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Sample records for encoded antigens recombinant

  1. Genes encoding homologous antigens in taeniid cestode parasites: Implications for development of recombinant vaccines produced in Escherichia coli.

    PubMed

    Gauci, Charles; Lightowlers, Marshall W

    2013-01-01

    Recombinant vaccine antigens are being evaluated for their ability to protect livestock animals against cysticercosis and related parasitic infections. Practical use of some of these vaccines is expected to reduce parasite transmission, leading to a reduction in the incidence of neurocysticercosis and hydatid disease in humans. We recently showed that an antigen (TSOL16), expressed in Escherichia coli, confers high levels of protection against Taenia solium cysticercosis in pigs, which provides a strategy for control of T. solium parasite transmission. Here, we discuss the characteristics of this antigen that may affect the utility of TSOL16 and related antigens for development as recombinant vaccines. We also report that genes encoding antigens closely related to TSOL16 from T. solium also occur in other related species of parasites. These highly homologous antigens have the potential to be used as vaccines and may provide protection against related species of Taenia that cause infection in other hosts.

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

    SciTech Connect

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

    1992-12-01

    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.

  3. Phase I trial of recombinant modified vaccinia ankara encoding Epstein-Barr viral tumor antigens in nasopharyngeal carcinoma patients.

    PubMed

    Hui, Edwin P; Taylor, Graham S; Jia, Hui; Ma, Brigette B Y; Chan, Stephen L; Ho, Rosalie; Wong, Wai-Lap; Wilson, Steven; Johnson, Benjamin F; Edwards, Ceri; Stocken, Deborah D; Rickinson, Alan B; Steven, Neil M; Chan, Anthony T C

    2013-03-15

    Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-γ ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.

  4. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen SO7.

    PubMed

    Konjufca, Vjollca; Jenkins, Mark; Wang, Shifeng; Juarez-Rodriguez, Maria Dolores; Curtiss, Roy

    2008-12-01

    Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella. For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain chi8879 (Delta phoP233 Delta sptP1033::xylE Delta asdA16), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella. The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain chi9242 (Delta phoP233 Delta asdA16 Delta araBAD23 Delta relA198::araC P(BAD) lacI TT [TT is the T4ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina, which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina. In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella.

  5. Immunogenicity of recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strains carrying a gene that encodes Eimeria tenella antigen S07

    USDA-ARS?s Scientific Manuscript database

    In an attempt to develop an efficacious vaccine against avian coccidiosis, research was conducted using Type III Secretion System (TTSS) of Salmonella to deliver Eimeria antigens into the cytoplasm of host cells. Once delivered, recombinant protein may enter the MHC I antigen processing pathway for...

  6. Discovery of STL polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique T antigen by alternative splicing.

    PubMed

    Lim, Efrem S; Reyes, Alejandro; Antonio, Martin; Saha, Debasish; Ikumapayi, Usman N; Adeyemi, Mitchell; Stine, O Colin; Skelton, Rebecca; Brennan, Daniel C; Mkakosya, Rajhab S; Manary, Mark J; Gordon, Jeffrey I; Wang, David

    2013-02-20

    The family Polyomaviridae is comprised of circular double-stranded DNA viruses, several of which are associated with diseases, including cancer, in immunocompromised patients. Here we describe a novel polyomavirus recovered from the fecal microbiota of a child in Malawi, provisionally named STL polyomavirus (STLPyV). We detected STLPyV in clinical stool specimens from USA and The Gambia at up to 1% frequency. Complete genome comparisons of two STLPyV strains demonstrated 5.2% nucleotide divergence. Alternative splicing of the STLPyV early region yielded a unique form of T antigen, which we named 229T, in addition to the expected large and small T antigens. STLPyV has a mosaic genome and shares an ancestral recombinant origin with MWPyV. The discovery of STLPyV highlights a novel alternative splicing strategy and advances our understanding of the complex evolutionary history of polyomaviruses.

  7. Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G

    SciTech Connect

    Templeton, D.; Voronova, A.; Eckhart, W.

    1984-02-01

    The authors constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middle-size tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.

  8. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  9. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  10. Dissection of Mycobacterium tuberculosis antigens using recombinant DNA.

    PubMed Central

    Young, R A; Bloom, B R; Grosskinsky, C M; Ivanyi, J; Thomas, D; Davis, R W

    1985-01-01

    A recombinant DNA strategy has been used systematically to survey the Mycobacterium tuberculosis genome for sequences that encode specific antigens detected by monoclonal antibodies. M. tuberculosis genomic DNA fragments with randomly generated endpoints were used to construct a large lambda gt11 recombinant DNA expression library. Sufficient numbers of recombinants were produced to contain inserts whose endpoints occur at nearly every base pair in the pathogen genome. Protein antigens specified by linear segments of pathogen DNA and produced by the recombinant phage of Escherichia coli were screened with monoclonal antibody probes. This approach was coupled with an improved detection method for gene isolation using antibodies to clonally isolate DNA sequences that specify polypeptide components of M. tuberculosis. The methodology described here, which is applicable to other pathogens, offers possibilities for the development of more sensitive and specific immunodiagnostic and seroepidemiological tests for tuberculosis and, ultimately, for the development of more effective vaccines. Images PMID:2581251

  11. Development of Prototype Filovirus Recombinant Antigen Immunoassays

    PubMed Central

    Boisen, Matt L.; Oottamasathien, Darin; Jones, Abigail B.; Millett, Molly M.; Nelson, Diana S.; Bornholdt, Zachary A.; Fusco, Marnie L.; Abelson, Dafna M.; Oda, Shun-ichiro; Hartnett, Jessica N.; Rowland, Megan M.; Heinrich, Megan L.; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W.; Geisbert, Joan B.; Geisbert, Thomas W.; Kulakosky, Peter C.; Grant, Donald S.; Shaffer, Jeffery G.; Schieffelin, John S.; Wilson, Russell B.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.; Khan, S. Humarr; Pitts, Kelly R.

    2015-01-01

    Background. Throughout the 2014–2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Methods. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Results. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus–specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. Conclusions. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. PMID:26232440

  12. A study of recombinant protective H. pylori antigens

    PubMed Central

    Jiang, Zheng; Tao, Xiao-Hong; Huang, Ai-Long; Wang, Pi-Long

    2002-01-01

    AIM: To construct a recombinant vector which can express Mr26000 outer membrane protein (OMP) from Helicobacter pylori (H. pylori), and to obtain the vaccine protecting against H. pylori infection and a diagnostic reagent kit quickly detecting H. pylori infection. METHODS: The gene encoding the structural Mr26000 outer membrane protein of H. pylori was amplified from H. pylori chromosomal DNA by PCR, and inserted in the prokaryotic expression vector pET32a(+), which was transformed into the Top10 E. coli strain. Recombinant vector was selected, identified and transformed into BL-21(DE3) E. coli strain. The recombinant fusion proteins were expressed. The antigenicity of recombinant protein was studied by ELISA or immunoblotting and immunized Balb/c mice. RESULTS: The gene of Mr26000 OMP was amplified to be 594 base pairs, 1.1% of the cloned genes was mutated and 1.51% of amino acid residues was changed, but there was homogeneity between them. The recombinant fusion protein encoded objective polypeptides of 198 amino acid residues, corresponding to calculated molecular masses of Mr26000. The level of soluble expression products was about 38.96% of the total cell protein. After purification by Ni-NTA agarose resin columniation, the purity of objective protein became about 90%. The ELISA results showed that recombinant fusion protein could be recognized by patient serum infected with H. pylori and rabbit serum immunized with the recombinant protein. Furthermore, Balb/c mice immunized with the recombinant protein were protected against H. pylori infection. CONCLUSION: Mr26000 OMP may be a candidate vaccine preventing H. pylori infection. PMID:11925614

  13. Development of Prototype Filovirus Recombinant Antigen Immunoassays.

    PubMed

    Boisen, Matt L; Oottamasathien, Darin; Jones, Abigail B; Millett, Molly M; Nelson, Diana S; Bornholdt, Zachary A; Fusco, Marnie L; Abelson, Dafna M; Oda, Shun-Ichiro; Hartnett, Jessica N; Rowland, Megan M; Heinrich, Megan L; Akdag, Marjan; Goba, Augustine; Momoh, Mambu; Fullah, Mohammed; Baimba, Francis; Gbakie, Michael; Safa, Sadiki; Fonnie, Richard; Kanneh, Lansana; Cross, Robert W; Geisbert, Joan B; Geisbert, Thomas W; Kulakosky, Peter C; Grant, Donald S; Shaffer, Jeffery G; Schieffelin, John S; Wilson, Russell B; Saphire, Erica Ollmann; Branco, Luis M; Garry, Robert F; Khan, S Humarr; Pitts, Kelly R

    2015-10-01

    Throughout the 2014-2015 Ebola outbreak in West Africa, major gaps were exposed in the availability of validated rapid diagnostic platforms, protective vaccines, and effective therapeutic agents. These gaps potentiated the development of prototype rapid lateral flow immunodiagnostic (LFI) assays that are true point-of-contact platforms, for the detection of active Ebola infections in small blood samples. Recombinant Ebola and Marburg virus matrix VP40 and glycoprotein (GP) antigens were used to derive a panel of monoclonal and polyclonal antibodies. Antibodies were tested using a multivariate approach to identify antibody-antigen combinations suitable for enzyme-linked immunosorbent assay (ELISA) and LFI assay development. Polyclonal antibodies generated in goats were superior reagents for capture and detection of recombinant VP40 in test sample matrices. These antibodies were optimized for use in antigen-capture ELISA and LFI assay platforms. Prototype immunoglobulin M (IgM)/immunoglobulin G (IgG) ELISAs were similarly developed that specifically detect Ebola virus-specific antibodies in the serum of experimentally infected nonhuman primates and in blood samples obtained from patients with Ebola from Sierra Leone. The prototype recombinant Ebola LFI assays developed in these studies have sensitivities that are useful for clinical diagnosis of acute ebolavirus infections. The antigen-capture and IgM/IgG ELISAs provide additional confirmatory assay platforms for detecting VP40 and other ebolavirus-specific immunoglobulins. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Strengthened tumor antigen immune recognition by inclusion of a recombinant Eimeria antigen in therapeutic cancer vaccination.

    PubMed

    Quiroga, Dionisia; Aldhamen, Yasser A; Appledorn, Daniel M; Godbehere, Sarah; Amalfitano, Andrea

    2015-04-01

    The need for novel, effective adjuvants that are capable of eliciting stronger cellular and humoral adaptive immune responses to antigenic targets is well understood in the vaccine development field. Unfortunately, many adjuvants investigated thus far are either too toxic for human application or too weak to induce a substantial response against difficult antigens, such as tumor-associated antigens (TAAs). In spite of this trend, clinical investigations of recombinant Eimeria antigen (rEA) have revealed this protein to be a non-toxic immunogenic agent with the ability to trigger a Th1-predominant response in both murine and human subjects. Our past studies have shown that the injection of a rEA-encoding adenovirus (rAd5-rEA) alongside an HIV antigen-encoding adenovirus greatly improves the adaptive immune response against this pathogen-derived transgene. In this report, we investigated whether rAd5-rEA could promote and/or alter cytotoxic memory responses toward carcinoembryonic antigen (CEA), a colorectal cancer-related TAA. We found that the addition of rAd5-rEA to an Ad-based CEA vaccine induced a dose-dependent increase in several anti-CEA T and B cell responses. Moreover, inclusion of rAd5-rEA increased the number of CEA-derived antigenic epitopes that elicited significant cell-mediated and IgG-mediated recognition. These enhanced anti-CEA immune responses also translated into superior CEA-targeted cell killing, as evaluated by an in vivo cytotoxic T lymphocyte assay. Overall, these results suggest that co-administration of rAd5-rEA with a tumor antigen vaccine can substantially boost and broaden the TAA-specific adaptive memory response, thereby validating the potential of rAd5-rEA to be a beneficial adjuvant during therapeutic cancer vaccination.

  15. Recombinant antigens for serodiagnosis of cysticercosis and echinococcosis.

    PubMed

    Sako, Yasuhito; Nakao, Minoru; Nakaya, Kazuhiro; Yamasaki, Hiroshi; Ito, Akira

    2006-01-01

    Diagnosis of cysticercosis/echinococcosis is primarily based on imaging techniques. These imaging techniques are sometimes limited by the small size of visualized lesions and atypical images, which are difficult to be distinguished from abscesses or neoplasms. Therefore, efforts have been directed toward identification and characterization of specific antigens of parasites for development of serodiagnostic method that can detect specific antibody. For cysticercosis, glycoproteins of 10-26 kDa in cyst fluid of Taenia solium have been widely accepted for serodiagnosis purpose. The glycoproteins consist of a very closely related family of 8-kDa proteins. We identified four genes (designated Ag1, Ag1V1, Ag2 and Ag2V1) encoding the 7- and 10-kDa polypeptides. Based on the antigenicities of these clones, Ag1V1 and Ag2 were chosen as ELISA antigens and the Ag1V1/Ag2 chimeric protein was expressed. The Ag1V1/Ag2 chimeric protein showed the similar sensitivity and specificity as the native glycoproteins. For alveolar echinococcosis, the 65-kDa protein of Echinococcus multilocularis protoscolices and Em18 has been considered as serodiagnostic antigens. The sensitivity and specificity of Em18 are very compatible to those of the recombinant 65-kDa protein. Recently, we demonstrated that Em18 was the proteolytic product of the 65-kDa protein following the action by cysteine proteinases. From the information of N-terminal amino acid sequences, molecular size and isoelectric point of Em18, recombinant Em18 ((349)K to (508)K of the 65-kDa protein, RecEm18) was expressed and evaluated for serodiagnostic value. RecEm18 has the potential for use in the differential serodiagnosis of alveolar echinococcosis.

  16. Cloning and expression of genes encoding Haemophilus somnus antigens.

    PubMed Central

    Corbeil, L B; Chikami, G; Yarnall, M; Smith, J; Guiney, D G

    1988-01-01

    A genomic library of Haemophilus somnus 2336, a virulent isolate from a calf with pneumonia (later used to reproduce H. somnus experimental pneumonia), was constructed in the cosmid vector pHC79. The gene bank in Escherichia coli DH1 was screened by filter immunoassay with convalescent-phase serum, which reacted with several outer membrane antigens of H. somnus. On Western blotting (immunoblotting) of immunoreactive colonies, five clones were found to express proteins which comigrated with H. somnus surface antigens. Three clones (DH1 pHS1, pHS3, and pHS4) expressed both a 120-kilodalton (kDa) antigen and a 76-kDa antigen, one clone (DH1 pHS2) expressed only the 76-kDa antigen, and the fifth clone (DH1 pHS5) expressed a 60-kDa antigen. The 120-kDa and 76-kDa antigens were found internally, whereas the 60-kDa protein was detected in the DH1 pHS5 culture supernatant as membrane blebs or insoluble protein. Both the H. somnus 120-kDa antigen and the recombinant 120-kDa antigen had immunoglobulin Fc-binding activity. Restriction endonuclease mapping demonstrated that the genomic DNA inserts of clones expressing the 76-kDa antigen shared a common 28.4-kilobase-pair region, and the three clones also expressing the 120-kDa antigen shared an additional 7.0-kilobase-pair region. The restriction endonuclease map of pHS5, which expressed the 60-kDa antigen, was not similar to the maps of the other four plasmids. Since these three H. somnus antigens reacted with protective convalescent-phase serum, the recombinants which express these proteins should be useful in further studies of protective immunity in bovine H. somnus disease. Images PMID:2843469

  17. The TL region gene 37 encodes a Qa-1 antigen

    PubMed Central

    1990-01-01

    Of all the biochemically defined mouse MHC class I molecules, the Qa-1 antigens are the only ones for which a gene has not been identified. Recent evidence has suggested that Qa-1 antigens are functional class I molecules and can function as restriction elements for gamma/delta T cells. We have examined the relationship between Qa-1 and the product of gene 37, a presumed novel class I antigen encoded within the TL region. Immunoprecipitation and polyacrylamide gel electrophoresis analysis of the molecules reactive with anti-Qa-1 and anti-37 sera show that the Qa-1 molecule of Qa-1b (Qa-1.2) mouse strains is identical to the product of gene 37 on the basis of molecular weight, pI, and strain distribution. Immunodepletion, biosynthetic labeling, and tunicamycin treatment confirm that the protein encoded by gene 37 in Qa-1b mice is Qa-1.2. In contrast, the anti-37 serum was unable to recognize the Qa-1 molecule in Qa-1a strains. Given the fact that the only allele to gene 37 thus far identified in a Qa-1a strain (A/J) has a termination codon in the alpha 3 domain, our data lead us to conclude that the Qa-1 molecule expressed in Qa-1a mice is not a true allelic product of the gene 37 encoded antigen of Qa-1b mouse strains. PMID:2258708

  18. Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines

    PubMed Central

    2012-01-01

    Background Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines. Result To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited. Conclusion Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally

  19. Use of Recombinant Antigens for the Diagnosis of Invasive Candidiasis

    PubMed Central

    Laín, Ana; Elguezabal, Natalia; Amutio, Elena; Fernández de Larrinoa, Iñigo; Moragues, María Dolores; Pontón, José

    2008-01-01

    Invasive candidiasis is a frequent and often fatal complication in immunocompromised and critically ill patients. Unfortunately, the diagnosis of invasive candidiasis remains difficult due to the lack of specific clinical symptoms and a definitive diagnostic method. The detection of antibodies against different Candida antigens may help in the diagnosis. However, the methods traditionally used for the detection of antibodies have been based on crude antigenic fungal extracts, which usually show low-reproducibility and cross-reactivity problems. The development of molecular biology techniques has allowed the production of recombinant antigens which may help to solve these problems. In this review we will discuss the usefulness of recombinant antigens in the diagnosis of invasive candidiasis. PMID:18382617

  20. Serological diagnosis with recombinant N antigen for hantavirus infection.

    PubMed

    Yoshimatsu, Kumiko; Arikawa, Jiro

    2014-07-17

    Hantaviruses are causative agents of two rodent-borne zoonoses, hemorrhagic fever with renal syndrome (HFRS) and nephropathia epidemica (NE) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. Serological examinations to detect hantavirus antibodies have been most widely used for surveillance among humans and rodent reservoirs. Here, we will review antigenic structure of nucleocapsid (N) protein of hantaviruses and application of recombinant N protein as diagnostic antigen for screening and serotyping.

  1. Enhancing the Immune Response to Recombinant Plague Antigens

    DTIC Science & Technology

    2007-05-01

    CONTRACT NUMBER Enhancing the Immune Response to Recombinant Plague Antigens 5b. GRANT NUMBER DAMD17-02-2-0058 5c. PROGRAM ELEMENT NUMBER 6...mally integrated copy of the Bacillus anthracis protective antigen gene protects mice against an anthrax spore challenge. Infect Im- mun 2003;71(7):3831...multiplying the empirically determined aerosol exposure concentration (CFU/liter air) in the chamber by the amount of air that was estimated to have been

  2. 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

  3. Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli.

    PubMed

    Yee, Siew Fung; Chu, Chia Huay; Poili, Evenni; Sum, Magdline Sia Henry

    2017-02-01

    Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes.

  4. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    PubMed Central

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G

    1997-01-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population. PMID:9060668

  5. Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

    PubMed

    Lin, S F; Sun, R; Heston, L; Gradoville, L; Shedd, D; Haglund, K; Rigsby, M; Miller, G

    1997-04-01

    We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

  6. Antigenic structures stably expressed by recombinant TGEV-derived vectors.

    PubMed

    Becares, Martina; Sanchez, Carlos M; Sola, Isabel; Enjuanes, Luis; Zuñiga, Sonia

    2014-09-01

    Coronaviruses (CoVs) are positive-stranded RNA viruses with potential as immunization vectors, expressing high levels of heterologous genes and eliciting both secretory and systemic immune responses. Nevertheless, its high recombination rate may result in the loss of the full-length foreign gene, limiting their use as vectors. Transmissible gastroenteritis virus (TGEV) was engineered to express porcine reproductive and respiratory syndrome virus (PRRSV) small protein domains, as a strategy to improve heterologous gene stability. After serial passage in tissue cultures, stable expression of small PRRSV protein antigenic domains was achieved. Therefore, size reduction of the heterologous genes inserted in CoV-derived vectors led to the stable expression of antigenic domains. Immunization of piglets with these TGEV vectors led to partial protection against a challenge with a virulent PRRSV strain, as immunized animals showed reduced clinical signs and lung damage. Further improvement of TGEV-derived vectors will require the engineering of vectors with decreased recombination rate.

  7. Vaccination with recombinant oncosphere antigens reduces the susceptibility of sheep to infection with Taenia multiceps☆

    PubMed Central

    Gauci, Charles; Vural, Gulay; Öncel, Taraneh; Varcasia, Antonio; Damian, Veronica; Kyngdon, Craig T.; Craig, Philip S.; Anderson, Garry A.; Lightowlers, Marshall W.

    2008-01-01

    Taenia multiceps is a cestode parasite, the larval stage of which encysts in the brain of sheep, goats and cattle causing an often fatal condition. The parasite also causes zoonotic infections in humans. Homologues of the recombinant oncosphere vaccine antigens from Taenia ovis and other Taenia species were identified in T. multiceps. Sequencing of the associated T. multiceps genes and cloning of the encoding mRNA has revealed conserved features in the genes and proteins. The T. multiceps oncosphere proteins, designated Tm16 and Tm18, contain a predicted secretory signal and fibronectin type III domain. The recombinant Tm16 and Tm18 proteins were successfully expressed in Escherichia coli as fusion proteins with GST. The antigens, formulated with Quil A adjuvant, were tested in a vaccine trial in sheep. The antigens stimulated immunity in sheep against challenge infection with T. multiceps eggs. Five of nine control sheep died due to a challenge infection with T. multiceps whereas none of 20 vaccinated animals died as a result of the parasite challenge (P = 0.001). In addition, vaccination with the Tm16 protein, or Tm16 plus Tm18, induced significant protection against the number of parasites encysting in the brain as a result of the challenge infection (P = 0.023, P = 0.015, respectively). No clear relationship was apparent between the level of specific serum antibody in vaccinated animals and either the presence or absence of parasites or the number of parasites that occurred in some of the vaccinated animals. We believe this study is the first description of recombinant vaccine-related investigations for T. multiceps. The recombinant oncosphere antigens identified may allow development of effective vaccination strategies against T. multiceps infection in sheep. They raise the potential for the development of a combined vaccine with the Echinococcus granulosus EG95 antigen for prevention of T. multiceps as well as preventing the transmission of cystic

  8. Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B.

    PubMed

    Fuchs, Manon; Kämpfer, Susanne; Helmsing, Saskia; Spallek, Ralf; Oehlmann, Wulf; Prilop, Wiebke; Frank, Ronald; Dübel, Stefan; Singh, Mahavir; Hust, Michael

    2014-07-17

    Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic. In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection. Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important.

  9. Novel human recombinant antibodies against Mycobacterium tuberculosis antigen 85B

    PubMed Central

    2014-01-01

    Background Tuberculosis is the leading cause of death due to bacterial infections worldwide, mainly caused by Mycobacterium tuberculosis. The antigen 85 complex comprises a set of major secreted proteins of M. tuberculosis, which are potential biomarkers for diagnostic. Results In this work, the first human single chain fragment variable (scFv) antibodies specific for the tuberculosis biomarker 85 B were selected by phage display from naïve antibody gene libraries (HAL7/8). Produced as scFv-Fc in mammalian cells, these antibodies were further characterized and analysed for specificity and applicability in different tuberculosis antigen detection assays. Sandwich detection of recombinant 85 B was successful in enzyme linked immunosorbent assay (ELISA), lateral flow immunoassay and immunoblot. Whereas detection of M. tuberculosis cell extracts and culture filtrates was only possible in direct ELISA and immunoblot assays. It was found that the conformation of 85 B, depending on sample treatment, influenced antigen detection. Conclusions Recombinant antibodies, selected by phage display, may be applicable for 85 B detection in various assays. These antibodies are candidates for the development of future point of care tuberculosis diagnostic kits. Using 85 B as a biomarker, the antigen conformation influenced by sample treatment is important. PMID:25033887

  10. 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.

  11. Mismatch repair regulates homologous recombination, but has little influence on antigenic variation, in Trypanosoma brucei.

    PubMed

    Bell, Joanna S; McCulloch, Richard

    2003-11-14

    Antigenic variation is critical in the life of the African trypanosome, as it allows the parasite to survive in the face of host immunity and enhance its transmission to other hosts. Much of trypanosome antigenic variation uses homologous recombination of variant surface glycoprotein (VSG)-encoding genes into specialized transcription sites, but little is known about the processes that regulate it. Here we describe the effects on VSG switching when two central mismatch repair genes, MSH2 and MLH1, are mutated. We show that disruption of the parasite mismatch repair system causes an increased frequency of homologous recombination, both between perfectly matched DNA molecules and between DNA molecules with divergent sequences. Mismatch repair therefore provides an important regulatory role in homologous recombination in this ancient eukaryote. Despite this, the mismatch repair system has no detectable role in regulating antigenic variation, meaning that VSG switching is either immune to mismatch selection or that mismatch repair acts in a subtle manner, undetectable by current assays.

  12. Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

    PubMed

    Chan, Kun-Wei; Hsieh, Hsien-Hua; Wang, Hsien-Chi; Lee, Ya-Jane; Sung, Ming-Hua; Wong, Min-Liang; Hsu, Wei-Li

    2009-01-01

    Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

  13. The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

    PubMed

    Griot-Wenk, M E; Cherpillod, P; Koch, A; Zurbriggen, R; Bruckner, L; Wittek, R; Zurbriggen, A

    2001-06-01

    This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

  14. Antigenicity of partial fragments of recombinant Pasteurella multocida toxin.

    PubMed

    Lee, Jeongmin; Woo, Hee-Jong

    2010-12-01

    Pasteurella multocida serogroup D strain, which produces P. multocida toxin (PMT), is a widespread and harmful pathogen of respiratory diseases such as pneumonia and progressive atrophic rhinitis (PAR) in swine. Vaccination has been considered the most desirable and effective approach for controlling the diseases caused by toxigenic P. multocida. To investigate the antigenicity and immunogenicity of partial fragments of recombinant PMT, recombinant proteins of the N-terminal (PMT-A), middle (PMT-B), Cterminal (PMT-C), and middle-C-terminal (PMT2.3) regions of PMT were successfully produced in an Escherichia coli expression system. The molecular masses of PMT-A, PMT-B, PMT-C, and PMT2.3 were ca. 53, 55, 35, and 84 kDa, respectively, purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. All the recombinant proteins except for PMT-A showed immune responses to antisera obtained from a swine showing symptoms of PAR. Moreover, high titers of PMT-specific antibodies were raised from mice immunized with each of the recombinant proteins; however, the immunoreactivities of the antibodies to authentic PMT and heat-inactivated whole bacteria were different, respectively. In the protection study, the highest protection against homologous challenge was shown in the case of PMT2.3; relatively poor protections occurred for the other PMT fragments.

  15. Indirect Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Reactive with a Recombinant Protein Expressed from the Gene Encoding the 116-Kilodalton Protein of Mycoplasma pneumoniae

    PubMed Central

    Duffy, Michael F.; Whithear, Kevin G.; Noormohammadi, Amir H.; Markham, Philip F.; Catton, Michael; Leydon, Jennie; Browning, Glenn F.

    1999-01-01

    Serology remains the method of choice for laboratory diagnosis of Mycoplasma pneumoniae infection. Currently available serological tests employ complex cellular fractions of M. pneumoniae as antigen. To improve the specificity of M. pneumoniae diagnosis, a recombinant protein was assessed as a serodiagnostic reagent. A panel of recombinant proteins were expressed from a cloned M. pneumoniae gene that encodes a 116-kDa surface protein antigen. The recombinant proteins were assessed for reactivity with patient sera and the most antigenic was further assessed for its serodiagnostic potential by indirect enzyme-linked immunosorbent assay (ELISA). The ELISA based on the recombinant protein was equivalent in sensitivity to the commercial test (Serodia Myco II; Fujirebio Inc.) to which it was compared. Southern and Western blotting data suggested that the recombinant protein derived from the 116-kDa protein of M. pneumoniae could provide a species-specific diagnostic tool, although further assessment is required. PMID:10074521

  16. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    PubMed Central

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Arinus, Shira B.; Dgany, Or; Shoseyov, Oded; Kaplan, David L.

    2011-01-01

    Resilin is an elastomeric protein found in specialized regions of the cuticle of most insects, providing outstanding material properties including high resilience and fatigue lifetime for insect flight and jumping needs. Two exons (1 and 3) from the resilin gene in Drosophila melanogaster were cloned and the encoded proteins expressed as soluble products in Escherichia coli. A heat and salt precipitation method was used for efficient purification of the recombinant proteins. The proteins were solution cast from water and formed into rubber-like biomaterials via horseradish peroxidase-mediated cross-linking. Comparative studies of the two proteins expressed from the two different exons were investigated by Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichrosim (CD) for structural features. Little structural organization was found, suggesting structural order was not induced by the enzyme-mediateed dityrosine cross-links. Atomic Force Microscopy (AFM) was used to study the elastomeric properties of the uncross-linked and cross-linked proteins. The protein from exon 1 exhibited 90% resilience in comparison to 63% for the protein from exon 3, and therefore may be the more critical domain for functional materials to mimic native resilin. Further, the cross-linking of the recombinant exon 1 via the citrate-modified photo-Fenton reaction was explored as an alternative dityrosine mediated polymerization method and resulted in both highly elastic and adhesive materials. The citrate-modified photo-Fenton system may be suitable for in-vivo applications of resilin biomaterials. PMID:21963157

  17. Immunogenic and antigenic properties of recombinant soluble glycoprotein of rabies virus.

    PubMed

    Gupta, Praveen K; Sharma, Sameer; Walunj, Sameer S; Chaturvedi, V K; Raut, Ashwin A; Patial, Sonika; Rai, A; Pandey, K D; Saini, M

    2005-07-01

    Rabies virus glycoprotein is a type I transmembrane protein exposed on the surface on the mature virus particle that induces virus neutralizing antibodies. In the present study, 60 amino acid C-terminal hydrophobic anchor (transmembrane) and cytoplasmic domains of glycoprotein were deleted from full-length glycoprotein and fused with polyhistidine tag. The N-terminal viral signal peptide was also replaced with CD33 signal peptide for efficient secretion in mammalian cells. Following transfection of Madin Darby bovine kidney (MDBK) cells with plasmid encoding this soluble form of glycoprotein, polyclonal populations of stably transfected resistant cells were obtained after G418 selection. The protein was expressed as a glycosylated protein and secreted outside the cells utilizing N-terminal CD33 signal peptide. The secreted soluble glycoprotein was purified from cell culture supernatant by Ni--agarose affinity chromatography utilizing C-terminal polyhistidine tag. Like full-length glycoprotein, the expressed recombinant soluble glycoprotein was found to be immunogenic when injected in rabbits. In this study, we have assessed the potential of recombinant soluble glycoprotein as diagnostic antigen in ELISA and found that this recombinant protein can be used as diagnostic antigen in ELISA for detecting anti-glycoprotein antibodies in immunized host.

  18. Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products

    PubMed Central

    1990-01-01

    A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well- characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as

  19. Intranasal Vaccination against Cutaneous Leishmaniasis with a Particulated Leishmanial Antigen or DNA Encoding LACK

    PubMed Central

    Pinto, Eduardo Fonseca; Pinheiro, Roberta Olmo; Rayol, Alice; Larraga, Vicente; Rossi-Bergmann, Bartira

    2004-01-01

    We have previously demonstrated that oral delivery of a disease-promoting particulated antigen of Leishmania amazonensis (LaAg) partially protects mice against cutaneous leishmaniasis. In the present work, we sought to optimize a mucosal vaccine by using the intranasal route for delivery of different antigen preparations, including (i) LaAg, (ii) soluble recombinant p36/LACK leishmanial antigen (LACK), and (iii) plasmid DNA encoding LACK (LACK DNA). BALB/c mice that received two intranasal doses of 10 μg of LaAg and were challenged 1 week postvaccination with L. amazonensis developed delayed but effective control of lesion growth. A diminished parasite burden was accompanied by enhancement of both gamma interferon (IFN-γ) and interleukin-10 levels in the lesion-draining lymph nodes. The vaccine efficacy improved with time. At 4 months postvaccination, when a strong parasite-specific TH1-type response was present in vivo, the infection was controlled for at least 5 months after challenge. In contrast to the particulated LaAg, soluble LACK (10 μg/dose) had no effect. Interestingly, LACK DNA (30 μg/dose), but not empty DNA, promoted rapid and durable protective immunity. Parasite growth was effectively controlled, and at 5 months after challenge LACK-reactive cells in both the mucosal and lesion-draining lymph nodes produced high levels of IFN-γ. These results demonstrate for the first time the feasibility of using the intranasal route for long-lived memory vaccination against cutaneous leishmaniasis with adjuvant-free crude antigens or DNA. PMID:15271911

  20. A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

    PubMed

    de Oliveira, Natasha Rodrigues; Jorge, Sérgio; Gomes, Charles Klazer; Rizzi, Caroline; Pacce, Violetta Dias; Collares, Thais Farias; Monte, Leonardo Garcia; Dellagostin, Odir Antônio

    2017-03-01

    Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP.

  1. Maturation of recombinant hepatitis B virus surface antigen particles.

    PubMed

    Zhao, Qinjian; Wang, Yang; Freed, Daniel; Fu, Tong-Ming; Gimenez, Juan A; Sitrin, Robert D; Washabaugh, Michael W

    2006-01-01

    The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immuno-genicity of this widely-used vaccine component.

  2. Polymorphism in the gene encoding the Pfs48/45 antigen of Plasmodium falciparum. XI. Asembo Bay Cohort Project.

    PubMed

    Escalante, Ananias A; Grebert, Heather M; Chaiyaroj, Sansanee C; Riggione, Flavia; Biswas, Sukla; Nahlen, Bernard L; Lal, Altaf A

    2002-01-01

    We have investigated the genetic diversity of the gene encoding the transmission-blocking vaccine antigen Pfs48/45 of Plasmodium falciparum parasites from western Kenya and compared it with parasite populations from Thailand, India, and Venezuela. We report 44 complete new sequences. Overall, the antigen is less polymorphic as compared with other pre-erythrocytic and blood stage antigens. Contrary to other P. falciparum antigens, the number of synonymous substitutions per synonymous site exceeds the number of non-synonymous substitutions per non-synonymous site. We have found that the Pfs48/45 gene of Kenyan parasites is more polymorphic than parasites from other geographic origins. Our analysis reveals that positive natural selection is involved in the maintenance of the observed polymorphism. No evidence of intragenic recombination was found. F(st) values reveal high levels of gene flow between India and Thailand, however, there are strong constraints in gene flow among Kenyan, Southeast Asian, and Venezuelan parasites. No alleles could be linked to a specific geographic region. The results of this study suggest that this gametocyte antigen, like other asexual blood stage antigens, is under selection pressure.

  3. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens

    PubMed Central

    1992-01-01

    The lymphocyte activation gene 3 (LAG-3), expressed in human activated T and natural killer (NK) cells, is closely related to CD4 at the gene and protein levels. We report here the initial characterization of the LAG-3-encoded protein. We have generated two monoclonal antibodies after immunization of mice with a 30-amino acid peptide that corresponds to an exposed extra loop region present in the LAG-3 immunoglobulin-like first domain. The reactivity of these reagents is directed against LAG-3 since they recognize both membrane-expressed and soluble recombinant LAG-3 molecules produced in a baculovirus expression system. The two antibodies are likely to react with the same or closely related epitope (termed LAG-3.1) exposed on the LAG-3 first domain extra loop, as assessed in competition experiments on LAG-3- expressing activated lymphocytes. Cellular distribution analysis indicated that the LAG-3.1 epitope is expressed on activated T (both CD4+ and CD8+ subsets) and NK cells, and not on activated B cells or monocytes. In immunoprecipitation experiments performed on activated T and NK cell lysates, a 70-kD protein was detected after SDS-PAGE analysis. 45-kD protein species were also immunoprecipitated. Both the 70- and 45-kD proteins were shown to be N-glycosylated. In Western blot analysis, only the former molecule was recognized by the anti-LAG-3 antibodies, demonstrating that it is LAG-3 encoded. These anti-LAG-3 antibodies were used to investigate whether the LAG-3 protein interacts with the CD4 ligands. By using a high-level expression cellular system based on COS-7 cell transfection with recombinant CDM8 vectors and a quantitative cellular adhesion assay, we demonstrate that rosette formation between LAG-3-transfected COS-7 cells and human leukocyte antigen (HLA) class II-bearing B lymphocytes is specifically dependent on LAG-3/HLA class II interaction. In contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120. This initial

  4. Detection of antibodies to Hypoderma lineatum in cattle by Western blotting with recombinant hypodermin C antigen.

    PubMed

    Boldbaatar, D; Xuan, X; Kimbita, E; Huang, X; Igarashi, I; Byambaa, B; Battsetseg, B; Battur, B; Battsetseg, G; Batsukh, Z; Nagasawa, H; Fujisaki, K; Mikami, T

    2001-08-01

    The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

  5. Production of recombinant botulism antigens: a review of expression systems.

    PubMed

    Moreira, G M S G; Cunha, C E P; Salvarani, F M; Gonçalves, L A; Pires, P S; Conceição, F R; Lobato, F C F

    2014-08-01

    Botulism is a paralytic disease caused by intoxication with neurotoxins produced by Clostridium botulinum. Despite their similar mechanism of action, the botulinum neurotoxins (BoNT) are classified in eight serotypes (A to H). As to veterinary medicine, the impact of this disease is essentially economic, since different species of production animals can be affected, especially by BoNT/C and D. In human health, botulism is feared in a possible biological warfare, what would involve mainly the BoNT/A, B, E and F. In both cases, the most effective way to deal with botulism is through prevention, which involves vaccination. However, the current vaccines against this disease have several drawbacks on their process of production and, besides this, can be dangerous to producers since it requires certain level of biosafety. This way, recombinant vaccines have been shown to be a great alternative for the development of vaccines against both animal and human botulism. All BoNTs have a 50-kDa light chain (LC) and a 100-kDa heavy chain (HC). The latter one presents two domains of 50 kDa, called the N-terminal (HN) and C-terminal (HC) halves. Among these regions, the HC alone seem to confer the proper immune response against intoxication. Since innumerous studies describe the expression of these distinct regions using different systems, strategies, and protocols, it is difficult to define the best option for a viable vaccine production. Thereby, the present review describes the problematic of botulism and discusses the main advances for the viable production of vaccines for both human and veterinary medicine using recombinant antigens. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. MAP1272c encodes an NlpC/P60 protein, an antigen detected in cattle with Johne's disease.

    PubMed

    Bannantine, John P; Lingle, Cari K; Stabel, Judith R; Ramyar, Kasra X; Garcia, Brandon L; Raeber, Alex J; Schacher, Pascal; Kapur, Vivek; Geisbrecht, Brian V

    2012-07-01

    The protein encoded by MAP1272c has been shown to be an antigen of Mycobacterium avium subsp. paratuberculosis that contains an NlpC/P60 superfamily domain found in lipoproteins or integral membrane proteins. Proteins containing this domain have diverse enzymatic functions that include peptidases, amidases, and acetyltransferases. The NlpC protein was examined in comparison to over 100 recombinant proteins and showed the strongest antigenicity when analyzed with sera from cattle with Johne's disease. To further localize the immunogenicity of NlpC, recombinant proteins representing defined regions were expressed and evaluated with sera from cattle with Johne's disease. The region from amino acids 74 to 279 was shown to be the most immunogenic. This fragment was also evaluated against a commercially available enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies were produced in mice immunized with the full-length protein, and each recognized a distinct epitope. These antibodies cross-reacted with proteins from other mycobacterial species and demonstrated variable sizes of the proteins expressed from these subspecies. Both antibodies were further analyzed, and their interaction with MAP1272c and MAP1204 was characterized by a solution-based, luminescent binding assay. These tools provide additional means to study a strong antigen of M. avium subsp. paratuberculosis.

