.7% were in PspA family 2 and clade 3/family 2, respectively. PspA clade types were correlated with genotypes using multilocus sequence typing and divided into several subclades based on diversity analysis of the N-terminal α-helical regions, which showed nucleotide sequence identities of 45.7-100% and amino acid sequence identities of 23.1-100%. Putative antigenicity plots were also diverse among individual clades and subclades. The differences in antigenicity patterns were concentrated within the N-terminal 120 amino acids. In conclusion, the N-terminal α-helical domain, which is known to be the major immunogenic portion of PspA, is genetically variable and should be further evaluated for antigenic differences and cross-reactivity between various PspA types from pneumococcal isolates.

Establishment of a Molecular Serotyping Scheme and a Multiplexed Luminex-Based Array for Enterobacter aerogenes

PubMed Central

Guo, Xi; Wang, Min; Wang, Lu; Wang, Yao; Chen, Tingting; Wu, Pan; Chen, Min; Liu, Bin; Feng, Lu

2018-01-01

Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens. Polysaccharide gene clusters (PSgcs) are typically responsible for the diversity of bacterial surface polysaccharides. Through whole-genome sequencing and analysis, eight putative PSgc types were identified in 23 Enterobacter aerogenes strains from several geographic areas, allowing us to present the first molecular serotyping system for E. aerogenes. A conventional antigenic scheme was also established and correlated well with the molecular serotyping system that was based on PSgc genetic variation, indicating that PSgc-based molecular typing and immunological serology provide equally valid results. Further, a multiplex Luminex-based array was developed, and a double-blind test was conducted with 97 clinical specimens from Shanghai, China, to validate our array. The results of these analyses indicated that strains containing PSgc4 and PSgc7 comprised the predominant groups. We then examined 86 publicly available E. aerogenes strain genomes and identified an additional seven novel PSgc types, with PSgc10 being the most abundant type. In total, our study identified 15 PSgc types in E. aerogenes, providing the basis for a molecular serotyping scheme. From these results, differing epidemic patterns were identified between strains that were predominant in different regions. Our study highlights the feasibility and reliability of a serotyping system based on PSgc diversity, and for the first time, presents a molecular serotyping system, as well as an antigenic scheme for E. aerogenes, providing the basis for molecular diagnostics and epidemiological surveillance of this important emerging pathogen. PMID:29616012

Establishment of a Molecular Serotyping Scheme and a Multiplexed Luminex-Based Array for Enterobacter aerogenes.

PubMed

Guo, Xi; Wang, Min; Wang, Lu; Wang, Yao; Chen, Tingting; Wu, Pan; Chen, Min; Liu, Bin; Feng, Lu

2018-01-01

Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens. Polysaccharide gene clusters (PSgcs) are typically responsible for the diversity of bacterial surface polysaccharides. Through whole-genome sequencing and analysis, eight putative PSgc types were identified in 23 Enterobacter aerogenes strains from several geographic areas, allowing us to present the first molecular serotyping system for E. aerogenes . A conventional antigenic scheme was also established and correlated well with the molecular serotyping system that was based on PSgc genetic variation, indicating that PSgc-based molecular typing and immunological serology provide equally valid results. Further, a multiplex Luminex-based array was developed, and a double-blind test was conducted with 97 clinical specimens from Shanghai, China, to validate our array. The results of these analyses indicated that strains containing PSgc4 and PSgc7 comprised the predominant groups. We then examined 86 publicly available E. aerogenes strain genomes and identified an additional seven novel PSgc types, with PSgc10 being the most abundant type. In total, our study identified 15 PSgc types in E. aerogenes , providing the basis for a molecular serotyping scheme. From these results, differing epidemic patterns were identified between strains that were predominant in different regions. Our study highlights the feasibility and reliability of a serotyping system based on PSgc diversity, and for the first time, presents a molecular serotyping system, as well as an antigenic scheme for E. aerogenes , providing the basis for molecular diagnostics and epidemiological surveillance of this important emerging pathogen.

Genetic diversity of three surface protein genes in Plasmodium malariae from three Asian countries.