  7. Protein expression in yeast as an approach to production of recombinant malaria antigens.

    PubMed

    Bathurst, I C

    1994-01-01

    The selection of a system suitable for expression of recombinant malaria antigens for vaccine development is, in the final analysis, empirical. However, experience gained with both malaria antigens and other recombinant proteins has provided helpful guidelines. Recombinant DNA technology has been successfully applied to the development of vaccines against a number of human diseases. For example, recombinant DNA-derived hepatitis B virus surface antigen has been produced from both prokaryotic and eukaryotic systems. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics, and vaccine production. Both intracellular and secretory systems have been developed and optimized for the production of high levels of recombinant proteins. Recombinant DNA technology, and in particular yeast expression systems, have been successfully used to produce malaria antigens, several of which have been protective in various animal models. In contrast, attempts to produce sufficient quantities of antigens for a malaria vaccine from in vitro cultures of the malaria parasite have been unsuccessful. Recombinant proteins can be produced and purified from yeast in large quantities and at low cost, each being requirements for a vaccine to be used in a global vaccination program against malaria.

  8. Immune Response to Plasmid- and Chromosome-Encoded Yersinia Antigens,

    DTIC Science & Technology

    The immune response of humans and mice to temperature-specific, plasmid- or chromosome-encoded proteins of Yersinia pestis and Yersinia ... enterocolitica was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Extracts from Y. pestis and Y. enterocolitica

  9. Induction of murine immune responses by DNA encoding a 23-kDa antigen of Cryptosporidium parvum.

    PubMed

    Ehigiator, Humphrey N; Romagnoli, Pablo; Priest, Jeffrey W; Secor, W Evan; Mead, Jan R

    2007-09-01

    Cp23 has been identified as one of the immunodominant antigens involved in the immune response to Cryptosporidium parvum infection. Thus, in this study, Cp23 antigen was investigated as a vaccine candidate using the DNA vaccine model in adult interleukin-12 (IL-12) knockout (KO) mice, which are susceptible to C. parvum infection. Our data show that subcutaneous immunization in the ear with DNA encoding Cp23 (Cp23-DNA) cloned into the pUMVCb4 vector induced a significant anti-Cp23 immunoglobulin G1 (IgG1) and IgG2a antibody response and specific in vitro spleen cell proliferation to recombinant Cp23 as compared to control mice. Long-term memory responses were also detected after administration of the Cp23-DNA vaccine. Furthermore, Cp23-DNA vaccination induced a 50-60% reduction in oocysts shedding, indicating a partial protection against C. parvum infection in IL-12 KO mice. However, it is possible that this protective response was nonspecific because mice immunized with vector only also exhibited lower oocyst shedding than the naive controls. These results suggest that DNA encoding for immunodominant C. parvum antigens may provide an effective means of eliciting humoral and cellular responses and possibly in generating protective immunity against C. parvum infections in mammals.

  10. Recombinant Carcinoembryonic Antigen as a Reporter Gene for Molecular Imaging

    PubMed Central

    Kenanova, Vania; Barat, Bhaswati; Olafsen, Tove; Chatziioannou, Arion; Herschman, Harvey R.; Braun, Jonathan; Wu, Anna M.

    2009-01-01

    Purpose Reporter genes can provide a way of non-invasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. Methods To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Results Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124I-labeled scFv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4h. MicroPET image quantitation showed tumor activity of 1.8(±0.2), 15.2(±1.3) and 4.6(±1.2) %ID/g at 4h, 20h and 48h, respectively. Biodistribution at 48h, demonstrated tumor uptake of 4.8(±0.8) %ID/g. Conclusion The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo. PMID:18719907

  11. Novel Treponema pallidum Recombinant Antigens for Syphilis Diagnostics: Current Status and Future Prospects

    PubMed Central

    Kubanov, Aleksey; Runina, Anastassia

    2017-01-01

    The recombinant protein technology considerably promoted the development of rapid and accurate treponema-specific laboratory diagnostics of syphilis infection. For the last ten years, the immunodominant recombinant inner membrane lipoproteins are proved to be sensitive and specific antigens for syphilis screening. However, the development of an enlarged T. pallidum antigen panel for diagnostics of early and late syphilis and differentiation of syphilis stages or cured syphilis remains as actual goal of multidisciplinary expertise. Current review revealed novel recombinant antigens: surface-exposed proteins, adhesins, and periplasmic and flagellar proteins, which are promising candidates for the improved syphilis serological diagnostics. The opportunities and limitations of diagnostic usage of these antigens are discussed and the criteria for selection of optimal antigens panel summarized. PMID:28523273

  12. Cloning and sequence analysis of a gene encoding a 67-kilodalton myosin-cross-reactive antigen of Streptococcus pyogenes reveals its similarity with class II major histocompatibility antigens.

    PubMed Central

    Kil, K S; Cunningham, M W; Barnett, L A

    1994-01-01

    The group A streptococcal sequela acute rheumatic fever (ARF) has been associated with immunological cross-reactivity between streptococcal and heart proteins. To identify Streptococcus pyogenes genes that encode a myosin cross-reactive antigen(s) recognized by ARF sera, a genomic library from an emm deletion strain (T28/51/4) was screened with a single ARF serum. A positively identified lambda EMBL3 clone (T.2.18) produced a protein which reacted with myosin-specific antibodies affinity purified from individual ARF sera. The recombinant protein was initially estimated to be 60 kDa in size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; however, upon sequence analysis it had a molecular mass equivalent to 67 kDa. Sera from patients with streptococcal infections, acute glomerulonephritis, and ARF were reactive with the recombinant 67-kDa protein. However, individual sera from healthy persons were negative or demonstrated low levels of reactivity with the 67-kDa antigen. The gene encoding the 67-kDa myosin-cross-reactive antigen was subcloned, and its nucleotide sequence was determined by using a combined strategy of DNA sequencing of the cloned gene and N-terminal amino acid sequencing of the protein expressed in Escherichia coli. The amino-terminal sequence deduced from the nucleotide sequence of an open reading frame was identical to that determined from the 67-kDa protein expressed in E. coli. The gene encoded 590 amino acids with a calculated molecular weight of 67,381. No cleavable signal peptide was detected with the 67-kDa protein expressed in E. coli. The deduced amino acid sequence of the 67-kDa protein did not exhibit significant similarity to any known streptococcal proteins. However, it was found to be 19% identical and 62% similar over 151 amino acid residues to the beta chain of mouse major histocompatibility complex class II antigen (I-Au). Similar degrees of homology to the beta chains of other murine and human class II haplotypes were

  13. Protection against Taenia pisiformis larval infection induced by a recombinant oncosphere antigen vaccine.

    PubMed

    Chen, L; Yang, D Y; Xie, Y; Nong, X; Huang, X; Fu, Y; Gu, X B; Wang, S X; Peng, X R; Yang, G Y

    2014-02-13

    Taenia pisiformis larvae cause significant health problems to rabbits. At present, it is not known whether the recombinant antigen from the T. pisiformis oncosphere is able to confer protective immunity against T. pisiformis larval infection. The full-length cDNA was cloned into a pET32a (+) vector, and the recombinant protein was then expressed in BL21 (DE3) cells. Vaccination with the purified rTpUbc2 coupled with QuilA was carried out in New Zealand rabbits to evaluate the immunoprotective effect against T. pisiformis infection. The full-length open reading frame of the TpUbc2 gene was 444 bp, and encoded a 16.63-kDa protein. Finally, rTpUbc2 was used to evaluate the ability to induce immunoprotective responses in rabbits. A 79.3-90.8% reduction (P < 0.01) in the recovery of larvae was observed in the experimental group compared to the control group. Specific anti-rTpUbc2 antibodies from immunized rabbits had significantly higher levels of IgG (P < 0.01) compared to the control group; however, no significant difference in IgA levels was found between groups (P > 0.05). Our data support the use of rTpUbc2 as a potential candidate to develop a vaccine against T. pisiformis larvae.

  14. A soluble recombinant form of human leucocyte antigen-G 6 (srHLA-G6).

    PubMed

    Pelá, Flávia Porto; Rustiguel, Joane Kathelen; Rodrigues, Lilian Cataldi; Mendonça, Jacqueline Nakau; Andrade, Camillo Del Cistia; Lopes, Norberto Peporine; Rosa, José Cesar; Nonato, Maria Cristina; Favier, Benoit; Donadi, Eduardo Antônio; Dias-Baruffi, Marcelo

    2017-05-20

    Human Leucocyte Antigen-G (HLA-G) is a non classical major histocompatibility complex (MHC) molecule that through RNA splicing can encode seven isoforms which are membrane bound (-G1, -G2, -G3 and -G4) and soluble (-G5, -G6 and -G7). HLA-G is described as important immune suppressor endogenous molecule to favor maternal-fetal tolerance, transplant survival and tumor immune scape. HLA-G shows low protein variability and a unique structural complexity that is related with the expression of different isoforms followed by biochemical processes, such as, proteolytic cleavage, molecular interactions, and protein ubiquitination. Studies with HLA-G have shown difficult to assess the role of the individual isoforms. Thus, the aim of this work was to obtain a HLA-G6 recombinant form. The results indicated the production of high homogeneous preparations of soluble recombinant HLA-G6 (srHLA-G6) with molecular mass 23,603.76 Da, determined by MALD-TOF/TOF. In addition, native and denatured srHLA-G6 were detected by ELISA, using commercial monoclonal antibodies. Finally, we developed a suitable methodology to express srHLA-G6 that could contribute in structural and functional studies involving specific isoforms. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Stable antigen is most effective for eliciting CD8+ T-cell responses after DNA vaccination and infection with recombinant vaccinia virus in vivo.

    PubMed

    Schliehe, Christopher; Bitzer, Annegret; van den Broek, Maries; Groettrup, Marcus

    2012-09-01

    The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.

  16. Protection of pigs against Taenia solium cysticercosis by immunization with novel recombinant antigens.

    PubMed

    Gauci, Charles G; Jayashi, César M; Gonzalez, Armando E; Lackenby, Julia; Lightowlers, Marshall W

    2012-06-06

    Recombinant antigens from the oncosphere stage of the parasite Taenia solium were expressed in Escherichia coli. The TSOL16, TSOL45-1A and TSOL45-1B recombinant antigens, each consisting of fibronectin type III (FnIII) domain S, were produced as fusion proteins with glutathione S-transferase (GST) and maltose binding protein (MBP). Groups of pigs were immunized twice with the GST fusions of the antigens and boosted a third time with the MBP fusions prior to receiving a challenge infection with T. solium eggs. The TSOL16 antigen was found to be capable of inducing high levels of immunity in pigs against a challenge infection with T. solium. Immunological investigations identified differences in immune responses in the pigs vaccinated with the various antigens. The results demonstrate that the TSOL16 antigen could be a valuable adjunct to current porcine vaccination approaches and may allow the further development of new vaccination strategies against T. solium cysticercosis.

  17. Toxoplasma gondii Recombinant Antigens as Tools for Serodiagnosis of Human Toxoplasmosis: Current Status of Studies

    PubMed Central

    2013-01-01

    Toxoplasma gondii is a parasitic protozoan which is the cause of toxoplasmosis. Although human toxoplasmosis in healthy adults is usually asymptomatic, serious disease can occur in the case of congenital infections and immunocompromised individuals. Furthermore, despite the exact recognition of its etiology, it still presents a diagnostic problem. Diagnosis of toxoplasmosis is mainly based on the results of serological tests detecting anti-T. gondii-specific antibodies in the patient's serum sample. The specificities and sensitivities of serology tests depend mostly on the diagnostic antigen(s) used. Most of the commercial serological kits currently available are based on Toxoplasma lysate antigens (TLAs). In recent years, many studies showed that recombinant antigenic proteins of T. gondii may be an alternative source of antigens which are very useful for the serodiagnosis of toxoplasmosis. This article presents a review of current studies on the application and usefulness of different T. gondii recombinant antigens in serological tests for the diagnosis of human toxoplasmosis. PMID:23784855

  18. Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

    PubMed

    Ortiz, B L; Garcia, A M; Restrepo, A; McEwen, J G

    1996-03-01

    We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.

  19. [Detection and antigenic characteristics of the recombinant nucleocapsid proteins of Lassa and Marburg viruses].

    PubMed

    Vladyko, A S; Scheslenok, E P; Fomina, E G; Semizhon, P A; Ignat'ev, G M; Shkolina, T V; Kras'ko, A G; Semenov, S F; Vinokurov, N V

    2012-01-01

    Two plasmid vectors, which allow the recombinant polypeptides of Lassa and Marburg viruses to be expressed in prokaryotic cells E. coli strain BL21 (DE3), were produced. The two recombinant polypeptides are able to bind specific antibodies. This provides an opportunity to use them as antigenic components of immunoassay diagnostic test kits.

  20. Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

    PubMed

    Marín-López, Alejandro; Ortego, Javier

    2016-01-01

    Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

  1. Immunogenicity of Novel DosR Regulon-Encoded Candidate Antigens of Mycobacterium tuberculosis in Three High-Burden Populations in Africa▿ †

    PubMed Central

    Black, Gillian F.; Thiel, Bonnie A.; Ota, Martin O.; Parida, Shreemanta K.; Adegbola, Richard; Boom, W. Henry; Dockrell, Hazel M.; Franken, Kees L. M. C.; Friggen, Annemiek H.; Hill, Philip C.; Klein, Michel R.; Lalor, Maeve K.; Mayanja, Harriet; Schoolnik, Gary; Stanley, Kim; Weldingh, Karin; Kaufmann, Stefan H. E.; Walzl, Gerhard; Ottenhoff, Tom H. M.

    2009-01-01

    Increasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosis antigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosis recombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosis infection responded to several novel DosR regulon-encoded M. tuberculosis proteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosis infection, active TB disease, and protective immunity. PMID:19553548

  2. Serological differentiation between cystic and alveolar echinococcosis by use of recombinant larval antigens.

    PubMed Central

    Helbig, M; Frosch, P; Kern, P; Frosch, M

    1993-01-01

    Two recombinant antigens of the larval stages of Echinococcus granulosus and Echinococcus multilocularis, termed EG55 and EM10, respectively, were applied for serodiagnosis and serological differentiation between parasitic infections caused by the metacestode tissue of both tapeworms. Antigen EM10 is synthesized by E. multilocularis larvae. Antigen EG55 represents the recombinant form of the low-molecular-weight subunit of antigen B, which is an Echinococcus genus-specific antigen. Both recombinant antigens were expressed as glutathione S-transferase fusion proteins. A sandwich enzyme-linked immunosorbent assay with monoclonal antibodies against EM10 and EG55 as capture reagents for the recombinant antigens was established and was evaluated with 74 serum samples from patients with histologically confirmed alveolar echinococcosis and 63 serum samples from patients with histologically confirmed cystic echinococcosis. A sensitivity of 93.2% and a specificity of 96.8% were achieved for the serodiagnosis of alveolar echinococcosis. Cystic echinococcosis could be detected with a sensitivity of 89.1% and a specificity of 98.6%. PMID:8308113

  3. A Recombinant Rabies Virus Encoding Two Copies of the Glycoprotein Gene Confers Protection in Dogs against a Virulent Challenge

    PubMed Central

    Sun, Zhaojin; Chen, Jing; Ai, Jun; Dun, Can; Fu, Zhen F.; Niu, Xuefeng; Guo, Xiaofeng

    2014-01-01

    The rabies virus (RABV) glycoprotein (G) is the principal antigen responsible for the induction of virus neutralizing antibodies (VNA) and is the major modality of protective immunity in animals. A recombinant RABV HEP-Flury strain was generated by reverse genetics to encode two copies of the G-gene (referred to as HEP-dG). The biological properties of HEP-dG were compared to those of the parental virus (HEP-Flury strain). The HEP-dG recombinant virus grew 100 times more efficiently in BHK-21 cell than the parental virus, yet the virulence of the dG recombinant virus in suckling mice was lower than the parental virus. The HEP-dG virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines PMID:24498294

  4. Evaluation of the Protective Effect of Deoxyribonucleic Acid Vaccines Encoding Granule Antigen 2 and 5 Against Acute Toxoplasmosis in BALB/c Mice.

    PubMed

    Ching, Xiao Teng; Fong, Mun Yik; Lau, Yee Ling

    2017-06-01

    AbstractToxoplasma gondii infects a broad range of warm-blooded hosts, including humans. Important clinical manifestations include encephalitis in immunocompromised patients as well as miscarriage and fetal damage during early pregnancy. Toxoplasma gondii dense granule antigen 2 and 5 (GRA2 and GRA5) are essential for parasitophorous vacuole development of the parasite. To evaluate the potential of GRA2 and GRA5 as recombinant DNA vaccine candidates, these antigens were cloned into eukaryotic expression vector (pcDNA 3.1C) and evaluated in vaccination experiments. Recombinant DNA vaccines constructed with genes encoding GRAs were validated in Chinese hamster ovary cells before evaluation using lethal challenge of the virulent T. gondii RH strain in BALB/c mice. The DNA vaccines of pcGRA2 and pcGRA5 elicited cellular-mediated immune response with significantly higher levels of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10 (P < 0.05) compared with controls. A mixed T-helper cell 1 (Th1)/Th2 response was associated with slightly prolonged survival. These findings provide evidence that DNA vaccination with GRA2 and GRA5 is associated with Th1-like cell-mediated immune responses. It will be worthwhile to construct recombinant multiantigen combining full-length GRA2 or/and GRA5 with various antigenic proteins such as the surface antigens and rhoptry antigens to improve vaccination efficacy.

  5. Use of Strep-tag II for rapid detection and purification of Mycobacterium tuberculosis recombinant antigens secreted by Streptomyces lividans.

    PubMed

    Ayala, Julio C; Pimienta, Elsa; Rodríguez, Caridad; Anné, Jozef; Vallín, Carlos; Milanés, María T; King-Batsios, Emmanuel; Huygen, Kris; Van Mellaert, Lieve

    2013-09-01

    Recent results with respect to the secretory production of bio-active Mycobacterium tuberculosis proteins in Streptomyces have stimulated the further exploitation of this host as a bacterial cell factory. However, the rapid isolation of a recombinant protein by conventional procedures can be a restrictive step. A previous attempt to isolate recombinant antigens fused to the widely used 6His-tag was found to be relatively incompatible with secretory production in the Streptomyces host. As an alternative, the eight-residue Strep-tag® II (WSHPQFEK), displaying intrinsic binding affinity towards streptavidin, was evaluated for the secretory production of two M. tuberculosis immunodominant antigens in Streptomyces lividans and their subsequent downstream processing. Therefore, the genes ag85A (Rv3804c, encoding the mycolyl-transferase Ag85A) and Rv2626c (encoding hypoxic response protein 1), were equipped with a 3'-Strep-tag® II-encoding sequence and placed under control of the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) transcriptional, translational and signal sequences. Strep-tagged Ag85A and Rv2626c proteins were detected in the spent medium of recombinant S. lividans cultures at 48h of growth, and purified using a Strep-Tactin Superflow® matrix. Recombinant Ag85A appeared as a 30-kDa protein of which the N-terminal amino acid sequence was identical to the expected one. Rv2626c was produced in two forms of 17 and 37kDa respectively, both with the same predicted N-terminal sequence, suggesting that the 37-kDa product is an Rv2626c dimer. The obtained results indicate that the Strep-tagII is proteolytically stable in Streptomyces and does not interfere with the membrane translocation of Ag85A and Rv2626c. A comparison of reactivity of serum from tuberculosis patients versus healthy persons by ELISA showed that both S. lividans-derived antigens were recognized by sera of individuals infected with M. tuberculosis, indicating that they remained

  6. Improved Serodiagnosis of Cystic Echinococcosis Using the New Recombinant 2B2t Antigen

    PubMed Central

    Hernández-González, Ana; Santivañez, Saúl; García, Héctor H.; Rodríguez, Silvia; Muñoz, Santiago; Ramos, Guillermo; Orduña, Antonio; Siles-Lucas, Mar

    2012-01-01

    A standardized test for the serodiagnosis of human cystic echinococcosis (CE) is still needed, because of the low specificity and sensitivity of the currently available commercial tools and the lack of proper evaluation of the existing recombinant antigens. In a previous work, we defined the new ELISA-B2t diagnostic tool for the detection of specific IgGs in CE patients, which showed high sensitivity and specificity, and was useful in monitoring the clinical evolution of surgically treated CE patients. Nevertheless, this recombinant antigen gave rise to false-negative results in a percentage of CE patients. Therefore, in an attempt to improve its sensitivity, we constructed B2t-derived recombinant antigens with two, four and eight tandem repeat of B2t units, and tested them by ELISA on serum samples of CE patients and patients with related parasites. The best diagnostic values were obtained with the two tandem repeat 2B2t antigen. The influence of several clinical variables on the performance of the tests was also evaluated. Finally, the diagnostic performance of the 2B2t-ELISA was compared with that of an indirect haemagglutination commercial test. The 2B2t recombinant antigen performed better than the HF and B2t antigens, and the IHA commercial kit. Therefore, this new 2B2t-ELISA is a promising candidate test for the serodiagnosis of CE in clinical settings. PMID:22802975

  7. Multiple antigens of Yersinia pestis delivered by live recombinant attenuated Salmonella vaccine strains elicit protective immunity against plague.

    PubMed

    Sanapala, Shilpa; Rahav, Hannah; Patel, Hetal; Sun, Wei; Curtiss, Roy

    2016-05-05

    Based on our improved novel Salmonella vaccine delivery platform, we optimized the recombinant attenuated Salmonella typhimurium vaccine (RASV) χ12094 to deliver multiple Yersinia pestis antigens. These included LcrV196 (amino acids, 131-326), Psn encoded on pYA5383 and F1 encoded in the chromosome, their synthesis did not cause adverse effects on bacterial growth. Oral immunization with χ12094(pYA5383) simultaneously stimulated high antibody titers to LcrV, Psn and F1 in mice and presented complete protection against both subcutaneous (s.c.) and intranasal (i.n.) challenges with high lethal doses of Y. pestis CO92. Moreover, no deaths or other disease symptoms were observed in SCID mice orally immunized with χ12094(pYA5383) over a 60-day period. Therefore, the trivalent S. typhimurium-based live vaccine shows promise for a next-generation plague vaccine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Isolation and characterization of recombinant antigens from Leishmania aethiopica that react with human antibodies.

    PubMed Central

    Osland, A; Beyene, D; Ashenafi, S; Beetsma, A

    1992-01-01

    A genomic expression library of Leishmania aethiopica was constructed in lambda gt11 and screened with patient sera and sera from healthy people living in an area of endemicity. Forty-five recombinant clones were isolated and partly characterized. Clone-specific antibodies were prepared and used with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis to estimate the molecular masses of the parasite-derived antigens containing the reactive epitope(s). Antigens with apparent molecular masses of 90, 85, 63, 50, 41, 25 and 24 kDa as well as several antigens with lower molecular masses were detected. The clone-specific antibodies from patients with diffuse cutaneous leishmaniasis reacted with high-molecular-weight antigens (30,000 less than Mr less than 90,000), whereas antibodies from patients with localized cutaneous leishmaniasis recognized low-molecular-weight antigens (Mr less than 25,000). Nine different purified recombinant antigens were obtained from lysogens in Escherichia coli Y1089 by immunoaffinity chromatography on anti-beta-galactosidase columns and were subsequently tested with patient sera. It is suggested that some of these recombinant antigens might be used for immunodiagnostic purposes. Images PMID:1372294

  9. Mucosal immunization with recombinant adenovirus encoding soluble globular head of hemagglutinin protects mice against lethal influenza virus infection.

    PubMed

    Kim, Joo Young; Choi, Youngjoo; Nguyen, Huan H; Song, Man Ki; Chang, Jun

    2013-12-01

    Influenza virus is one of the major sources of respiratory tract infection. Due to antigenic drift in surface glycoproteins the virus causes annual epidemics with severe morbidity and mortality. Although hemagglutinin (HA) is one of the highly variable surface glycoproteins of the influenza virus, it remains the most attractive target for vaccine development against seasonal influenza infection because antibodies generated against HA provide virus neutralization and subsequent protection against the virus infection. Combination of recombinant adenovirus (rAd) vector-based vaccine and mucosal administration is a promising regimen for safe and effective vaccination against influenza. In this study, we constructed rAd encoding the globular head region of HA from A/Puerto Rico/8/34 virus as vaccine candidate. The rAd vaccine was engineered to express high level of the protein in secreted form. Intranasal or sublingual immunization of mice with the rAd-based vaccine candidates induced significant levels of sustained HA-specific mucosal IgA and IgG. When challenged with lethal dose of homologous virus, the vaccinated mice were completely protected from the infection. The results demonstrate that intranasal or sublingual vaccination with HA-encoding rAd elicits protective immunity against infection with homologous influenza virus. This finding underlines the potential of our recombinant adenovirus-based influenza vaccine candidate for both efficacy and rapid production.

  10. Evaluation of recombinant antigens in combination and single formula for diagnosis of feline toxoplasmosis.

    PubMed

    Abdelbaset, Abdelbaset Eweda; Alhasan, Hend; Salman, Doaa; Karram, Mohamed Hassan; Ellah Rushdi, Mahmoud Abd; Xuenan, Xuan; Igarashi, Makoto

    2017-01-01

    Cats are the only definitive hosts of Toxoplasma gondii and constitute an essential source of infection to all warm blooded animals and humans. Diagnosis of T. gondii infection in cats is fundamental for proper management and control of infection in humans and animals. In the current study, we have evaluated the diagnostic performance of tachyzoite lysate antigen (TLA) and different T. gondii recombinant antigens including surface antigen 2 (SAG2), dense granule proteins 2, 6, 7, 15 (GRA2, GRA6, GRA7, GRA15) and microneme 10 protein (MIC10) in immunoglobulin G enzyme linked-immunosorbent assay (IgG ELISA) using cat serum samples, with reference to latex agglutination test (LAT). Remarkably, TLA showed better performance than other recombinant antigens in IgG ELISAs as compared to LAT, with concordance and Kappa values of 94.27% and 0.93, respectively. Furthermore, to improve the reactivity of the recombinant antigens, we have developed IgG ELISAs using different combinations with these recombinant antigens. Strikingly, a combination of SAG2 and GRAs has relatively similar performance as TLA evidenced by concordance and Kappa values of 94.27% and 0.81, respectively. The developed ELISA with a combination of recombinant antigens can be used as a promising diagnostic tool for routine testing of T. gondii infection and mass screening in cats. The major advantages of this assay are the high sensitivity and specificity, lower cost, safer production and easiness of standardization in various laboratories worldwide. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

    PubMed

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-11-01

    Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

  12. Recombinant antigen-based dipstick ELISA for the diagnosis of leptospirosis in dogs.

    PubMed

    Dey, S; Mohan, C Madhan; Ramadass, P; Nachimuthu, K

    2007-02-10

    A recombinant LipL 32 antigen-based dipstick ELISA was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change in the colour of the substrate solution when the recombinant antigen-coated dipsticks were dipped into it. The relative sensitivity, specificity and accuracy of the test, compared with the standard microscopic agglutination test, were 95.9 per cent, 93.8 per cent and 94.8 per cent, respectively.

  13. [The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

    PubMed

    Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

    2014-08-01

    The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

  14. Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis.

    PubMed Central

    Ortiz, B L; Garcia, A M; Restrepo, A; McEwen, J G

    1996-01-01

    We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis. PMID:8991645

  15. Recombinant measles AIK-C vaccine strain expressing heterologous virus antigens.

    PubMed

    Nakayama, Tetsuo; Sawada, Akihito; Yamaji, Yoshiaki; Ito, Takashi

    2016-01-04

    Further attenuated measles vaccines were developed more than 50 years ago and have been used throughout the world. Recombinant measles vaccine candidates have been developed and express several heterologous virus protective antigens. Immunogenicity and protective actions were confirmed using experimental animals: transgenic mice, cotton rats, and primates. The recent development of measles vaccine-based vectored vaccine candidates has been reviewed and some information on recombinant measles vaccines expressing respiratory syncytial virus proteins has been shown and discussed.

  16. Evaluation of three recombinant Leishmania infantum antigens in human and canine visceral leishmaniasis diagnosis.

    PubMed

    Fonseca, Aliani Moura; Faria, Angélica Rosa; Rodrigues, Fernandes Tenório Gomes; Nagem, Ronaldo Alves Pinto; Magalhães, Rubens Daniel Miserani; Cunha, João Luís Reis; Bartholomeu, Daniella Castanheira; de Andrade, Hélida Monteiro

    2014-09-01

    Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens' sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.

  17. The human mRNA encoding the Goodpasture antigen is alternatively spliced.

    PubMed

    Bernal, D; Quinones, S; Saus, J

    1993-06-05

    The noncollagenous (NC1) domain of the human collagen alpha 3(IV)-chain is the primary target of autoantibodies produced in Goodpasture syndrome and, therefore, has been designated as the Goodpasture antigen. In this report, we show that Goodpasture antigen mRNA undergoes processing to at least two alternatively spliced forms in a variety of human tissues, resulting in the exclusion of sequence encoded by either one or two exons. Interestingly, no alternatively spliced forms were observed in bovine or rat tissues. The derived amino acid sequences of the two variant mRNA forms are identical and significantly shorter than that arising from the complete Goodpasture antigen mRNA. They lack the carboxyl-terminal region contributing to the formation of the Goodpasture epitope and all but one of the cysteines found in the complete form. These sequence characteristics suggest that, if translated, the variant Goodpasture antigen is likely to be defective in triple helix formation and no longer reactive with Goodpasture autoantibodies. Although each tissue expressing Goodpasture antigen displayed a specific mRNA pattern, the complete form was always the most abundant and was present at levels apparently unrelated to whether or not the organ of origin is a potential target in Goodpasture syndrome. Furthermore, the antigen sequence was identical in the kidneys of normal and Goodpasture-affected individuals, and no major differences in the expression of the complete and spliced forms were observed.

  18. Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes

    PubMed Central

    Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan; Frock, Richard L.; Du, Zhou; Meyers, Robin M.; Meng, Fei-long; Schatz, David G.; Alt, Frederick W.

    2015-01-01

    SUMMARY RAG initiates antibody V(D)J recombination in developing lymphocytes by generating “on-target” DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically-inserted RSS pairs to identify huge numbers of RAG “off-target” breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage-site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation-dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them. PMID:26593423

  19. Chromosomal Loop Domains Direct the Recombination of Antigen Receptor Genes.

    PubMed

    Hu, Jiazhi; Zhang, Yu; Zhao, Lijuan; Frock, Richard L; Du, Zhou; Meyers, Robin M; Meng, Fei-long; Schatz, David G; Alt, Frederick W

    2015-11-05

    RAG initiates antibody V(D)J recombination in developing lymphocytes by generating "on-target" DNA breaks at matched pairs of bona fide recombination signal sequences (RSSs). We employ bait RAG-generated breaks in endogenous or ectopically inserted RSS pairs to identify huge numbers of RAG "off-target" breaks. Such breaks occur at the simple CAC motif that defines the RSS cleavage site and are largely confined within convergent CTCF-binding element (CBE)-flanked loop domains containing bait RSS pairs. Marked orientation dependence of RAG off-target activity within loops spanning up to 2 megabases implies involvement of linear tracking. In this regard, major RAG off-targets in chromosomal translocations occur as convergent RSS pairs at enhancers within a loop. Finally, deletion of a CBE-based IgH locus element disrupts V(D)J recombination domains and, correspondingly, alters RAG on- and off-target distributions within IgH. Our findings reveal how RAG activity is developmentally focused and implicate mechanisms by which chromatin domains harness biological processes within them. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Characterization of a recombinant immunodiagnostic antigen (NIE) from Strongyloides stercoralis L3-stage larvae.

    PubMed

    Ravi, Varatharajalu; Ramachandran, Srinivasan; Thompson, Robert W; Andersen, John F; Neva, Franklin A

    2002-01-01

    Due to the process of internal autoinfection, even chronic asymptomatic infections with Strongyloides stercoralis have the potential to become severe disseminated disease with fatal outcome. Intermittent and scanty larval excretion makes parasitologic diagnosis difficult. Serodiagnosis is helpful, but antigen preparation from infective larvae requires access to patients or immunosuppressed experimental animals. For these reasons, attention has turned to recombinant antigens for immunodiagnosis. A 31-kDa candidate antigen (NIE) derived from an L3 cDNA library is described in this report. Multiple alignment of the deduced amino acid sequence of NIE showed approximately 12-18% identity with various other organisms, including 17.9% of Asp1 of Ancylostoma caninum, 12.6% of Hemonchus contortus, and 17.6% of insect venom allergen 5 of yellow jacket. By ELISA, antibodies to the purified recombinant NIE antigen were demonstrated in 87.5% of 48 sera from strongyloides-infected patients and in only 6.5% of sera from presumed normal controls. Immunoreactivity of purified NIE antigen with parasite-specific IgE from sera of strongyloides-infected patients indicated its potential use as an immediate sensitivity skin test antigen. This application of the NIE antigen was supported by its capacity to trigger release of histamine upon in vitro exposure to blood from strongyloides-infected patients and its failure to produce histamine release from blood of normal controls.

  1. Murine responses to recombinant MVA versus ALVAC vaccines against tumor-associated antigens, gp100 and 5T4.

    PubMed

    Hanwell, David G; McNeil, Bryan; Visan, Lucian; Rodrigues, Lauren; Dunn, Pamela; Shewen, Patricia E; Macallum, Grace E; Turner, Patricia V; Vogel, Thorsten U

    2013-05-01

    Virally vectored cancer vaccines comprise a new form of immunotherapy that aim to generate anti-tumor immune responses with potential for tumor clearance and enhanced patient survival. Here, we compared 2 replication-deficient poxviruses modified vaccinia Ankara (MVA) and ALVAC(2) in their ability to induce antigen expression and immunogenicity of the tumor-associated antigens (TAAs) 5T4 and gp100. To facilitate the comparison, recombinant MVA-gp100M and ALVAC(2)-5T4 were constructed to complement existing ALVAC(2)-gp100M and MVA-5T4 vectors. Recombinant TAA expression in chicken embryo fibroblast cells was confirmed by Western blot analysis. 5T4 expression was approximately equal for both viruses, whereas ALVAC-derived gp100 was quickly degraded, at a time point when MVA-derived gp100 was still stable and expressed at high levels. Human leukocyte antigen-A2 transgenic mice were vaccinated with recombinant viruses and the CD8 T-cell responses elicited against each TAA were monitored by interferon-γ enzyme-linked immunospot. No 5T4 peptide responses were detected using splenocytes from mice vaccinated with either vector, whereas vaccination with MVA elicited a significantly higher gp100-specific response than ALVAC(2) at 10 PFU (P<0.001). In CD-1 mice, each vector elicited similar 5T4 antibody responses, whereas MVA was more potent and induced gp100 antibody responses at a lower immunization dose than ALVAC (P<0.001). In this study, immunogenicity varied depending on the viral vector used and reflected vector-associated differences in in vitro TAA expression and stability. These findings suggest that novel vector-transgene combinations must be assessed individually when designing vaccines, and that stability of vector-encoded proteins produced in vitro may be useful as a predictor for in vitro immunogenicity.

  2. [Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

    PubMed

    Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

    2013-08-01

    To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

  3. Infectious vaccinia virus recombinants that express hepatitis B virus surface antigen

    NASA Astrophysics Data System (ADS)

    Smith, Geoffrey L.; Mackett, Michael; Moss, Bernard

    1983-04-01

    Potential live vaccines against hepatitis B virus have been produced. The coding sequence for hepatitis B virus surface antigen (HBsAg) has been inserted into the vaccinia virus genome under control of vaccinia virus early promoters. Cells infected with these vaccinia virus recombinants synthesize and excrete HBsAg and vaccinated rabbits rapidly produce antibodies to HBsAg.

  4. Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

    PubMed Central

    Cho, Jung-Hwa; Chung, Woo-Suk; Song, Kyoung-Ju; Na, Byoung-Kuk; Kang, Seung-Won; Song, Chul-Yong

    2005-01-01

    Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N.caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo. PMID:15793355

  5. Several recombinant capsid proteins of equine rhinitis a virus show potential as diagnostic antigens.

    PubMed

    Li, Fan; Stevenson, Rachel A; Crabb, Brendan S; Studdert, Michael J; Hartley, Carol A

    2005-06-01

    Equine rhinitis A virus (ERAV) is a significant pathogen of horses and is also closely related to Foot-and-mouth disease virus (FMDV). Despite these facts, knowledge of the prevalence and importance of ERAV infections remains limited, largely due to the absence of a simple, robust diagnostic assay. In this study, we compared the antigenicities of recombinant full-length and fragmented ERAV capsid proteins expressed in Escherichia coli by using sera from experimentally infected and naturally exposed horses. We found that, from the range of antigens tested, recombinant proteins encompassing the C-terminal region of VP1, full-length VP2, and the N-terminal region of VP2 reacted specifically with antibodies present in sera from each of the five experimentally infected horses examined. Antibodies to epitopes on VP2 (both native and recombinant forms) persisted longer postinfection (>105 days) than antibodies specific for epitopes on other fragments. Our data also suggest that B-cell epitopes within the C terminus of VP1 and N terminus of VP2 contribute to a large proportion of the total reactivity of recombinant VP1 and VP2, respectively. Importantly, the reactivity of these VP1 and VP2 recombinant proteins in enzyme-linked immunosorbent assays (ELISAs) correlated well with the results from a range of native antigen-based serological assays using sera from 12 field horses. This study provides promising candidates for development of a diagnostic ERAV ELISA.

  6. Protection of gerbils from amebic liver abscess by immunization with a recombinant Entamoeba histolytica antigen.

    PubMed Central

    Zhang, T; Cieslak, P R; Stanley, S L

    1994-01-01

    Amebiasis, infection by the intestinal protozoan parasite Entamoeba histolytica, is a leading parasitic cause of death. As a step in the development of a recombinant antigen vaccine to prevent E. histolytica infection, we looked at the ability of a recombinant version of the serine-rich E. histolytica protein (SREHP) to elicit a protective immune response against invasive amebic disease. Gerbils, a standard model for amebic liver abscess, were immunized with either a recombinant SREHP/maltose-binding protein (MBP) fusion, recombinant MBP alone, or phosphate-buffered saline (PBS), all combined with complete Freund's adjuvant. In the first trial (group 1), gerbils received a primary and two booster immunizations intraperitoneally; in the second trial (group 2), gerbils were immunized by a single intradermal injection. SREHP/MBP-immunized gerbils in both groups produced antibody to native SHEHP and developed delayed-type hypersensitivity responses to recombinant SREHP. All gerbils were challenged by an intrahepatic injection with 5 x 10(4) virulent E. histolytica HM1-IMSS trophozoites. Complete protection from amebic liver abscess was seen in 64% of the SHEHP/MBP-immunized gerbils in group 1 and in 100% of the SREHP/MBP-immunized gerbils in group 2. There was no protection observed in MBP- or PBS-immunized gerbils in either group. Our results indicate that the SREHP molecule has potential as a vaccine to prevent amebic infection and demonstrate that successful vaccination of animals with recombinant E. histolytica antigen vaccines is possible. Images PMID:8132322

  7. Expression and purification of recombinant HTLV-I/-II linear epitopes antigen and its application for screening of suspected patients.

    PubMed

    Faramarzi, Roxana; Dolatabadi, Samaneh

    2017-02-01

    The linear epitopes of gp46-I, gp46-II, gp21 and p19 are used in diagnosis of HTLV-I/-II infections. The aims of this study was to obtain high-level expression and purification of recombinant antigen (RA) containing these epitopes. Large-scale preparation of such antigen probably worths for diagnostic purpose. The synthetic DNA encoding RA was synthesized and over-expressed as soluble in Escherichia coli BL21 (DE3) cells. Expression and distribution of the His-GST-RA protein were evaluated using SDS-PAGE. The soluble RA was purified utilizing Ni-NTA agarose beads under native conditions and was concentrated by ultra filtration. Using 20 sera specimens from HTLV infected patients, the antigenicity of the purified protein was confirmed in ELISA and western blotting analysis. SDS-PAGE revealed that the purified protein was more than 90% pure. The final yield was approximately 25 mg per liter of culture medium. ELISA results showed that RA could specifically bind to anti-HTLV-I/-II antibodies in infected sera. RA could be a candidate for HTLV-I/-II screening and the strategy presented in this study could be used for easy production of this diagnostic protein.

  8. Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

    PubMed

    Magalhães, Franklin B; Castro Neto, Artur L; Nascimento, Marilia B; Santos, Wagner J T; Medeiros, Zulma M; Lima Neto, Adelino S; Costa, Dorcas L; Costa, Carlos H N; Dos Santos, Washington L C; Pontes de Carvalho, Lain C; Oliveira, Geraldo G S; de Melo Neto, Osvaldo P

    2017-01-01

    Current strategies for the control of zoonotic visceral leishmaniasis (VL) rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay) with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity) although their performance was increased in the canine sera (48-91% sensitivity), with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84%) and canine (88%) sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

  9. Serum and colostrum antibody responses induced by jet-injection of sheep with DNA encoding a Cryptosporidium parvum antigen.

    PubMed

    Jenkins, M; Kerr, D; Fayer, R; Wall, R

    1995-12-01

    In an effort to generate high titer colostrum for immunotherapy of cryptosporidiosis, a study was conducted to test the efficacy of immunizing sheep with recombinant plasmid DNA (pCMV-CP15/60) encoding epitopes of 15 and 60 kDa surface antigens of Cryptosporidium parvum sporozoites. The plasmid DNA was used to immunize preparturient ewes at three dose levels by jet-injection into either hind limb muscle (IM) or mammary tissue (IMAM). Regardless of route of injection, a dose-dependent anti-CP15/60 immunoglobulin response was observed in sera and colostrum from sheep immunized with pCMV-CP15/60 plasmid DNA. High titer antibody responses were observed in one of three animals per group receiving an IM injection of 100 or 1000 micrograms pCMV-CP15/60. IMAM immunization with 100 or 1000 micrograms pCMV-CP15/60 plasmid DNA elicited higher titer colostrum responses and more consistent serum responses compared to IM injections. A negligible serum and colostrum anti-CP15/60 response was observed in ewes injected IM with 10 micrograms pCMV-CP15/60 or 1000 micrograms control plasmid DNA. Immunoblotting of native C. parvum sporozoite/oocyst protein with hyperimmune serum and colostrum corroborated the increased titers against CP15/60 antigen. Serum and colostrum antibodies from pCMV-CP15/60-immunized sheep were eluted from native CP15 protein and bound a surface antigen of C. parvum sporozoites as indicated by indirect immunofluorescence staining.

  10. Isolation of the gene encoding the Hin recombinational enhancer binding protein.

    PubMed Central

    Johnson, R C; Ball, C A; Pfeffer, D; Simon, M I

    1988-01-01

    In vitro DNA inversion mediated by the protein Hin requires the presence of a recombinational enhancer sequence located in cis relative to the recombination sites and a protein, Fis, which binds to the enhancer. We have cloned and determined the primary sequence of the gene encoding Fis. The deduced amino acid sequence of Fis indicates that the protein is 98 amino acids long and contains a potential helix-turn-helix DNA binding motif at its carboxyl terminus. The gene encoding Fis maps at 72 min on the Escherichia coli chromosome. The construction of mutant strains of E. coli that lack a functional fis gene demonstrates that Fis is not essential for cell growth under laboratory conditions but is required for high rates of Hin-mediated site-specific inversion in vivo. PMID:2835774

  11. A Gene Encoding Antigenic Peptides of Human Squamous Cell Carcinoma Recognized by Cytotoxic T Lymphocytes

    PubMed Central

    Shichijo, Shigeki; Nakao, Masanobu; Imai, Yasuhisa; Takasu, Hideo; Kawamoto, Mayumi; Niiya, Fumihiko; Yang, Damu; Toh, Yuji; Yamana, Hideaki; Itoh, Kyogo

    1998-01-01

    Except for melanomas, tumor antigens recognized by cytotoxic T lymphocytes (CTLs) are yet unidentified. We have identified a gene encoding antigenic peptides of human squamous cell carcinomas (SCCs) recognized by human histocompatibility leukocyte antigens (HLA)- A2601–restricted CTLs. This gene showed no similarity to known sequences, and encoded two (125- and 43-kilodalton [kD]) proteins. The 125-kD protein with the leucine zipper motif was expressed in the nucleus of the majority of proliferating cells tested, including normal and malignant cells. The 43-kD protein was expressed in the cytosol of most SCCs from various organs and half of lung adenocarcinomas, but was not expressed in other cancers nor in a panel of normal tissues. The three nonapeptides shared by the two proteins were recognized by the KE4 CTLs, and one of the peptides induced in vitro from peripheral blood mononuclear cells (PBMCs) the CTLs restricted to the autologous tumor cells. The 43-kD protein and this nonapeptide (KGSGKMKTE) may be useful for the specific immunotherapy of HLA-A2601+ epithelial cancer patients. PMID:9449708

  12. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

    PubMed Central

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A.; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S.; Greenblatt, Jack F.; Marcon, Edyta; Arrowsmith, Cheryl H.; Edwards, Aled M.; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols. PMID:26437229

  13. Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins.

    PubMed

    Zhong, Nan; Loppnau, Peter; Seitova, Alma; Ravichandran, Mani; Fenner, Maria; Jain, Harshika; Bhattacharya, Anandi; Hutchinson, Ashley; Paduch, Marcin; Lu, Vincent; Olszewski, Michal; Kossiakoff, Anthony A; Dowdell, Evan; Koide, Akiko; Koide, Shohei; Huang, Haiming; Nadeem, Vincent; Sidhu, Sachdev S; Greenblatt, Jack F; Marcon, Edyta; Arrowsmith, Cheryl H; Edwards, Aled M; Gräslund, Susanne

    2015-01-01

    We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.

  14. Toxoplasma gondii: cloning, expression and immunoreactivity of recombinant ROP5 and ROP18 antigens.

    PubMed

    Grzybowski, Marcin M; Dziadek, Bożena; Dziadek, Jarosław; Gatkowska, Justyna; Dzitko, Katarzyna; Długońska, Henryka

    2015-03-01

    Early diagnosis and determining the infective stage are critical for effective therapy of toxoplasmosis. Owing to the progress in biotechnology, commonly used native, non-standardized diagnostic antigens should be replaced by genetically engineered antigens. The recombinant proteins are also promising components of subunit vaccines against Toxoplasma gondii infections. A strategic biological role of rhoptry proteins (ROP) in parasitophorous vacuole biogenesis and virulence of the parasite creates a necessity for an intensive study on the serological activity and immunogenicity of newly developed recombinant ROP antigens. Our findings indicate that all generated preparations of recombinant ROP5 and ROP18 antigens, expressed in Escherichia coli bacteria, are recognized by specific antibodies produced during acute and chronic infections in inbred laboratory mice. We noticed, for the first time, that ROP5 IgM antibodies are an early and sensitive marker of T. gondii infection. The proven immunoreactivity of the obtained preparations has become a premise for a further study on their utility in routine diagnosis of human and animal toxoplasmosis as well as in the immunoprevention of T. gondii infection (as the main or supplementary component of the vaccine).

  15. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis

    PubMed Central

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-01-01

    Background & objectives: Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Methods: Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. Results: For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Interpretation & conclusions: Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB. PMID:23287127

  16. Vaccination with a cocktail of Ancylostoma ceylanicum recombinant antigens leads to worm burden reduction in hamsters.

    PubMed

    Wiśniewski, Marcin; Łapiński, Maciej; Daniłowicz-Luebert, Emilia; Jaros, Sławomir; Długosz, Ewa; Wędrychowicz, Halina

    2016-09-01

    Hookworms, a group to which Ancylostoma ceylanicum belongs, are gastrointestinal nematodes that infect more than 700 million people around the world. They are a leading cause of anemia in developing countries. In order to effectively prevent hookworm infections research is conducted to develop an effective vaccine using recombinant antigens of the parasite. The aim of this study was to examine the influence of the hosts' on protection against ancylostomiasis and the shaping of the humoral immune response among Syrian hamsters after immunization with a cocktail of five A. ceylanicum recombinant antigens. Ace-ASP-3, Ace-ASP-4, Ace-APR-1, Ace-MEP-6 and Ace-MEP-7 were obtained in the pET expression system. Immunization with a vaccine cocktail resulted in a 33.5% worm burden reduction. The immunogenicity of the recombinant proteins were determined using ELISA. Statistical analysis showed that vaccinated hamsters developed stronger humoral responses to four of five recombinant antigens (the exception being Ace-ASP-3) compared to hamsters from the control group.

  17. Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis

    PubMed Central

    Obuchowski, Michał; Nidzworski, Dawid

    2016-01-01

    Effective vaccination against influenza virus infection is a serious problem mainly due to antigenic variability of the virus. Among many of investigated antigens, the extracellular domain of the M2 protein (M2e) features high homology in all strains of influenza A viruses and antibodies against M2e and is protective in animal models; this makes it a potential candidate for generation of a universal influenza vaccine. However, due to the low immunogenicity of the M2e, formulation of a vaccine based on this antigen requires some modification to induce effective immune responses. In this work we evaluated the possible use of Bacillus subtilis spores as a carrier of the Influenza A M2e antigen in mucosal vaccination. A tandem repeat of 4 consensus sequences coding for human—avian—swine—human M2e (M2eH-A-S-H) peptide was fused to spore coat proteins and stably exposed on the spore surface, as demonstrated by the immunostaining of intact, recombinant spores. Oral immunization of mice with recombinant endospores carrying M2eH-A-S-H elicited specific antibody production without the addition of adjuvants. Bacillus subtilis endospores can serve as influenza antigen carriers. Recombinant spores constructed in this work showed low immunogenicity although were able to induce antibody production. The System of influenza antigen administration presented in this work is attractive mainly due to the omitting time-consuming and cost-intensive immunogen production and purification. Therefore modification should be made to increase the immunogenicity of the presented system. PMID:27902762

  18. Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

    PubMed

    Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

    2015-12-01

    Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

  19. DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice.

    PubMed

    Cao, Jun; Jin, Yiqi; Li, Wei; Zhang, Bin; He, Yang; Liu, Hongqiang; Xia, Ning; Wei, Huafeng; Yan, Jian

    2013-08-14

    Although DNA vaccine holds a great potential for cancer immunotherapy, effective long-lasting antitumoral immunity sufficient to induce durable responses in cancer patients remains to be achieved. Considering the pivotal role of dendritic cells (DC) in the antigen processing and presentation, we prepared DC-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragment (scFv) from NLDC-145 antibody specific for DC-restricted surface molecule DEC-205 (scFvNLDC-145), and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models. In vivo targeting assay demonstrated that scFvNLDC-145 specifically delivered DNA vaccine-encoded antigen to DC. Compared with untargeted HER2/neu DNA vaccines, vaccination with scFvNLDC-145-HER2/neu markedly promoted the HER2/neu-specific cellular and humoral immune responses with long-lasting immune memory, resulting in effective protection against challenge of HER2/neu-positive D2F2/E2 breast tumor while ineffective in parental HER2/neu-negative D2F2 breast tumor. More importantly, in combination with temporary depletion of regulatory T cells (Treg) by low-dose cyclophosphamide, vaccination with scFvNLDC-145-HER2/neu induced the regression of established D2F2/E2 breast tumor and significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice. Our findings demonstrate that DC-targeted DNA vaccines for in vivo direct delivery of tumor antigens to DC could induce potent antigen-specific cellular and humoral immune responses and, if additional combination with systemic Treg depletion, was able to elicit an impressively therapeutic antitumoral activity, providing a rationale for further development of this approach for cancer treatment.

  20. Duration of Protection of Rabbits after Vaccination with Bacillus anthracis Recombinant Protective Antigen Vaccine

    DTIC Science & Technology

    2005-12-27

    against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival...vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the...Vaccine 24 (2006) 2530–2536 Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine S.F

  1. Duration of Protection of Rabbits after Vaccination with Bacillus anthracis Recombinant Protective Antigen Vaccine

    DTIC Science & Technology

    2005-12-13

    against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the primary injection, survival...rPA) vaccine was examined against an aerosol spore challenge with the Ames isolate of Bacillus anthracis at 6 and 12 months. At 6 months after the...Vaccine 24 (2006) 2530–2536 Duration of protection of rabbits after vaccination with Bacillus anthracis recombinant protective antigen vaccine S.F

  2. A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease

    PubMed Central

    VIRGINIO, V G; HERNÁNDEZ, A; ROTT, M B; MONTEIRO, K M; ZANDONAI, A F; NIETO, A; ZAHA, A; FERREIRA, H B

    2003-01-01

    Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients’ sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93·1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99·5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58·6% and 89·7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins. PMID:12699422

  3. Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors

    PubMed Central

    Hart, Bryan E.; Asrican, Rose; Lim, So-Yon; Sixsmith, Jaimie D.; Lukose, Regy; Souther, Sommer J. R.; Rayasam, Swati D. G.; Saelens, Joseph W.; Chen, Ching-ju; Seay, Sarah A.; Berney-Meyer, Linda; Magtanong, Leslie; Vermeul, Kim; Pajanirassa, Priyadharshini; Jimenez, Amanda E.; Ng, Tony W.; Tobin, David M.; Porcelli, Steven A.; Larsen, Michelle H.; Schmitz, Joern E.; Haynes, Barton F.; Jacobs, William R.; Lee, Sunhee

    2015-01-01

    The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches. PMID:25924766

  4. MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans.

    PubMed

    Cunha-Neto, E

    1999-02-01

    The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

  5. Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system.

    PubMed

    Yousefi-Rad, Narges; Shokrgozar, Mohammad Ali; Behdani, Mahdi; Moradi-Kalbolandi, Shima; Motamedi-Rad, Mahdieh; Habibi-Anbouhi, Mahdi

    2015-12-01

    Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Induction of Protection against Porcine Cysticercosis by Vaccination with Recombinant Oncosphere Antigens

    PubMed Central

    Flisser, Ana; Gauci, Charles G.; Zoli, André; Martinez-Ocaña, Joel; Garza-Rodriguez, Adriana; Dominguez-Alpizar, Jose Luis; Maravilla, Pablo; Rodriguez-Canul, Rossana; Avila, Guillermina; Aguilar-Vega, Laura; Kyngdon, Craig; Geerts, Stanny; Lightowlers, Marshall W.

    2004-01-01

    Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis. PMID:15322025

  7. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    PubMed

    Miura, Ryuichi; Kooriyama, Takanori; Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

  8. An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

    PubMed Central

    Katz, David; Shi, Wei; Patrusheva, Irina; Perelygina, Ludmila; Gowda, Manjunath S; Krug, Peter W; Filfili, Chadi N; Ward, John A; Hilliard, Julia K

    2012-01-01

    B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV. PMID:23561887

  9. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

    PubMed Central

    Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs. PMID:26162094

  10. Evaluation of a Recombinant Trypanosoma cruzi Mucin-Like Antigen for Serodiagnosis of Chagas' Disease ▿

    PubMed Central

    De Marchi, Claudia R.; Di Noia, Javier M.; Frasch, Alberto C. C.; Amato Neto, Vicente; Almeida, Igor C.; Buscaglia, Carlos A.

    2011-01-01

    Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi and is one of the most important endemic problems in Latin America. Lately, it has also become a health concern in the United States and Europe. Currently, a diagnosis of Chagas' disease and the screening of blood supplies for antiparasite antibodies are achieved by conventional serological tests that show substantial variation in the reproducibility and reliability of their results. In addition, the specificity of these assays is curtailed by antigenic cross-reactivity with sera from patients affected by other endemic diseases, such as leishmaniasis. Here we used a highly sensitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) to evaluate a recombinant protein core of a mucin-like molecule (termed trypomastigote small surface antigen [TSSA]) for the detection of specific serum antibodies in a broad panel of human sera. The same samples were evaluated by CL-ELISA using as the antigen either a mixture of native T. cruzi trypomastigote mucins or an epimastigote extract and, for further comparison, by conventional serologic tests, such as an indirect hemagglutination assay and indirect immunofluorescence assay. TSSA showed ∼87% sensitivity among the seropositive Chagasic panel, a value which was increased up to >98% when only parasitologically positive samples were considered. More importantly, TSSA showed a significant increase in specificity (97.4%) compared to those of currently used assays, which averaged 80 to 90%. Overall, our data demonstrate that recombinant TSSA may be a useful antigen for the immunodiagnosis of Chagas' disease. PMID:21880857

  11. MVA vaccine encoding CMV antigens safely induces durable expansion of CMV-specific T cells in healthy adults.

    PubMed

    La Rosa, Corinna; Longmate, Jeff; Martinez, Joy; Zhou, Qiao; Kaltcheva, Teodora I; Tsai, Weimin; Drake, Jennifer; Carroll, Mary; Wussow, Felix; Chiuppesi, Flavia; Hardwick, Nicola; Dadwal, Sanjeet; Aldoss, Ibrahim; Nakamura, Ryotaro; Zaia, John A; Diamond, Don J

    2017-01-05

    Attenuated poxvirus modified vaccinia Ankara (MVA) is a useful viral-based vaccine for clinical investigation, because of its excellent safety profile and property of inducing potent immune responses against recombinant (r) antigens. We developed Triplex by constructing an rMVA encoding 3 immunodominant cytomegalovirus (CMV) antigens, which stimulates a host antiviral response: UL83 (pp65), UL123 (IE1-exon4), and UL122 (IE2-exon5). We completed the first clinical evaluation of the Triplex vaccine in 24 healthy adults, with or without immunity to CMV and vaccinia virus (previous DryVax smallpox vaccination). Three escalating dose levels (DL) were administered IM in 8 subjects/DL, with an identical booster injection 28 days later and 1-year follow-up. Vaccinations at all DL were safe with no dose-limiting toxicities. No vaccine-related serious adverse events were documented. Local and systemic reactogenicity was transient and self-limiting. Robust, functional, and durable Triplex-driven expansions of CMV-specific T cells were detected by measuring T-cell surface levels of 4-1BB (CD137), binding to CMV-specific HLA multimers, and interferon-γ production. Marked and durable CMV-specific T-cell responses were also detected in Triplex-vaccinated CMV-seronegatives, and in DryVax-vaccinated subjects. Long-lived memory effector phenotype, associated with viral control during CMV primary infection, was predominantly found on the membrane of CMV-specific and functional T cells, whereas off-target vaccine responses activating memory T cells from the related herpesvirus Epstein-Barr virus remained undetectable. Combined safety and immunogenicity results of MVA in allogeneic hematopoietic stem cell transplant (HCT) recipients and Triplex in healthy adults motivated the initiation of a placebo-controlled multicenter trial of Triplex in HCT patients. This trial was registered at www.clinicaltrials.gov as #NCT02506933. © 2017 by The American Society of Hematology.

  12. Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane.

    PubMed Central

    Fehniger, T E; Radolf, J D; Walfield, A M; Cunningham, T M; Miller, J N; Lovett, M A

    1986-01-01

    A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy. Images PMID:3516880

  13. Clinical and parasitological protection in a Leishmania infantum-macaque model vaccinated with adenovirus and the recombinant A2 antigen.

    PubMed

    Grimaldi, Gabriel; Teva, Antonio; Porrozzi, Renato; Pinto, Marcelo A; Marchevsky, Renato S; Rocha, Maria Gabrielle L; Dutra, Miriam S; Bruña-Romero, Oscar; Fernandes, Ana-Paula; Gazzinelli, Ricardo T

    2014-06-01

    Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. The remarkable clinical protection induced by A2 in an animal model that is

  14. Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

    PubMed Central

    Grimaldi, Gabriel; Teva, Antonio; Porrozzi, Renato; Pinto, Marcelo A.; Marchevsky, Renato S.; Rocha, Maria Gabrielle L.; Dutra, Miriam S.; Bruña-Romero, Oscar; Fernandes, Ana-Paula; Gazzinelli, Ricardo T.

    2014-01-01

    Background Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs. Methodology/Principal Findings Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol. Conclusions/Significance The

  15. Antigen-specific induction of peripheral T cell tolerance in vivo by codelivery of DNA vectors encoding antigen and Fas ligand.

    PubMed

    Georgantas, R W; Leong, K W; August, J T

    2000-04-10

    Fas ligand (FasL, CD95L) induces apoptosis in activated T cells with upregulated Fas (CD95) expression through the process termed activation-induced cell death (AICD). We postulated that coexpression of antigen and FasL within individual antigen-presenting cells would lead to antigen-specific activation of T cells and to their consequent deletion by FasL-mediated AICD. A DNA-gelatin coacervate containing transferrin cell ligand, calcium, and the lysosomatropic agent chloroquine, a formulation previously shown to achieve high-level transfection of immune and muscle cells in vivo, was used to codeliver plasmids encoding FasL and antigen. Mice developed a strong cytolytic T cell response to beta-Gal when injected with DNA encoding beta-galactosidase (LacZ) model antigen, either as naked DNA or DNA nanoparticles, but failed to respond when there was concomitant injection of nanoparticles containing both the LacZ and murine FasL DNA vectors. This loss of T cell response was systemic, specific for beta-Gal, complete when nanoparticles were administered before antigen challenge, and decreased the T cell response from prior immunization with LacZ DNA. In effect, this "tolerization" injection induced antigen-specific peripheral tolerance in study mice, and represents a possible approach to the treatment of autoimmune diseases and transplantation rejection.

  16. Evaluation of Babesia bigemina 200 kDa recombinant antigen in enzyme-linked immunosorbent assay.

    PubMed

    Altangerel, Khukhuu; Alhassan, Andy; Iseki, Hiroshi; Sivakumar, Thillaiampalam; Boldbaatar, Damdinsuren; Yokoyama, Naoaki; Igarashi, Ikuo

    2009-07-01

    A truncated fragment of the gene encoding the 200-kDa protein (P200) of Babesia bigemina was cloned into a plasmid vector, pGEX-4 T-1 and expressed in Escherichia coli as a glutathione-S-transferase fused protein. An indirect enzyme-linked immunosorbent assay (ELISA) using the rp200/CT detected specific antibodies in cattle experimentally infected with B. bigemina. Furthermore, the antigen did not cross-react with antibodies to Babesia bovis, a closely related Babesia parasite indicating that rp200/CT is a specific antigen for the diagnosis of B. bigemina infection. Additionally, ELISA using p200/CT and polymerase chain reaction were conducted on serum and corresponding DNA samples obtained from field cattle to evaluate the diagnostic utility of the p200/CT antigen. Results from the current study suggest that p200/CT ELISA is a sensitive and specific method for improved serodiagnosis of B. bigemina infection.

  17. Characterization and expression of multiple alternatively spliced transcripts of the Goodpasture antigen gene region. Goodpasture antibodies recognize recombinant proteins representing the autoantigen and one of its alternative forms.

    PubMed

    Penadés, J R; Bernal, D; Revert, F; Johansson, C; Fresquet, V J; Cervera, J; Wieslander, J; Quinones, S; Saus, J

    1995-05-01

    Collagen IV, the major component of basement membranes, is composed of six distinct alpha chains (alpha 1-alpha 6). Atypically among the collagen IV genes, the exons encoding the carboxyl-terminal region of the human alpha 3(IV) chain undergo alternative splicing. This region has been designated as the Goodpasture antigen because of its reactivity in the kidney and lung with the pathogenic autoantibodies causing Goodpasture syndrome. The data presented in this report demonstrate that, in human kidney, the gene region encompassing the Goodpasture antigen generates at least six alternatively spliced transcripts predicting five distinct proteins that differ in their carboxyl-terminus and retain, except in one case, the exon that harbors the characteristic amino-terminus of the antigen. Goodpasture antibodies specifically recognize recombinant proteins representing the antigen and the alternative form that retains the amino-half of the antigen, suggesting that this moiety could be involved in the in vivo binding of the pathogenic antibodies. Furthermore, the sera of control individuals contain autoantibodies against the antigen that can be differentiated from those causing the syndrome based on their specific reactivities, suggesting that the binding of the pathogenic autoantibodies to a specific determinant likely trigger a distinct and unique cascade of events causing the disease.

  18. Construction and expression of recombinant plasmids encoding type 1 fimbriae of a urinary Klebsiella pneumoniae isolate.

    PubMed Central

    Purcell, B K; Clegg, S

    1983-01-01

    The type 1 fimbriae of Klebsiella pneumoniae have been implicated as important virulence factors in mediating Klebsiella urinary infections. The chromosomally encoded fimbrial genes were cloned by a cosmid cloning technique. Further subcloning was performed with the cloning vehicles pBR322 and pACYC184, and a recombinant plasmid containing the fimbrial genes was constructed. After transformation by this plasmid, both Escherichia coli and Salmonella typhimurium were shown to express fimbriae which reacted with Klebsiella fimbrial antiserum. The approximate location of the relevant genes on the chimeric plasmid was determined by insertion of the transposable element Tn5. Hemagglutination-negative phenotypes were used to estimate the minimum size of the DNA fragment necessary to encode fimbrial biosynthesis and expression. The size of the coding region of this fragment was found to be 5.5 kilobase pairs. PMID:6132874

  19. Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture.

    PubMed

    Mousli, Mohamed; Turki, Imène; Kharmachi, Habib; Saadi, Mohamed; Dellagi, Koussay

    2007-12-01

    The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.

  20. Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays.

    PubMed Central

    Magnarelli, L A; Flavell, R A; Padula, S J; Anderson, J F; Fikrig, E

    1997-01-01

    Serum samples from dogs and equids suspected of having canine or equine borreliosis, respectively, were analyzed in polyvalent enzyme-linked immunosorbent assays (ELISAs) with whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Purified preparations of recombinant antigens included outer surface protein A (OspA), OspB, OspC, OspE, OspF, and p41-G (a fragment of flagellin). Of the 36 dog sera that reacted positively to whole-cell antigen, 32 (88.9%) contained antibodies to one or more recombinant antigens. Reactivities to OspF (88.9% positive) and p41-G (75% positive) were most prevalent. In analyses of 30 equid sera positive in an ELISA with whole cells, 24 (80%) contained antibodies to one or more recombinant antigens. Seropositivities in ELISAs with p41-G (50% positive) and OspF (46.7% positive) were more than twofold greater than in ELISAs with OspA, OspB, or OspC (10 to 20% positive). In parallel tests of eight canine and three equine sera, there was good agreement in results of Western blot (immunoblot) analyses and ELISAs. Although dog and equid sera with antibodies to whole-cell B. burgdorferi frequently reacted positively to one or more recombinant antigens, the inclusion of OspF and p41-G antigens in ELISAs was most useful in the serologic diagnosis of canine and equine borreliosis. PMID:8968901

  1. Antigen-capture blocking enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen to differentiate Transmissible gastroenteritis virus from Porcine respiratory coronavirus antibodies.

    PubMed

    López, Lissett; Venteo, Angel; García, Marga; Camuñas, Ana; Ranz, Ana; García, Julia; Sarraseca, Javier; Anaya, Carmen; Rueda, Paloma

    2009-09-01

    A new commercially available antigen-capture, blocking enzyme-linked immunosorbent assay (antigen-capture b-ELISA), based on baculovirus truncated-S recombinant protein of Transmissible gastroenteritis virus (TGEV) and 3 specific monoclonal antibodies, was developed and evaluated by examining a panel of 453 positive Porcine respiratory coronavirus (PRCoV), 31 positive TGEV, and 126 negative field sera by using another commercially available differential coronavirus b-ELISA as the reference technique to differentiate TGEV- from PRCoV-induced antibodies. The recombinant S protein-based ELISA appeared to be 100% sensitive for TGEV and PRCoV detection and highly specific for TGEV and PRCoV detection (100% and 92.06%, respectively), when qualitative results (positive or negative) were compared with those of the reference technique. In variability experiments, the ELISA gave consistent results when the same serum was evaluated on different wells and different plates. These results indicated that truncated recombinant S protein is a suitable alternative to the complete virus as antigen in ELISA assays. The use of recombinant S protein as antigen offers great advantages because it is an easy-to-produce, easy-to-standardize, noninfectious antigen that does not require further purification or concentration. Those advantages represent an important improvement for antigen preparation, in comparison with other assays in which an inactivated virus from mammalian cell cultures is used.

  2. Usefulness of the recombinant liver stage antigen-3 for an early serodiagnosis of Plasmodium falciparum infection

    PubMed Central

    Lee, Hyeong-Woo; Moon, Sung-Ung; Ryu, Hye-Sun; Kim, Yeon-Joo; Cho, Shin-Hyeong; Chung, Gyung-Tae; Lin, Khin; Na, Byoung-Kuk; Kong, Yoon; Chung, Kyung-Suk

    2006-01-01

    In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection. PMID:16514282

  3. Usefulness of the recombinant liver stage antigen-3 for an early serodiagnosis of Plasmodium falciparum infection.

    PubMed

    Lee, Hyeong-Woo; Moon, Sung-Ung; Ryu, Hye-Sun; Kim, Yeon-Joo; Cho, Shin-Hyeong; Chung, Gyung-Tae; Lin, Khin; Na, Byoung-Kuk; Kong, Yoon; Chung, Kyung-Suk; Kim, Tong-Soo

    2006-03-01

    In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection.

  4. Recombinant Jembrana disease virus proteins as antigens for the detection of antibody to bovine lentiviruses.

    PubMed

    Burkala, E J; Narayani, I; Hartaningsih, N; Kertayadnya, G; Berryman, D I; Wilcox, G E

    1998-09-01

    Jembrana disease virus (JDV) is a recently identified bovine lentivirus causing an acute severe disease syndrome in banteng cattle (Bos javanicus) and a milder disease syndrome in Bos taurus cattle in Indonesia. The virus is closely related genetically to the previously identified bovine lentivirus, bovine immunodeficiency virus (BIV). Recombinant clones were produced which contained the capsid (CA) and transmembrane (TM) subunits of the respective gag and env open reading frames of JDV. The proteins were expressed as fusions to the glutathione-s-transferase (GST) enzyme in Escherichia coli and purification was achieved using affinity chromatography via immobilized reduced glutathione. The soluble recombinant CA and TM antigens of JDV were reacted in western immunoblots with both serum antibodies from JDV-infected Bos javanicus cattle and Bos taurus cattle immunized with BIV. The recombinant CA protein of JDV reacted equally well with both the JDV and BIV antisera. The recombinant TM protein of JDV also reacted with antibody from the JDV infected cattle and with the BIV antisera. The results indicated conservation of immunogenic epitopes of the CA and TM proteins of the two viruses. The production of the recombinant proteins should enable the development of rapid and sensitive serological tests for JDV and BIV, and tools for further study of the immune response to JDV and the differential epidemiology of JDV infections in cattle.

  5. Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients with type-1 autoimmune hepatitis

    PubMed Central

    Costa, M; Rodríguez-Sánchez, J L; Czaja, A J; Gelpí, C

    2000-01-01

    We previously described autoantibodies against a UGA serine tRNA–protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti‐tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47·5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48·8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35·9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec. PMID:10931155

  6. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate.

    PubMed

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen.

  7. Experimental studies of a vaccine formulation of recombinant human VEGF antigen with aluminum phosphate

    PubMed Central

    Pérez Sánchez, Lincidio; Morera Díaz, Yanelys; Bequet-Romero, Mónica; Ramses Hernández, Gerardo; Rodríguez, Yadira; Castro Velazco, Jorge; Puente Pérez, Pedro; Ayala Avila, Marta; Gavilondo, Jorge V

    2015-01-01

    CIGB-247 is a cancer vaccine that is a formulation of a recombinant protein antigen representative of the human vascular endothelial growth factor (VEGF) with a bacterially-derived adjuvant (VSSP). The vaccine has shown an excellent safety profile in mice, rats, rabbits, not-human primates and in recent clinical trials in cancer patients. Response to the vaccine is characterized by specific antibody titers that neutralize VEGF/VEGFR2 binding and a cytotoxic tumor-specific response. To expand our present anti-VEGF active immunotherapy strategies, we have now studied in mice and non-human primates the effects of vaccination with a formulation of our recombinant VEGF antigen and aluminum phosphate adjuvant (hereafter denominated CIGB-247-A). Administered bi-weekly, CIGB-247-A produces high titers of anti-VEGF IgG blocking antibodies in 2 mice strains. Particularly in BALB/c, the treatment impaired subcutaneous F3II mammary tumor growth and reduced the number of spontaneous lung macro metastases, increasing animals' survival. Spleen cells from specifically immunized mice directly killed F3II tumor cells in vitro. CIGB-247-A also showed to be immunogenic in non-human primates, which developed anti-VEGF blocking antibodies and the ability for specific direct cell cytotoxic responses, all without impairing the healing of deep skin wounds or other side effect. Our results support consideration of aluminum phosphate as a suitable adjuvant for the development of new vaccine formulations using VEGF as antigen. PMID:25891359

  8. Durable cytotoxic immune responses against gp120 elicited by recombinant SV40 vectors encoding HIV-1 gp120 +/- IL-15.

    PubMed

    McKee, Hayley J; T'sao, Patricia Y; Vera, Maria; Fortes, Puri; Strayer, David S

    2004-08-23

    BACKGROUND: A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency. METHODS: SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120. RESULTS: Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with >/= 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses. CONCLUSIONS: Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV.

  9. Contrasting Population Structures of the Genes Encoding Ten Leading Vaccine-Candidate Antigens of the Human Malaria Parasite, Plasmodium falciparum

    PubMed Central

    Barry, Alyssa E.; Schultz, Lee; Buckee, Caroline O.; Reeder, John C.

    2009-01-01

    The extensive diversity of Plasmodium falciparum antigens is a major obstacle to a broadly effective malaria vaccine but population genetics has rarely been used to guide vaccine design. We have completed a meta-population genetic analysis of the genes encoding ten leading P. falciparum vaccine antigens, including the pre-erythrocytic antigens csp, trap, lsa1 and glurp; the merozoite antigens eba175, ama1, msp's 1, 3 and 4, and the gametocyte antigen pfs48/45. A total of 4553 antigen sequences were assembled from published data and we estimated the range and distribution of diversity worldwide using traditional population genetics, Bayesian clustering and network analysis. Although a large number of distinct haplotypes were identified for each antigen, they were organized into a limited number of discrete subgroups. While the non-merozoite antigens showed geographically variable levels of diversity and geographic restriction of specific subgroups, the merozoite antigens had high levels of diversity globally, and a worldwide distribution of each subgroup. This shows that the diversity of the non-merozoite antigens is organized by physical or other location-specific barriers to gene flow and that of merozoite antigens by features intrinsic to all populations, one important possibility being the immune response of the human host. We also show that current malaria vaccine formulations are based upon low prevalence haplotypes from a single subgroup and thus may represent only a small proportion of the global parasite population. This study demonstrates significant contrasts in the population structure of P. falciparum vaccine candidates that are consistent with the merozoite antigens being under stronger balancing selection than non-merozoite antigens and suggesting that unique approaches to vaccine design will be required. The results of this study also provide a realistic framework for the diversity of these antigens to be incorporated into the design of next

  10. Making an avipoxvirus encoding a tumor-associated antigen and a costimulatory molecule.

    PubMed

    Howley, Paul M; Diener, Kerrilyn R; Hayball, John D

    2014-01-01

    Fowlpox virus (FPV) is a double-stranded DNA virus with a history of use as a live attenuated vaccine in commercial poultry production systems. FPV is also highly amenable to genetic engineering, with a large cloning capacity and many nonessential sites available for integration, meaning that in recombinant form, several transgenes can be expressed simultaneously. Recombinant FPV has proven an effective prophylactic vaccine vector for other diseases of birds, as well as other animal species (Brun et al., Vaccine 26:6508-6528, 2008). These vectors do not integrate into the host genome nor do they undergo productive replication in mammalian cells; thus they have a proven and impeccable safety profile and have been progressed as prophylactic and therapeutic vaccine vectors for use in humans (Beukema et al., Expert Rev Vaccines 5:565-577, 2006; Lousberg et al., Expert Rev Vaccines 10:1435-1449, 2011). Furthermore, repeated immunization with FPV does not blunt subsequent vaccine responses, presumably because it is replication-defective, and thus larger doses can be routinely administered (Brun et al., Vaccine 26:6508-6528, 2008). This strengthens the case for FPV as a viable platform vaccine vector, as it means it can be used repeatedly in an individual to achieve different immunological outcomes. Here we describe in detail the construction of a recombinant variant of FPV expressing the prostate tumor-associated antigen prostatic acid phosphatase (PAP) in conjunction with the immunostimulatory cytokine, interleukin-2 (IL-2), which, if undertaken under the appropriate regulatory conditions and with approvals in place, would theoretically be amenable to clinical trial applications.

  11. High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

    PubMed

    Kamenetzky, L; Muzulin, P M; Gutierrez, A M; Angel, S O; Zaha, A; Guarnera, E A; Rosenzvit, M C

    2005-12-01

    Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

  12. Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Halsey, Eric S.; Guevara, Carolina; Canal, Enrique; Hall, Eric; Maves, Ryan; Tilley, Drake H.; Kochel, Tadeusz J.; Ching, Wei-Mei

    2013-01-01

    We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia, where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies. PMID:24166046

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

    DTIC Science & Technology

    1986-11-26

    SIE cop AD nCloning and Expression of Genes for Dengue Virus ,4. CJ Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 0ANNUAL...Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 12. PERSONAL AUTHOR(S) Radha K. Padmanabhan, Ph.D. 13a. TYPE OF REPORT 13b. TIME...pVVI and pVVI7 cDNA clones, synthetic peptides homologous to NS5 and NSI regions were synthesized. These peptides are being used at Walter Reed Army

  14. Recombinant measles viruses expressing single or multiple antigens of human immunodeficiency virus (HIV-1) induce cellular and humoral immune responses.

    PubMed

    Liniger, Matthias; Zuniga, Armando; Morin, Teldja Neige Azzouz; Combardiere, Behazine; Marty, Rene; Wiegand, Marian; Ilter, Orhan; Knuchel, Marlyse; Naim, Hussein Y

    2009-05-26

    Recombinant measles viruses (rMV) based on the live attenuated measles vaccine strain (MVb) expressing antigens of HIV-1 clade B were generated by reverse genetics. Recombinants expressing single or double antigens of HIV-1 (rMV-HIV) were genetically highly stable on human diploid cells. The production process of these viruses was essentially similar to the parental MV strain, yielding comparative end titers. Immunization of tg-mice by different regimens and formulations showed potent humoral and cellular immune responses against MV and HIV antigens. Recombinant MV-HIV expressing Gag protein conferred protective immunity in tg-mice after a high-dose pseudochallenge with recombinant vaccinia virus. In addition, rMV-HIV boosted anti-HIV antibodies, in the presence of pre-existing anti-vector antibodies.

  15. Characterisation of antibody responses in pigs induced by recombinant oncosphere antigens from Taenia solium.

    PubMed

    Jayashi, César M; Gonzalez, Armando E; Castillo Neyra, Ricardo; Kyngdon, Craig T; Gauci, Charles G; Lightowlers, Marshall W

    2012-12-14

    Recombinant antigens cloned from the oncosphere life cycle stage of the cestode parasite Taenia solium (T. solium) have been proven to be effective as vaccines for protecting pigs against infections with T. solium. Previous studies have defined three different host protective oncosphere antigens, TSOL18, TSOL16 and TSOL45. In this study, we evaluated the potential for combining the antigens TSOL16 and TSOL18 as a practical vaccine. Firstly, in a laboratory trial, we compared the immunogenicity of the combined antigens (TSOL16/18) versus the immunogenicity of the antigens separately. Secondly, in a field trial, we tested the ability of the TSOL16/18 vaccine to induce detectable antibody responses in animals living under environmental stress and traditionally reared in areas where T. solium cysticercosis is endemic; and finally, we characterised the immune response of the study population. Pigs of 8-16 weeks of age were vaccinated with 200 μg each of TSOL16 and TSOL18, plus 5mg of Quil-A. Specific total IgG, IgG(1) and IgG(2) antibody responses induced by TSOL16 and TSOL18 were determined with ELISA. The immunogenicity of both antigens was retained in the combined TSOL16/18 vaccine. The combined vaccine TSOL16/18 induced detectable specific anti-TSOL18 antibody responses in 100% (113/113) and specific anti-TSOL16 in 99% (112/113) of the vaccinated animals measured at 2 weeks following the booster vaccination. From the two IgG antibody subtypes analysed we found there was stronger response to IgG(2).