PubMed

Srisutham, Suttipat; Saralamba, Naowarat; Sriprawat, Kanlaya; Mayxay, Mayfong; Smithuis, Frank; Nosten, Francois; Pukrittayakamee, Sasithon; Day, Nicholas P J; Dondorp, Arjen M; Imwong, Mallika

2018-01-11

Genetic diversity of the three important antigenic proteins, namely thrombospondin-related anonymous protein (TRAP), apical membrane antigen 1 (AMA1), and 6-cysteine protein (P48/45), all of which are found in various developmental stages of Plasmodium parasites is crucial for targeted vaccine development. While studies related to the genetic diversity of these proteins are available for Plasmodium falciparum and Plasmodium vivax, barely enough information exists regarding Plasmodium malariae. The present study aims to demonstrate the genetic variations existing among these three genes in P. malariae by analysing their diversity at nucleotide and protein levels. Three surface protein genes were isolated from 45 samples collected in Thailand (N = 33), Myanmar (N = 8), and Lao PDR (N = 4), using conventional polymerase chain reaction (PCR) assay. Then, the PCR products were sequenced and analysed using BioEdit, MEGA6, and DnaSP programs. The average pairwise nucleotide diversities (π) of P. malariae trap, ama1, and p48/45 were 0.00169, 0.00413, and 0.00029, respectively. The haplotype diversities (Hd) of P. malariae trap, ama1, and p48/45 were 0.919, 0.946, and 0.130, respectively. Most of the nucleotide substitutions were non-synonymous, which indicated that the genetic variations of these genes were maintained by positive diversifying selection, thus, suggesting their role as a potential target of protective immune response. Amino acid substitutions of P. malariae TRAP, AMA1, and P48/45 could be categorized to 17, 20, and 2 unique amino-acid variants, respectively. For further vaccine development, carboxyl terminal of P48/45 would be a good candidate according to conserved amino acid at low genetic diversity (π = 0.2-0.3). High mutational diversity was observed in P. malariae trap and ama1 as compared to p48/45 in P. malariae samples isolated from Thailand, Myanmar, and Lao PDR. Taken together, these results suggest that P48/45 might be a good vaccine candidate against P. malariae infection because of its sufficiently low genetic diversity and highly conserved amino acids especially on the carboxyl end.

Molecular characterisation and the protective immunity evaluation of Eimeria maxima surface antigen gene.

PubMed

Liu, Tingqi; Huang, Jingwei; Li, Yanlin; Ehsan, Muhammad; Wang, Shuai; Zhou, Zhouyang; Song, Xiaokai; Yan, Ruofeng; Xu, Lixin; Li, Xiangrui

2018-05-30

Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated. Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA. The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4 + and CD8 + T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P < 0.05). The EmSAG-specific antibody concentrations of both the rEmSAG and pVAX1-EmSAG groups were much higher than those of the negative controls (P < 0.05). Higher concentrations of IL-4, IFN-γ, TGF-β1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P < 0.05). Our findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima.

Co-infection with human polyomavirus BK enhances gene expression and replication of human adenovirus.

PubMed

Bil-Lula, Iwona; Woźniak, Mieczysław

2018-03-26

Immunocompromised patients are susceptible to multiple viral infections. Relevant interactions between co-infecting viruses might result from viral regulatory genes which trans-activate or repress the expression of host cell genes as well as the genes of any co-infecting virus. The aim of the current study was to show that the replication of human adenovirus 5 is enhanced by co-infection with BK polyomavirus and is associated with increased expression of proteins including early region 4 open reading frame 1 and both the large tumor antigen and small tumor antigen. Clinical samples of whole blood and urine from 156 hematopoietic stem cell transplant recipients were tested. We also inoculated adenocarcinomic human alveolar basal epithelial cells with both human adenovirus 5 and BK polyomavirus to evaluate if co-infection of viruses affected their replication. Data showed that adenovirus load was significantly higher in the plasma (mean 7.5 x 10 3  ± 8.5 x 10 2 copies/ml) and urine (mean 1.9 x 10 3  ± 8.0 x 10 2 copies/ml) of samples from patients with co-infections, in comparison to samples from patients with isolated adenovirus infection. In vitro co-infection led to an increased (8.6 times) expression of the adenovirus early region 4 open reading frame gene 48 hours post-inoculation. The expression of the early region 4 open reading frame gene positively correlated with the expression of BK polyomavirus large tumor antigen (r = 0.90, p < 0.0001) and small tumor antigen (r = 0.83, p < 0.001) genes. The enhanced expression of the early region 4 open reading frame gene due to co-infection with BK polyomavirus was associated with enhanced adenovirus, but not BK polyomavirus, replication. The current study provides evidence that co-infection of adenovirus and BK polyomavirus contributes to enhanced adenovirus replication. Data obtained from this study may have significant importance in the clinical setting.

Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

PubMed Central

Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

2008-01-01

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails.

PubMed

Adel, Amany; Arafa, Abdelsatar; Hussein, Hussein A; El-Sanousi, Ahmed A

2017-06-01

The LPAI viruses of H9N2 subtype became widely distributed in Middle Eastern countries, causing great economic losses in poultry industry especially when complicated with other pathogens. The H9N2 viruses in Egypt have a wide spread nature since its first occurrence in 2011. In this study, we collected cloacal and tracheal samples from 19 flocks for detection and propagation of H9N2 virus using real-time RT-PCR and egg inoculation. We studied the molecular evolution of the Hemagglutinin gene of H9N2 viruses by full HA gene sequencing, then the antigenic characterization was implemented using the cross HI assay and analyzed using 3D Bioinformatics cartography software. The phylogenetic analysis of the HA gene of Egyptian H9N2 viruses clearly points out the presence of only one group (Egy/G1) of originally introduced viruses in 2011 related to the G1 lineage within group B, with the presence of multiple minor clusters includes viruses from 2011 to 2015. However, a new variant (Egy/G1var) cluster was detected in quails since 2012. Genetically, Egy/G1var viruses characterized by presence of 20 amino acid substitutions within and adjacent to the antigenic sites in comparison to other Egyptian viruses. In addition, two glycosylation sites at amino acid residues 127 and 189 were determined in close to the receptor binding and antigenic sites. The antigenic analysis based on 3D antigenic mapping showed that the Egy/G1var cluster was clearly distinct from the original Egy/G1 viruses. In conclusion, Egy/G1var is shown to be a new escape mutant variant cluster with an adaptive evolution in quails. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters.

PubMed

Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

2017-01-01

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains.

The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters

PubMed Central

Delannoy, Sabine; Beutin, Lothar; Mariani-Kurkdjian, Patricia; Fleiss, Aubin; Bonacorsi, Stéphane; Fach, Patrick

2017-01-01

Escherichia coli strains belonging to serogroups O1 and O2 are frequently associated with human infections, especially extra-intestinal infections such as bloodstream infections or urinary tract infections. These strains can be associated with a large array of flagellar antigens. Because of their frequency and clinical importance, a reliable detection of E. coli O1 and O2 strains and also the frequently associated K1 capsule is important for diagnosis and source attribution of E. coli infections in humans and animals. By sequencing the O-antigen clusters of various O1 and O2 strains we showed that the serogroups O1 and O2 are encoded by different sets of O-antigen encoding genes and identified potentially new O-groups. We developed qPCR-assays to detect the various O1 and O2 variants and the K1-encoding gene. These qPCR assays proved to be 100% sensitive and 100% specific and could be valuable tools for the investigations of zoonotic and food-borne infection of humans with O1 and O2 extra-intestinal (ExPEC) or Shiga toxin-producing E. coli (STEC) strains. PMID:28224115

Evolution of the hemagglutinin gene of H3N8 canine influenza virus in dogs.