  16. Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia virus vaccine.

    PubMed

    Kantor, J; Irvine, K; Abrams, S; Kaufman, H; DiPietro, J; Schlom, J

    1992-07-15

    Human carcinoembryonic antigen (CEA) is a 180-kd glycoprotein expressed in human colorectal, gastric, pancreatic, breast, and non-small-cell lung carcinomas. Previous studies have demonstrated enhanced immune responses to other antigens presented with vaccinia virus proteins via a recombinant vaccinia virus construct. In addition, we have developed a recombinant CEA-vaccinia virus construct, designated rV(WR)-CEA, and have demonstrated humoral anti-CEA responses in mice after immunization with that virus. The goals of this study were (a) to construct a recombinant CEA-vaccinia vaccine in a less virulent vaccinia strain that is potentially safe and effective for treatment of patients whose tumors express CEA and (b) to evaluate the ability of the recombinant CEA-vaccinia vaccine to prevent and reverse tumor growth in mice and to elicit cell-mediated and humoral anti-CEA immune responses. Using the New York City strain of vaccinia virus, which is used in smallpox vaccination and is more attenuated for humans than rV(WR), we derived a recombinant CEA-vaccinia construct, designated rV(NYC)-CEA. The ability of this construct to induce antitumor immunity was evaluated in mice receiving subcutaneous injections of murine colon adenocarcinoma cells expressing the human CEA gene. Administration of rV(NYC)-CEA in mice induced strong anti-CEA antibody responses, as well as CEA-specific cell-mediated responses, including delayed-type hypersensitivity, lymphoproliferative, and cytotoxic responses. Vaccination of mice with the rV(NYC)-CEA rendered them resistant to the growth of subsequently transplanted CEA-expressing tumors. Moreover, when mice were vaccinated 7 days after tumor cell injection, tumor growth was either greatly reduced or eliminated. No toxic effects were observed in any of the mice. These studies demonstrate that antitumor activity can be induced with the use of a recombinant CEA-vaccinia virus construct derived from an attenuated vaccinia strain, and they

  17. Bovine immunoprotection against Rhipicephalus (Boophilus) microplus with recombinant Bm86-Campo Grande antigen.

    PubMed

    Cunha, Rodrigo Casquero; Pérez de León, Adalberto Angel; Leite, Fábio Pereira Leivas; Pinto, Luciano da Silva; Dos Santos Júnior, Alceu Gonçalves; Andreotti, Renato

    2012-01-01

    The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul.

  18. Recombinant antigen-based enzyme-linked immunosorbent assay for diagnosis of Baylisascaris procyonis larva migrans.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Ndao, Momar; Kazacos, Kevin R

    2011-10-01

    Baylisascaris larva migrans is an important zoonotic disease caused by Baylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although a B. procyonis excretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis of Baylisascaris larva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies to Toxocara infection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology of Baylisascaris larva migrans by ELISA.

  19. Evaluation of a recombinant parasite antigen for the diagnosis of lymphatic filariasis.

    PubMed

    Dissanayake, S; Zheng, H; Dreyer, G; Xu, M; Watawana, L; Cheng, G; Wang, S; Morin, P; Deng, B; Kurniawan, L

    1994-06-01

    We evaluated the usefulness of a recombinant parasite antigen (recSXP1) for the serologic diagnosis of lymphatic filariasis. A large proportion of sera from microfilaremic donors living in five different endemic countries (356 of 446 [80%]) contained IgG antibodies to recSXP1, as do sera from approximately 33% of amicrofilaremic patients with acute filarial disease and/or indirect evidence of active filarial infection. Exposure to filarial worms per se does not appear sufficient to elicit an anti-SXP1 antibody response. Thus, this serologic test identifies a large proportion of persons with active lymphatic filariasis among residents of endemic areas.

  20. Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis

    PubMed Central

    Sripa, Jittiyawadee; Brindley, Paul J.; Sripa, Banchob; Loukas, Alex; Kaewkes, Sasithorn; Laha, Thewarach

    2011-01-01

    A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis. PMID:21704728

  1. Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis.

    PubMed

    Sripa, Jittiyawadee; Brindley, Paul J; Sripa, Banchob; Loukas, Alex; Kaewkes, Sasithorn; Laha, Thewarach

    2012-03-01

    A cathepsin B-like cysteine protease belonging to family C1 is abundantly expressed in the transcriptome and proteome of the carcinogenic liver fluke of humans, Opisthorchis viverrini. This enzyme is present in excretory/secretory (ES) products released by parasites cultured in vitro. This study evaluated the performance of recombinant O. viverrini cathepsin B1 (rOv-CB-1) as an antigen for immunodiagnosis of opisthorchiasis. The full length Ov-CB-1 cDNA was cloned and recombinant protein was produced in catalytically active form in Pichia pastoris. The recombinant Ov-CB-1 (rOv-CB-1) was affinity purified via nickel-NTA chromatography and tested in enzyme-linked immunosorbent assays (ELISA) with human sera from an opisthorchiasis endemic area. Sera from egg-positive O. viverrini infections produced a strong IgG antibody response to rOv-CB-1 both in ELISA and immunoblot analysis. The sensitivity and specificity of the ELISA test was 67% and 81%, respectively. These findings support the feasibility of using recombinant Ov-CB-1 in ELISA for the serodiagnosis of human opisthorchiasis. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Glennon, Erin; Ching, Wei-Mei

    2014-01-01

    Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA) using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1) which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33) of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156) of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies. PMID:26904739

  3. Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines.

    PubMed

    Choi, Yejin; Kwon, Seong Yi; Oh, Ho Jung; Shim, Sunbo; Chang, Seokkee; Chung, Hye Joo; Kim, Do Keun; Park, Younsang; Lee, Younghee

    2017-09-01

    The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed. We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months. The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

  4. Antikinetochore and antitopoisomerase I antibodies in systemic scleroderma: comparative study using immunoblotted recombinant antigens, immunofluorescence, and double immunodiffusion.

    PubMed

    Jarzabek-Chorzelska, M; Blaszczyk, M; Kolacinska-Strasz, Z; Chorzelski, T; Jabłońska, S; Maul, G G

    1990-01-01

    In 135 patients with systemic scleroderma, we compared three different methods to determine antinuclear autoantibody (ANA) specificity: indirect immunofluorescence, double immunodiffusion, and, employing recombinant antigens, immunoblotting using both marker autoantigens of this disease. A characteristic Scl-70 antibody pattern was found on HEp-2 cells in 83.8% of the patients, double immunodiffusion was positive for the Scl-70 antibodies in 81.9%, and immunoblot with the recombinant topoisomerase I (Topo I) was positive in 71% of the patients. For the centromere autoantibodies we found a high concordance between the anticentromere antibody (ACA) pattern on HEp-2 cells (27 patients positive) and the detection of recombinant kinetochore in immunoblotting (26 patients positive). The three testing techniques gave comparable results, except that the Topo I recombinant antigen used in immunoblotting reacted strongly with fewer than expected of the known Scl-70-positive sera. However, a method using recombinant antigens expressing all epitopes (rather than one of the epitopes of Topo I) will undoubtedly become the method of choice for detecting antibodies in systemic scleroderma. Using the immunoblotting technique with the recombinant antigens we detected in four patients antibodies against both Topo I and kinetochore. More severe symptoms of systemic scleroderma were found in patients who had both antibodies. The combined presence of both marker autoantibodies is therefore not as rare as previously reported and may predict severe disease.

  5. Recombinant adenovirus encoding NLRP3 RNAi attenuate inflammation and brain injury after intracerebral hemorrhage.

    PubMed

    Yuan, Bangqing; Shen, Hanchao; Lin, Li; Su, Tonggang; Zhong, Shanchuan; Yang, Zhao

    2015-10-15

    Numerous evidence have shown that microglia mediated inflammation plays a pivotal role in the development of brain injury after intracerebral hemorrhage (ICH). Therefore anti-inflammation therapy represents a potentially promising approach to ICH. Recently, NLRP3 inflammasome was discovered to facilitate the inflammatory response. However, the effect of NLRP3 inflammasome after ICH has not been fully studied. To explore the potential of NLRP3 inflammasome, we detected NLRP3 expression, inflammation, brain edema and neurological functions in vitro and in vivo. We found that ICH activated the NLRP3 inflammasome and inflammation. However, NLRP3 RNAi could attenuate inflammation and brain injury after ICH. Therefore, the findings suggested that recombinant adenovirus encoding NLRP3 RNAi might be valuable as a potential strategy for anti-inflammation therapy in ICH. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Expression, purification, immunogenicity, and protective efficacy of a recombinant Tc24 antigen as a vaccine against Trypanosoma cruzi infection in mice.

    PubMed

    Martinez-Campos, Viridiana; Martinez-Vega, Pedro; Ramirez-Sierra, Maria Jesus; Rosado-Vallado, Miguel; Seid, Christopher A; Hudspeth, Elissa M; Wei, Junfei; Liu, Zhuyun; Kwityn, Cliff; Hammond, Molly; Ortega-López, Jaime; Zhan, Bin; Hotez, Peter J; Bottazzi, Maria Elena; Dumonteil, Eric

    2015-08-26

    The Tc24 calcium binding protein from the flagellar pocket of Trypanosoma cruzi is under evaluation as a candidate vaccine antigen against Chagas disease. Previously, a DNA vaccine encoding Tc24 was shown to be an effective vaccine (both as a preventive and therapeutic intervention) in mice and dogs, as evidenced by reductions in T. cruzi parasitemia and cardiac amastigotes, as well as reduced cardiac inflammation and increased host survival. Here we developed a suitable platform for the large scale production of recombinant Tc24 (rTc24) and show that when rTc24 is combined with a monophosphoryl-lipid A (MPLA) adjuvant, the formulated vaccine induces a Th1-biased immune response in mice, comprised of elevated IgG2a antibody levels and interferon-gamma levels from splenocytes, compared to controls. These immune responses also resulted in statistically significant decreased T. cruzi parasitemia and cardiac amastigotes, as well as increased survival following T. cruzi challenge infections, compared to controls. Partial protective efficacy was shown regardless of whether the antigen was expressed in Escherichia coli or in yeast (Pichia pastoris). While mouse vaccinations will require further modifications in order to optimize protective efficacy, such studies provide a basis for further evaluations of vaccines comprised of rTc24, together with alternative adjuvants and additional recombinant antigens. Copyright © 2015. Published by Elsevier Ltd.

  7. Western Immunoblotting with Five Treponema pallidum Recombinant Antigens for Serologic Diagnosis of Syphilis

    PubMed Central

    Sambri, Vittorio; Marangoni, Antonella; Eyer, Christina; Reichhuber, Christine; Soutschek, Erwin; Negosanti, Massimo; D'Antuono, Antonietta; Cevenini, Roberto

    2001-01-01

    Five immunodominant Treponema pallidum recombinant polypeptides (rTpN47, rTmpA, rTpN37, rTpN17, and rTpN15) were blotted onto strips, and 450 sera (200 from blood donors, 200 from syphilis patients, and 50 potentially cross-reactive) were tested to evaluate the diagnostic performance of recombinant Western blotting (recWB) in comparison with in-house whole-cell lysate antigen-based immunoblotting (wclWB) and T. pallidum hemagglutination (MHA-TP) for the laboratory diagnosis of syphilis. None of the serum specimens from blood donors or from potential cross-reactors gave a positive result when evaluated by recWB, wclWB, or MHA-TP. The evaluation of the immunoglobulin G immune response by recWB in sera from patients with different stages of syphilis showed that rTmpA was the most frequently identified antigen (95%), whereas only 41% of the specimens were reactive to rTpN37. The remaining recombinant polypeptides were recognized as follows: rTpN47, 92.5%; rTpN17, 89.5%; and rTpN15, 67.5%. The agreement between recWB and MHA-TP was 95.0% (100% with sera from patients with latent and late disease), and the concordance between wclWB and MHA-TP was 92.0%. The overall concordance between recWB and wclWB was 97.5% (100% with sera from patients with secondary and late syphilis and 94.6 and 98.6% with sera from patients with primary and latent syphilis, respectively). The overall sensitivity of recWB was 98.8% and the specificity was 97.1% with MHA-TP as the reference method. These values for sensitivity and specificity were slightly superior to those calculated for wclWB (sensitivity, 97.1%, and specificity, 96.1%). With wclWB as the standard test, the sensitivity and specificity of recWB were 98.9 and 99.3%, respectively. These findings suggest that the five recombinant polypeptides used in this study could be used as substitutes for the whole-cell lysate T. pallidum antigens and that this newly developed recWB test is a good, easy-to-use confirmatory method for the

  8. Antigen Presentation by Dendritic Cells after Immunization with DNA Encoding a Major Histocompatibility Complex Class II–restricted Viral Epitope

    PubMed Central

    Casares, Sofia; Inaba, Kayo; Brumeanu, Teodor-Doru; Steinman, Ralph M.; Bona, Constantin A.

    1997-01-01

    Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines. PMID:9348305

  9. Expression and antigenicity of recombinant human respiratory syncytial virus glycoproteins having different affinity tags.

    PubMed

    Lee, Han Saem; Kim, A-Reum; Kim, Kisoon; Lee, Wan-Ji; Kim, Sung Soon; Kim, You-Jin

    2017-04-01

    Human respiratory syncytial virus (HRSV) is a main cause of lower respiratory tract infections in infants and the elderly. Glycoprotein (G) is major antigen on the viral surface, and plays a key role for virus entry. Therefore, purification of the glycoprotein of HRSV is critical for the development of HRSV vaccine and serological diagnosis. In this study, we report the design and characterization of glycoprotein engineered rationally to enhance the protein solubility and to facilitate efficient purification. We permuted HRSV glycoproteins with two tags: (i) an immunoglobulin (Ig) M signal peptide and a protein A B domain tag to render HRSV glycoprotein secret into the culture media and (ii) a foldon and 6 × histidine tag with or without transmembrane domain. Three recombinant baculoviruses were constructed: (i) transmembrane-truncated HRSV glycoprotein (amino acid positions 66-298) inserted with the N-terminal IgM signal peptide and protein A B domain (MG-GΔTM), (ii) truncated HRSV glycoprotein (amino acid positions 66-298) fused with a C-terminal foldon and 6 × histidine tag (GΔTM-FH), and (iii) full-length HRSV glycoprotein (amino acid positions 1-298) fused with a C-terminal foldon and 6 × histidine tag (G-FH). Highly soluble recombinant MG-GΔTM protein was clearly purified using one-step affinity chromatography with IgG-sepharose resin, whereas the recombinant G-FH protein and truncated GΔTM-FH were purified partially using nickel-resin. Although, the antigenicity of GΔTM-FH was stronger than highly mannose-rich MG-GΔTM protein, MG-GΔTM induced neutralizing antibodies efficiently in the mice to protect from infectious HRSV. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii.

    PubMed

    Tomás, A L; Cardoso, F; Esteves, F; Matos, O

    2016-11-08

    Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis.

  11. Serological diagnosis of pneumocystosis: production of a synthetic recombinant antigen for immunodetection of Pneumocystis jirovecii

    PubMed Central

    Tomás, A. L.; Cardoso, F.; Esteves, F.; Matos, O.

    2016-01-01

    Diagnosis of Pneumocystis pneumonia (PcP) relies on the detection of P. jirovecii in respiratory specimens obtained by invasive techniques. Thus, the development of a serological test is urgently needed as it will allow the diagnosis of PcP using blood, an inexpensive and non-invasive specimen. This study aims to combine the production of a multi-epitope synthetic recombinant antigen (RSA) and an ELISA test for detection of anti-P. jirovecii antibodies, in order to develop a new approach for PcP diagnosis. The RSA was selected and designed based on the study of the immunogenicity of the carboxyl-terminal domain of the major surface glycoprotein. This antigen was purified and used as an antigenic tool in an ELISA technique for detection of Ig, IgG and IgM antibodies anti-P. jirovecii (patent-pending no. PT109078). Serum specimens from 88 patients previously categorized in distinct clinical subgroups and 17 blood donors, were analysed. The IgM anti-P. jirovecii levels were statistically increased in patients with PcP (p = 0.001) and the ELISA IgM anti-P. jirovecii test presented a sensitivity of 100% and a specificity of 80.8%, when associated with the clinical diagnosis criteria. This innovative approach, provides good insights about what can be done in the future serum testing for PcP diagnosis. PMID:27824115

  12. Sero-diagnosis of surra exploiting recombinant VSG antigen based ELISA for surveillance.

    PubMed

    Sengupta, P P; Rudramurthy, G R; Ligi, M; Roy, M; Balamurugan, V; Krishnamoorthy, P; Nagalingam, M; Singh, L; Rahman, H

    2014-10-15

    Trypanosoma evansi, a haemoflagellate, causes "surra" an important chronic wasting disease of a wide range of wild and domestic herbivorous and carnivorous animals including cattle, buffaloes, camels, horses, etc. The untreated recovered animal can act as a carrier without exhibiting the disease symptoms and can be a source of infection to healthy animals. The diagnosis and subsequent treatment of the carrier animals is helpful to curb the disease. As the parasitaemia in carrier animals is very scanty, the conventional blood smear examination, which is widely practiced in the field, cannot detect such condition. For this purpose improved diagnostics are very much useful for mass sero-screening test such as ELISA. In the present study, the VSG of T. evansi was expressed in prokaryotic system (E. coli) and thereafter its immunoreactivity has been evaluated in immuno blot and enzyme immuno assay. The expressed protein showed 95.6% sensitivity, 98.0% specificity and 0.93 Cohen's kappa value, when compared with standard antigens. The developed antigen has also been validated with field serum samples from bovine, camel and horse collected from different states of India. The data showed that the developed recombinant antigen can be a diagnostic tool to detect carrier animals as well as control of the disease.

  13. Recombinant Antigens from Phlebotomus perniciosus Saliva as Markers of Canine Exposure to Visceral Leishmaniases Vector

    PubMed Central

    Sumova, Petra; Rohousova, Iva; Jimenez, Maribel; Molina, Ricardo; Volf, Petr

    2014-01-01

    Background Phlebotomus perniciosus is the main vector in the western Mediterranean area of the protozoan parasite Leishmania infantum, the causative agent of canine and human visceral leishmaniases. Infected dogs serve as a reservoir of the disease, and therefore measuring the exposure of dogs to sand fly bites is important for estimating the risk of L. infantum transmission. In bitten hosts, sand fly saliva elicits a specific antibody response that reflects the intensity of sand fly exposure. As screening of specific anti-saliva antibodies is limited by the availability of salivary gland homogenates, utilization of recombinant salivary proteins is a promising alternative. In this manuscript we show for the first time the use of recombinant salivary proteins as a functional tool for detecting P. perniciosus bites in dogs. Methodology/Principal Findings The reactivity of six bacterially-expressed recombinant salivary proteins of P. perniciosus, yellow-related protein rSP03B, apyrases rSP01B and rSP01, antigen 5-related rSP07, ParSP25-like protein rSP08 and D7-related protein rSP04, were tested with sera of mice and dogs experimentally bitten by this sand fly using immunoblots and ELISA. In the immunoblots, both mice and canine sera gave positive reactions with yellow-related protein, both apyrases and ParSP25-like protein. A similar reaction for recombinant salivary proteins was observed by ELISA, with the reactivity of yellow-related protein and apyrases significantly correlated with the antibody response of mice and dogs against the whole salivary gland homogenate. Conclusions/Significance Three recombinant salivary antigens of P. perniciosus, yellow-related protein rSP03B and the apyrases rSP01B and rSP01, were identified as the best candidates for evaluating the exposure of mice and dogs to P. perniciosus bites. Utilization of these proteins, or their combination, would be beneficial for screening canine sera in endemic areas of visceral leishmaniases for vector

  14. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems.

  15. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  16. Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

    PubMed

    Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S

    2015-09-01

    The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

  17. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    NASA Astrophysics Data System (ADS)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  18. Recombinant 35-kDa inclusion membrane protein IncA as a candidate antigen for serodiagnosis of Chlamydophila pecorum.

    PubMed

    Mohamad, Khalil Yousef; Rekiki, Abdessalem; Berri, Mustapha; Rodolakis, Annie

    2010-07-14

    Chlamydophila pecorum strains are commonly found in the intestine and vaginal mucus of asymptomatic ruminants and may therefore induce a positive serological response when the animals are tested for C. abortus. They have also been associated with different pathological diseases in ruminants, swine and koala. The aim of this study was to identify specific C. pecorum immunodominant antigens which could be used in ELISA tests allowing to distinguish between animals infected with C. pecorum and those infected with other chlamydial species. A gene encoding 35-kDa inclusion membrane protein incA of C. pecorum was isolated by immunoscreening of the C. pecorum DNA library using ovine anti-C. pecorum antibodies. The recombinant IncA protein did not react with a murine serum directed against C. abortus but did react with a specific monoclonal antibody of C. pecorum and toward several ovine serum samples obtained after experimental infection with different C. pecorum strains. This protein could be a good candidate for specific diagnosis of C. pecorum infection.

  19. Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva

    SciTech Connect

    Toye, P.G.; Metzelaar, M.J.; Wijngaard, P.L.J.

    1995-08-01

    Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5{prime} and 3{prime} regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3{prime} region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

  20. Analysis of Mycobacterium tuberculosis genoma and production of a recombinant protein containing specific B and T cell antigenic determinants--new approaches to second generation antituberculosis vaccines.

    PubMed

    Colizzi, V; Vismara, D; D'Urso, C; Mezzopreti, M F; Lombardi, G; Piccolella, E; Damiani, G; Marelli, P; Campa, M

    1989-01-01

    Tuberculosis is still a major health problem in almost all over the world. Thus, new directions in basic and applied research of tuberculosis are under investigation in several laboratories. In this paper, we provide recent data obtained in our laboratory with the recombinant DNA technology which allow a systematic survey of the microbial genome. Screening of the M. tuberculosis genomic DNA library in the phage lambda gt11 expression vector, using E. coli as surrogate host, has evidenced the possibility of producing recombinant M. tuberculosis proteins recognized by sera from tuberculosis patients and by specific monoclonal antibodies. Using this technology, we have isolated a recombinant protein (molecular weight 130 kilodaltons) which is identified by a murine monoclonal antibody recognizing a mycobacterial cell wall antigenic determinant present on a mycobacterial protein. This protein is only present on the mycobacterial species related to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) and does not crossreact with nonpathogenic Mycobacteria. Since the immune response to mycobacterial infections is cell-mediated, the question arises about the use of M. tuberculosis-specific T lymphocytes to screen this gene bank. Thus, the recombinant mycobacterial protein isolated by antibodies has been then used to stimulate the proliferation of T lymphocytes from patients with tubercular pleuritis. This experiment indicates that the recombinant protein contains antigenic determinants recognized by T cells. Moreover, such protein is able to elicit delayed type hypersensitivity skin reaction in mice immunized or infected with M. tuberculosis and M. bovis. Finally, gene mapping and hybridization studies with native M. tuberculosis DNA confirme the mycobacterial nature of the recombinant DNA insert. Thus a good candidate for the prophylaxis and the immunodiagnosis of tuberculosis has been identified. The identification and selection of genes encoding antigenic

  1. Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

    PubMed

    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

    2004-03-01

    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.

  2. Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen

    PubMed Central

    Zhang, Wei; Dong, Sheng-Fu; Sun, Shu-Hui; Wang, Yuan; Li, Guang-Di; Qu, Di

    2006-01-01

    AIM: To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine. METHODS: We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8+ T cells (CD8+IFN-γ+ T cells) were detected by intracellular cytokine staining at different time points. RESULTS: After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8+ cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8+ Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8+ T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8+ T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8+ T cells induced by hepatitis B virus core gene DNA vaccine. CONCLUSION: DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8+ T cells. PMID:16937447

  3. Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain.

    PubMed

    Shim, Byoung-Shik; Stadler, Konrad; Nguyen, Huan Huu; Yun, Cheol-Heui; Kim, Dong Wook; Chang, Jun; Czerkinsky, Cecil; Song, Man Ki

    2012-09-21

    Sublingual (s.l.) administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs) infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S) (rADV-S) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV. Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n.) administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb. Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV.

  4. Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain

    PubMed Central

    2012-01-01

    Background Sublingual (s.l.) administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs) infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S) (rADV-S) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV. Results Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n.) administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb. Conclusion Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV. PMID:22995185

  5. Crosstalk between ABO and Forssman (FORS) blood group systems: FORS1 antigen synthesis by ABO gene-encoded glycosyltransferases

    PubMed Central

    Yamamoto, Miyako; Cid, Emili; Yamamoto, Fumiichiro

    2017-01-01

    A and B alleles at the ABO genetic locus specify A and B glycosyltransferases that catalyze the biosynthesis of A and B oligosaccharide antigens, respectively, of blood group ABO system which is important in transfusion and transplantation medicine. GBGT1 gene encodes Forssman glycolipid synthase (FS), another glycosyltransferase that produces Forssman antigen (FORS1). Humans are considered to be Forssman antigen-negative species without functional FS. However, rare individuals exhibiting Apae phenotype carry a dominant active GBGT1 gene and express Forssman antigen on RBCs. Accordingly, FORS system was recognized as the 31st blood group system. Mouse ABO gene encodes a cis-AB transferase capable of producing both A and B antigens. This murine enzyme contains the same GlyGlyAla tripeptide sequence as FSs at the position important for the determination of sugar specificity. We, therefore, transfected the expression construct into appropriate recipient cells and examined whether mouse cis-AB transferase may also exhibit FS activity. The result was positive, confirming the crosstalk between the ABO and FORS systems. Further experiments have revealed that the introduction of this tripeptide sequence to human A transferase conferred some, although weak, FS activity, suggesting that it is also involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugars. PMID:28134301

  6. Evidence of Localized Prophage-Host Recombination in the lytA Gene, Encoding the Major Pneumococcal Autolysin ▿

    PubMed Central

    Morales, María; García, Pedro; de la Campa, Adela G.; Liñares, Josefina; Ardanuy, Carmen; García, Ernesto

    2010-01-01

    According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages. PMID:20304992

  7. Generation and Characterization of Human Monoclonal Antibodies Targeting Anthrax Protective Antigen following Vaccination with a Recombinant Protective Antigen Vaccine.

    PubMed

    Chi, Xiangyang; Li, Jianmin; Liu, Weicen; Wang, Xiaolin; Yin, Kexin; Liu, Ju; Zai, Xiaodong; Li, Liangliang; Song, Xiaohong; Zhang, Jun; Zhang, Xiaopeng; Yin, Ying; Fu, Ling; Xu, Junjie; Yu, Changming; Chen, Wei

    2015-05-01

    The anthrax protective antigen (PA) is the central component of the three-part anthrax toxin, and it is the primary immunogenic component in the approved AVA anthrax vaccine and the "next-generation" recombinant PA (rPA) anthrax vaccines. Animal models have indicated that PA-specific antibodies (AB) are sufficient to protect against infection with Bacillus anthracis. In this study, we investigated the PA domain specificity, affinity, mechanisms of neutralization, and synergistic effects of PA-specific antibodies from a single donor following vaccination with the rPA vaccine. Antibody-secreting cells were isolated 7 days after the donor received a boost vaccination, and 34 fully human monoclonal antibodies (hMAb) were identified. Clones 8H6, 4A3, and 22F1 were able to neutralize lethal toxin (LeTx) both in vitro and in vivo. Clone 8H6 neutralized LeTx by preventing furin cleavage of PA in a dose-dependent manner. Clone 4A3 enhanced degradation of nicked PA, thereby interfering with PA oligomerization. The mechanism of 22F1 is still unclear. A fourth clone, 2A6, that was protective only in vitro was found to be neutralizing in vivo in combination with a toxin-enhancing antibody, 8A7, which binds to domain 3 of PA and PA oligomers. These results provide novel insights into the antibody response elicited by the rPA vaccine and may be useful for PA-based vaccine and immunotherapeutic cocktail design.

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

    DTIC Science & Technology

    1990-12-12

    Della-Porta and Westaway, 1977; Kitano et al., 1974; Heinz et al., 1981). In order to develop a subunit vaccine against dengue virus, it is important to...Antigens for Rapid Diagnosis and Vaccine Development DPC TAB 0 A .. asin]o ANNUAL PROGRESS REPORT ’Q;-10.v&,,,d 0 by By SAv.ailability Caote# Radha Krishnan...Type 2 Encoded Antigens for Rapid Diagnosis and Vaccine Development 12. PERSONAL AUTHOR(S) Radha K. Padmanabhan 13a. TYPE OF REPORT 13b. TIME COVERED

  9. Host Immunization with Recombinant Proteins to Screen Antigens for Tick Control.

    PubMed

    Galay, Remil Linggatong; Miyata, Takeshi; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

    2016-01-01

    Ticks (Parasitiformes: Ixodida) are known for their obligate blood feeding habit and their role in transmitting pathogens to various vertebrate hosts. Tick control using chemical acaricides is extensively used particularly in livestock management, but several disadvantages arise from resistance development of many tick species, and concerns on animal product and environmental contamination. Vaccination offers better protection and more cost-effective alternative to application of chemical acaricides, addressing their disadvantages. However, an ideal anti-tick vaccine targeting multiple tick species and all the tick stages is still wanting. Here, we describe the procedures involved in the evaluation of a vaccine candidate antigen against ticks at the laboratory level, from the preparation of recombinant proteins, administration to the rabbit host and monitoring of antibody titer, to tick infestation challenge and determination of the effects of immunization to ticks.

  10. Robust antigen-specific humoral immune responses to sublingually delivered adenoviral vectors encoding HIV-1 Env: association with mucoadhesion and efficient penetration of the sublingual barrier.

    PubMed

    Domm, William; Brooks, Lauren; Chung, Hung Li; Feng, Changyong; Bowers, William J; Watson, Gene; McGrath, James L; Dewhurst, Stephen

    2011-09-16

    The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) - unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery.

  11. Robust Antigen-Specific Humoral Immune Responses to Sublingually Delivered Adenoviral Vectors Encoding HIV-1 Env: Association with Mucoadhesion and Efficient Penetration of the Sublingual Barrier

    PubMed Central

    Domm, William; Brooks, Lauren; Chung, Hung Li; Feng, Changyong; Bowers, William J.; Watson, Gene; McGrath, James L.; Dewhurst, Stephen

    2011-01-01

    The efficient induction of virus-specific mucosal antibodies is an important unmet objective in Human Immunodeficiency Virus Type-1 (HIV-1) vaccine research. One promising approach is sublingual (SL) immunization. We examined the effectiveness of SL delivery of two different viral vectors: (i) a recombinant adenovirus (rAd5), and (ii) a Herpes Simplex Virus Type-1 amplicon vector (HSV-1). Initial in vitro videomicroscopy experiments showed that rAd5 particles were trapped in saliva (i.e., that Ad5 was mucoadhesive) - unlike HSV-1 virions, which migrated freely in both saliva and water. In vivo imaging studies in mice revealed that only the rAd5 vector efficiently transduced the SL epithelium. Consistent with this, SL delivery of an rAd5 encoding HIV-1 envelope glycoprotein (Env) resulted in robust antigen-specific antibody responses in plasma and in vaginal washes, whereas SL delivery of a HSV-1 amplicon vector encoding HIV-1 Env failed to elicit Env-specific antibodies. In contrast, both vectors elicited equivalent humoral responses following intramuscular (IM) delivery. Finally, SL delivery of the rAd5:Env vector resulted in elevated levels of Env-specific serum IgA, and vaginal IgA and IgG, when compared to IM delivery of the same vector. These results findings shed light on vector properties (mucoadhesion, penetration of the sublingual barrier) which may be important for the induction of potent humoral immune responses following sublingual vector administration. Our data also show that SL delivery of an Env-encoding rAd5 vector can elicit a potent antigen-specific mucosal antibody response in the absence of adjuvant. Overall, these findings support the further exploration of the SL delivery route for HIV-1 vaccine delivery. PMID:21801777

  12. Vaccination with a recombinant Saccharomyces cerevisiae expressing a tumor antigen breaks immune tolerance and elicits therapeutic antitumor responses

    PubMed Central

    Wansley, Elizabeth K.; Chakraborty, Mala; Hance, Kenneth W.; Bernstein, Michael B.; Boehm, Amanda L.; Guo, Zhimin; Quick, Deborah; Franzusoff, Alex; Greiner, John W.; Schlom, Jeffrey; Hodge, James W.

    2009-01-01

    Purpose Saccharomyces cerevisiae, a nonpathogenic yeast, has previously been used as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumorburden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic mice (where CEA is a self-antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity. Experimental Design CEA-transgenic mice were vaccinated with yeast-CEA, and CD4+ and CD8+ T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and subcutaneous pancreatic tumor models. Results These studies demonstrate that recombinant yeast can break tolerance and that a) yeast-CEA constructs elicit both CEA-specific CD4+ and CD8+ T-cell responses; b) repeated yeast-CEA administration causes increased antigen-specific T-cell responses after each vaccination; c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and subcutaneous pancreatic tumor models. Conclusions Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-transgenic mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols. PMID:18594015

  13. Effects of vector fusion peptides on the conformation and immune reactivity of epitope-shuffled, recombinant multi-epitope antigens.

    PubMed

    Wang, Jian; Lin, Yahui; Cai, Pengfei; Wang, Heng

    2011-01-01

    The use of multi-epitopes has been considered as a promising strategy to overcome the obstacle of antigenic variation in malarial vaccine development. Previously, we constructed a multi-epitope artificial antigen, Malaria Random Constructed Antigen-1(M.RCAg-1), to optimize expression of the antigen, and we subcloned the gene into three prokaryotic expression vectors that contain different fusion tags at the N-terminus. Three recombinant proteins expressed by these vectors, named M.RCAg-1/Exp.V-1, V-2, and V-3, were purified after the cleavage of the fusion tag. All three recombinant proteins were able to induce similar levels of antigenicity in BALB/c murine models. However, the antibody responses against the individual epitope peptides of the recombinant products were dramatically different. Additionally, the different epitopes elicited various CD4(+) T-cell responses, as shown by the resulting lymphocyte proliferation and varied IFN-γ and IL-4 levels determined by EILSPOT; however, each could be distinctly recognized by sera derived from malaria patients. Additionally, the rabbit antibody induced by these proteins showed diverse efficacy in malaria parasite growth inhibition assays in vitro. Furthermore, analysis via circular dichroism spectroscopy confirmed that the secondary structure was different among these recombinant proteins. These results suggest that the expressed multi-epitope artificial antigens originating from the different vector fusion peptides indeed affect the protein folding and, subsequently, the epitope exposure. Thus, these proteins are able to induce both distinct humoral and cellular immune responses in animal models, and they affect the efficacy of immune inhibition against the parasite. This work should lead to a further understanding of the impact of vector fusion peptides on the conformation and immune reactivity of recombinant proteins and could provide a useful reference for the development of artificial multi-epitope vaccines.

  14. Specific immunoassays for endocrine disruptor monitoring using recombinant antigens cloned by degenerated primer PCR.

    PubMed

    Ferraz, Natalia; Carnevia, Daniel; Nande, Gonzalo; Rossotti, Martin; Miguez, María N; Last, Jerold A; Gonzalez-Sapienza, Gualberto

    2007-12-01

    Vitellogenin (VTG) and choriogenin (CHO) are valuable biomarkers of endocrine-disrupting compound (EDC) exposure in fish. Existing immunoassays are limited to a few species, which restricts their use for the analysis of local wildlife sentinels. Using C. facetum as a relevant South American model fish, this work presents a new strategy for the preparation of antibodies to VTG and CHO, with zero cross-reactivity with fish serum components. Recombinant fragments of Cichlasoma facetum VTG (280-mer) and CHO (223-mer) were prepared by degenerate primer RT-PCR and expression in E. coli. Polyclonal and monoclonal antibodies prepared with these antigens were used to develop rapid dotblot assays for VTG and CHO. Both the polyclonal and monoclonal antibodies prepared with the recombinant antigens reacted against the native proteins adsorbed on to nitrocellulose allowing the set up of sensitive dotblot assays. The VTG assay was further validated with spiked samples and purified native VTG. Exposure experiments with several estrogenic compounds revealed the potential of C. facetum as a sensitive biomonitor that produced measurable responses at concentrations of 100 ng L(-1) of 17-beta-estradiol, 100 ng L(-1) of ethynylestradiol, and 6.6 microg L(-1) of nonylphenol. The approach described here may be applied to other native species to produce highly specific and sensitive rapid tests. It may be particularly advantageous for species that cannot be kept in captivity or when homogeneous purification of the immunizing proteins is particularly challenging. In conclusion, we present a novel approach to develop a strategy for the generation of immunoassay reagents for vitellogenin (VTG) and choriogenin (CHO), which will facilitate regional studies on the impact of endocrine-disrupting chemicals on local wildlife.

  15. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

    PubMed Central

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  16. Partial protective of chickens against Eimeria tenella challenge with recombinant EtMIC-1 antigen.

    PubMed

    Qi, N S; Wang, Y Y; Liao, S Q; Wu, C Y; Lv, M N; Li, J; Tong, Z X; Sun, M F

    2013-06-01

    Eimeria tenella microneme protein 1 (EtMIC-1) is highly conserved with TgMIC-2, which is involved in parasite binding specifically to host cells. Little is known about the immune responses and protective efficacy against E. tenella infection with EtMIC-1 antigen. In the present study, the recombinant proteins of E. tenella mature MIC-1 and adhesive domain (von Willebrand factor type A domain, EtMIC-1-VD) were obtained, protective efficacy against E.tenella infection and the mucosal immune response, which is induced in broilers was evaluated. The antibody levels and the transcription profiles of cytokine of chickens, such as interleukin-12 (IL-12) and interferon-γ (IFN-γ), were detected after being immunized three times with the recombinant EtMIC-1 and EtMIC-1-VD by ELISA assay and quantitative real-time PCR, respectively. The results showed that both groups of chickens, after being immunized with 100 μg EtMIC-1 or EtMIC-1-VD antigen, induced about tenfold higher IgG levels compared to the nonimmune groups. The transcription profiles of IL-12 and IFN-γ of the immunized groups were significantly higher than the control groups as well. The anticoccidial index of the group immunized with 100 μg EtMIC-1 and the group immunized with 100 μg EtMIC-1-VD were 167.2 and 165.5, respectively, which are significantly higher than low-dose immunized groups and challenged control groups. Our data suggests that VD domain is the key functional structure of EtMIC-1 that could trigger a significant humoral and cellular response against E. tenella infection, and EtMIC-1 had the potential in imparting partial protection in chickens against homologous challenge.

  17. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    PubMed

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner; Denner, Joachim

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  18. Comparison of Recombinant Trypanosoma cruzi Peptide Mixtures versus Multiepitope Chimeric Proteins as Sensitizing Antigens for Immunodiagnosis▿

    PubMed Central

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S.; Pujato, Nazarena; Ribone, María E.; Lagier, Claudia M.; Marcipar, Iván S.

    2009-01-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp. PMID:19339486

  19. Comparison of recombinant Trypanosoma cruzi peptide mixtures versus multiepitope chimeric proteins as sensitizing antigens for immunodiagnosis.

    PubMed

    Camussone, Cecilia; Gonzalez, Verónica; Belluzo, María S; Pujato, Nazarena; Ribone, María E; Lagier, Claudia M; Marcipar, Iván S

    2009-06-01

    The aim of this work was to determine the best strategy to display antigens (Ags) on immunochemical devices to improve test selectivity and sensitivity. We comparatively evaluated five Trypanosoma cruzi antigenic recombinant peptides, chose the three more sensitive ones, built up chimeras bearing these selected Ags, and systematically compared by enzyme-linked immunosorbent assay the performance of the assortments of those peptides with that of the multiepitope constructions bearing all those peptides lineally fused. The better-performing Ags that were compared included peptides homologous to the previously described T. cruzi flagellar repetitive Ag (here named RP1), shed acute-phase Ag (RP2), B13 (RP5), and the chimeric recombinant proteins CP1 and CP2, bearing repetitions of RP1-RP2 and RP1-RP2-RP5, respectively. The diagnostic performances of these Ags were assessed for discrimination efficiency by the formula +OD/cutoff value (where +OD is the mean optical density value of the positive serum samples tested), in comparison with each other either alone, in mixtures, or as peptide-fused chimeras and with total parasite homogenate (TPH). The discrimination efficiency values obtained for CP1 and CP2 were 25% and 52% higher, respectively, than those of their individual-Ag mixtures. CP2 was the only Ag that showed enhanced discrimination efficiency between Chagas' disease-positive and -negative samples, compared with TPH. This study highlights the convenience of performing immunochemical assays using hybrid, single-molecule, chimeric Ags instead of peptide mixtures. CP2 preliminary tests rendered 98.6% sensitivity when evaluated with a 141-Chagas' disease-positive serum sample panel and 99.4% specificity when assessed with a 164-Chagas' disease-negative serum sample panel containing 15 samples from individuals infected with Leishmania spp.

  20. Successful immunization of naturally reared pigs against porcine cysticercosis with a recombinant oncosphere antigen vaccine

    PubMed Central

    Jayashi, César M.; Kyngdon, Craig T.; Gauci, Charles G.; Gonzalez, Armando E.; Lightowlers, Marshall W.

    2012-01-01

    Taenia solium causes cysticercosis in pigs and taeniasis and neurocysticercosis in humans. Oncosphere antigens have proven to be effective as vaccines to protect pigs against an experimental infection with T. solium. A pair-matched vaccination trial field, using a combination of two recombinant antigens, TSOL16 and TSOL18, was undertaken in rural villages of Peru to evaluate the efficacy of this vaccine under natural conditions. Pairs of pigs (n = 137) comprising one vaccinated and one control animal, were allocated to local villagers. Animals received two vaccinations with 200 μg of each of TSOL16 and TSOL18, plus 5 mg Quil-A. Necropsies were performed 7 months after the animals were distributed to the farmers. Vaccination reduced 99.7% and 99.9% (p < 0.01) the total number of cysts and the number of viable cysts, respectively. Immunization with the TSOL16–TSOL18 vaccines has the potential to control T. solium transmission in areas where the disease is endemic, reducing the source for tapeworm infections in humans. PMID:22541797

  1. Successful immunization of naturally reared pigs against porcine cysticercosis with a recombinant oncosphere antigen vaccine.

    PubMed

    Jayashi, César M; Kyngdon, Craig T; Gauci, Charles G; Gonzalez, Armando E; Lightowlers, Marshall W

    2012-09-10

    Taenia solium causes cysticercosis in pigs and taeniasis and neurocysticercosis in humans. Oncosphere antigens have proven to be effective as vaccines to protect pigs against an experimental infection with T. solium. A pair-matched vaccination trial field, using a combination of two recombinant antigens, TSOL16 and TSOL18, was undertaken in rural villages of Peru to evaluate the efficacy of this vaccine under natural conditions. Pairs of pigs (n=137) comprising one vaccinated and one control animal, were allocated to local villagers. Animals received two vaccinations with 200 μg of each of TSOL16 and TSOL18, plus 5mg Quil-A. Necropsies were performed 7 months after the animals were distributed to the farmers. Vaccination reduced 99.7% and 99.9% (p<0.01) the total number of cysts and the number of viable cysts, respectively. Immunization with the TSOL16-TSOL18 vaccines has the potential to control T. solium transmission in areas where the disease is endemic, reducing the source for tapeworm infections in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Vaccination with Brucella abortus recombinant in vivo-induced antigens reduces bacterial load and promotes clearance in a mouse model for infection.

    PubMed

    Lowry, Jake E; Isaak, Dale D; Leonhardt, Jack A; Vernati, Giulia; Pate, Jessie C; Andrews, Gerard P

    2011-03-11

    Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5 x 10⁴ CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of

  3. Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen

    PubMed Central

    Burman, Alison; Jackson, Terry; Ren, Jingshan; Loureiro, Silvia; Jones, Ian M.; Fry, Elizabeth E.; Stuart, David I.; Charleston, Bryan

    2013-01-01

    Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals. PMID:23544011

  4. Control of Boophilus microplus populations in grazing cattle vaccinated with a recombinant Bm86 antigen preparation.

    PubMed

    Rodríguez, M; Penichet, M L; Mouris, A E; Labarta, V; Luaces, L L; Rubiera, R; Cordovés, C; Sánchez, P A; Ramos, E; Soto, A

    1995-04-01

    Current methods for the control of cattle tick Boophilus microplus infestations are not effective and the parasite remains a serious problem for the cattle industry in tropical and subtropical areas. Recently, we developed a vaccine against B. microplus employing a recombinant Bm86 (rBm86) antigen preparation (Gavac, Heber Biotec) and it was shown to induce a protective response in vaccinated animals under controlled conditions. Here we show that, under field conditions in grazing cattle, the vaccine is able to control B. microplus populations. Two parasite-free farms were employed for the study. In the first farm, animals were vaccinated with the recombinant vaccine, while, in the second, animals received a saline injection in adjuvant. After immunization, animals were artificially infected and the infestation rate was recorded. Over the 33 weeks of the experiment, the infestation rate was lower in the vaccinated group compared with the control group. At the end of the experiment it was necessary to use chemicals in the control farm after serious losses in production and animals.

  5. Evaluation of recombinant Lig antigen-based ELISA for detection of leptospiral antibodies in canine sera.

    PubMed

    La-Ard, Anchalee; Amavisit, Patamaporn; Sukpuaram, Thavajchai; Wajjwalku, Worawidh

    2011-01-01

    Abstract. The objectives of this study were to clone the conserved region of leptospiral immunoglobulin-like protein (lig) gene and evaluate the utility of the recombinant Lig as an ELISA antigen for detection of leptospiral antibodies in canine sera. Leptospira kirschneri serovar Grippotyposa strain Moskva V was chosen to be a target for cloning the conserved region of Lig gene. This assay was evaluated with canine sera (n = 91) that were MAT-negative (< 1:100 dilution) and sera (n = 103) that were MAT-positive (> or = 1:100 dilution) using 24 serovars. The ELISA showed a relative sensitivity as compared to MAT of 84.5% whereas the specificity was 76.9%. This assay is simple and can be routinely prepared in large amounts. It was concluded that the GST.Lig recombinant protein-based ELISA could be used as a screening test for serodiagnosis of canine leptospirosis with also for confirmation of MAT-positive test results.

  6. Further development of a recombinant feline herpesvirus type 1 vector expressing feline calicivirus immunogenic antigen.

    PubMed

    Yokoyama, N; Fujita, K; Damiani, A; Sato, E; Kurosawa, K; Miyazawa, T; Ishiguro, S; Mochizuki, M; Maeda, K; Mikami, T

    1998-06-01

    We previously reported the attenuation of thymidine kinase (TK) deficient mutant (C7301dlTK) of feline herpesvirus type 1 (FHV-1) in cats and the construction of a recombinant FHV-1 (C7301dlTK-Cap) inserted a precursor capsid gene of feline calcivirus (FCV) into the TK deletion locus of the C7301dlTK. In this study, we constructed a further improved recombinant FHV-1 (dlTK(gCp)-Cap) carrying a putative FHV-1 gC promoter sequence upstream of the FCV precursor capsid gene of the C7301dlTK-Cap. Growth kinetics of the dlTK(gCp)-Cap in cell cultures was similar to those of C7301dlTK and C7301dlTK-Cap. A strong expression of FCV immunogenic antigen by dlTK(gCp)-Cap was confirmed by indirect immunofluorescence and enzyme-linked immunosorbent assays. In addition, one vaccination with dlTK(gCp)-Cap protected cats more effective against subsequent virulent FCV challenge than that with C7301dlTK-Cap.

  7. Messenger RNA Sequence Rather than Protein Sequence Determines the Level of Self-synthesis and Antigen Presentation of the EBV-encoded Antigen, EBNA1

    PubMed Central

    Tellam, Judy T.; Lekieffre, Lea; Zhong, Jie; Lynn, David J.; Khanna, Rajiv

    2012-01-01

    Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to

  8. Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1.

    PubMed

    Tellam, Judy T; Lekieffre, Lea; Zhong, Jie; Lynn, David J; Khanna, Rajiv

    2012-12-01

    Unique purine-rich mRNA sequences embedded in the coding sequences of a distinct group of gammaherpesvirus maintenance proteins underlie the ability of the latently infected cell to minimize immune recognition. The Epstein-Barr virus nuclear antigen, EBNA1, a well characterized lymphocryptovirus maintenance protein has been shown to inhibit in cis antigen presentation, due in part to a large internal repeat domain encoding glycine and alanine residues (GAr) encoded by a purine-rich mRNA sequence. Recent studies have suggested that it is the purine-rich mRNA sequence of this repeat region rather than the encoded GAr polypeptide that directly inhibits EBNA1 self-synthesis and contributes to immune evasion. To test this hypothesis, we generated a series of EBNA1 internal repeat frameshift constructs and assessed their effects on cis-translation and endogenous antigen presentation. Diverse peptide sequences resulting from alternative repeat reading frames did not alleviate the translational inhibition characteristic of EBNA1 self-synthesis or the ensuing reduced surface presentation of EBNA1-specific peptide-MHC class I complexes. Human cells expressing the EBNA1 frameshift variants were also poorly recognized by antigen-specific T-cells. Furthermore, a comparative analysis of the mRNA sequences of the corresponding repeat regions of different viral maintenance homologues highlights the high degree of identity between the nucleotide sequences despite very little homology in the encoded amino acid sequences. Based on these combined observations, we propose that the cis-translational inhibitory effect of the EBNA1 internal repeat sequence operates mechanistically at the nucleotide level, potentially through RNA secondary structural elements, and is unlikely to be mediated through the GAr polypeptide. The demonstration that the EBNA1 repeat mRNA sequence and not the encoded protein sequence underlies immune evasion in this class of virus suggests a novel approach to

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

    DTIC Science & Technology

    1988-10-31

    and the Administrative Practices Suppliments, as indicated in the Memorandum of Understanding and Agreement, approved by the Institutional Biosafety...00 0 Cloning and Expression of Genes for Dengue Virus (Type-2 Encoded-Antigens for Rapid ODiagnosis and Vaccine DevelopmentN| ANNUAL PROGRESS REPORT...CI DOD DISTRIBUTION STATEMENT Approved for Public Release; distribution unlimited The findings in this report are not to be construed as an official

  10. 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.

  11. 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

  12. The Cryptococcus neoformans Gene DHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

    PubMed Central

    Mandel, M. Alejandra; Grace, Greg G.; Orsborn, Kris I.; Schafer, Fredda; Murphy, Juneann W.; Orbach, Marc J.; Galgiani, John N.

    2000-01-01

    When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic

  13. A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV) antigens

    PubMed Central

    2010-01-01

    Background Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells. Results We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68). These were rearranged in line format and validated with pre-characterized sera. Conclusions The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens. PMID:20646309

  14. A systematic approach for the identification of novel, serologically reactive recombinant Varicella-Zoster Virus (VZV) antigens.

    PubMed

    Vizoso Pinto, Maria G; Pfrepper, Klaus-Ingmar; Janke, Tobias; Noelting, Christina; Sander, Michaela; Lueking, Angelika; Haas, Juergen; Nitschko, Hans; Jaeger, Gundula; Baiker, Armin

    2010-07-20

    Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells. We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens (ORFs 1, 4, 14, 49, and 68). These were rearranged in line format and validated with pre-characterized sera. The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens.

  15. Expression of VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes of Epstein-Barr virus in Pichia pastoris and application of the products in a screening test for patients with nasopharyngeal carcinoma.

    PubMed

    Hu, Bo; Hong, Guoqiang; Li, Zhaoxia; Xu, Jue; Zhu, Zhenyu; Li, Lin

    2007-05-01

    EBV (Epstein-Barr virus) serological tests have been used for many years as accessory diagnostic predictors of NPC (nasopharyngeal carcinoma). To date, IF (indirect immunofluorescence) assays still serve as the 'gold standard' for EBV serodiagnosis. However, IF assays are time-consuming, unsuitable for automatic handling and difficult to standardize. This makes their application in mass screening of populations inconvenient. Some of the technical difficulties associated with IF have been overcome by the development of specific ELISAs, but, at present, high sensitivity and specificity cannot be achieved simultaneously by using recombinant protein-based ELISAs, as the diagnostic value of different fragments of EBV in NPC is different. In an attempt to determine a suitable recombinant EBV protein for diagnostic purposes, fragments of EBV VCA (viral capsid antigen) and EBNA1 (Epstein-Barr-virus-encoded nuclear antigen 1) genes were expressed in the methylotrophic yeast Pichia pastoris, and a novel ELISA was established using P. pastoris-expressed VCA-BALF4 [aa (amino acids) 287-623; the BALF4 gene encodes the EBV glycoprotein gp125], EBNA1 (aa 390-641) and VCA-BFRF3 (the gene BFRF3 encodes a viral structural capsid protein or tegument protein VCA p18) proteins. Serum samples were collected from patients with NPC and healthy controls and were tested using this ELISA. The sensitivity of VCA-BFRF3, VCA-BALF4 and EBNA1 tests in the NPC sera were 65.0 (195/300), 76.3 (229/300) and 81.4% (244/300) respectively, whereas the specificity of normal individuals were 92 (460/500), 96 (480/500) and 95.8% (479/500). The optimum combination is VCA-BALF4 plus EBNA1, which identified 90.3% (271/300) of the NPC patients and had a specificity of 92.8% (464/500) for normal individuals. The results obtained from the evaluation of three antibodies to EBV as markers for detecting NPC suggests that a combination of EBNA1 (aa 390-641) and VCA-BALF4 (aa 287-623) assays would give better results

  16. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites.

    PubMed

    Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

    2016-03-01

    Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody-mediated reactions also in other host species.

  17. Recombinant Salivary Proteins of Phlebotomus orientalis are Suitable Antigens to Measure Exposure of Domestic Animals to Sand Fly Bites

    PubMed Central

    Sima, Michal; Ferencova, Blanka; Warburg, Alon; Rohousova, Iva; Volf, Petr

    2016-01-01

    Background Certain salivary proteins of phlebotomine sand flies injected into the host skin during blood-feeding are highly antigenic and elicit strong antibody-mediated immune responses in repeatedly-exposed hosts. These antibodies can be measured by enzyme-linked immuno sorbent assays (ELISAs) using salivary gland homogenates (SGHs) as the source of antigens and serve as a markers for exposure to biting sand flies. Large-scale screening for anti-sand fly saliva antibodies requires replacement of SGH with recombinant salivary proteins. In East Africa, Phlebotomus orientalis is the main vector of Leishmania donovani, a trypanosomatid parasite causing visceral leishmaniasis. We tested recombinant salivary proteins derived from Ph. orientalis saliva to study exposure of domestic animals to this sand fly species. Methodology/Principal Findings Antigenic salivary proteins from Ph. orientalis were identified by immunoblot and mass spectrometry. Recombinant apyrase rPorSP15, yellow-related protein rPorSP24, ParSP25-like protein rPorSP65, D7-related protein rPorSP67, and antigen 5-related protein rPorSP76 were tested using ELISA with sera of domestic animals from L. donovani foci in Ethiopia where Ph. orientalis is present. Our results highlighted recombinant yellow-related protein rPorSP24 as the most promising antigen, displaying a high positive correlation coefficient as well as good sensitivity and specificity when compared to SGH. This recombinant protein was the most suitable one for testing sera of dogs, sheep, and goats. In addition, a different antigen, rPorSP65 was found efficacious for testing canine sera. Conclusions/Significance Recombinant salivary proteins of Ph. orientalis, specifically rPorSP24, were shown to successfully substitute SGH in serological experiments to measure exposure of domestic animals to Ph. orientalis, the vector of L. donovani. The results suggest that rPorSP24 might be a suitable antigen for detecting anti-Ph. orientalis antibody

  18. Oral vaccination with attenuated Salmonella enterica strains encoding T-cell epitopes from tumor antigen NY-ESO-1 induces specific cytotoxic T-lymphocyte responses.

    PubMed

    Meng, Jia-Zi; Dong, Yu-Jun; Huang, He; Li, Shuang; Zhong, Yi; Liu, Shu-Lin; Wang, Yue-Dan

    2010-06-01

    Bacterial fimbriae can accept foreign peptides and display them on the cell surface. A highly efficient gene replacement method was used to generate peptide vaccines based on Salmonella enterica serovar Typhimurium SL3261. The T-cell epitopes (NY-ESO-1 p157-165 and p157-167) from NY-ESO-1, which is a promising target antigen in patients for the specific immune recognition of cancer, were incorporated into the gene encoding AgfA (the major subunit protein of thin aggregative fimbriae of Salmonella) by replacing an equal length of the DNA segment. To improve cytotoxic T-lymphocyte recognition, both termini of the peptide were flanked by double alanine (AA) residues. Immunofluorescence microscopy with AgfA-specific antiserum verified the expression of chimeric AgfA, which was also proved by a Congo red binding assay. Oral immunizations of HLA-A*0201 transgenic mice with recombinant SL3261 strains encoding NY-ESO-1 p157-165 or p157-167 induced NY-ESO-1 p157-165-specific CD8(+) T cells, detected by an HLA-A*0201 pentamer, and induced a T-cell response detected by an enzyme-linked immunospot assay. The Salmonella fimbrial display system was efficient at the induction of an antitumor cellular immune response in vivo, providing a new strategy for the development of efficient cancer vaccinations.

  19. T cell tolerance and activation to a transgene-encoded tumor antigen.

    PubMed

    Antoniou, A; McCormick, D; Scott, D; Yeoman, H; Chandler, P; Mellor, A; Dyson, J

    1996-05-01

    Much has been learned in recent years concerning the nature of tumor antigens recognized by T cells. To apply this knowledge clinically, the nature of the host response to individual and multiple tumor antigens has to be characterized. This will help to define the efficacy of immune surveillance and the immune status of the host following exposure to tumor antigens expressed on pre-neoplastic tissue. To approach these questions, we have developed a transgenic mouse which expresses the tumor-specific antigen P91A. The single amino acid substitution in P91A results in the expression of a new MHC class I (H-2Ld)-binding peptide. In transgenic tissue, the H-2Ld/P91A complex is expressed in isolation from other tumor-associated antigens, allowing definition of the immune response to a single defined tumor antigen, a situation closely analogous to events during tumorigenesis. We show that CD8+ T cell immune surveillance of P91A is ineffective without the introduction of a helper determinant operating through stimulation of CD4+ T cells. Recognition of the isolated P91A tumor antigen on normal tissue by CD8+ T cells is a tolerogenic process. Induction of T cell tolerance suggests tumor antigen-T cell interactions occurring during tumorigenesis may elicit T cell tolerance and hence confound some immunotherapeutic approaches.

  20. Cross-Reactive Immunity to Mycobacterium tuberculosis DosR Regulon-Encoded Antigens in Individuals Infected with Environmental, Nontuberculous Mycobacteria▿ †

    PubMed Central

    Lin, May Young; Reddy, T. B. K.; Arend, Sandra M.; Friggen, Annemieke H.; Franken, Kees L. M. C.; van Meijgaarden, Krista E.; Verduyn, Marleen J. C.; Schoolnik, Gary K.; Klein, Michel R.; Ottenhoff, Tom H. M.

    2009-01-01

    Mycobacterium tuberculosis DosR regulon-encoded antigens are highly immunogenic in M. tuberculosis-infected humans and are associated with latent tuberculosis infection. We have investigated the hypothesis that infection with or exposure to nontuberculous mycobacteria (NTM) can induce cross-reactive immunity to M. tuberculosis DosR regulon-encoded antigens since responsiveness has been observed in non-M. tuberculosis-exposed but purified protein derivative-responsive individuals. M. tuberculosis DosR regulon-encoded antigen-specific T-cell responses were studied in peripheral blood mononuclear cells (PBMCs) of NTM-infected/exposed individuals. BLASTP was used to determine the presence of M. tuberculosis DosR regulon-encoded protein orthologs among environmental mycobacteria and nonmycobacteria. Significant gamma interferon production was observed in PBMCs from NTM-infected/exposed individuals in response to M. tuberculosis DosR regulon-encoded antigens. DosR regulon-encoded protein orthologs were prominently present in tuberculous and environmental mycobacteria and surprisingly also in nonmycobacteria. The ubiquitous presence of the highly conserved DosR master regulator protein Rv3133c suggests that this is a general adaptive bacterial response regulator. We report a first series of M. tuberculosis antigens to which cross-reactive immunity is induced by NTM infection/exposure. The high conservation of M. tuberculosis DosR regulon-encoded antigens most likely enables them to induce cross-reactive T-cell responses. PMID:19737909

  1. ERCC4 (XPF) encodes a human nucleotide excision repair protein with eukaryotic recombination homologs.

    PubMed Central

    Brookman, K W; Lamerdin, J E; Thelen, M P; Hwang, M; Reardon, J T; Sancar, A; Zhou, Z Q; Walter, C A; Parris, C N; Thompson, L H

    1996-01-01

    ERCC4 is an essential human gene in the nucleotide excision repair (NER) pathway, which is responsible for removing UV-C photoproducts and bulky adducts from DNA. Among the NER genes, ERCC4 and ERCC1 are also uniquely involved in removing DNA interstrand cross-linking damage. The ERCC1-ERCC4 heterodimer, like the homologous Rad10-Rad1 complex, was recently found to possess an endonucleolytic activity that incises on the 5' side of damage. The ERCC4 gene, assigned to chromosome 16p13.1-p13.2, was previously isolated by using a chromosome 16 cosmid library. It corrects the defect in Chinese hamster ovary (CHO) mutants of NER complementation group 4 and is implicated in complementation group F of the human disorder xeroderma pigmentosum. We describe the ERCC4 gene structure and functional cDNA sequence encoding a 916-amino-acid protein (104 kDa), which has substantial homology with the eukaryotic DNA repair and recombination proteins MEI-9 (Drosophila melanogaster), Rad16 (Schizosaccharomyces pombe), and Rad1 (Saccharomyces cerevisiae). ERCC4 cDNA efficiently corrected mutants in rodent NER complementation groups 4 and 11, showing the equivalence of these groups, and ERCC4 protein levels were reduced in mutants of both groups. In cells of an XP-F patient, the ERCC4 protein level was reduced to less than 5%, consistent with XPF being the ERCC4 gene. The considerable identity (40%) between ERCC4 and MEI-9 suggests a possible involvement of ERCC4 in meiosis. In baboon tissues, ERCC4 was expressed weakly and was not significantly higher in testis than in nonmeiotic tissues. PMID:8887684

  2. Stable integration vector for nutrient broth-based selection of attenuated Listeria monocytogenes strains with recombinant antigen expression.

    PubMed

    Lenz, Laurel L; Huang, William A; Zhou, Chenghui; Li, Zhongxia; Calendar, Richard

    2008-09-01

    Recombinant Listeria monocytogenes strains induce strong cellular immune responses and may prove useful for antigen delivery for the vaccination of humans. However, the genetic systems currently available for the stable expression of recombinant antigens by L. monocytogenes rely on the use of antibiotic resistance genes. We report on a derivative, pPL2dalGlnA, of the Listeria monocytogenes pPL2 integration vector that completely lacks drug resistance genes. The selectable markers in pPL2dalGlnA are glutamine synthetase (GlnA) and alanine racemase (Dal). This novel vector was stably maintained in auxotropic L. monocytogenes strains that normally require d-alanine. The pPL2dalGlnA vector also partially restored the ability of an L. monocytogenes Deltadal Deltadat strain to colonize the spleens and livers of infected mice. A novel, highly attenuated strain of L. monocytogenes with quadruple deletions was also engineered by deleting the L. monocytogenes actA and plcB virulence genes from a Deltadal Deltadat strain. Infection of mice with recombinants of this mutant strain that express the antigen from pPL2dalGlnA were shown to elicit CD8(+) T-cell responses to human immunodeficiency virus Tat. This vector system is thus useful for stable antigen expression and vaccination studies.

  3. Targeting TARP, a novel breast and prostate tumor-associated antigen, with T cell receptor-like human recombinant antibodies.

    PubMed

    Epel, Malka; Carmi, Irit; Soueid-Baumgarten, Sharon; Oh, Sang Kon; Bera, Tapan; Pastan, Ira; Berzofsky, Jay; Reiter, Yoram

    2008-06-01

    MHC class I molecules are important components of immune surveillance. There are no available methods to directly visualize and determine the quantity and distribution of MHC/peptide complexes on individual cells or to detect such complexes on antigen-presenting cells in tissues. MHC-restricted recombinant antibodies with the same specificity of T cell receptors (TCR) may become a valuable tool to address these questions. They may also serve as valuable targeting molecules that mimic the specificity of cytotoxic T cells. We isolated by phage display a panel of human recombinant Fab antibodies with peptide-specific, MHC-restricted TCR-like reactivity directed toward HLA-A2-restricted T cell epitopes derived from a novel antigen termed TCRgamma alternative reading frame protein (TARP) which is expressed on prostate and breast cancer cells. We have characterized one of these recombinant antibodies and demonstrated its capacity to directly detect specific HLA-A2/TARP T cell epitopes on antigen-presenting cells that have complexes formed by naturally occurring active intracellular processing of the antigen, as well as on the surface of tumor cells. Moreover, by genetic fusion we armed the TCR-like antibody with a potent toxin and demonstrated that it can serve as a targeting moiety killing tumor cells in a peptide-specific, MHC-restricted manner similar to cytotoxic T lymphocytes.

  4. Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

    PubMed Central

    Galli, Vanessa; Simionatto, Simone; Marchioro, Silvana Beutinger; Klabunde, Gustavo Henrique Ferrero; Conceição, Fabricio Rochedo

    2013-01-01

    Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae. PMID:23803903

  5. Prostate-specific antigen-retargeted recombinant newcastle disease virus for prostate cancer virotherapy.

    PubMed

    Shobana, Raghunath; Samal, Siba K; Elankumaran, Subbiah

    2013-04-01

    Oncolytic virus (OV) therapies of cancer are based on the use of replication-competent, tumor-selective viruses with limited toxicity. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising OV and is inherently tumor selective and cytotoxic only to tumor cells. Replication is restricted in normal cells. Despite encouraging phase I/II clinical trials with NDV, further refinements for tumor-specific targeting are needed to enhance its therapeutic index. Systemically delivered NDV fails to reach solid tumors in therapeutic concentrations and also spreads poorly within the tumors due to barriers including complement, innate immunity, and the extracellular matrix. Overcoming these hurdles is paramount to realizing the exceptional oncolytic efficacy of NDV. We engineered the F protein of NDV and generated a recombinant NDV (rNDV) whose F protein is cleavable exclusively by prostate-specific antigen (PSA). The rNDV replicated efficiently and specifically in prostate cancer (CaP) cells and 3-dimensional prostaspheres but failed to replicate in the absence of PSA. Induction of intracellular PSA production by a synthetic androgen analog (R1881) enhanced fusogenicity in androgen-responsive CaP cells. Further, PSA-cleavable rNDV caused specific lysis of androgen-independent and androgen-responsive/nonresponsive CaP cells and prostaspheres, with a half-maximal effective concentration (EC50) ranging from a multiplicity of infection of 0.01 to 0.1. PSA-retargeted NDV efficiently lysed prostasphere tumor mimics, suggesting efficacy in vivo. Also, PSA-cleavable NDV failed to replicate in chicken embryos, indicating no pathogenicity for chickens. Prostate-specific antigen targeting is likely to enhance the therapeutic index of rNDV owing to tumor-restricted replication and enhanced fusogenicity.

  6. The Drosophila Meiotic Recombination Gene Mei-9 Encodes a Homologue of the Yeast Excision Repair Protein Rad1

    PubMed Central

    Sekelsky, J. J.; McKim, K. S.; Chin, G. M.; Hawley, R. S.

    1995-01-01

    Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cerevisiae, many genes required to repair DNA double-strand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cerevisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cerevisiae excision repair protein Rad1, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cerevisiae, in that it requires an NER protein. The biochemical properties of the Rad1 protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion. PMID:8647398

  7. Improved diagnosis of Strongyloides stercoralis using recombinant antigen-based serologies in a community-wide study in northern Argentina.

    PubMed

    Krolewiecki, Alejandro J; Ramanathan, Roshan; Fink, Valeria; McAuliffe, Isabel; Cajal, Silvana P; Won, Kimberly; Juarez, Marisa; Di Paolo, Adriana; Tapia, Laura; Acosta, Norma; Lee, Rogan; Lammie, Patrick; Abraham, David; Nutman, Thomas B

    2010-10-01

    The serodiagnosis of Strongyloides stercoralis infection by enzyme-linked immunosorbent assays based on crude antigen (CrAg-ELISA), while useful, has been limited by the reliance on crude parasite extracts. Newer techniques such as the luciferase immunoprecipitation system assay (LIPS), based on a 31-kDa recombinant antigen (termed NIE) from S. stercoralis and/or the recombinant antigen S. stercoralis immunoreactive antigen (SsIR), or the NIE-ELISA have shown promise in controlled settings. We compared each of these serologic assays in individuals from both regions of the world in which S. stercoralis is endemic and those in which it is not. A comprehensive stool evaluation (sedimentation concentration, Baermann concentration with charcoal cultures, agar plate, and Harada-Mori) and four different serologic techniques using CrAg-ELISA or recombinant NIE-ELISA as well as LIPS using NIE alone or in combination with a second recombinant antigen (NIE/SsIR-LIPS) were compared among individuals with parasitologically proven infection (n = 251) and healthy controls from regions of the world in which the infection is nonendemic (n = 11). Accuracy was calculated for each assay. The prevalence of S. stercoralis infection was 29.4% among Argentinean stool samples (n = 228). Sedimentation concentration and Baermann were the most sensitive stool-based methods. NIE-LIPS showed the highest sensitivity (97.8%) and specificity (100%) of the serologic assays. The calculated negative predictive value was highest for both the NIE-LIPS and CrAg-ELISA (>97%) irrespective of disease prevalence. No cross-reactivity with soil-transmitted helminths was noted. NIE-LIPS compares favorably against the current CrAg-ELISA and stool evaluation, providing additional accuracy and ease of performance in the serodiagnosis of S. stercoralis infections irrespective of disease prevalence.

  8. Inclusion bodies of recombinant Epstein-Barr virus capsid antigen p18 as potential immobilized antigens in enzyme immunoassays for detection of nasopharyngeal carcinoma.

    PubMed

    Lim, Chun Shen; Goh, Siang Ling; Kariapper, Leena; Krishnan, Gopala; Lim, Yat-Yuen; Ng, Ching Ching

    2015-08-25

    Development of indirect enzyme-linked immunosorbent assays (ELISAs) often utilizes synthetic peptides or recombinant proteins from Escherichia coli as immobilized antigens. Because inclusion bodies (IBs) formed during recombinant protein expression in E. coli are commonly thought as misfolded aggregates, only refolded proteins from IBs are used to develop new or in-house diagnostic assays. However, the promising utilities of IBs as nanomaterials and immobilized enzymes as shown in recent studies have led us to explore the potential use of IBs of recombinant Epstein-Barr virus viral capsid antigen p18 (VCA p18) as immobilized antigens in ELISAs for serologic detection of nasopharyngeal carcinoma (NPC). Thioredoxin fusion VCA p18 (VCA-Trx) and IBs of VCA p18 without fusion tags (VCA-IBs) were purified from E. coli. The diagnostic performances of IgG/VCA-IBs, IgG/VCA-Denat-IBs (using VCA-IBs coated in 8mol/l urea), IgG/VCA-Trx, and IgG/VCA-Peptide assays were compared by screening 100 NPC case-control pairs. The IgG/VCA-Denat-IBs assay showed the best area under the receiver operating characteristic curve (AUC: 0.802; p<0.05), while the AUCs for the IgG/VCA-IBs, IgG/VCA-Trx, and IgG/VCA-Peptide assays were comparable (AUC: 0.740, 0.727, and 0.741, respectively). We improved the diagnostic performance of the ELISA significantly using IBs of recombinant VCA p18. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Design and evaluation of protein expression in a recombinant plasmid encoding epitope gp 350/220 of the Epstein-Barr virus (EBV)

    NASA Astrophysics Data System (ADS)

    Himmah, Karimatul; Dluha, Nurul; Anyndita, Nadya V. M.; Rifa'i, Muhaimin; Widodo

    2017-05-01

    The Epstein - Barr virus (EBV) causes severe infections that may lead to cancers such as nasopharyngeal carcinoma. Development of effective EBV vaccines is necessary to prevent the virus spreading throughout the community. TheEBV has a surface protein gp 350/220, which serves as an antigen to help interact with host cells. Epitopes of the protein can potentially serve as bases for a vaccine. In a previous study, we have found a conserved epitope of gp 350/220 from all strains EBV through an in silico approach. The aim of this study is to design and overproduce a recombinant peptide of epitope gp 350/220 in E. coli. DNA encoding the conserved epitope was synthesized and cloned into plasmid pET-22b(+); the recombinant plasmid was transformed into E. coli strains DH5α and BL21. The transformed plasmid DNA was isolated and confirmed by restriction using XbaI and PstI enzymes followed by DNA sequencing. Protein expression was induced by isopropyl-D-thiogalactopyranoside (IPTG) with final concentrations of 0.1, 0.2, 1, and 2 mM in consecutive times. An osmotic shock method was used to isolate protein from periplasmic fraction of E. coli DH5α and BL21. The SDS-PAGE analysis was carried out to detect peptide target (3.4 kDa). Based on this result, the induction process did not work properly, and thus needs further investigation.

  10. Simultaneous electrochemical immunoassay using graphene-Au grafted recombinant apoferritin-encoded metallic labels as signal tags and dual-template magnetic molecular imprinted polymer as capture probes.

    PubMed

    Wang, De; Gan, Ning; Zhang, Huairong; Li, Tianhua; Qiao, Li; Cao, Yuting; Su, Xiurong; Jiang, Shan

    2015-03-15

    A novel electrochemical multiplexed immunoassay was designed for simultaneous determination of alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) using recombinant apoferritin-encoded metallic nanoparticles (rApo-M) as labels and dual-template magnetic molecularly imprinted polymers (MMIPs) as capture probes. The labels were prepared by loading recombinant apoferritin (r-Apo) and separately immobilize primary antibodies (anti-AFP and anti-CEA) via Au nanoparticles of in site growth on graphene (G). The capture probes were synthesized by self-polymerization of dopamine (DA) on the Fe3O4 nanoparticles (Fe3O4 NPs) and using AFP and CEA as the template proteins, which were used to enrich the targets simultaneously. After a sandwich-type immunoreaction, the labels were captured to the surface of MMIPs. The subsequent electrochemical stripping analysis of the metal components from the immunocomplex provide a means for quantification of targets based on the peak currents of Cd and Pb. Experimental results showed the immunoassay enabled the simultaneous determination of AFP and CEA in a single run with wide dynamic ranges of 0.001-5ngmL(-1). And the detection limits of AFP and CEA were 0.3 and 0.35pgmL(-1) (S/N=3), respectively. These results suggested that the proposed multiplexed immunoassay would be applied for clinical screening of other biomarkers.

  11. Cryogenic transmission electron microscopy of recombinant tuberculosis vaccine antigen with anionic liposomes reveals formation of flattened liposomes.

    PubMed

    Fox, Christopher B; Mulligan, Sean K; Sung, Joyce; Dowling, Quinton M; Fung, H W Millie; Vedvick, Thomas S; Coler, Rhea N

    2014-01-01

    Development of lipid-based adjuvant formulations to enhance the immunogenicity of recombinant vaccine antigens is a focus of modern vaccine research. Characterizing interactions between vaccine antigens and formulation excipients is important for establishing compatibility between the different components and optimizing vaccine stability and potency. Cryogenic transmission electron microscopy (TEM) is a highly informative analytical technique that may elucidate various aspects of protein- and lipid-based structures, including morphology, size, shape, and phase structure, while avoiding artifacts associated with staining-based TEM. In this work, cryogenic TEM is employed to characterize a recombinant tuberculosis vaccine antigen, an anionic liposome formulation, and antigen-liposome interactions. By performing three-dimensional tomographic reconstruction analysis, the formation of a population of protein-containing flattened liposomes, not present in the control samples, was detected. It is shown that cryogenic TEM provides unique information regarding antigen-liposome interactions not detectable by light-scattering-based methods. Employing a suite of complementary analytical techniques is important to fully characterize interactions between vaccine components.

  12. Local Production of Tumor Necrosis Factor Encoded by Recombinant Vaccinia Virus is Effective in Controlling Viral Replication in vivo

    NASA Astrophysics Data System (ADS)

    Sambhi, Sharan K.; Kohonen-Corish, Maija R. J.; Ramshaw, Ian A.

    1991-05-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-α. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-α during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo.

  13. Local production of tumor necrosis factor encoded by recombinant vaccinia virus is effective in controlling viral replication in vivo.

    PubMed Central

    Sambhi, S K; Kohonen-Corish, M R; Ramshaw, I A

    1991-01-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-alpha. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-alpha during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo. Images PMID:2023951

  14. Small-angle neutron scattering study of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particle

    NASA Astrophysics Data System (ADS)

    Sato, M.; Ito, Y.; Kameyama, K.; Imai, M.; Ishikawa, N.; Takagi, T.

    1995-02-01

    The overall and internal structure of recombinant yeast-derived human hepatitis B virus surface antigen vaccine particles was investigated by small-angle neutron scattering using the contrast variation method. The vaccine is a nearly spherical particle, and its contrast-matching point was determined to be at about 24% D 2O content, indicating that a large part of the vaccine particle is occupied by lipids and carbohydrates from the yeast. The Stuhrmann plot suggests that the surface antigens exist predominantly in the peripheral region of the particle, which is favorable to the induction of anti-virus antibodies.

  15. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay.

    PubMed

    Van der Poel, C L; Cuypers, H T; Reesink, H W; Weiner, A J; Quan, S; Di Nello, R; Van Boven, J J; Winkel, I; Mulder-Folkerts, D; Exel-Oehlers, P J

    1991-02-09

    A new four-antigen recombinant immunoblot assay (4-RIBA) for confirmation of hepatitis C virus (HCV) C-100 enzyme-linked immunosorbent assay (ELISA) reactivity was tested in stored serum samples (1984-86) of blood donors and recipients and compared with results from polymerase chain reaction (PCR) analysis of fresh (1990) plasma samples in donors and recipients from the original study. Of 37 HCV C-100 ELISA-positive blood products, 8 were 4-RIBA positive, of which 7 were implicated in post-transfusion non-A, non-B hepatitis (PT-NANBH) and/or PCR confirmed recipient HCV infection. Of 9 recipients with PT-NANBH, 8 were reactive in 4-RIBA (6 positive and 2 indeterminate). With fresh plasma samples, 3 donors and 6 recipients who were 4-RIBA positive were also PCR positive. 4 4-RIBA indeterminate and 78 4-RIBA negative samples of donors and recipients were PCR negative. Of 6 4-RIBA positive recipients, 5 were PCR positive four to six years later. 1.6% of the 383 recipients became chronically infected with HCV. The new 4-RIBA represents a candidate confirmation test to discriminate between infective and non-infective HCV C-100 ELISA-positive blood donors.

  16. Molecular cloning of MER-2, a human chromosome-11-encoded red blood cell antigen, using linkage of cotransfected markers.