PubMed

Pecoraro, Heidi L; Bennett, Susi; Spindel, Miranda E; Landolt, Gabriele A

2014-12-01

With the widespread use of a recently developed canine influenza virus (CIV) H3N8 vaccine, continual molecular evaluation of circulating CIVs is necessary for monitoring antigenic drift. The aim of this project was to further describe the genetic evolution of CIV, as well as determine any genetic variation within potential antigenic regions that might result in antigenic drift. To this end, the hemagglutinin gene of 19 CIV isolates from dogs residing in Colorado, New York, and South Carolina humane shelters was sequenced and compared to CIV strains isolated during 2003-2012. Phylogenetic analysis suggests that CIV might be diverging into two geographically distinct lineages. Using a mixed-effects model for evolution and single likelihood ancestor counting methods, several amino acid sites were found to be undergoing selection pressure. Additionally, a total of six amino acid changes were observed in two possible antigenic sites for CIVs isolated from Colorado and New York humane shelters between 2009 and 2011. As CIV isolates might be diverging into geographically distinct lineages, further experiments are warranted to determine the extent of antigenic drift occurring within circulating CIV.

Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei brucei.

PubMed Central

Lheureux, M; Lheureux, M; Vervoort, T; Van Meirvenne, N; Steinert, M

1979-01-01

Polyadenylated RNA isolated from total polyribosomes of two variable antigen types (VATs) of T. brucei brucei were shown to program the synthesis, in mRNA-dependant reticulocyte lysates, of a wide variety of polypeptides. After immunoprecipitation of these cell-free products with an homologous antiserum raised against purified variant-specific surface antigen (VSSA), a major electrophoretic band was apparent on fluorography. It was confirmed that this band corresponds to the variable antigen since only an excess of purified homologous antigen will provoke competition. The apparent molecular weight of the in vitro synthesized antigen is about 63,000 daltons. The VSSA mRNA has been found in membrane-bound polyribosomes and a 15 fold immunological purification of this mRNA has been obtained, using partially purified anti-VSSA IgG in conjunction with inactivated Staphylococcus aureus. Images PMID:116191

Strategies to induce broadly protective antibody responses to viral glycoproteins.

PubMed

Krammer, F

2017-05-01

Currently, several universal/broadly protective influenza virus vaccine candidates are under development. Many of these vaccines are based on strategies to induce protective antibody responses against the surface glycoproteins of antigenically and genetically diverse influenza viruses. These strategies might also be applicable to surface glycoproteins of a broad range of other important viral pathogens. Areas covered: Common strategies include sequential vaccination with divergent antigens, multivalent approaches, vaccination with glycan-modified antigens, vaccination with minimal antigens and vaccination with antigens that have centralized/optimized sequences. Here we review these strategies and the underlying concepts. Furthermore, challenges, feasibility and applicability to other viral pathogens are discussed. Expert commentary: Several broadly protective/universal influenza virus vaccine strategies will be tested in humans in the coming years. If successful in terms of safety and immunological readouts, they will move forward into efficacy trials. In the meantime, successful vaccine strategies might also be applied to other antigenically diverse viruses of concern.

Functional expression of a cattle MHC class II DR-like antigen on mouse L cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraser, D.C.; Craigmile, S.; Campbell, J.D.M.

1996-09-01

Cattle DRA and DRB genes, cloned by reverse-transcription polymerase chain reaction, were transfected into mouse L cells. The cattle DR-expressing L-cell transfectant generated was analyzed serologically, biochemically, and functionally. Sequence analysis of the transfected DRB gene clearly showed showed that it was DRB3 allele DRB3*0101, which corresponds to the 1D-IEF-determined allele DRBF3. 1D-IEF analysis of the tranfectant confirmed that the expressed DR product was DRBF3. Functional integrity of the transfected gene products was demonstrated by the ability of the transfectant cell line to present two antigens (the foot-and-mouth disease virus-derived peptide FMDV15, and ovalbumin) to antigen-specific CD4{sup +} T cellsmore » from both the original animal used to obtain the genes, and also from an unrelated DRBF3{sup +} heterozygous animal. Such transfectants will be invaluable tools, allowing us to dissect the precise contributions each locus product makes to the overall immune response in heterozygous animals, information essential for rational vaccine design. 45 refs., 5 figs., 1 tab.« less

Cooperation between Epstein-Barr Virus Immune Evasion Proteins Spreads Protection from CD8+ T Cell Recognition across All Three Phases of the Lytic Cycle

PubMed Central

Quinn, Laura L.; Zuo, Jianmin; Abbott, Rachel J. M.; Shannon-Lowe, Claire; Tierney, Rosemary J.; Hislop, Andrew D.; Rowe, Martin

2014-01-01

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

In vitro and in vivo evidence of a potential A(H1N1)pdm09 antigenic drift mediated by escape mutations in the haemagglutinin Sa antigenic site.

PubMed

Retamal, Miguel; Abed, Yacine; Rhéaume, Chantal; Baz, Mariana; Boivin, Guy

2017-06-01

Influenza A(H1N1)pdm09 virus continues to circulate worldwide without evidence of significant antigenic drift between 2009 and 2016. By using escape mutants, we previously identified six haemagglutinin (HA) changes (T80R, G143E, G158E, N159D, K166E and A198E) that were located within antigenic sites. Combinations of these mutations were introduced into the A(H1N1)pdm09 HA plasmid by mutagenesis. Reassortant 6 : 2 viruses containing both the HA and NA genes of the A(H1N1)pdm09 and the six internal gene segments of A/PR/8/34 were rescued by reverse genetics. In vitro, HA inhibition and microneutralization assays showed that the HA hexa-mutant reassortant virus (RG1) escaped A(H1N1)pdm09 hyper-immune ferret antiserum recognition. C57Black/6 mice that received the vaccine formulated with A/California/07/09 were challenged with 2×104 p.f.u. of either the 6 : 2 wild-type (WT) or RG1 viruses. Reductions in body weight loss, mortality rate and lung viral titre were observed in immunized animals challenged with the 6 : 2 WT virus compared to non-immunized mice. However, immunization did not protect mice challenged with RG1 virus. To further characterize the mutations causing this antigenic change, 11 additional RG viruses whose HA gene contained single or combinations of mutations were evaluated in vitro. Although the RG1 virus was still the least reactive against hyper-immune serum by HAI testing, mutations G158E and N159D within the Sa antigenic site appeared to play the major role in the altered antigenicity of the A(H1N1)pdm09 virus. These results show that the Sa antigenic site contains the most prominent epitopes susceptible to cause an antigenic drift, escaping actual vaccine protection.

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

PubMed Central

Yamanaka, Nobuko; Wong, Christine J.; Gertsenstein, Marina; Casper, Robert F.; Nagy, Andras; Rogers, Ian M.

2009-01-01

Background Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100% of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cells and that trogocytosis is evident in non-blood cells in chimera mice. PMID:20046883

A novel interdigitated capacitor based biosensor for detection of cardiovascular risk marker.

PubMed

Quershi, Anjum; Gurbuz, Yasar; Kang, Weng P; Davidson, Jimmy L

2009-12-15

C-reactive protein (CRP) is a potential biomarker whose elevated levels in humans determine cardiovascular disease risk and inflammation. In this study, we have developed a novel capacitive biosensor for detection of CRP-antigen using capacitor with interdigitated gold (GID) electrodes on nanocrystalline diamond (NCD) surface. The NCD surface served as a dielectric layer between the gold electrodes. GID-surface was functionalized by antibodies and the immobilization was confirmed by Fourier transform spectroscopy (FT-IR) and contact angle measurements. The CRP-antigen detection was performed by capacitive/dielectric-constant measurements. The relaxation time and polarizability constants were estimated using Cole-Cole model. Our results showed that the relaxation time constant (tau) of only CRP-antibody was within 10(-16)-10(-13)s, which was increased to 10(-11)s after the incubation with CRP-antigen, suggesting that the CRP-antigen was captured by the antibodies on GID-surface. In addition, polarizability constant (m) of CRP was also increased upon incubation with increasing concentration of CRP-antigen. Our results showed that the response of GID-NCD-based capacitive biosensor for CRP-antigen was dependent on both concentration (25-800ng/ml) as well as frequency (50-350MHz). Furthermore, using optimized conditions, the GID-NCD based capacitive biosensor developed in this study can potentially be used for detection of elevated levels of protein risk markers in suspected subjects for early diagnosis of disease.