    PubMed

    Bill, J; Palmer, E; Jones, C

    1987-09-01

    We report the molecular cloning of a human gene MER-2 located on chromosome 11 that encodes a cell surface antigen which is polymorphic on red blood cells. An essential element of the cloning strategy was cotransfection-induced linkage of pSV2-neo, which encodes resistance to the antibiotic G418, to the human MER-2 gene. An important feature of the pSV2-neo construct is that the same gene (the transposon, Tn5) that encodes G418 resistance in eukaryotic cells confers neomycin resistance in bacteria. Chinese hamster ovary (CHO) cells were cotransfected with pSV2-neo and genomic DNA from a CHO X human cell hybrid containing a single human chromosome (chromosome 11). Transfectants expressing both the human MER-2 gene and G418 resistance were isolated by selection in the antibiotic G418, followed by indirect immunofluorescence using the monoclonal antibody 1D12, which recognizes the MER-2 antigen, manual enrichment, and single-cell cloning. Genomic DNA from a primary transfectant positive for MER-2 expression and G418 resistance was used to construct a cosmid library and cosmid clones able to grow in neomycin were isolated. Of 150,000 cosmid clones screened, 90 were resistant to neomycin and of these, 11 contained human repetitive sequences. Five neomycin-resistant cosmid clones containing human repetitive DNA were able to transfect CHO cells for G418 resistance and MER-2 expression.

  17. A Novel Plasmid-Encoded Serotype Conversion Mechanism through Addition of Phosphoethanolamine to the O-Antigen of Shigella flexneri

    PubMed Central

    Senchenkova, Sof’ya N.; Jin, Dong; Shashkov, Alexander S.; Xia, Shengli; Perepelov, Andrei V.; Chen, Qiang; Wang, Yan; Wang, Haiyin; Xu, Jianguo

    2012-01-01

    Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O–antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes. PMID:23049947

  18. A novel plasmid-encoded serotype conversion mechanism through addition of phosphoethanolamine to the O-antigen of Shigella flexneri.

    PubMed

    Sun, Qiangzheng; Knirel, Yuriy A; Lan, Ruiting; Wang, Jianping; Senchenkova, Sof'ya N; Jin, Dong; Shashkov, Alexander S; Xia, Shengli; Perepelov, Andrei V; Chen, Qiang; Wang, Yan; Wang, Haiyin; Xu, Jianguo

    2012-01-01

    Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6) share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037) epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN) group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O-antigen), mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.

  19. Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure.

    PubMed

    Gorny, Miroslaw K; Sampson, Jared; Li, Huiguang; Jiang, Xunqing; Totrov, Maxim; Wang, Xiao-Hong; Williams, Constance; O'Neal, Timothy; Volsky, Barbara; Li, Liuzhe; Cardozo, Timothy; Nyambi, Phillipe; Zolla-Pazner, Susan; Kong, Xiang-Peng

    2011-01-01

    Preferential usage of immunoglobulin (Ig) genes that encode antibodies (Abs) against various pathogens is rarely observed and the nature of their dominance is unclear in the context of stochastic recombination of Ig genes. The hypothesis that restricted usage of Ig genes predetermines the antibody specificity was tested in this study of 18 human anti-V3 monoclonal Abs (mAbs) generated from unrelated individuals infected with various subtypes of HIV-1, all of which preferentially used pairing of the VH5-51 and VL lambda genes. Crystallographic analysis of five VH5-51/VL lambda-encoded Fabs complexed with various V3 peptides revealed a common three dimensional (3D) shape of the antigen-binding sites primarily determined by the four complementarity determining regions (CDR) for the heavy (H) and light (L) chains: specifically, the H1, H2, L1 and L2 domains. The CDR H3 domain did not contribute to the shape of the binding pocket, as it had different lengths, sequences and conformations for each mAb. The same shape of the binding site was further confirmed by the identical backbone conformation exhibited by V3 peptides in complex with Fabs which fully adapted to the binding pocket and the same key contact residues, mainly germline-encoded in the heavy and light chains of five Fabs. Finally, the VH5-51 anti-V3 mAbs recognized an epitope with an identical 3D structure which is mimicked by a single mimotope recognized by the majority of VH5-51-derived mAbs but not by other V3 mAbs. These data suggest that the identification of preferentially used Ig genes by neutralizing mAbs may define conserved epitopes in the diverse virus envelopes. This will be useful information for designing vaccine immunogen inducing cross-neutralizing Abs.

  20. Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2

    PubMed Central

    2010-01-01

    Background The capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in E. coli, for diagnosis of PCV infection. Results The ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%. Conclusions This ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera. PMID:20958981

  1. Cloning, expression and evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag3 antigen in mice.

    PubMed

    Wang, Tao; He, Guangzhi; Yang, Guangyou; Fei, Yuxiang; Zhang, Zhihe; Wang, Chengdong; Yang, Zhi; Lan, Jingchao; Luo, Li; Liu, Li

    2008-12-09

    The gene Bs-Ag3 enconding a antigen of 37kDa from Baylisascaris schroederi (giant panda isolates), as well as the recombinant Bs-Ag3, obtained by cloning and expression of the Bs-Ag3 gene in heterologous host Escherichia coli BL-21 (DE3), were used to evaluate their ability to induce immune protective responses in BALB/c mice against L3-challenge infection in a mouse-B. schroederi model. There was a significant reduction (62.91%) of mice vaccinated with rBs-Ag3 coupled with Freund's complete adjuvant (FCA) in recovery of challenged B. schroederi L3 compared with either controls or mice vaccinated with FCA alone. Our data indicate recombinant Bs-Ag3 may be a potential target as a vaccine antigen for giant panda ascariasis.

  2. The Use of Directed Evolution to Create a Stable and Immunogenic Recombinant BCG Expressing a Modified HIV-1 Gag Antigen

    PubMed Central

    Shephard, Enid; Stutz, Helen; Douglass, Nicola; Mgwebi, Thandi; Meyers, Ann; Chin'ombe, Nyasha; Williamson, Anna-Lise

    2014-01-01

    Numerous features make Mycobacterium bovis BCG an attractive vaccine vector for HIV. It has a good safety profile, it elicits long-lasting cellular immune responses and in addition manufacturing costs are affordable. Despite these advantages it is often difficult to express viral antigens in BCG, which results in genetic instability and low immunogenicity. The aim of this study was to generate stable recombinant BCG (rBCG) that express high levels of HIV antigens, by modification of the HIV genes. A directed evolution process was applied to recombinant mycobacteria that expressed HIV-1 Gag fused to the green fluorescent protein (GFP). Higher growth rates and increased GFP expression were selected for. Through this process a modified Gag antigen was selected. Recombinant BCG that expressed the modified Gag (BCG[pWB106] and BCG[pWB206]) were more stable, produced higher levels of antigen and grew faster than those that expressed the unmodified Gag (BCG[pWB105]). The recombinant BCG that expressed the modified HIV-1 Gag induced 2 to 3 fold higher levels of Gag-specific CD4 T cells than those expressing the unmodified Gag (BCG[pWB105]). Mice primed with 107 CFU BCG[pWB206] and then boosted with MVA-Gag developed Gag-specific CD8 T cells with a frequency of 1343±17 SFU/106 splenocytes, 16 fold greater than the response induced with MVA-Gag alone. Levels of Gag-specific CD4 T cells were approximately 5 fold higher in mice primed with BCG[pWB206] and boosted with MVA-Gag than in those receiving the MVA-Gag boost alone. In addition mice vaccinated with BCG[pWB206] were protected from a surrogate vaccinia virus challenge. PMID:25061753

  3. [DNA vaccines and recombinant antigens in prevention of Toxoplasma gondii infections--current status of the studies].

    PubMed

    Hiszczyńska-Sawicka, Elzbieta; Holec-Gasior, Lucyna; Kur, Józef

    2009-01-01

    Toxoplasmosis caused by an intracellular parasite Toxoplasma gondii is still one of major medical and veterinary problems and there is still need for a vaccine for human toxoplasmosis. Despite years of research much remains to be done to develop effective vaccine. The article presents the current status of vaccine strategies against toxoplasmosis with focus on the most developed approaches using naked DNA and recombinant antigens.

  4. Immunization with recombinant DNA and modified vaccinia virus Ankara (MVA) vectors delivering PSCA and STEAP1 antigens inhibits prostate cancer progression.

    PubMed

    Krupa, Magdalena; Canamero, Marta; Gomez, Carmen E; Najera, Jose L; Gil, Jesus; Esteban, Mariano

    2011-02-04

    Despite recent advances in early detection and improvement of conventional therapies, there is an urgent need for development of additional approaches for prevention and/or treatment of prostate cancer, and the use of immunotherapeutic modalities, such as cancer vaccines, is one of the most promising strategies. In this study, we evaluated the prophylactic efficacy of an active immunization protocol against prostate cancer associated antigens mPSCA and mSTEAP1 in experimental prostate cancer. Two antigen delivery platforms, recombinant DNA and MVA vectors, both encoding either mPSCA or mSTEAP1 were used in diversified DNA prime/MVA boost vaccination protocol. Antitumour activity was evaluated in TRAMP-C1 subcutaneous syngeneic tumour model and TRAMP mice. DNA prime/MVA boost immunization against either mPSCA or mSTEAP1, delayed tumour growth in TRAMP-C1 cells-challenged mice. Furthermore, simultaneous vaccination with both antigens produced a stronger anti-tumour effect against TRAMP-C1 tumours than vaccination with either mPSCA or mSTEAP1 alone. Most importantly, concurrent DNA prime/MVA boost vaccination regimen with those antigens significantly decreased primary tumour burden in TRAMP mice without producing any apparent adverse effects. Histopathological analysis of prostate tumours from vaccinated and control TRAMP mice revealed also that mPSCA/mSTEAP1 based-vaccination was effective at reducing the severity of prostatic lesions and incidence of high-grade poorly differentiated prostate cancer. Suppression of the disease progression in TRAMP mice was correlated with decreased proliferation index and increased infiltration of T-cells in prostate tissue. Active immunization against PSCA and STEAP1 using DNA prime/MVA boost strategy is a promising approach for prevention and/or treatment of prostate cancer.

  5. A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

    USGS Publications Warehouse

    Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  6. Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag

    PubMed Central

    Xu, Wanghui; Zhao, Qing; Xing, Lei; Lin, Zhanglin

    2016-01-01

    The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggregating peptide ELK16. In this scheme, the target proteins were fused in the C-termini with cSAT and expressed as insoluble aggregates. After intein self-cleavage, target proteins were released into the soluble fraction with high yield and reasonable purity. We demonstrated the applicability of this scheme by preparing seven model viral peptides, with lengths ranging from 32 aa to 72 aa. By adding an N-terminal thioredoxin tag, we enhanced the yield of target peptides released from the aggregates. The purified viral peptides demonstrated high antigenic activities in ELISA and were successfully applied to dissecting the antigenic regions of influenza hemagglutinin. The cSAT scheme described here allows for the rapid and low-cost preparation of multiple antigenic peptides for immunological screening of a broad range of viral antigens. PMID:27808126

  7. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague

    PubMed Central

    Rocke, Tonie E.; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E.

    2014-01-01

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

  8. A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague.

    PubMed

    Rocke, Tonie E; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E

    2014-10-27

    In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

  9. Distinct immune responses of recombinant plasmid DNA replicon vaccines expressing two types of antigens with or without signal sequences.

    PubMed

    Yu, Yun-Zhou; Li, Na; Wang, Wen-Bin; Wang, Shuang; Ma, Yao; Yu, Wei-Yuan; Sun, Zhi-Wei

    2010-11-03

    Here, DNA replicon vaccines encoding the Hc domain of botulinum neurotoxin serotype A (AHc) or the receptor binding domain of anthrax protective antigen (PA4) with or without signal sequences were evaluated in mice. Strong antibody and protective responses were elicited only from AHc DNA vaccines with an Ig κ signal sequence or tissue plasminogen activator signal sequence. Meanwhile, there were no differences in total antibody responses or isotypes, lymphocyte proliferative responses, cytokine profiles and protective immune responses with the PA4 DNA vaccines with or without a signal sequence. Therefore, use of targeting sequences in designing DNA replicon vaccines depends on the specific antigen.

  10. Functional Recombinant Extra Membrane Loop of Human CD20, an Alternative of the Full Length CD20 Antigen

    PubMed Central

    Anbouhi, Mahdi Habibi; Baraz, Aida Feiz; Bouzari, Saeid; Abolhassani, Mohsen; Khanahmad, Hossein; Golkar, Majid; Aghasadeghi, Mohammad Reza; Behdani, Mahdi; Najafabadi, Ali Jahanian; Shokrgozar, Mohammad Ali

    2012-01-01

    Background: Targeting of CD20 antigen with monoclonal antibodies has become the mainstay in the treatment of non-Hodgkin's lymphomas and immunotherapeutic depletion of malignant B cells. Accessibility of antigen is one of the crucial factors in development of monoclonal antibodies against this antigen. One major problem in expression of full length CD20 is aggregation and misfolding. Therefore, production of an alternative polypeptide is easer and favorable comparing to that of a full length transmembrane protein CD20. Methods: In this study, we expressed the extra membrane loop of hCD20 (exCD20) consisting of a non-glycosylated 47-amino acids region. The exCD20 coding sequence was amplified by PCR and cloned in pET32a(+) expression vector. The desired protein was expressed in fusion with thioredoxin and 6× His tag in E. coli Origami strain. ELISA and Western-blotting data were performed to indicate the functionality of this protein. Results: We have obtained the exCD20 recombinant protein which can be detected in ELISA and Western-blot experiments. This recombinant fusion protein was soluble and stable without aggregation and misfolding problems. Conclusion: The recombinant extra membrane loop of human CD20 protein in fusion with thioredoxin (exCD20) can be used in function assays and some applications such as ELISA, immuneblotting, affinity purification, immunization, screening, and development of anti-CD20 antibodies. PMID:23023212

  11. A Plasmodium vivax plasmid DNA- and adenovirus-vectored malaria vaccine encoding blood stage antigens AMA1 and MSP142 in a prime/boost heterologous immunization regimen partially protects Aotus monkeys against blood stage challenge.

    PubMed

    Obaldia, Nicanor; Stockelman, Michael G; Otero, William; Cockrill, Jennifer A; Ganeshan, Harini; Abot, Esteban N; Zhang, Jianfeng; Limbach, Keith; Charoenvit, Yupin; Doolan, Denise L; Tang, De-Chu C; Richie, Thomas L

    2017-02-08

    Malaria is caused by parasites of the genus Plasmodium that are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of P. falciparum it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside of Africa, stressing the importance of developing a vaccine against malaria. In this study we assess the immunogenicity and protective efficacy of two P. vivax antigens, AMA1 and MSP142 in a recombinant DNA plasmid prime/adenoviral vector (Ad) boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with DNA alone, Ad alone, prime/boost regimens of each antigen, prime/boost with both antigens, and empty vector controls, and then subjected to blood stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, based on their ability to induced the longest pre-patent period and time to peak parasitemia; the lowest peak and mean parasitemia; the smallest area under the parasitemia curve and the highest self-cured rate. Overall, pre-challenge MSP1 antibody titers strongly correlated with decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, P. vivax plasmid DNA/Ad5 vaccine encoding blood stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen, provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and regimen for further development.

  12. Molecular characterization of KatY (antigen 5), a thermoregulated chromosomally encoded catalase-peroxidase of Yersinia pestis.

    PubMed

    Garcia, E; Nedialkov, Y A; Elliott, J; Motin, V L; Brubaker, R R

    1999-05-01

    The first temperature-dependent proteins (expressed at 37 degrees C, but not 26 degrees C) to be identified in Yersinia pestis were antigens 3 (fraction 1), 4 (pH 6 antigen), and 5 (hereafter termed KatY). Antigens 3 and 4 are now established virulence factors, whereas little is known about KatY, except that it is encoded chromosomally, produced in abundance, possesses modest catalase activity, and is shared by Yersinia pseudotuberculosis, but not Yersinia enterocolitica. We report here an improved chromatographic method (DEAE-cellulose, calcium hydroxylapatite, and Sephadex G-150) that yields enzymatically active KatY (2,423 U/mg of protein). Corresponding mouse monoclonal antibody 1B70.1 detected plasminogen activator-mediated hydrolysis of KatY, and a polyclonal rabbit antiserum raised against outer membranes of Y. pestis was enriched for anti-KatY. A sequenced approximately 16-kb Y. pestis DNA insert of a positive pLG338 clone indicated that katY encodes an 81.4-kDa protein (pI 6.98) containing a leader sequence of 2.6 kDa; the deduced molecular mass and pI of processed KatY were 78.8 kDa and 6. 43, respectively. A minor truncated variant (predicted molecular mass of 53.6 kDa) was also expressed. KatY is similar (39 to 59% identity) to vegetative bacterial catalase-peroxidases (KatG in Escherichia coli) and is closely related to plasmid-encoded KatP of enterohemorrhagic E. coli O157:H7 (75% identity). katY encoded a putative Ca2+-binding site, and its promoter contained three homologues to the consensus recognition sequence of the pCD-encoded transcriptional activator LcrF. rbsA was located upstream of katY, and cybB, cybC, dmsABC, and araD were mapped downstream. These genes are not linked to katG or katP in E. coli.

  13. Variable performance of a human derived Sarcoptes scabiei recombinant antigen ELISA in swine mange diagnosis.

    PubMed

    Casais, R; Goyena, E; Martínez-Carrasco, C; Ruiz de Ybáñez, R; Alonso de Vega, F; Ramis, G; Prieto, J M; Berriatua, E

    2013-10-18

    The performance of an indirect ELISA test based on Sarcoptes scabiei var hominis recombinant antigen Ssλ20ΔB3 (rec-ELISA), to diagnose pig mange was investigated in 15 experimentally infected and non-infected pigs and 692 commercial pigs from 16 herds in southeast Spain. These latter animals included 6-7 month old fatteners (13 herds), 11-12 month old replacement sows (1 herd) and ≥24 month old breeding sows (7 herds). All pigs were examined for mites in ear skin scrapings and the presence of S. scabiei-associated macroscopic dermatitis; moreover, fatteners were also tested for antibodies against porcine viruses including: Aujeszky disease virus (ADV), swine influenza virus (SIV), type 2 porcine circovirus (PCV2) and porcine respiratory and reproductive syndrome virus (PRRSV). S. scabiei and chronic hyperkeratotic dermatitis were detected in breeding sows from 6 herds. Mite prevalence in other pigs was 83% in replacement sows, 0% in 7 fattener's herds and 3-82% in other fattener's herds. All fattener herds had pigs with acute hypersensitivity dermatitis and the percentage of affected pigs and lesion area was significantly greater in S. scabiei infected ones. Rec-ELISA relative optical densities (RODs) were greater in older than in young pigs, as well as in infected compared to non-infected pigs. However, RODs differed significantly between infected individuals, regardless of age and origin (commercial or experimental) and the herd prevalence of S. scabiei. Low repeatability between ELISA microtiter plates, suggesting variable specific antibody binding to antigen, are likely partly responsible for ROD variation. Other potential causes of variation were examined in fatteners using random effects logistic regression analysis, after defining a seropositivity threshold value with receiver-operating characteristics (ROC) analysis. The logistic model indicated that seropositivity was associated with large dermatitis areas and with the only herd with low PCV2

  14. Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70

    PubMed Central

    DONG, LEI; ZHANG, XIAOPENG; YU, CHANGMING; REN, JUN; HOU, LIHUA; FU, LING; YI, SHAOQIONG; CHEN, WEI

    2013-01-01

    The aim of this study was to express and purify recombinant proteins based on human prostate stem cell antigen (PSCA) and heat shock protein-70 (HSP70). The PSCA gene and various structural domains of HSP70 were amplified by polymerase chain reaction (PCR) with the respective primers. Then, the PSCA was cloned into the prokaryotic expression vector pET21a(+) with the amino-terminus, carboxyl-terminus and overall length of HSP70, by enzyme digestion to construct the recombinant plasmids pET21-PSCA-HSPN, pET21-PSCA-HSPC and pET21-PSCA-HSP, respectively. After being expressed in Escherichia coli (E. coli) by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, recombinant fusion proteins were purified. Western blotting was performed to confirm the expression of the recombinant proteins. The results revealed that recombinant plasmids were successfully constructed. The PSCA-HSPC and PSCA-HSP expressed in E. coli existed in soluble form, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purity of the recombinant proteins PSCA-HSPC and PSCA-HSP reached >95% following purification with the nickel-nitrilotriacetic acid (Ni-NTA) resin, Phenyl-Sepharose Fast Flow and Superdex 75, which lays a foundation for the development of vaccines for prostate cancer. PMID:23596484

  15. Inclusion bodies from recombinant bacteria as a novel system for delivery of vaccine antigen by the oral route.

    PubMed

    Kesik, Małgorzata; Saczyńska, Violetta; Szewczyk, Bogusław; Płucienniczak, Andrzej

    2004-02-15

    A fragment of non-glycosylated E2 antigen of classical swine fever virus (CSFV), lacking the trans-membrane anchor (TM-) of the native glycoprotein, was produced in recombinant Escherichia coli strain BL21(DE3) in the form of inclusion bodies. These inclusion bodies isolated from the bacteria cells were administrated orally to mice twice at either 10 or 50 microg per dose. Each mouse fed with inclusion bodies carrying the E2 antigen responded with plasma antibodies and/or fecal IgA at least once during the entire investigation. Our study showed the capacity of inclusion bodies to induce both systemic and mucosal responses as well as to evoke relatively-long mucosal memory when fed to mice at low-number vaccination schedule and without any adjuvant. We propose the use of inclusion bodies for oral vaccination as an alternative to artificial systems for delivery of recombinant antigens by the oral route. Very few steps are needed to obtain an antigen ready for use as a vaccine. The procedure is easy and inexpensive and can be used for development of vaccine against classical swine fever.

  16. Expression, Polyubiquitination, and Therapeutic Potential of Recombinant E6E7 from HPV16 Antigens Fused to Ubiquitin.

    PubMed

    de Oliveira, Liliane M Fernandes; Morale, Mirian G; Chaves, Agtha A M; Demasi, Marilene; Ho, Paulo L

    2017-01-01

    Ubiquitin-proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8(+) T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies. The antigens were solubilized using urea and purified by nickel affinity chromatography in denatured condition. Fusion of ubiquitin to E6E7 resulted in marked polyubiquitination in vitro mainly when fused to the E6E7 N-terminal. When tested in a therapeutic scenario, the fusion of ubiquitin to E6E7 reinforced the anti-tumor protection and increased the E6/E7-specific cellular immune responses. Present results encourage the investigation of the adjuvant potential of the ubiquitin fusion to recombinant vaccines requiring CD8(+) T cells.

  17. Effects of solution conditions and surface chemistry on the adsorption of three recombinant botulinum neurotoxin antigens to aluminum salt adjuvants.

    PubMed

    Vessely, Christina; Estey, Tia; Randolph, Theodore W; Henderson, Ian; Nayar, Rajiv; Carpenter, John F

    2007-09-01

    Botulinum neurotoxin (BoNT) is a biological warfare threat. Protein antigens have been developed against the seven major BoNT serotypes for the development of a recombinant protein vaccine. This study is an evaluation of adsorption profiles for three of the recombinant protein antigens to aluminum salt adjuvants in the development of a trivalent vaccine against BoNT. Adsorption profiles were obtained over a range of protein concentrations. The results document that charge-charge interactions dominate the adsorption of antigen to adjuvant. Optimal conditions for adsorption were determined. However, potency studies and solution stability studies indicated the necessity of using aluminum hydroxide adjuvant at low pH. To improve the adsorption profiles to AlOH adjuvant, phosphate ions were introduced into the adsorption buffers. The resulting change in the adjuvant chemistry led to an improvement of adsorption of the BoNT antigens to aluminum hydroxide adjuvant while maintaining potency. Competitive adsorption profiles were also determined, and showed changes in maximum adsorption from mixed solutions compared to adsorption from individual protein solutions. The adsorption profiles for each protein varied due to differences in adsorption mechanism and affinity for the adjuvant surface. These results emphasize the importance of evaluating competitive adsorption in the development of multivalent vaccine products. (c) 2007 Wiley-Liss, Inc. and the American Pharmacists Association.

  18. Development of Ss-NIE-1 Recombinant Antigen Based Assays for Immunodiagnosis of Strongyloidiasis

    PubMed Central

    Rascoe, Lisa N.; Price, Courtney; Shin, Sun Hee; McAuliffe, Isabel; Priest, Jeffrey W.; Handali, Sukwan

    2015-01-01

    Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States. PMID

  19. Adjuvant activity of chicken interleukin-12 co-administered with infectious bursal disease virus recombinant VP2 antigen in chickens.

    PubMed

    Su, Bor Sheu; Chiu, Hua Hsien; Lin, Cheng Chung; Shien, Jui Hung; Yin, Hsien Sheng; Lee, Long Huw

    2011-02-15

    A recombinant fowlpox virus (rFPV/VP2) expressing infectious bursal diseases virus (IBDV) VP2 gene has been constructed. After purification and identification of rFPV/VP2, the adjuvant activity of the recombinant chicken IL-12 (rchIL-12), synthesized by our previous construct of rFPV/chIL-12, in rFPV/VP2-expressed rVP2 antigen was assessed in one-week-old specific-pathogen free chickens. The results indicated that rchIL-12 alone or rchIL-12 plus mineral oil (MO) co-administered with rVP2 antigen significantly enhanced the production of serum neutralization (SN) antibody against IBDV, compared to those with MO alone. The SN titers in groups receiving rVP2 antigen with MO alone were more inconsistent after vaccination. On the other hand, rchIL-12 significantly stimulated IFN-γ production in serum and in splenocyte cultured supernatant, suggesting that rchIL-12 alone or plus MO significantly induced a cell-mediated immune response. Finally, bursal lesion protection from very virulent IBDV (vvIBDV) challenge in chickens receiving rVP2 antigen with rchIL-12 alone or plus MO was much more effective than that with MO alone at two weeks after boosting. Taken together, rchIL-12 alone augmented in vivo the induction of a primary and also a secondary SN antibody production and a cell-mediated immunity against IBDV rVP2 antigen, which conferred the enhancement of bursal lesion protective efficacy from vvIBDV challenge. These data indicated that a potential for chIL-12 as immunoadjuvant for chicken vaccine development such as IBDV rVP2 antigen.

  20. Recombinative events of the T cell antigen receptor delta gene in peripheral T cell lymphomas.

    PubMed Central

    Kanavaros, P; Farcet, J P; Gaulard, P; Haioun, C; Divine, M; Le Couedic, J P; Lefranc, M P; Reyes, F

    1991-01-01

    Recombinative events of the T cell antigen receptor (TCR) delta-chain gene were studied in 37 cases of peripheral T cell lymphoma (PTCL) and related to their clinical presentation and the expression of the alpha beta or gamma delta heterodimers as determined by immunostaining of frozen tissue samples. There were 22 cases of alpha beta, 5 cases of gamma delta, and 10 cases of silent TCR expressing neither the alpha beta nor gamma delta TCR. 5 different probes were used to examine the delta locus. The 22 cases of alpha beta PTCL displayed biallelic and monoallelic deletions; a monoallelic V delta 1 J delta 1 rearrangement was observed in 1 case and a monoallelic germ line configuration in 7 cases. The 5 cases of gamma delta PTCL displayed biallelic rearrangements: the productive rearrangements could be ascribed to V delta 1J delta 1 joining in 3 cases and VJ delta 1 joining in 2 cases according to the combined pattern of DNA hybridization with the appropriate probes and of cell reactivity with the TCR delta-1, delta TCS-1, and anti-V delta 2 monoclonal antibodies. In the VJ delta 1 joining, the rearranged V segments were located between V delta 1 and V delta 2. Interestingly, in the third group of 10 cases of silent PTCL, 5 cases were found to have a TCR gene configuration identical to that in the TCR alpha beta PTCL, as demonstrated by biallelic delta gene deletion. These 5 cases were CD3 positive. The 5 remaining cases showed a monoallelic delta gene rearrangement with a monoallelic germ line configuration in 4 and a monoallelic deletion in 1. Four of these cases were CD3 negative, which was consistent with an immature genotype the TCR commitent of which could not be ascertained. Finally, TCR gamma delta PTCL consisted of a distinct clinical morphological and molecular entity whereas TCR alpha beta and silent PTCL had a similar presentation. Images PMID:1991851

  1. Structural and Immunological Analysis of Anthrax Recombinant Protective Antigen Adsorbed to Aluminum Hydroxide Adjuvant

    PubMed Central

    Wagner, Leslie; Verma, Anita; Meade, Bruce D.; Reiter, Karine; Narum, David L.; Brady, Rebecca A.; Little, Stephen F.

    2012-01-01

    New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies. PMID:22815152

  2. Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice.

    PubMed

    Xie, Yue; Chen, Sijie; Yan, Yubo; Zhang, Zhihe; Li, Desheng; Yu, Hua; Wang, Chengdong; Nong, Xiang; Zhou, Xuan; Gu, Xiaobin; Wang, Shuxian; Peng, Xuerong; Yang, Guangyou

    2013-10-03

    The intestinal nematode Baylisascaris schroederi is an important cause of death for wild and captive giant pandas. Inorganic pyrophosphatases (PPases) are critical for development and molting in nematode parasites and represent potential targets for vaccination. Here, a new PPase homologue, Bsc-PYP-1, from B. schroederi was identified and characterized, and its potential as a vaccine candidate was evaluated in a mouse challenge model. Sequence alignment of PPases from nematode parasites and other organisms show that Bsc-PYP-1 is a nematode-specific member of the family I soluble PPases. Immunohistochemistry revealed strong localization of native Bsc-PYP-1 to the body wall, gut epithelium, ovary and uterus of adult female worms. Additionally, Bsc-PYP-1 homologues were found in roundworms infecting humans (Ascaris lumbricoides), swine (Ascaris suum) and dogs (Toxocara canis). In two vaccine trials, recombinant Bsc-PYP-1 (rBsc-PYP-1) formulated with Freund complete adjuvant induced significantly high antigen-specific immunoglobulin (Ig)G but no IgE or IgM responses. Analysis of IgG-subclass profiles revealed a greater increase of IgG1 than IgG2a. Splenocytes from rBsc-PYP-1/FCA-immunized mice secreted low levels of T helper (Th)1-type cytokines, interferon-γ and interleukin (IL)-2, while producing significantly high levels of IL-10 and significantly elevated levels of IL-4 (Th2 cytokines) after stimulation with rBsc-PYP-1 in vitro. Finally, vaccinated mice had 69.02-71.15% reductions (in 2 experiments) in larval recovery 7 days post-challenge (dpc) and 80% survival at 80 dpc. These results suggest that Th2-mediated immunity elicited by rBsc-PYP-1 provides protection against B. schroederi, and the findings should contribute to further development of Bsc-PYP-1 as a candidate vaccine against baylisascariasis.

  3. Physicochemical properties of recombinant hepatitis B surface antigen expressed in mammalian cell (C127).

    PubMed

    Lee, Y S; Kim, B K; Choi, E C

    1998-10-01

    The physicochemical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10-15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAg by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCl density gradient method was 1.19 g/ml and the isoelectric point by Mono P HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. Fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and its major component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derived r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.

  4. Prevention and Immunotherapy of Secondary Murine Alveolar Echinococcosis Employing Recombinant EmP29 Antigen

    PubMed Central

    Boubaker, Ghalia; Hemphill, Andrew; Huber, Cristina Olivia; Spiliotis, Markus; Babba, Hamouda; Gottstein, Bruno

    2015-01-01

    Alveolar echinococcosis (AE) is caused by infection with the larval stage of the tapeworm Echinococcus multilocularis. An increasing understanding of immunological events that account for the metacestode survival in human and murine AE infection prompted us to undertake explorative experiments tackling the potential of novel preventive and/or immunotherapeutic measures. In this study, the immunoprotective and immunotherapeutic ability of recombinant EmP29 antigen (rEmP29) was assessed in mice that were intraperitoneally infected with E. multilocularis metacestodes. For vaccination, three intraperitoneal injections with 20μg rEmP29 emulsified in saponin adjuvants were applied over 6 weeks. 2 weeks after the last boost, mice were infected, and at 90 days post-infection, rEmP29-vaccinated mice exhibited a median parasite weight that was reduced by 75% and 59% when compared to NaCl- or saponin–treated control mice, respectively. For immunotherapeutical application, the rEmP29 (20μg) vaccine was administered to experimentally infected mice, starting at 1 month post-infection, three times with 2 weeks intervals. Mice undergoing rEmP29 immunotherapy exhibited a median parasite load that was reduced by 53% and 49% when compared to NaCl- and saponin–treated control mice, respectively. Upon analysis of spleen cells, both, vaccination and treatment with rEmP29, resulted in low ratios of Th2/Th1 (IL-4/IFN-γ) cytokine mRNA and low levels of mRNA coding for IL-10 and IL-2. These results suggest that reduction of the immunosuppressive environment takes place in vaccinated as well as immunotreated mice, and a shift towards a Th1 type of immune response may be responsible for the observed increased restriction of parasite growth. The present study provides the first evidence that active immunotherapy may present a sustainable route for the control of AE. PMID:26053794

  5. Effect of homologous and heterologous prime-boost on the immune response to recombinant plague antigens.

    PubMed

    Glynn, Audrey; Freytag, Lucy C; Clements, John D

    2005-03-14

    Among the pathogens that have been identified as potential agents of biological warfare or bioterrorism, Yersinia pestis is one of the main concerns due to the severity and potential transmissibility of the pneumonic form of the disease in humans. There are no approved vaccines for protection against pneumonic plague, but a Y. pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen in murine studies. In the current study, we examine different prime-boost regimens, including parenteral, mucosal, and transcutaneous delivery, in order to explore the effect of changing the route of prime and boost on the ability of recombinant F1-V to promote the development of long-lasting, high-titer antibodies. The most significant findings of the study reported here are that (1) intranasal and subcutaneous immunizations are both effective and essentially equivalent for induction of serum and bronchioalveolar anti-F1-V IgG1 responses when a single booster dose is administered by the same (homologous) route, (2) heterologous boosting can be as or more effective than homologous boosting for induction of either serum or bronchioalveolar anti-F1-V IgG1 responses, and (3) anti-F1 and anti-V total IgG responses were highest in animals primed intranasally and boosted by any route when compared to animals primed transcutaneously or subcutaneously. As with previously published studies, there were still significant levels of circulating anti-F1-V antibodies 1 year post-primary immunization. These studies provide important insights into the development of new-generation biodefense vaccines.

  6. Potential of recombinant inorganic pyrophosphatase antigen as a new vaccine candidate against Baylisascaris schroederi in mice

    PubMed Central

    2013-01-01

    The intestinal nematode Baylisascaris schroederi is an important cause of death for wild and captive giant pandas. Inorganic pyrophosphatases (PPases) are critical for development and molting in nematode parasites and represent potential targets for vaccination. Here, a new PPase homologue, Bsc-PYP-1, from B. schroederi was identified and characterized, and its potential as a vaccine candidate was evaluated in a mouse challenge model. Sequence alignment of PPases from nematode parasites and other organisms show that Bsc-PYP-1 is a nematode-specific member of the family I soluble PPases. Immunohistochemistry revealed strong localization of native Bsc-PYP-1 to the body wall, gut epithelium, ovary and uterus of adult female worms. Additionally, Bsc-PYP-1 homologues were found in roundworms infecting humans (Ascaris lumbricoides), swine (Ascaris suum) and dogs (Toxocara canis). In two vaccine trials, recombinant Bsc-PYP-1 (rBsc-PYP-1) formulated with Freund complete adjuvant induced significantly high antigen-specific immunoglobulin (Ig)G but no IgE or IgM responses. Analysis of IgG-subclass profiles revealed a greater increase of IgG1 than IgG2a. Splenocytes from rBsc-PYP-1/FCA-immunized mice secreted low levels of T helper (Th)1-type cytokines, interferon-γ and interleukin (IL)-2, while producing significantly high levels of IL-10 and significantly elevated levels of IL-4 (Th2 cytokines) after stimulation with rBsc-PYP-1 in vitro. Finally, vaccinated mice had 69.02–71.15% reductions (in 2 experiments) in larval recovery 7 days post-challenge (dpc) and 80% survival at 80 dpc. These results suggest that Th2-mediated immunity elicited by rBsc-PYP-1 provides protection against B. schroederi, and the findings should contribute to further development of Bsc-PYP-1 as a candidate vaccine against baylisascariasis. PMID:24090087

  7. Screening of recombinant proteins as antigens in indirect ELISA for diagnosis of bovine tuberculosis.

    PubMed

    Souza, Ingrid If; Melo, Elaine Sp; Ramos, Carlos An; Farias, Thaís A; Osório, Ana Luiza Ar; Jorge, Klaudia Sg; Vidal, Carlos Es; Silva, Altino S; Silva, Márcio R; Pellegrin, Aiesca O; Araújo, Flábio R

    2012-12-01

    Bovine tuberculosis is an important infectious disease caused by Mycobacterium bovis, which is responsible for considerable economic losses. This disease constitutes a serious public health problem. Control programs in most countries, including Brazil, are based on the identification and slaughter of infected animals, as defined by the skin tuberculin test, which has its constraints. In the present study, the recombinant proteins CFP-10, ESAT-6, Mb0143, MPB83, PE5, PE13, TB10.4, TB15.3 and a chimera of ESAT-6/MPB70/MPB83 (fusion protein) were tested as ELISA antigens for the diagnosis of bovine tuberculosis. The proteins were produced in Escherichia coli, purified and tested in ELISAs with sera from 126 cattle having tested negative in the comparative intradermal tuberculin test (CITT) and 107 sera from cattle having tested positive in the CITT. Also, 236 sera from two BTB-free beef cattle herds were tested. Among the proteins tested, only the ESAT-6/MPB70/MPB83 chimera demonstrated satisfactory agreement with the CITT (kappa index: 0.688), reflecting in 83.2% sensitivity and 86.5% specificity. The ELISA absorbances of the cattle sera from BTB-free herds showed similar levels to those of CITT positive cattle, probably as the result of successive skin tuberculinizations to define the BTB-free status of the herds. However, the ELISA with the ESAT-6/MPB70/MPB83 chimera was useful to discriminate BTB positive and negative cattle in herds prior to the tuberculin skin test.

  8. Evolution and Recombination of Genes Encoding HIV-1 Drug Resistance and Tropism during Antiretroviral Therapy

    PubMed Central

    Shi, Binshan; Kitchen, Christina; Weiser, Barbara; Mayers, Douglas; Foley, Brian; Kemal, Kimdar; Anastos, Kathryn; Suchard, Marc; Parker, Monica; Brunner, Cheryl; Burger, Harold

    2010-01-01

    Characterization of residual plasma virus during antiretroviral therapy (ART) is a high priority to improve understanding of HIV-1 pathogenesis and therapy. To understand the evolution of HIV-1 pol and env genes in viremic patients under selective pressure of ART, we performed longitudinal analyses of plasma-derived pol and env sequences from single HIV-1 genomes. We tested the hypotheses that drug resistance in pol was unrelated to changes in coreceptor usage (tropism), and that recombination played a role in evolution of viral strains. Recombinants were identified by using Bayesian and other computational methods. High-level genotypic resistance was seen in ~70% of X4 and R5 strains during ART. There was no significant association between resistance and tropism. Each patient displayed at least one recombinant encompassing env and representing a change in predicted tropism. These data suggest that, in addition to mutation, recombination can play a significant role in shaping HIV-1 evolution. PMID:20451945

  9. Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

    PubMed

    Pereira, Vanessa Bastos; Saraiva, Tessália Diniz Luerce; Souza, Bianca Mendes; Zurita-Turk, Meritxell; Azevedo, Marcela Santiago Pacheco; De Castro, Camila Prósperi; Mancha-Agresti, Pamela; Dos Santos, Janete Soares Coelho; Santos, Ana Cristina Gomes; Faria, Ana Maria Caetano; Leclercq, Sophie; Azevedo, Vasco; Miyoshi, Anderson

    2015-02-01

    The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

  10. Inhibition of mouse alkali burn induced-corneal neovascularization by recombinant adenovirus encoding human vasohibin-1

    PubMed Central

    Xie, Zhao-lian; Xiao, Ou; Yang, Xiao-ru; Heng, Boon Chin; Sato, Yasufumi

    2010-01-01

    Purpose To evaluate the activity of recombinant adenovirus encoding human vasohibin-1 (Ad-Vasohibin-1) on mouse corneal neovasularization induced by alkali burn. Methods For the treatment group, 50 mice each received subconjunctival injection (5 μl) of 109 plaque forming units of replication-defective Ad-Vasohibin-1. Control group mice received the same dosage of blank adenoviral vector (AdNull). Five days after injection, corneal neovascularization (CNV) was induced by placing 2.5 μl of 0.1 M NaOH on the right cornea for 30 s. Subsequently, CNV was observed and photographed every 3 days for a total duration of 9 days after the alkali burn. The percentage of neovascularized area was measured and compared with the AdNull control. The expression of human vasohibin-1 protein was detected by immunohistochemistry and western blotting at 5, 8, and 14 days after injection. The mRNA expression levels of murine vascular endothelial growth factor (Vegf), VEGF receptor 1 and 2 (Vegfr1, Vegfr2), and vasohibin-1 (Vash1) were analyzed and compared by real time quantitative reverse-transcription polymerase chain reaction. Results The percentage of neovascularized area within the cornea was significantly reduced in mice treated with Ad-Vasohibin-1 compared to mice treated with AdNull at every time point after alkali-induced injury (7.11%±3.91% and 15.48%±1.79% of corneal area in the treatment and control groups, respectively, on day 3; 31.64%±4.71% and 43.93%±6.15% on day 6, and 45.02%±9.98% and 66.24%±7.17% on day 9, all p<0.001). Human vasohibin-1 protein was detected at the injection sites on day 3 after corneal burn and was highly expressed in the central subepithelial stroma and co-localized with neovascularized vessels within the alkali-treated cornea on day 6. On day 9, the peripheral cornea exhibited a similar staining pattern as the central cornea, but a more intense vasohibin-1 immunostaining signal was detected in the deep stroma. Some of the vasohibin-1 stain

  11. Antibody Responses to Recombinant Epstein-Barr Virus Antigens in Nasopharyngeal Carcinoma Patients: Complementary Test of ZEBRA Protein and Early Antigens p54 and p138

    PubMed Central

    Dardari, R.; Hinderer, W.; Lang, D.; Benider, A.; El Gueddari, B.; Joab, I.; Benslimane, A.; Khyatti, M.