Rh blood phenotyping (D, E, e, C, c) microarrays using multichannel surface plasmon resonance imaging.

PubMed

Pipatpanukul, Chinnawut; Takeya, Sasaki; Baba, Akira; Amarit, Ratthasart; Somboonkaew, Armote; Sutapun, Boonsong; Kitpoka, Pimpun; Kunakorn, Mongkol; Srikhirin, Toemsak

2018-04-15

The application of Surface Plasmon Resonance Imaging (SPRi) for the detection of transmembrane antigen of the Rhesus (Rh) blood group system is demonstrated. Clinically significant Rh blood group system antigens, including D, C, E, c, and e, can be simultaneously identified via solid phase immobilization assay, which offers significant time savings and assay simplification. Red blood cells (RBCs) flowed through the micro-channel, where a suitable condition for Rh blood group detection was an RBC dilution of 1:10 with a stop-flow condition. Stop flow showed an improvement in specific binding compared to continuous flow. Rh antigens required a longer incubation time to react with the immobilized antibody than A and B antigens due to the difference in antigen type and their location on the RBC. The interaction between the immobilized antibodies and their specific antigenic counterpart on the RBC showed a significant difference in RBC removal behavior using shear flow, measured from the decay of the SPR signal. The strength of the interaction between the immobilized antibody and RBC antigen was determined from the minimum wall shear stress required to start the decay process in the SPR signal. For a given range of immobilized antibody surface densities, the Rh antigen possesses a stronger interaction than A, B, and AB antigens. Identification of 82 samples of ABO and Rh blood groups using SPRi showed good agreement with the standard micro-column agglutination technique. A wider coverage of antigenic recognition for RBC when using the solid phase immobilization assay was demonstrated for the RBC with the antigenic site located on the transmembrane protein of the clinically significant Rh antigen. Given the level of accuracy and precision, the technique showed potential for the detection of the Rh minor blood group system. Copyright © 2017 Elsevier B.V. All rights reserved.

Effectiveness of the combined evaluation of KLK3 genetics and free-to-total prostate specific antigen ratio for prostate cancer diagnosis.

PubMed

Zambon, Carlo-Federico; Prayer-Galetti, Tommaso; Basso, Daniela; Padoan, Andrea; Rossi, Elisa; Secco, Silvia; Pelloso, Michela; Fogar, Paola; Navaglia, Filippo; Moz, Stefania; Zattoni, Filiberto; Plebani, Mario

2012-10-01

Of serum prostate specific antigen variability 40% depends on inherited factors. We ascertained whether the knowledge of KLK3 genetics would enhance prostate specific antigen diagnostic performance in patients with clinical suspicion of prostate cancer. We studied 1,058 men who consecutively underwent prostate biopsy for clinical suspicion of prostate cancer. At histology prostate cancer was present in 401 cases and absent in 657. Serum total prostate specific antigen and the free-to-total prostate specific antigen ratio were determined. Four polymorphisms of the KLK3 gene (rs2569733, rs2739448, rs925013 and rs2735839) and 1 polymorphism of the SRD5A2 gene (rs523349) were studied. The influence of genetics on prostate specific antigen variability was evaluated by multivariate linear regression analysis. The performance of total prostate specific antigen and the free-to-total prostate specific antigen ratio alone or combined with a genetically based patient classification were defined by ROC curve analyses. For prostate cancer diagnosis the free-to-total prostate specific antigen ratio index alone (cutoff 11%) was superior to total prostate specific antigen (cutoff 4 ng/ml) and to free-to-total prostate specific antigen ratio reflex testing (positive predictive value 61%, 43% and 54%, respectively). Prostate specific antigen correlated with KLK3 genetics (rs2735839 polymorphism p = 0.001, and rs2569733, rs2739448 and rs925013 haplotype combination p = 0.003). In patients with different KLK3 genetics 2 optimal free-to-total prostate specific antigen ratio cutoffs (11% and 14.5%) were found. For free-to-total prostate specific antigen ratio values between 11% and 14.5% the prostate cancer probability ranged from 30.0% to 47.4% according to patient genetics. The free-to-total prostate specific antigen ratio is superior to total prostate specific antigen for prostate cancer diagnosis, independent of total prostate specific antigen results. Free-to-total prostate specific antigen ratio findings below 11% are positively associated with prostate cancer and those above 14.5% are negatively associated with prostate cancer, while the interpretation of those between 11% and 14.5% is improved by patient KLK3 genetic analysis. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a