    2001-01-01

    Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with older NPC patients. This is a problem for the diagnosis of NPC, especially among Maghrebians, among whom young people are also affected, and emphasizes the necessity to search for more reliable markers. The present study reports results of immunoglobulin G (IgG) and IgA responses of NPC patients to recombinant EA antigens p54 (BMRF1) and p138 (BALF2), VCA complex antigens p18 (BFRF3) and p23 (BLRF2), and EBNA antigen p72 (BKRF1). Our results show that IgA-EA-p54 and -p138 (IgA-EA-p54+138) antibodies have a diagnostic value for detection of NPC (70%), compared with IgA-VCA-p18+23 and IgA-EBNA-p72, which have limited diagnostic value, especially in young patients. It is also noteworthy that IgA-EA-p54+138 can detect a high percentage (64%) of NPC cases negative by immunofluorescence. These results, however, clearly show that a single test cannot achieve the objective of detecting all NPC patients, and it seems advisable to combine different tests for the diagnosis of NPC. The combination of IgG-ZEBRA with IgA-EA-p54+138 improved the sensitivity of detection of NPC to 95% in the overall NPC population. The use of IgA-EA-p54+138 in combination with IgG-ZEBRA will facilitate detailed studies on the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC. PMID:11526145

  12. Multiple HLA A11-restricted cytotoxic T-lymphocyte epitopes of different immunogenicities in the Epstein-Barr virus-encoded nuclear antigen 4.

    PubMed

    Gavioli, R; Kurilla, M G; de Campos-Lima, P O; Wallace, L E; Dolcetti, R; Murray, R J; Rickinson, A B; Masucci, M G

    1993-03-01

    Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA A11 allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA A11-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14- or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA A11-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by A11-restricted CTL clones at concentrations as low as 5 x 10(-14) M.

  13. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

    PubMed

    Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

    2013-02-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

  14. Evaluation of Protective Potential of Yersinia pestis Outer Membrane Protein Antigens as Possible Candidates for a New-Generation Recombinant Plague Vaccine

    PubMed Central

    Erova, Tatiana E.; Rosenzweig, Jason A.; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C.; Kirtley, Michelle L.; van Lier, Christina J.; Telepnev, Maxim V.; Motin, Vladimir L.

    2013-01-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1− strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1− mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1− CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains. PMID:23239803

  15. Cloning and Characterization of Multigenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the Recombinant Protein for Serodiagnosis

    PubMed Central

    Ohashi, Norio; Unver, Ahmet; Zhi, Ning; Rikihisa, Yasuko

    1998-01-01

    A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30

  16. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  17. Immunogenicity and protective efficacy of recombinant Modified Vaccinia virus Ankara candidate vaccines delivering West Nile virus envelope antigens.

    PubMed

    Volz, Asisa; Lim, Stephanie; Kaserer, Martina; Lülf, Anna; Marr, Lisa; Jany, Sylvia; Deeg, Cornelia A; Pijlman, Gorben P; Koraka, Penelope; Osterhaus, Albert D M E; Martina, Byron E; Sutter, Gerd

    2016-04-07

    West Nile virus (WNV) cycles between insects and wild birds, and is transmitted via mosquito vectors to horses and humans, potentially causing severe neuroinvasive disease. Modified Vaccinia virus Ankara (MVA) is an advanced viral vector for developing new recombinant vaccines against infectious diseases and cancer. Here, we generated and evaluated recombinant MVA candidate vaccines that deliver WNV envelope (E) antigens and fulfil all the requirements to proceed to clinical testing in humans. Infections of human and equine cell cultures with recombinant MVA demonstrated efficient synthesis and secretion of WNV envelope proteins in mammalian cells non-permissive for MVA replication. Prime-boost immunizations in BALB/c mice readily induced circulating serum antibodies binding to recombinant WNV E protein and neutralizing WNV in tissue culture infections. Vaccinations in HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice elicited WNV E-specific CD8+ T cell responses. Moreover, the MVA-WNV candidate vaccines protected C57BL/6 mice against lineage 1 and lineage 2 WNV infection and induced heterologous neutralizing antibodies. Thus, further studies are warranted to evaluate these recombinant MVA-WNV vaccines in other preclinical models and use them as candidate vaccine in humans.

  18. Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite

    PubMed Central

    Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

    2014-01-01

    Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. PMID:24727440

  19. Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite.

    PubMed

    Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

    2014-07-01

    Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

    PubMed

    Bassi, Maria R; Larsen, Mads A B; Kongsgaard, Michael; Rasmussen, Michael; Buus, Søren; Stryhn, Anette; Thomsen, Allan R; Christensen, Jan P

    2016-02-01

    The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

  1. Antigenicity in sheep of synthetic peptides derived from stress-regulated Mycobacterium avium subsp. paratuberculosis proteins and comparison with recombinant protein and complex native antigens.

    PubMed

    Gurung, Ratna B; Begg, Douglas J; Purdie, Auriol C; Whittington, Richard J

    2014-03-15

    Serum antibody enzyme-linked immunosorbent assay is the most commonly used test for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in ruminants. However, the assay requires serum preabsorption with Mycobacterium phlei proteins to reduce cross reactions potentially contributed by the exposure of livestock to environmental mycobacteria. To trial the discovery of novel antigens which do not require serum absorption, synthetic MAP-specific peptides were selected based on in silico research to identify putative B cell epitopes. Four peptides from previously identified stress-regulated proteins were synthesized and evaluated using enzyme linked immunosorbent assay to detect Mycobacterium avium subsp. paratuberculosis specific antibodies in sheep. Two peptides were from hypothetical MAP proteins (MAP3567 and MAP1168c) and two were from proteins with known function (MAP2698c, an acyl-acyl carrier protein desaturase-DesA2 and MAP2487c a carbonic anhydrase). The ability of each peptide to discriminate between unexposed and MAP exposed (infected and vaccinated) animals was similar to that of the parent recombinant MAP antigen, with area under receiver operating curve values of 0.86-0.93. Assays run with a combination of two peptides showed slightly higher reactivity than those of individual peptides. Peptides evaluated in this study had diagnostic potential similar to corresponding recombinant proteins but not superior to a complex native MAP antigen or a commercial assay. Further study is required to investigate other peptides for their diagnostic potential, and this may be simpler and cheaper than subunit protein-based research. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. G-quadruplexes regulate Epstein-Barr virus–encoded nuclear antigen 1 mRNA translation

    PubMed Central

    Murat, Pierre; Zhong, Jie; Lekieffre, Lea; Cowieson, Nathan P; Clancy, Jennifer L; Preiss, Thomas; Balasubramanian, Shankar; Khanna, Rajiv; Tellam, Judy

    2014-01-01

    Viruses that establish latent infections have evolved unique mechanisms to avoid host immune recognition. Maintenance proteins of these viruses regulate their synthesis to levels sufficient for maintaining persistent infection but below threshold levels for host immune detection. The mechanisms governing this finely tuned regulation of viral latency are unknown. Here we show that mRNAs encoding gammaherpesviral maintenance proteins contain within their open reading frames clusters of unusual structural elements, G-quadruplexes, which are responsible for the cis-acting regulation of viral mRNA translation. By studying the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1) mRNA, we demonstrate that destabilization of G-quadruplexes using antisense oligonucleotides increases EBNA1 mRNA translation. In contrast, pretreatment with a G-quadruplex-stabilizing small molecule, pyridostatin, decreases EBNA1 synthesis, highlighting the importance of G-quadruplexes within virally encoded transcripts as unique regulatory signals for translational control and immune evasion. Furthermore, these findings suggest alternative therapeutic strategies focused on targeting RNA structure within viral ORFs. PMID:24633353

  3. Expression and refolding of the protective antigen of Bacillus anthracis: A model for high-throughput screening of antigenic recombinant protein refolding.

    PubMed

    Pavan, María Elisa; Pavan, Esteban Enrique; Cairó, Fabián Martín; Pettinari, María Julia

    2016-01-01

    Bacillus anthracis protective antigen (PA) is a well known and relevant immunogenic protein that is the basis for both anthrax vaccines and diagnostic methods. Properly folded antigenic PA is necessary for these applications. In this study a high level of PA was obtained in recombinant Escherichia coli. The protein was initially accumulated in inclusion bodies, which facilitated its efficient purification by simple washing steps; however, it could not be recognized by specific antibodies. Refolding conditions were subsequently analyzed in a high-throughput manner that enabled nearly a hundred different conditions to be tested simultaneously. The recovery of the ability of PA to be recognized by antibodies was screened by dot blot using a coefficient that provided a measure of properly refolded protein levels with a high degree of discrimination. The best refolding conditions resulted in a tenfold increase in the intensity of the dot blot compared to the control. The only refolding additive that consistently yielded good results was L-arginine. The statistical analysis identified both cooperative and negative interactions between the different refolding additives. The high-throughput approach described in this study that enabled overproduction, purification and refolding of PA in a simple and straightforward manner, can be potentially useful for the rapid screening of adequate refolding conditions for other overexpressed antigenic proteins. Copyright © 2015 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

    PubMed Central

    2011-01-01

    Background Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS). Methods In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in E. coli. Results The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in E. coli showed that both were equally effective in terms of sensitivity and specificity. Conclusions Our results therefore indicate that either of these proteins can be used in serological tests in Brazil. PMID:21569341

  5. Immune responses to a recombinant attenuated Salmonella typhimurium strain expressing a Taenia solium oncosphere antigen TSOL18.

    PubMed

    Ding, Juntao; Zheng, Yadong; Wang, Ying; Dou, Yongxi; Chen, Xiaoyu; Zhu, Xueliang; Wang, Shuai; Zhang, Shaohua; Liu, Zhenyong; Hou, Junling; Zhai, Junjun; Yan, Hongbin; Luo, Xuenong; Cai, Xuepeng

    2013-01-01

    A tapeworm, Taenia solium, remains a great threat to human health, particularly in developing countries. The life cycle of T. solium is thought to be terminated via vaccination of intermediate hosts. In this study, we constructed a recombinant attenuated Salmonella typhimurium live vaccine strain χ4558 expressing a TSOL18 antigen. SDS-PAGE and Western blot confirmed the expression of the interest protein and its antigenic property. The recombinant strain stably propagated in vitro, of which the growth was not reversely influenced by TSOL18 protein expressed. It was also shown that mice survived 10(12) CFU of S. typhimurium χ4558, while all mice infected with 10(7) CFU of the wild-type died within five days. The mouse experiment indicated that vaccine strain χ4558 induced a high titer of specific antibody for a long time. In contrast to the controls, the vaccinated mice had an obvious augment of CD4(+) and CD8(+) T lymphocytes and the percentage of helper CD4(+)/CD8(+) T lymphocytes was significantly increased (p<0.01). After oral administration, S. typhimurium χ4558 was first colonized mainly in the Peyer's patches and then predominantly in the mesenteric lymph nodes and spleens in the vaccinated mice. In addition, the high levels of specific anti-TSOL18 antibodies were also observed in pigs administrated with S. typhimurium χ4558. Collectively, these results demonstrate the possibility of use of an attenuated S. typhimurium strain as a vector to deliver protective antigens of T. solium.

  6. Immune responses against recombinant tick antigen, Bm95, for the control of Rhipicephalus (Boophilus) microplus ticks in cattle.

    PubMed

    Kumar, Amit; Garg, Rajat; Yadav, C L; Vatsya, Stuti; Kumar, R R; Sugumar, Parthasarthy; Chandran, Dev; Mangamoorib, Lakshmi Narasu; Bedarkar, S N

    2009-10-28

    Immune responses against Bm95 recombinant cattle tick antigen and its protective efficacy for control of Rhipicephalus (Boophilus) microplus ticks were determined in experimental crossbred cow calves. Anti-Bm95 antibody titers, as assessed by indirect ELISA, in immunized calves ranged from 196.1+/-13.7 on day 0 to 7979.9+/-312.5 on day 110 post-primary immunization. The rise in antibody titer was statistically significant (p<0.01) throughout the study period. Besides this, constantly higher lymphoproliferative response (LPR), as assessed by lymphocyte stimulation test, was observed from 10 days post-immunization, but a positive LPR of antigen stimulated cells in immunized animals was recorded only on day 50 and day 70 post-immunization. Following challenge of immunized calves with larvae of R. microplus, significant increase (p<0.01) in rejection percentage, mean number of damaged ticks, mean percentage of dead ticks, and decrease in engorgement weight were recorded in immunized animals. Also, there were significant differences (p<0.01) in preoviposition period, oviposition period, egg mass weight and percent hatchability between the immunized and control calves. The percent reduction in number of adult females in vaccinated calves, reduction in mean weight of egg masses, percent reduction in mean weight and reduction in fertility of engorged females collected from vaccinated calves were determined and the efficacy of Bm95 recombinant cattle tick antigen was 81.27%.

  7. Cryogenic transmission electron microscopy of recombinant tuberculosis vaccine antigen with anionic liposomes reveals formation of flattened liposomes

    PubMed Central

    Fox, Christopher B; Mulligan, Sean K; Sung, Joyce; Dowling, Quinton M; Fung, H W Millie; Vedvick, Thomas S; Coler, Rhea N

    2014-01-01

    Development of lipid-based adjuvant formulations to enhance the immunogenicity of recombinant vaccine antigens is a focus of modern vaccine research. Characterizing interactions between vaccine antigens and formulation excipients is important for establishing compatibility between the different components and optimizing vaccine stability and potency. Cryogenic transmission electron microscopy (TEM) is a highly informative analytical technique that may elucidate various aspects of protein- and lipid-based structures, including morphology, size, shape, and phase structure, while avoiding artifacts associated with staining-based TEM. In this work, cryogenic TEM is employed to characterize a recombinant tuberculosis vaccine antigen, an anionic liposome formulation, and antigen–liposome interactions. By performing three-dimensional tomographic reconstruction analysis, the formation of a population of protein-containing flattened liposomes, not present in the control samples, was detected. It is shown that cryogenic TEM provides unique information regarding antigen–liposome interactions not detectable by light-scattering-based methods. Employing a suite of complementary analytical techniques is important to fully characterize interactions between vaccine components. PMID:24648734

  8. Construction of a complementary DNA library for Parelaphostrongylus tenuis and identification of a potentially sero-diagnostic recombinant antigen.

    PubMed

    Ogunremi, Oladele; Benjamin, Jane; MacDonald, Lily; Schimpf, Robert

    2008-12-01

    Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

  9. Recombinant Salmonella typhimurium strains that invade nonphagocytic cells are resistant to recognition by antigen-specific cytotoxic T lymphocytes.

    PubMed Central

    Gao, X M; Tite, J P; Lipscombe, M; Rowland-Jones, S; Ferguson, D J; McMichael, A J

    1992-01-01

    To address the question of whether Salmonella-infected nonphagocytic cells could serve as target cells for recognition by antigen-specific, major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL), four recombinant Salmonella typhimurium constructs that expressed full-length, or fragments of, influenza A virus nucleoprotein (NP) were made. The bacteria were shown to infect Chinese hamster ovary (CHO) cells. Appropriate major histocompatibility complex restriction molecules, HLA-B27 and H-2 Db, were transfected into CHO cells, which were then infected with recombinant S. typhimurium and used as targets for NP-specific CTL. The cells in which NP was expressed by intracellularly replicating bacteria were not lysed by NP-specific CTL, although they were killed when appropriate influenza A virus or peptides were used. Thus, S.typhimurium bacteria within nonphagocytic cells were resistant to CTL recognition. In contrast to these results, mice infected with recombinant S.typhimurium that expressed fragments of NP in the periplasm were primed for NP-specific CTL responses. The results indicate that CTL responses specific to Salmonella antigens can be generated, but the bacteria may be safe from the CTL attack once they have entered the nonphagocytic cells. Images PMID:1500187

  10. A novel hepatitis C virus vaccine approach using recombinant Bacillus Calmette-Guerin expressing multi-epitope antigen.

    PubMed

    Wei, S-H; Yin, W; An, Q-X; Lei, Y-F; Hu, X-B; Yang, J; Lu, X; Zhang, H; Xu, Z-K

    2008-01-01

    Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV infection is associated with high morbidity and has become a major problem in public health. Until now, there has been no effective prophylactic or therapeutic vaccine. BCG, a live vaccine typically used for tuberculosis prevention, has been increasingly utilized as a vector for the expression of recombinant proteins that will induce specific humoral and cellular immune responses. In this study, recombinant BCG (rBCG) was engineered to express a HCV multi-epitope antigen CtEm, and HLA-A2.1 transgenic mice were immunized with rBCG-CtEm. High levels of specific anti-HCV antibodies targeted to mimotopes of HVR1 were detected in the serum. HCV-specific lymphocyte proliferation assay, cytokine determination and cytotoxicity assay indicated that HCV epitope-specific cellular immune responses were elicited in vitro. The rBCG-CtEm immunization conferred protection against infection with the recombinant vaccinia virus (rVV-HCV-CNS) in vivo. These results suggest that rBCG expressing multi-epitope antigen may serve as an effective vaccine against HCV infection.

  11. Recombinant Interferon Alpha-2b is a High-Affinity Antigen for Type 1 Diabetes Autoantibodies.

    PubMed

    Khan, Wahid Ali

    2017-04-01

    Type 1 diabetes results from T-cell-mediated destruction of the beta cells of the pancreas and is associated with several autoimmune phenomena. Many studies have suggested the involvement of interferon alpha (IFN α) in the development of type 1 diabetes, but the exact mechanism remains unclear. In this study, the binding of type 1 diabetes antibodies with recombinant interferon alpha-2b (hrIFN α-2b), their gene (cIFN α-2b gene) and commercially available interferon α-2b (IFN α-2b) were assessed. Furthermore, we also sought to use anti-hrIFN α-2b antibodies as a probe for the estimation of plasma IFN α in patients with type 1 diabetes. The binding specificity of antibodies was analyzed by direct binding, inhibition ELISA and quantitative precipitin titration in 45 patients with type 1 diabetes and 30 control subjects. Competition ELISA was also used to estimate INF α in the serum of patients with type 1 diabetes. Antibodies from type 1 diabetes sera, purified in a protein A-agarose matrix, exhibited greater recognition of hrIFN α-2b than IFN α-2b (p<0.05) and cIFN α-2b gene (p<0.001). The relative affinity of type 1 diabetes antibodies for the hrIFN α-2b, IFN α-2b and cIFN α-2b genes was found to be 1.34×10(-7), 1.28×10(-6) and 1.13×10(-6), respectively. The concentration of plasma INF α evaluated by induced antibodies was found to be significantly higher than in controls (p<0.05). High binding of hrIFN α-2b with IgG from patients with type 1 diabetes might suggest involvement of hrIFN α-2b in type 1 diabetes, especially as an antigenic agent. Anti-hrIFN α-2b antibodies were shown to be good probes for estimation of plasma INF α in patients with type 1 diabetes. Copyright © 2016 Canadian Diabetes Association. Published by Elsevier Inc. All rights reserved.

  12. A recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) for lungworm detection in seals.

    PubMed

    Ulrich, Sophia Arlena; Lehnert, Kristina; Siebert, Ursula; Strube, Christina

    2015-09-02

    Pinnipeds are frequently infected by the lungworms Otostrongylus circumlitus and Parafilaroides gymnurus (Metastrongyloidea). Infections are frequently associated with secondary bacterial bronchopneumonia and are often lethal. To date, a reliable lungworm diagnosis in individual seals is only possible during necropsy as examination of faeces collected from resting places does not allow assignment to individuals. Therefore, a diagnostic tool for lungworm detection in living seals is desirable for monitoring health of seals in the wild and in captivity. Previously, an ELISA based on recombinant bovine lungworm major sperm protein (MSP) as diagnostic antigen was developed for lungworm diagnosis in cattle. In the present study, this test was adapted for detection of antibodies against lungworms in harbour (Phoca vitulina) and grey seals (Halichoerus grypus). Furthermore, sera of northern elephant seals (Mirounga angustirostris) were tested to evaluate whether the harbour/grey seal ELISA is suitable for this seal species as well. For ELISA evaluation, lungworm-positive and -negative sera of harbour and grey seals were analysed using horseradish peroxidase (HRP)-conjugated Protein A as secondary antibody. Optical density was measured and a receiver operating characteristic (ROC) analysis was performed to determine a cut-off value. Potential cross-reactions were examined by testing serum of seals positive for gastrointestinal and heart nematodes, but negative for lungworm infections. In addition, sera of northern elephant seals were analysed. Harbour and grey seal serum samples showed significant differences in optical density (OD) between serum of infected and uninfected animals resulting in a cut-off value of 0.422 OD with a specificity of 100% (95% CI: 87.23-100%) and a sensitivity of 97.83% (95% CI: 88.47-99.94%). Cross-reactions with heart or gastrointestinal nematodes were not observed. Analysis of northern elephant seal samples resulted in detection of antibodies

  13. Immunogenicity, protective efficacy and safety of a recombinant DNA vaccine encoding truncated Plasmodium yoelii sporozoite asparagine-rich protein 1 (PySAP1).

    PubMed

    Zhao, Jia; Deng, Shu; Liang, Jiayuan; Cao, Yaming; Liu, Jun; Du, Feng; Shang, Hong; Cui, Liwang; Luo, Enjie

    2013-05-01

    Although great efforts have been undertaken for the development of malaria vaccines, no completely effective malaria vaccines are available yet. Despite being clinically silent, the pre-erythrocytic stage is considered an ideal target for the development of malaria vaccines. Sporozoite asparagine-rich protein 1 (SAP1) is a sporozoite-localized protein that regulates the expression of UIS (upregulated in infectious sporozoites) genes, which are essential for the infectivity of sporozoites. In this study, a recombinant DNA vaccine encoding a predicted antigenic determinant region of Plasmodium yoelii SAP1 (PySAP1) was constructed. Immunization of mice with this DNA vaccine construct resulted in significant elevation of cytokines such as IFN-γ, IL-2, IL-4 and IL-10, and total IgG as compared with control groups immunized with either the empty DNA vector or saline. After challenge with sporozoites, the group receiving the DNA vaccine showed delayed development of parasitemia and prolonged survival time compared with the control group. The DNA vaccine provided partial protection against P. yoelii 17XL infection, with an overall protection rate of 20%. In addition, the DNA vaccine did not show integration into the host genome. Further studies of SAP1 are needed to test whether it can be used as subunit vaccine candidate.

  14. R46 encodes a site-specific recombination system interchangeable with the resolution function of TnA.

    PubMed

    Dodd, H M; Bennett, P M

    1983-05-01

    Transposition of Tn4 onto the IncN plasmic R46 generates unstable DNA molecules. The R46::TnA recombinant plasmids undergo further DNA rearrangements which depend on the orientation in which the TnA element is inserted into the plasmid, and deletions and inversions of R46 and TnA sequences have been observed. Both types of rearrangement have the same specific endpoints, one within TnA and one located between the R46 coordinates, 36.0 and 37.0. The results are consistent with the operation of a recA-independent, site-specific recombination system utilizing, at least in part, the transposon cointegrate resolution system of TnA, together with R46-encoded functions. Data are presented that indicate that R46 encodes analogs of both the res site of TnA and its tnpR gene, although little homology between this element and the plasmid is apparent. Models for the TnA-induced generation of site-specific deletions and inversions upon transposition of TnA to R46 are presented.

  15. Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination.

    PubMed Central

    Lemon, S M; Murphy, P C; Shields, P A; Ping, L H; Feinstone, S M; Cromeans, T; Jansen, R W

    1991-01-01

    Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype. Images PMID:1705995

  16. Identification, characterization, and application of a recombinant antigen for the serological investigation of feline hemotropic Mycoplasma infections.

    PubMed

    Wolf-Jäckel, Godelind A; Jäckel, Christian; Museux, Kristina; Hoelzle, Katharina; Tasker, Séverine; Lutz, Hans; Hofmann-Lehmann, Regina

    2010-12-01

    In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, "Candidatus Mycoplasma haemominutum," and "Candidatus Mycoplasma turicensis." In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, size-exclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, "Ca. Mycoplasma haemominutum," and "Ca. Mycoplasma turicensis," respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.

  17. Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen

    PubMed Central

    Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

    2014-01-01

    Background Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis. PMID:24675711

  18. Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses.

    PubMed

    Buriani, Giampaolo; Mancini, Camillo; Benvenuto, Eugenio; Baschieri, Selene

    2012-04-01

    Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that pHSP70 derived from plants producing recombinant antigens may be used to formulate multiepitope vaccines.

  19. Recombinant Salmonella Expressing Burkholderia mallei LPS O Antigen Provides Protection in a Murine Model of Melioidosis and Glanders

    PubMed Central

    Moustafa, Dina A.; Scarff, Jennifer M.; Garcia, Preston P.; Cassidy, Sara K. B.; DiGiandomenico, Antonio; Waag, David M.; Inzana, Thomas J.; Goldberg, Joanna B.

    2015-01-01

    Burkholderia pseudomallei and Burkholderia mallei are the etiologic agents of melioidosis and glanders, respectively. These bacteria are highly infectious via the respiratory route and can cause severe and often fatal diseases in humans and animals. Both species are considered potential agents of biological warfare; they are classified as category B priority pathogens. Currently there are no human or veterinary vaccines available against these pathogens. Consequently efforts are directed towards the development of an efficacious and safe vaccine. Lipopolysaccharide (LPS) is an immunodominant antigen and potent stimulator of host immune responses. B. mallei express LPS that is structurally similar to that expressed by B. pseudomallei, suggesting the possibility of constructing a single protective vaccine against melioidosis and glanders. Previous studies of others have shown that antibodies against B. mallei or B. pseudomallei LPS partially protect mice against subsequent lethal virulent Burkholderia challenge. In this study, we evaluated the protective efficacy of recombinant Salmonella enterica serovar Typhimurium SL3261 expressing B. mallei O antigen against lethal intranasal infection with Burkholderia thailandensis, a surrogate for biothreat Burkholderia spp. in a murine model that mimics melioidosis and glanders. All vaccine-immunized mice developed a specific antibody response to B. mallei and B. pseudomallei O antigen and to B. thailandensis and were significantly protected against challenge with a lethal dose of B. thailandensis. These results suggest that live-attenuated SL3261 expressing B. mallei O antigen is a promising platform for developing a safe and effective vaccine. PMID:26148026

  20. Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Baylisascaris procyonis Larva Migrans ▿

    PubMed Central

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Ndao, Momar; Kazacos, Kevin R.

    2011-01-01

    Baylisascaris larva migrans is an important zoonotic disease caused by Baylisascaris procyonis, the raccoon roundworm, and is being increasingly considered in the differential diagnosis of eosinophilic meningoencephalitis in children and young adults. Although a B. procyonis excretory-secretory (BPES) antigen-based enzyme-linked immunosorbent assay (ELISA) and a Western blot assay are useful in the immunodiagnosis of this infection, cross-reactivity remains a major problem. Recently, a recombinant B. procyonis antigen, BpRAG1, was reported for use in the development of improved serological assays for the diagnosis of Baylisascaris larva migrans. In this study, we tested a total of 384 human patient serum samples in a BpRAG1 ELISA, including samples from 20 patients with clinical Baylisascaris larva migrans, 137 patients with other parasitic infections (8 helminth and 4 protozoan), and 227 individuals with unknown/suspected parasitic infections. A sensitivity of 85% and a specificity of 86.9% were observed with the BpRAG1 ELISA, compared to only 39.4% specificity with the BPES ELISA. In addition, the BpRAG1 ELISA had a low degree of cross-reactivity with antibodies to Toxocara infection (25%), while the BPES antigen showed 90.6% cross-reactivity. Based on these results, the BpRAG1 antigen has a high degree of sensitivity and specificity and should be very useful and reliable in the diagnosis and seroepidemiology of Baylisascaris larva migrans by ELISA. PMID:21832102

  1. Salivary sIg-A response against the recombinant Ag38 antigen of Mycobacterium tuberculosis Indonesian strain.

    PubMed

    Raras, Tri Yudani Mardining; Sholeh, Gamal; Lyrawati, Diana

    2014-01-01

    An evaluation of the humoral response based on secretory immunoglobulin A levels in the saliva of pulmonary tuberculosis (TB) acid-fast bacillus-positive (TB-AFB+) patients against a recombinant 38 kDa antigen (Ag38-rec) is reported. A total of 60 saliva samples consist of 30 TB-AFB+ patients and 30 healthy controls were tested against 500 ng of semi-purified antigen using the dot blot method. Results showed that the protein antigen could differentiate between healthy individuals and TB-AFB(+) patients. Whole saliva demonstrated better reactivity than centrifuged saliva. The Ag38-rec protein indicated statistically comparable sensitivity (80% versus 90%), but lower specificity (36.6% versus 70%) compared with purified protein derivative (PPD). Surprisingly, both antigens similarly recognized secretory immunoglobulin A in the saliva of the healthy group (50% versus 50%, respectively). These findings suggest that the Ag38-rec protein originating from a local strain of Mycobacterium tuberculosis may be used for TB screening, however require purity improvement.

  2. Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

    PubMed

    Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

    2012-01-01

    Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

  3. C19orf48 Encodes a Minor Histocompatibility Antigen Recognized by CD8+ Cytotoxic T Cells from Renal Cell Carcinoma Patients

    PubMed Central

    Tykodi, Scott S.; Fujii, Nobuharu; Vigneron, Nathalie; Lu, Sharon M.; Mito, Jeffrey K.; Miranda, Maureen X.; Chou, Jeffrey; Voong, Lilien N.; Thompson, John A.; Sandmaier, Brenda M.; Cresswell, Peter; Van den Eynde, Benoît; Riddell, Stanley R.; Warren, Edus H.

    2009-01-01

    Purpose Tumor regression has been observed in some patients with metastatic renal cell carcinoma (RCC) after nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Cellular and molecular characterization of antigens recognized by tumor-reactive T cells isolated from responding patients could potentially provide insight into the mechanisms of tumor regression. Experimental Design CD8+ cytotoxic T lymphocyte (CTL) clones that recognized a novel RCC-associated minor histocompatibility (H) antigen presented by HLA-A*0201 were isolated from two patients with metastatic RCC who experienced tumor regression or stable disease following nonmyeloablative allogeneic HCT. These clones were used to screen a cDNA library and isolate the unique cDNA encoding the antigen. Results An alternative open reading frame in the C19orf48 gene located on chromosome 19q13 encodes the HLA-A*0201-restricted minor H antigen recognized by the RCC-reactive T cells. Differential T cell recognition of donor- and recipient-derived target cells is attributable to a nonsynonymous single nucleotide polymorphism within the nucleotide interval that encodes the antigenic peptide. Assays for gene expression and CTL recognition demonstrated that the C19orf48-encoded peptide is widely expressed in renal tumors and solid tumors of other histologies. The antigenic peptide can be processed for CTL recognition via both TAP-dependent and TAP-independent pathways. Conclusions Donor T cell responses against the HLA-A*0201-restricted minor H antigen encoded by C19orf48 may contribute to RCC regression after MHC-matched allogeneic HCT. PMID:18698046

  4. Protection Against Aerosolized Yersinia pestis Challenge Following Homologous and Heterologous Prime-Boost With Recombinant Plague Antigens

    DTIC Science & Technology

    2005-08-01

    INFECTION AND IMMUNITY , Aug. 2005, p. 5256–5261 Vol. 73, No. 8 0019-9567/05/$08.000 doi:10.1128/IAI.73.8.5256–5261.2005 Copyright © 2005, American...and heterologous prime-boost with recombinant plague antigens, Infection and Immunity 73:5256 - 5261 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM... infection following aerosol challenge with Y. pestis with a median survival time (MST) of 72 h. By con- trast, 9/10 positive-control animals immunized

  5. Full length nucleotide sequences of 30 common SLC44A2 alleles encoding human neutrophil antigen-3 (HNA-3)

    PubMed Central

    Chen, Qing; Srivastava, Kshitij; Ardinski, Stefanie C.; Lam, Kevin; Huvard, Michael J.; Schmid, Pirmin; Flegel, Willy A.

    2015-01-01

    Background HNA-3a alloantibodies can cause severe transfusion-related acute lung injury (TRALI). The frequency of the single nucleotide polymorphisms (SNPs) indicative of the two clinically relevant HNA-3a/b antigens are known in many populations. In the present study, we determined the full length nucleotide sequence of common SLC44A2 alleles encoding the choline transporter-like protein-2 (CTL2) that harbors HNA-3a/b antigens. Study design and methods A method was devised to determine the full length coding sequence and adjacent intron sequences from genomic DNA by 8 polymerase chain reaction (PCR) amplifications covering all 22 SLC44A2 exons. Samples from 200 African American, 96 Caucasian, 2 Hispanic and 4 Asian blood donors were analyzed. We developed a decision tree to determine alleles (confirmed haplotypes) from the genotype data. Results A total of 10 SNPs were detected in the SLC44A2 coding sequence. The non-coding sequences harbored an additional 28 SNPs (1 in the 5’-untranslated region (UTR); 23 in the introns; and 4 in the 3’-UTR). No SNP indicative of a non-functional allele was detected. The nucleotide sequences for 30 SLC44A2 alleles (haplotypes) were confirmed. There may be 66 haplotypes among the 604 chromosomes screened. Conclusions We found 38 SNPs, including 1 novel SNP, in 8192 nucleotides covering the coding sequence of the SLC44A2 gene among 302 blood donors. Population frequencies of these SNPs were established for African Americans and Caucasians. Because alleles encoding HNA-3b are more common than non-functional SLC44A2 alleles, we confirmed our previous postulate that African American donors are less likely to form HNA-3a antibodies compared to Caucasians. PMID:26437811

  6. Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2017-03-28

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.

    PubMed

    Olvera, Alejandro; Ramos-Cerrillo, Blanca; Estévez, Judith; Clement, Herlinda; de Roodt, Adolfo; Paniagua-Solís, Jorge; Vázquez, Hilda; Zavaleta, Alfonso; Arruz, María Salas; Stock, Roberto P; Alagón, Alejandro

    2006-07-01

    We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.

  8. Serodiagnosis of Toxoplasma gondii infection in bovines from Kerala, India using a recombinant surface antigen 1 ELISA.

    PubMed

    Sudan, Vikrant; Tewari, Anup Kumar; Singh, Harkirat

    2015-07-01

    Data on the prevalence of toxoplasmosis in farm animals from India is scanty. Though a few reports exist on prevalence of toxoplasmosis in small ruminants, information on toxoplasmosis in large ruminants is virtually nonexistent from India. An antibody detection recombinant ELISA specific for Toxoplasma gondii was laboratory standardized using recombinant surface antigen 1 (SAG1) protein. A 958-bp truncated sequence coding for tachyzoite stage specific SAG1 protein was amplified and expressed in Escherichia coli BL21(DE3) cells. A high-level expression of the histidine-tagged thioredoxin fusion protein was obtained after 8 h of incubation. The recombinant protein was affinity purified by Ni-NTA agarose chromatography and characterized by SDS-PAGE and Western blot. Subsequently, the diagnostic potential of the recombinant protein was assessed with 258 cattle sera samples from field by a laboratory standardized recSAG1 ELISA. Sera from 71.8% of the cattle showed sero positivity for T. gondii specific IgG. The sensitivity and specificity of the recSAG1 ELISA were 84.38% and 87.88%, respectively in comparison to indirect fluorescent antibody test (IFAT). This is the first report on sensitive serodetection of Toxoplasma infection in bovines from India. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  9. Definition of the gene encoding the minor histocompatibility antigen HA-1 and typing for HA-1 from genomic DNA.

    PubMed

    Tseng, L H; Lin, M T; Martin, P J; Pei, J; Smith, A G; Hansen, J A

    1998-10-01

    Recipient mismatching for the minor histocompatibility antigen HA-1 has been associated with acute graft-versus-host disease after allogeneic marrow transplantation. Two polymorphic nucleotides near an exon-intron junction of the gene encoding this minor histocompatibility antigen have been identified. In this study, we determined the genomic DNA sequence of the intron downstream from this polymorphic exon. Based on this sequence, primers were designed to amplify the genomic HA-1 gene sequence, and analysis of restriction fragment length polymorphisms was used to assign the HA-1 genotypes of 160 unrelated probands and a paired sibling for each proband. Among probands, the HA-1H allele frequency was 0.441, and the HA-1R allele frequency was 0.559. The distribution of HA-1 genotypes showed close fit with Hardy-Weinberg equilibrium. Likewise, the number of sibling pairs with disparity for HA-1 alleles showed close fit with predictions based on Hardy-Weinberg equilibrium. These results provide a simple and well validated method for future studies correlating HA-1 disparity with clinical outcome after allogeneic marrow transplantation.