PubMed Central

Osorio, Manuel; Takeda, Kazuyo; Stibitz, Scott; Kopecko, Dennis J.

2017-01-01

ABSTRACT We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S. Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S. Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever. PMID:29046309

Genetic Mapping Identifies Novel Highly Protective Antigens for an Apicomplexan Parasite

PubMed Central

Blake, Damer P.; Billington, Karen J.; Copestake, Susan L.; Oakes, Richard D.; Quail, Michael A.; Wan, Kiew-Lian; Shirley, Martin W.; Smith, Adrian L.

2011-01-01

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed ‘immune mapped protein-1’ (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult. PMID:21347348

Identification of variant-specific surface proteins in Giardia muris trophozoites.

PubMed

Ropolo, Andrea S; Saura, Alicia; Carranza, Pedro G; Lujan, Hugo D

2005-08-01

Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

Antigen Specific Responses and ANA production in B6.Sle1b mice: A role for SAP

PubMed Central

Jennings, Paula; Chan, Alice; Schwartzberg, Pamela; Wakeland, Edward K.; Yuan, Dorothy

2010-01-01

B6.Sle1b mice, which contain the Sle1b gene interval derived from lupus prone NZM2410 mice on a C57BL/6 background, present with gender-biased, highly penetrant anti-nuclear antibody (ANA) production. To obtain some insight into the possible induction mechanism of autoantibodies in these mice we compared antigen specific T dependent (TD) and T independent (TI-II) responses between B6.Sle1b and B6 mice before the development of high ANA titers. Our results show that B6.Sle1b mice mount enhanced responses to a TI-II antigen. Additionally, the memory T cell response generated by a TD antigen was also increased. This enhancement correlates with the greater ability of B cells from B6.Sle1b mice to present antigen to T cells. The SLAM Associated Protein (SAP) is critical for signaling of many of the molecules encoded by the SLAM/CD2 gene cluster, candidates for mediating the Sle1b phenotype; therefore, we also investigated the effect of sap deletion in these strains on the TD and TI-II responses as well as on ANA production. The results of these studies of responses to non-self antigens provide further insight for the mechanism by which responses to self-antigens might be initiated in the context of specific genetic alterations. PMID:18845419

Mechanisms of Chemical Modulation and Toxicity of the Immune System.

DTIC Science & Technology

1988-05-15

expressing the L3T4 surface antigens and suppressor/cytotoxic cells bearing Lyt-2 surface antigens. Autoimmune or immunodeficient diseases have been...Ill. Written Publications (cumulative list) A. Suppression of mitogen-induced blastogenesis of feline lymphocytes by in vitro incubation with

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2011 CFR

2011-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

21 CFR 660.41 - Processing.

Code of Federal Regulations, 2010 CFR

2010-04-01

... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Hepatitis B Surface Antigen § 660.41 Processing. (a... that will reduce the risk of transmitting type B viral hepatitis. (b) Ancillary reagents and materials... its dating period. (d) Date of manufacture. The date of manufacture of Hepatitis B Surface Antigen...

Transcription factor NF-kappaB regulates inducible CD83 gene expression in activated T lymphocytes.

PubMed

McKinsey, T A; Chu, Z; Tedder, T F; Ballard, D W

2000-01-01

The immunoglobulin superfamily member CD83 is expressed on the surface of mature dendritic cells that present processed antigens to T lymphocytes. In addition, T cells acquire CD83 expression following mitogenic stimulation in vitro. Here we report two lines of evidence demonstrating that this inducible lymphocyte response is genetically programmed by transcription factor NF-kappaB and contingent upon proteolytic breakdown of its cytoplasmic inhibitor IkappaBalpha. First, signal-dependent induction of CD83 mRNA expression is blocked in both transformed and primary T cells harboring a degradation-resistant mutant of IkappaBalpha that constitutively represses NF-kappaB. Second, as revealed in gel retardation assays, the IkappaBalpha constitutive repressor prevents the inducible interaction of NF-kappaB with consensus recognition sites identified in the CD83 promoter. Given that IkappaBalpha is functionally coupled to the T-cell antigen receptor, these findings suggest that the downstream transcription unit for CD83 is triggered by NF-kappaB during an adaptive immune response.

Piezoelectric cantilever sensors

NASA Technical Reports Server (NTRS)

Shih, Wan Y. (Inventor); Shih, Wei-Heng (Inventor); Shen, Zuyan (Inventor)

2008-01-01

A piezoelectric cantilever with a non-piezoelectric, or piezoelectric tip useful as mass and viscosity sensors. The change in the cantilever mass can be accurately quantified by monitoring a resonance frequency shift of the cantilever. For bio-detection, antibodies or other specific receptors of target antigens may be immobilized on the cantilever surface, preferably on the non-piezoelectric tip. For chemical detection, high surface-area selective absorbent materials are coated on the cantilever tip. Binding of the target antigens or analytes to the cantilever surface increases the cantilever mass. Detection of target antigens or analytes is achieved by monitoring the cantilever's resonance frequency and determining the resonance frequency shift that is due to the mass of the adsorbed target antigens on the cantilever surface. The use of a piezoelectric unimorph cantilever allows both electrical actuation and electrical sensing. Incorporating a non-piezoelectric tip (14) enhances the sensitivity of the sensor. In addition, the piezoelectric cantilever can withstand damping in highly viscous liquids and can be used as a viscosity sensor in wide viscosity range.

Molecular characterization of phosphorylcholine expression on the lipooligosaccharide of Histophilus somni.

PubMed

Elswaifi, Shaadi F; St Michael, Frank; Sreenivas, Avula; Cox, Andrew; Carman, George M; Inzana, Thomas J

2009-10-01

Histophilus somni (Haemophilus somnus) is an important pathogen of cattle that is responsible for respiratory disease, septicemia, and systemic diseases such as thrombotic meningoencephalitis, myocarditis, and abortion. A variety of virulence factors have been identified in H. somni, including compositional and antigenic variation of the lipooligosaccharide (LOS). Phosphorylcholine (ChoP) has been identified as one of the components of H. somni LOS that undergoes antigenic variation. In this study, five genes (lic1ABCD(Hs) and glpQ) with homology to genes responsible for ChoP expression in Haemophilus influenzae LOS were identified in the H. somni genome. An H. somni open reading frame (ORF) with homology to H. influenzae lic1A (lic1A(Hi)) contained a variable number of tandem repeats (VNTR). However, whereas the tetranucleotide repeat 5'-CAAT-3' is present in lic1A(Hi), the VNTR in H. somni lic1A (lic1A(Hs)) consisted of 5'-AACC-3'. Due to the propensity of VNTR to vary during replication and cause the ORF to shift in and out of frame with the upstream start codon, the VNTR were deleted from lic1A(Hs) to maintain the gene constitutively on. This construct was cloned into Escherichia coli, and functional enzyme assays confirmed that lic1A(Hs) encoded a choline kinase, and that the VNTR were not required for expression of a functional gene product. Variation in the number of VNTR in lic1A(Hs) correlated with antigenic variation of ChoP expression in H. somni strain 124P. However, antigenic variation of ChoP expression in strain 738 predominately occurred through variable extension/truncation of the LOS outer core. These results indicated that the lic1(Hs) genes controlled expression of ChoP on the LOS, but that in H. somni there are two potential mechanisms that account for antigenic variation of ChoP.