  10. A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes

    PubMed Central

    1996-01-01

    A number of antigens recognized by tumor-reactive T cells have recently been identified. The antigens identified in mouse model systems appear, with one exception, to represent the products of mutated genes. In contrast, most of the antigens recognized by human tumor-reactive T cells reported to date appear to represent the products of non-mutated genes. Here we report the isolation of a cDNA clone encoding beta- catenin, which was shown to be recognized by the tumor-infiltrating lymphocyte (TIL) 1290, a HLA-A24 restricted melanoma-specific CTL line from patient 888. The cDNA clone, which was isolated from the autologous melanoma cDNA library, differed by a single base pair from the published beta-catenin sequence, resulting in a change from a serine to a phenylalanine residue at position 37. Normal tissues from this patient did not express the altered sequence, nor did 12 allogeneic melanomas, indicating that this represented a unique mutation in this patient's melanoma. A peptide corresponding to the sequence between amino acids 29 and 37 of the mutant gene product was identified as the T cell epitope recognized by TIL 1290. The observation that HLA-A24 binding peptides contain an aromatic or hydrophobic residue at position 9 suggested that the change at position 37 may have generated a peptide (SYLDSGIHF) which was capable of binding to HLA-A24, and a competitive binding assay confirmed this hypothesis. The beta-catenin protein has been shown previously to be involved in cell adhesion mediated through the cadherin family of cell surface adhesion molecules. The high frequency of mutations found in members of cellular adhesion complexes in a variety of cancers suggests that these molecules may play a role in development of the malignant phenotype. PMID:8642260

  11. Gene encoding Duffy antigen/receptor for chemokines is associated with asthma and IgE in three populations.

    PubMed

    Vergara, Candelaria; Tsai, Yuhjung J; Grant, Audrey V; Rafaels, Nicholas; Gao, Li; Hand, Tracey; Stockton, Maria; Campbell, Monica; Mercado, Dilia; Faruque, Mezbah; Dunston, Georgia; Beaty, Terri H; Oliveira, Ricardo Riccio; Ponte, Eduardo V; Cruz, Alvaro A; Carvalho, Edgar; Araujo, Maria Ilma; Watson, Harold; Schleimer, Robert P; Caraballo, Luis; Nickel, Renate G; Mathias, Rasika A; Barnes, Kathleen C

    2008-11-15

    Asthma prevalence and severity are high among underserved minorities, including those of African descent. The Duffy antigen/receptor for chemokines is the receptor for Plasmodium vivax on erythrocytes and functions as a chemokine-clearing receptor. Unlike European populations, decreased expression of the receptor on erythrocytes is common among populations of African descent, and results from a functional T-46C polymorphism (rs2814778) in the promoter. This variant provides an evolutionary advantage in malaria-endemic regions, because Duffy antigen/receptor for chemokines-negative erythrocytes are more resistant to infection by P. vivax. To determine the role of the rs2814778 polymorphism in asthma and atopy as measured by total serum IgE levels among four populations of African descent (African Caribbean, African American, Brazilian, and Colombian) and a European American population. Family-based association tests were performed in each of the five populations to test for association between the rs2814778 polymorphism and asthma or total IgE concentration. Asthma was significantly associated with the rs2814778 polymorphism in the African Caribbean, Colombian, and Brazilian families (P < 0.05). High total IgE levels were associated with this variant in African Caribbean and Colombian families (P < 0.05). The variant allele was not polymorphic among European Americans. Susceptibility to asthma and atopy among certain populations of African descent is influenced by a functional polymorphism in the gene encoding Duffy antigen/receptor for chemokines. This genetic variant, which confers resistance to malarial parasitic infection, may also partially explain ethnic differences in morbidity of asthma.

  12. Molecular cloning, expression, and chromosomal localization of the gene encoding a human myeloid membrane antigen (gp150).

    PubMed Central

    Look, A T; Peiper, S C; Rebentisch, M B; Ashmun, R A; Roussel, M F; Lemons, R S; Le Beau, M M; Rubin, C M; Sherr, C J

    1986-01-01

    DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells. Images PMID:2428842

  13. Novel prostate cancer immunotherapy with a DNA-encoded anti-prostate-specific membrane antigen monoclonal antibody.

    PubMed

    Muthumani, Kar; Marnin, Liron; Kudchodkar, Sagar B; Perales-Puchalt, Alfredo; Choi, Hyeree; Agarwal, Sangya; Scott, Veronica L; Reuschel, Emma L; Zaidi, Faraz I; Duperret, Elizabeth K; Wise, Megan C; Kraynyak, Kimberly A; Ugen, Kenneth E; Sardesai, Niranjan Y; Joseph Kim, J; Weiner, David B

    2017-08-17

    Prostate-specific membrane antigen (PSMA) is expressed at high levels on malignant prostate cells and is likely an important therapeutic target for the treatment of prostate carcinoma. Current immunotherapy approaches to target PSMA include peptide, cell, vector or DNA-based vaccines as well as passive administration of PSMA-specific monoclonal antibodies (mAb). Conventional mAb immunotherapy has numerous logistical and practical limitations, including high production costs and a requirement for frequent dosing due to short mAb serum half-life. In this report, we describe a novel strategy of antibody-based immunotherapy against prostate carcinoma that utilizes synthetic DNA plasmids that encode a therapeutic human mAb that target PSMA. Electroporation-enhanced intramuscular injection of the DNA-encoded mAb (DMAb) plasmid into mice led to the production of functional and durable levels of the anti-PSMA antibody. The anti-PSMA produced in vivo controlled tumor growth and prolonged survival in a mouse model. This is likely mediated by antibody-dependent cellular cytotoxicity (ADCC) effect with the aid of NK cells. Further study of  this novel approach for treatment of human prostate disease and other malignant conditions is warranted.

  14. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of Streptococcus pneumontae

    DOEpatents

    Lacks, S.A.

    1990-10-02

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252. 9 figs.

  15. Recombinant plasmids for encoding restriction enzymes DpnI and DpnII of streptococcus pneumontae

    DOEpatents

    Lacks, Sanford A.

    1990-01-01

    Chromosomal DNA cassettes containing genes encoding either the DpnI or DpnII restriction endonucleases from Streptococcus pneumoniae are cloned into a streptococcal vector, pLS101. Large amounts of the restriction enzymes are produced by cells containing the multicopy plasmids, pLS202 and pLS207, and their derivatives pLS201, pLS211, pLS217, pLS251 and pLS252.

  16. Murine T-Cell Response to Native and Recombinant Protein Antigens of Rickettsia Tsutsugamushi

    DTIC Science & Technology

    1993-02-01

    Wright, and J. Sadoff. 1985. 18-kilodalton protein of Mycobacterium leprae recognized by Immunoenzymatic analysis by monoclonal antibodies of bacte- Vo...determinants and closely resembles T-cell antigenic determinants, Rothbard and Taylor, by the GroEL homolog (65 kDa) of Mycobacterium tuberculo- analysis of...not be completely present in protein that is recognized by 20% of the mycobacterium - peptide 91-110. If this were the core of the antigenic deter

  17. Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis.

    PubMed

    Spaapen, Robbert M; Lokhorst, Henk M; van den Oudenalder, Kelly; Otterud, Brith E; Dolstra, Harry; Leppert, Mark F; Minnema, Monique C; Bloem, Andries C; Mutis, Tuna

    2008-11-24

    Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)-matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4(+) T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19(L)-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19(L)-specific CD4(+) T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8(+) mHag-specific T cells. They also lysed CD19(L)-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19(L)-derived HLA class II-restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings.

  18. Toward targeting B cell cancers with CD4+ CTLs: identification of a CD19-encoded minor histocompatibility antigen using a novel genome-wide analysis

    PubMed Central

    Spaapen, Robbert M.; Lokhorst, Henk M.; van den Oudenalder, Kelly; Otterud, Brith E.; Dolstra, Harry; Leppert, Mark F.; Minnema, Monique C.; Bloem, Andries C.; Mutis, Tuna

    2008-01-01

    Some minor histocompatibility antigens (mHags) are expressed exclusively on patient hematopoietic and malignant cells, and this unique set of antigens enables specific targeting of hematological malignancies after human histocompatability leucocyte antigen (HLA)–matched allogeneic stem cell transplantation (allo-SCT). We report the first hematopoietic mHag presented by HLA class II (HLA-DQA1*05/B1*02) molecules to CD4+ T cells. This antigen is encoded by a single-nucleotide polymorphism (SNP) in the B cell lineage-specific CD19 gene, which is an important target antigen for immunotherapy of most B cell malignancies. The CD19L-encoded antigen was identified using a novel and powerful genetic strategy in which zygosity-genotype correlation scanning was used as the key step for fine mapping the genetic locus defined by pairwise linkage analysis. This strategy was also applicable for genome-wide identification of a wide range of mHags. CD19L-specific CD4+ T cells provided antigen-specific help for maturation of dendritic cells and for expansion of CD8+ mHag-specific T cells. They also lysed CD19L-positive malignant cells, illustrating the potential therapeutic advantages of targeting this novel CD19L-derived HLA class II–restricted mHag. The currently available immunotherapy strategies enable the exploitation of these therapeutic effects within and beyond allo-SCT settings. PMID:19001137

  19. [Application of recombinant 38000 protein antigen of Mycobacterium tuberculosis in screening Mycobacterium tuberculosis infection].

    PubMed

    He, Xiu-yun; Zhuang, Yu-hui; Zhang, Xiao-gang

    2009-08-01

    To evaluate the potential of recombinant 38000 protein of Mycobacterium tuberculosis (38000 protein) as a tuberculosis-specific tuberculin for screening M. tuberculosis infection. A total of 1342 subjects (706 men and 636 women, age 18-60 years) from several communities in Kazuo County and Xidaziying Town, Chaoyang, Liaoning Province, and Hongdong County, Linfen, Shanxi Province were enrolled from September 2004 to February 2005. The skin tests were performed with double-blinded and the intradermal injections were administered on both forearms with 0.1 ml solution of PPD and 38000 protein at the right side and the left side, respectively. The vertical and transverse diameters of induration or erythema were measured following 24 h for 38000 protein and 48 h for PPD, respectively. The diameters of the the skin test reactions were defined as the means of the vertical and transverse diameters, and positive skin reactions were identified when the diameter was greater than or equal to 5 mm. The comparison of the positive rate was performed via chi(2) test and the consistency of positive skin test reactions between 38000 protein and TB-PPD was analyzed through calculating Kappa coefficients. The positive rate was 55.1% (740/1342) and 28.6% (384/1342) for PPD and 38000 protein, respectively; the difference being significant (chi(2) = 190.6, P < 0.01). The consistency of positive skin test reactions between 38000 protein and PPD was low due to a negative Kappa coefficient. The positive rates induced by PPD and 38000 protein tended to increase with age except for the 33-37 year group. For a given age group, the positive rate of PPD was much higher than that of 38000 protein. The subjects without BCG scar had a lower positive rate for 38000 protein (24.3%, 137/566) than those with BCG scar (31.9%, 247/776) (chi(2) = 4.7, P < 0.05). The subjects with tuberculosis contact history had a higher positive rate for 38000 protein (74.4%, 32/43) than those without tuberculosis contact

  20. Bioinformatic prediction of the antigenic epitopes of recombinant ferritin of Echinococcus granulosus.

    PubMed

    Liu, Xuelei; Zhao, Hui; Cao, Wenyan; Liu, Yumei; Zhang, Chuntao; Lan, Xi; Peng, Shanshan; Wen, Hao; Ding, Jianbing; Ma, Xiumin

    2016-01-01

    Echinococcosis is a zoonotic parasitic disease affecting humans and other mammals, which is mainly caused Echinococcus at larval stages. It is predominantly endemic in Chinese pasture regions, including Xinjiang, Qinghai, Gansu and Ningxia. The aim of the present study was to predict the T‑ and B‑combined epitopes of Echinococcus granulosus (Eg). ferritin, and to analyze its secondary structure using online software. Prediction of the T‑ and B‑combined epitopes of Eg. ferritin was performed using IEDB, SYFPEITHI and LEPS software, which are used to identify common areas of T‑ and B‑cells. The results of the present study identified several potential antigenic epitopes of Eg. ferritin, including seven B‑cell antigen epitope amino acid sequences with high values: 8‑16, 54‑61, 70‑75, 80‑90, 103‑109, 117‑124 and 167‑173; and four T‑cell antigen epitope amino acid sequences with high values: 85‑93, 105‑113, 133‑141 and 157‑165. Furthermore, a combined epitope region comprising an 105‑109 amino acid sequence was identified. In conclusion, using bioinformatic methods, the present study confirmed the existence of Eg. ferritin on four T‑cell antigen epitopes, seven B‑cell antigen epitopes, and one T‑ and B‑combined epitope region. These findings provide significant information for further investigation of the antigenicity of Eg. ferritin and the development of highly efficient epitope vaccines.

  1. Production and evaluation of the recombinant antigen TES-30 of Toxocara canis for the immunodiagnosis of toxocariasis.

    PubMed

    Olave, Ana M; Mesa, Jairo A; Botero, Jorge H; Patiño, Edwin B; García, Gisela M; Alzate, Juan F

    2016-03-03

    Toxocara canis is a pathogenic nematode of canines which can be accidentally transmitted to humans. Although serology is the most important diagnostic tool for this zoonosis, diagnostic kits use crude excretion/secretion antigens, most of them being glycoproteins which are not species-specific and may cross-react with antibodies generated against other parasites.  To produce the rTES-30 recombinant antigen of Toxocara canis and evaluate it in the immunodiagnosis of toxocariasis.  The gene that codes for TES-30 was cloned in the expression vector pET28a (+) using single-stranded oligonucleotides united by PCR. The protein rTES-30 was purified by Ni2+ affinity chromotography. Seroreactivity of rTES-30 was evaluated by immunoblot. Given that there is no gold standard test, the behaviour of the antigen was compared with the method that is routinely used to immunodiagnose toxocariasis, i.e., the conventional ELISA technique using excretion/secretion antigens.  The rTES-30 was produced from an Escherichia coli LB culture which yielded 2.25 mg/L of the antigen with a purity of 95%. The results obtained showed 73% (46/63) concordance of reactivity between the rTES-30 immunoblot and the conventional ELISA, and 100% concordance with the nonreactive sera (21). Nineteen of the 21 sera positive for other parasitoses reacted with ELISA, while only seven of these were positive with the rTES-30 immunoblot. Concordance between the ELISA and the immunoblot was moderate (kappa coefficient: 0.575; 95% CI: 0.41- 0.74).  The data presented show the potential of the rTES-30 inmunoblot for confirmation of possible ELISA positives, not only in epidemiological studies, but also as a candidate for the development of diagnostic tests for ocular toxocariasis in Colombia.

  2. A novel recombinant BCG vaccine encoding eimeria tenella rhomboid and chicken IL-2 induces protective immunity against coccidiosis.

    PubMed

    Wang, Qiuyue; Chen, Lifeng; Li, Jianhua; Zheng, Jun; Cai, Ning; Gong, Pengtao; Li, Shuhong; Li, He; Zhang, Xichen

    2014-06-01

    A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4(+) and CD8(+) cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

  3. The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL

    PubMed Central

    Brickner, Anthony G.; Evans, Anne M.; Mito, Jeffrey K.; Xuereb, Suzanne M.; Feng, Xin; Nishida, Tetsuya; Fairfull, Liane; Ferrell, Robert E.; Foon, Kenneth A.; Hunt, Donald F.; Shabanowitz, Jeffrey; Engelhard, Victor H.; Riddell, Stanley R.; Warren, Edus H.

    2006-01-01

    Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A*0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19+ B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19+ cells, and identify PANE1 as a potential therapeutic target in B-CLL. PMID:16391015

  4. Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field.

    PubMed

    Sentsui, H; Inoshima, Y; Murakami, K; Akashi, H; Purevtseren, B; Pagmajav, O; Sugiura, T

    2001-01-01

    Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337-V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test. Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombinant antigens were essentially the same as those with the virion antigen prepared from horse cell cultures both in the AGID test and ELISA. The reactivities of the antigens were further compared using serum samples collected from horses in 1999 in certain districts of Mongolia where equine infectious anemia has been prevalent, and from horses in Japan in 1973 when EIA had not been eliminated completely from Japanese horses. These results were completely concurrent. Generally, recombinant antigens have high specificity but low cross reactivity to heterologous strains. However, the present study showed that the recombinant EIAV p26 antigen has cross reactivity to the heterologous strain and is useful for diagnosis of EIA in the field.

  5. Dot-ELISA Rapid Test Using Recombinant 56-kDa Protein Antigens for Serodiagnosis of Scrub Typhus

    PubMed Central

    Rodkvamtook, Wuttikon; Zhang, Zhiwen; Chao, Chien-Chung; Huber, Erin; Bodhidatta, Dharadhida; Gaywee, Jariyanart; Grieco, John; Sirisopana, Narongrid; Kityapan, Manerat; Lewis, Michael; Ching, Wei-Mei

    2015-01-01

    We developed a rapid dot–enzyme-linked immunosorbent assay (dot-ELISA) using the combination of recombinant 56-kDa protein antigens that exhibited broad reactivity with serum antibodies against the four most prevalent strains (Karp, Kato, Gilliam, and TA763) of Orientia tsutsugamushi. The assay is rapid (30 minutes), and can be done at room temperature, and results can be read by the naked eye. Only a simple shaker is required to wash the membrane. Sera from 338 patients suspected of being ill with scrub typhus from rural hospitals around Thailand were tested using this dot-ELISA. Seventy-five (22.2%) patients were found to be positive. The sensitivity and specificity of dot-ELISA were determined using the indirect immunofluorescent assay (IFA) test as the gold standard, with the cutoff titer of immunoglobulin peroxidase conjugate M (IgM)/G (IgG) greater than 1:400/1:400. The dot-ELISA had a sensitivity of 98.5%, a specificity of 96.3%, a positive predictive value of 86.7%, and a negative predictive value of 99.6% for the acute-phase specimens. The results indicate that dot-ELISA rapid test using recombinant 56-kDa protein antigen was comparable with the IFA test and may be very useful for the diagnosis of scrub typhus in rural hospitals, where IFA is not available. PMID:25802430

  6. Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

    PubMed

    Arévalo-Herrera, Myriam; Vallejo, Andrés F; Rubiano, Kelly; Solarte, Yezid; Marin, Catherin; Castellanos, Angélica; Céspedes, Nora; Herrera, Sócrates

    2015-01-01

    Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

  7. Recombinant Leishmania (Leishmania) infantum Ecto-Nucleoside Triphosphate Diphosphohydrolase NTPDase-2 as a new antigen in canine visceral leishmaniasis diagnosis.

    PubMed

    de Souza, Ronny Francisco; Dos Santos, Yaro Luciolo; de Souza Vasconcellos, Raphael; Borges-Pereira, Lucas; Caldas, Ivo Santana; de Almeida, Márcia Rogéria; Bahia, Maria Terezinha; Fietto, Juliana Lopes Rangel

    2013-01-01

    Canine visceral leishmaniasis is an important public health concern. In the epidemiological context of human visceral leishmaniasis, dogs are considered the main reservoir of Leishmania parasites; therefore, dogs must be epidemiologically monitored constantly in endemic areas. Furthermore, dog to human transmission has been correlated with emerging urbanization and increasing rates of leishmaniasis infection worldwide. Leishmania (Leishmania) infantum (L. chagasi) is the etiologic agent of visceral leishmaniasis in the New World. In this work, a new L. (L.) infantum (L. chagasi) recombinant antigen, named ATP diphosphohydrolase (rLic-NTPDase-2), intended for use in the immunodiagnosis of CVL was produced and validated. The extracellular domain of ATP diphosphohydrolase was cloned and expressed in the pET21b-Escherichia coli expression system. Indirect ELISA assays were used to detect the purified rLic-NTPDase-2 antigen using a standard canine sera library. This library contained CVL-positive samples, leishmaniasis-negative samples and samples from Trypanosoma cruzi-infected dogs. The results show a high sensitivity of 100% (95% CI=92.60-100.0%) and a high specificity of 100% (95% CI=86.77-100.0%), with a high degree of confidence (k=1). These findings demonstrate the potential use of this recombinant protein in immune diagnosis of canine leishmaniasis and open the possibility of its application to other diagnostic approaches, such as immunochromatography fast lateral flow assays and human leishmaniasis diagnosis.

  8. Recombinant Pvs48/45 Antigen Expressed in E. coli Generates Antibodies that Block Malaria Transmission in Anopheles albimanus Mosquitoes

    PubMed Central

    Arévalo-Herrera, Myriam; Vallejo, Andrés F.; Rubiano, Kelly; Solarte, Yezid; Marin, Catherin; Castellanos, Angélica; Céspedes, Nora; Herrera, Sócrates

    2015-01-01

    Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential. PMID:25775466

  9. Development and comparative evaluation of a plate enzyme-linked immunosorbent assay based on recombinant outer membrane antigens Omp28 and Omp31 for diagnosis of human brucellosis.

    PubMed

    Tiwari, Sapana; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-08-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  10. Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

    PubMed Central

    Tiwari, Sapana; Kumar, Ashu; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-01-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  11. DDX3Y encodes a class I MHC–restricted H-Y antigen that is expressed in leukemic stem cells

    PubMed Central

    Rosinski, Kellie V.; Fujii, Nobuharu; Mito, Jeffrey K.; Koo, Kevin K. W.; Xuereb, Suzanne M.; Sala-Torra, Olga; Gibbs, James S.; Radich, Jerald P.; Akatsuka, Yoshiki; Van den Eynde, Benoît J.; Riddell, Stanley R.

    2008-01-01

    The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8+ cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705–restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)–identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705+ patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8+ T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354. PMID:18299450

  12. DDX3Y encodes a class I MHC-restricted H-Y antigen that is expressed in leukemic stem cells.

    PubMed

    Rosinski, Kellie V; Fujii, Nobuharu; Mito, Jeffrey K; Koo, Kevin K W; Xuereb, Suzanne M; Sala-Torra, Olga; Gibbs, James S; Radich, Jerald P; Akatsuka, Yoshiki; Van den Eynde, Benoît J; Riddell, Stanley R; Warren, Edus H

    2008-05-01

    The Y chromosome encodes male-specific minor histocompatibility (H-Y) antigens that stimulate T- and B-lymphocyte responses after sex-mismatched allogeneic hematopoietic cell transplantation (HCT). A CD8(+) cytotoxic T lymphocyte (CTL) clone that recognizes a novel HLA-B*2705-restricted H-Y antigen encoded by the DDX3Y gene was isolated from a male who had received a hematopoietic cell graft from his human leukocyte antigen (HLA)-identical sister. The antigenic peptide is a decamer that differs from the homologous DDX3X-encoded peptide at 4 positions. Expression of DDX3Y and of the H-Y epitope that it encodes was examined by quantitative polymerase chain reaction (PCR) and by CTL recognition assays. Expression of DDX3Y is detected in all myeloid and lymphoid leukemic cells that carry an intact Y chromosome. Moreover, the DDX3Y-encoded H-Y epitope is presented on the surface of both myeloid and lymphoid leukemic cells from male HLA-B*2705(+) patients. DDX3Y-specific CTLs prevent engraftment of human acute leukemia in nonobese diabetic/severe combined immune deficient mice, demonstrating that the DDX3Y-encoded H-Y antigen is also expressed in leukemic stem cells. These results demonstrate that CD8(+) T-cell responses against DDX3Y have the potential to contribute to graft-versus-leukemia (GVL) activity after female into male allogeneic HCT. This study is registered at http://clinicaltrials.gov as NCT00107354.

  13. Homologous recombination with linear DNA to insert antigenic protein in the flagellin: improvement of the Th1 immune response.

    PubMed

    Le Moigne, Vincent; Robreau, Georges; Mahana, Wahib

    2006-01-01

    Bacterial flagellin is a surface protein with numerous advantages for the presentation of exogenous peptides. However, the production of recombinant bacteria and the expression of fusion proteins is laborious and time consuming. Here, we present a simple way to produce modified bacteria. Partially deleted, non-functional, chromosomal flagellin gene (fliC ) was changed using homologous recombination by a functional linear fliC gene in which we introduced an exogenous oligonucleotide encoding for the peptide of interest. The modified fliC gene was produced by polymerase chain amplification. Linear amplicons were introduced into the non-motile E. coli by electroporation. The formation of functional flagellar filaments allowed the discrimination of motile transformants from non-motile, non-transformed cells. Thus antibiotic selection and gene expression inductors are not required since transformed bacteria can be easily isolated and used as a vector and adjuvant for immunization. To validate this hypothesis, we studied the immune response against the N-terminal peptide of Clostridium tyrobutyricum flagellin fragment. BALB/c mice were immunized either with the protein displayed as flagellin fusion protein on the surface of E. coli, with the recombinant protein in Freund's adjuvant (FA), or with the pcDNA3 vector bearing the DNA fragment encoding this protein. Immunization with the flagellin recombinant bacteria induced a strong Th1 response as measured by high level of IFN-gamma production and the lack of IL-4 production. The results indicate that the flagellar filament protein carrying a specific epitope can be a potent inducer of the Th1 cellular response.

  14. Recombinant Forms of Leishmania amazonensis Excreted/Secreted Promastigote Surface Antigen (PSA) Induce Protective Immune Responses in Dogs

    PubMed Central

    Petitdidier, Elodie; Pagniez, Julie; Papierok, Gérard; Vincendeau, Philippe; Lemesre, Jean-Loup; Bras-Gonçalves, Rachel

    2016-01-01

    Preventive vaccination is a highly promising strategy for interrupting leishmaniasis transmission that can, additionally, contribute to elimination. A vaccine formulation based on naturally excreted secreted (ES) antigens was prepared from L. infantum promastigote culture supernatant. This vaccine achieved successful results in Phase III trials and was licensed and marketed as CaniLeish. We recently showed that newly identified ES promastigote surface antigen (PSA), from both viable promastigotes and axenically-grown amastigotes, represented the major constituent and the highly immunogenic antigen of L. infantum and L. amazonensis ES products. We report here that three immunizations with either the recombinant ES LaPSA-38S (rPSA) or its carboxy terminal part LaPSA-12S (Cter-rPSA), combined with QA-21 as adjuvant, confer high levels of protection in naive L. infantum-infected Beagle dogs, as checked by bone marrow parasite absence in respectively 78.8% and 80% of vaccinated dogs at 6 months post-challenge. The parasite burden in infected vaccinated dogs was significantly reduced compared to placebo group, as measured by q-PCR. Moreover, our results reveal humoral and cellular immune response clear-cut differences between vaccinated and control dogs. An early increase in specific IgG2 antibodies was observed in rPSA/QA-21- and Cter-rPSA/QA-21-immunized dogs only. They were found functionally active in vitro and were highly correlated with vaccine protection. In vaccinated protected dogs, IFN-γ and NO productions, as well as anti-leishmanial macrophage activity, were increased. These data strongly suggest that ES PSA or its carboxy-terminal part, in recombinant forms, induce protection in a canine model of zoonotic visceral leishmaniasis by inducing a Th1-dominant immune response and an appropriate specific antibody response. These data suggest that they could be considered as important active components in vaccine candidates. PMID:27223609

  15. Recovery and purification of recombinant 503 antigen of Leishmania infantum chagasi using expanded bed adsorption chromatography.

    PubMed

    de Sousa Junior, Francisco Caninde; Vaz, Michelle Rossana Ferreira; de Araújo Padilha, Carlos Eduardo; Chibério, Abimaelle Silva; Martins, Daniella Regina Arantes; de Macedo, Gorete Ribeiro; dos Santos, Everaldo Silvino

    2015-04-01

    Visceral leishmaniasis, a disease caused by Leishmania infantum chagasi, represents a major public health problem in many areas of the world. However, there is currently no vaccine for human use. The aim of this work was to purify the 503 antigen of Leishmania i. chagasi directly from unclarified Escherichia coli feedstock through expanded bed adsorption (EBA) chromatography. Batch experiments were performed to optimize the adsorption and elution conditions of the antigen onto a STREAMLINE Chelating resin using two central composite rotatable designs (CCRD). The results showed that the optimal binding conditions of the 503 antigen were pH 8.0 in the presence of 2.4 M NaCl. For the elution of the target protein, the optimized conditions included the presence of 600.0 mM imidazole. The adsorption isothermal data of the 503 antigen were fitted to the Langmuir adsorption isotherm. The EBA experiment successfully recovered 59.2% of the 503 antigen from the unclarified E. coli homogenate with a purification factor of 6.0. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis.

    PubMed

    Zahabiun, Farzaneh; Sadjjadi, Seyed Mahmoud; Yunus, Muhammad Hafiznur; Rahumatullah, Anizah; Moghaddam, Mohammad Hosein Falaki; Saidin, Syazwan; Noordin, Rahmah

    2015-08-01

    Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis.

  17. Regeneration of recombinant antigen microarrays for the automated monitoring of antibodies against zoonotic pathogens in swine sera.

    PubMed

    Meyer, Verena K; Kober, Catharina; Niessner, Reinhard; Seidel, Michael

    2015-01-23

    The ability to regenerate immobilized proteins like recombinant antigens (rAgs) on surfaces is an unsolved problem for flow-based immunoassays on microarray analysis systems. The regeneration on microarray chip surfaces is achieved by changing the protein structures and desorption of antibodies. Afterwards, reactivation of immobilized protein antigens is necessary for reconstitution processes. Any backfolding should be managed in a way that antibodies are able to detect the protein antigens in the next measurement cycle. The regeneration of rAg microarrays was examined for the first time on the MCR3 flow-based chemiluminescence (CL) microarray analysis platform. The aim was to reuse rAg microarray chips in order to reduce the screening effort and costs. An antibody capturing format was used to detect antibodies against zoonotic pathogens in sera of slaughtered pigs. Different denaturation and reactivation buffers were tested. Acidic glycine-SDS buffer (pH 2.5) and 8 M guanidinium hydrochloride showed the best results in respect of denaturation efficiencies. The highest CL signals after regeneration were achieved with a carbonate buffer containing 10 mM DTT and 0.1% BSA for reactivation. Antibodies against Yersinia spp. and hepatitis E virus (HEV) were detected in swine sera on one immunochip over 4 days and 25 measurement cycles. Each cycle took 10 min for detection and regeneration. By using the rAg microarray chip, a fast and automated screening of antibodies against pathogens in sera of slaughtered pigs would be possible for zoonosis monitoring.

  18. Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis

    PubMed Central

    Zahabiun, Farzaneh; Sadjjadi, Seyed Mahmoud; Yunus, Muhammad Hafiznur; Rahumatullah, Anizah; Moghaddam, Mohammad Hosein Falaki; Saidin, Syazwan; Noordin, Rahmah

    2015-01-01

    Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory–secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis. PMID:26033026

  19. Mycobacterium avium ssp. paratuberculosis recombinant heat shock protein 70 interaction with different bovine antigen-presenting cells.

    PubMed

    Langelaar, M F M; Hope, J C; Rutten, V P M G; Noordhuizen, J P T M; van Eden, W; Koets, A P

    2005-03-01

    Abstract Heat shock proteins (Hsp) can deliver antigen into the major histocompatibility complex class I presentation pathway of antigen-presenting cells (APC), a process called cross priming, thus stimulating antigen-specific CD8+ T-cell reactions. Hsp were shown to elicit proinflammatory responses in APC. Both processes require interaction of Hsp with APC via specific receptors. This study describes the interaction of recombinant Hsp70 (rHsp70) of Mycobacterium avium subspecies paratuberculosis with bovine peripheral blood mononuclear cells that was restricted to CD14+ cells. Characterized monocyte-derived macrophages, monocyte-derived dendritic cells (DC) and BoMac, an immortalized bovine macrophage cell line, were used to investigate the interaction of rHsp70 with different bovine APC. Saturation of immature DC with high concentrations of rHsp70 is demonstrated, and it was found that interaction of rHsp70 with DC was related to the maturation stage of the DC. Involvement of CD91 as a cellular receptor for rHsp70 was demonstrated; however, competition studies with immature DC demonstrated that other receptors exist on bovine APC. These data suggest that rHsp70-based vaccines may be useful for the successful immunization of cattle.

  20. Effect of context and adjuvant on the immunogenicity of recombinant proteins and peptide conjugates derived from the polymorphic malarial surface antigen MSA2.

    PubMed

    Jones, G L; Spencer, L; Lord, R; Saul, A J

    1996-01-01

    We have identified a 51 kDa glycosylated myristylated merozoite surface antigen (MSA2) as the target of a number of monoclonal antibodies which inhibit in vitro invasion of the human malarial parasite Plasmodium falciparum. This antigen has been shown to exist in a limited number of strain specific forms but despite wide variation in the sequences of the internal repeat regions both N and C terminal elements of the protein are almost totally conserved. Accordingly, we prepared a large number of overlapping peptide constructs and demonstrated that one peptide SNTFINNA (E71) from the N terminus and two peptides, QHGHMHGS (G5) and NTSDSQKE (G12) from the C terminus could, when suitably conjoined to the carrier protein diphtheria toxoid (DT), elicit antibodies reactive with MSA2 from diverse strains of P. falciparum. Here we compare the immunogenicity of these peptide constructs with two recombinant proteins containing the entire amino acid sequence of MSA2 from the FCQ-27/PNG strain (1609) and the 3D7 strain (1623). We have formulated these recombinant and peptide antigens with Freund's adjuvant, Alum and Algammulin. Both recombinant and peptide antigens elicit high titre antibodies when tested by ELISA against the immunogens themselves. Although both recombinant proteins include the constant region peptide sequences E71, G5 and G12, the extent of ELISA cross reaction between antibody raised against recombinant and peptide antigen or antibody raised against peptide and recombinant antigen is small and sporadic, and depends to an extent on the adjuvant employed. Antisera against both recombinant proteins 1609 and 1623 detected either recombinant on Western blots, as well as detecting native MSA2 in whole protein extracts from both FCQ-27/PNG and 3D7 strains. Antisera against peptide construct E71 recognized recombinant 1609 but not 1623 but recognized the native MSA2 in both strains studied. Antisera against peptide construct G5 showed a similar pattern of recognition

  1. Complete DNA Sequence and Detailed Analysis of the Yersinia pestis KIM5 Plasmid Encoding Murine Toxin and Capsular Antigen

    PubMed Central

    Lindler, Luther E.; Plano, Gregory V.; Burland, Valerie; Mayhew, George F.; Blattner, Frederick R.

    1998-01-01

    Yersinia pestis, the causative agent of plague, harbors at least three plasmids necessary for full virulence of the organism, two of which are species specific. One of the Y. pestis-specific plasmids, pMT1, is thought to promote deep tissue invasion, resulting in more acute onset of symptoms and death. We determined the entire nucleotide sequence of Y. pestis KIM5 pMT1 and identified potential open reading frames (ORFs) encoded by the 100,990-bp molecule. Based on codon usage for known yersinial genes, homology with known proteins in the databases, and potential ribosome binding sites, we determined that 115 of the potential ORFs which we considered could encode polypeptides in Y. pestis. Five of these ORFs were genes previously identified as being necessary for production of the classic virulence factors, murine toxin (MT), and the fraction 1 (F1) capsule antigen. The regions of pMT1 encoding MT and F1 were surrounded by remnants of multiple transposition events and bacteriophage, respectively, suggesting horizontal gene transfer of these virulence factors. We identified seven new potential virulence factors that might interact with the mammalian host or flea vector. Forty-three of the remaining 115 putative ORFs did not display any significant homology with proteins in the current databases. Furthermore, DNA sequence analysis allowed the determination of the putative replication and partitioning regions of pMT1. We identified a single 2,450-bp region within pMT1 that could function as the origin of replication, including a RepA-like protein similar to RepFIB, RepHI1B, and P1 and P7 replicons. Plasmid partitioning function was located ca. 36 kb from the putative origin of replication and was most similar to the parABS bacteriophage P1 and P7 system. Y. pestis pMT1 encoded potential genes with a high degree of similarity to a wide variety of organisms, plasmids, and bacteriophage. Accordingly, our analysis of the pMT1 DNA sequence emphasized the mosaic nature of

  2. Detection of anti-PL-12 autoantibodies by ELISA using a recombinant antigen; study of the immunoreactive region

    PubMed Central

    García-Lozano, J R; González-Escribano, M F; Rodríguez, R; Rodriguez-Sanchez, J L; Targoff, I N; Wichmann, I; Núñez-Roldán, A

    1998-01-01

    Autoantibodies to aminoacyl-tRNA synthetases are highly associated with myositis and detection is important in clinical diagnosis; however, current methods of screening limit its clinical utility. In the present study, alanyl-tRNA synthetase (PL-12) recombinant protein was obtained by immunological screening of a HeLa expression library and used in an ELISA with 22 anti-PL-12 sera, 200 autoimmune sera negative for PL-12 and 100 healthy individual sera. Sensitivity of the method was 95% (21/22) and specificity 100%. Mapping of the immunoreactive region was carried out using three anti-PL-12 sera and different recombinant protein-derived peptides. Results show that the same conformational epitope located within amino acids 730–951 of the PL-12 antigen outside the catalytic region was recognized by the three anti-PL-12 sera tested. We conclude that ELISA using recombinant protein is an effective and useful method for routine screening for anti-PL-12 autoantibodies. PMID:9822271

  3. Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

    PubMed

    Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

    2013-11-15

    The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

  4. Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

    PubMed Central

    Green, K Y; Kapikian, A Z; Valdesuso, J; Sosnovtsev, S; Treanor, J J; Lew, J F

    1997-01-01

    The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent. PMID:9196224

  5. A novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses.

    PubMed

    Durso, Robert J; Andjelic, Sofija; Gardner, Jason P; Margitich, Dennis J; Donovan, Gerald P; Arrigale, Robert R; Wang, Xinning; Maughan, Maureen F; Talarico, Todd L; Olmsted, Robert A; Heston, Warren D W; Maddon, Paul J; Olson, William C

    2007-07-01

    Prostate-specific membrane antigen (PSMA) is an attractive target for active immunotherapy. Alphavirus vaccines have shown promise in eliciting immunity to tumor antigens. This study investigated the immunogenicity of alphavirus vaccine replicon particles (VRP) that encode PSMA (PSMA-VRP). Cells were infected with PSMA-VRP and evaluated for PSMA expression and folate hydrolase activity. Mice were immunized s.c. with PSMA-VRP or purified PSMA protein. Sera, splenocytes, and purified T cells were evaluated for the magnitude, durability, and epitope specificity of the anti-PSMA response. Antibodies were measured by flow cytometry, and cellular responses were measured by IFN-gamma enzyme-linked immunospot and chromium release assays. Cellular responses in BALB/c and C57BL/6 mice were mapped using overlapping 15-mer PSMA peptides. A Good Laboratory Practice-compliant toxicology study was conducted in rabbits. PSMA-VRP directed high-level expression of active PSMA. Robust T-cell and B-cell responses were elicited by a single injection of 2 x 10(5) infectious units, and responses were boosted following repeat immunizations. Anti-PSMA responses were detected following three immunizations with 10(2) infectious units and increased with increasing dose. PSMA-VRP was more immunogenic than adjuvanted PSMA protein. Responses to PSMA-VRP were characterized by Th-1 cytokines, potent CTL activity, and IgG2a/IgG2b antibodies. T-cell responses in BALB/c and C57BL/6 mice were directed toward different PSMA peptides. Immunogenic doses of PSMA-VRP were well tolerated in mice and rabbits. PSMA-VRP elicited potent cellular and humoral immunity in mice, and specific anti-PSMA responses were boosted on repeat dosing. PSMA-VRP represents a promising approach for immunotherapy of prostate cancer.

  6. Discovery of African bat polyomaviruses and infrequent recombination in the large T antigen in the Polyomaviridae.

    PubMed

    Carr, Michael; Gonzalez, Gabriel; Sasaki, Michihito; Ito, Kimihito; Ishii, Akihiro; Hang'ombe, Bernard M; Mweene, Aaron S; Orba, Yasuko; Sawa, Hirofumi

    2017-04-01

    Bat species represent natural reservoirs for a number of high-consequence human pathogens. The present study investigated the diversity of polyomaviruses (PyVs) in Zambian insectivorous and fruit bat species. We describe the complete genomes from four newly proposed African bat PyV species employing the recently recommended criteria provided by the Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses. A comprehensive phylogenetic and recombination analysis was performed to determine genetic relationships and the distribution of recombination events in PyV from mammalian and avian species. The novel species of PyV from Zambian bats segregated with members of the genera Alphapolyomavirus and Betapolyomavirus, forming monophyletic clades with bat and non-human primate PyVs. Miniopterus schreibersii polyomavirus 1 and 2 segregated in a clade with South American bat PyV species, Old World monkey and chimpanzee PyVs and Human polyomavirus 13 (New Jersey PyV). Interestingly, the newly described Egyptian fruit bat PyV, tentatively named Rousettus aegyptiacus polyomavirus 1, had the highest nucleotide sequence identity to species of PyV from Indonesian fruit bats, and Rhinolophus hildebrandtii polyomavirus 1 was most closely related to New World monkey PyVs. The distribution of recombination events in PyV genomes was non-random: recombination boundaries existed in the intergene region between VP1 and LTAg and also at the 3' end of VP2/3 in the structural genes, whereas infrequent recombination was present within the LTAg gene. These findings indicate that recombination within the LTAg gene has been negatively selected against during polyomaviral evolution and support the recent proposal for taxonomic classification based on LTAg to define novel PyV species.

  7. Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

    PubMed Central

    Potocki De Montalk, G.; Remaud-Simeon, M.; Willemot, R. M.; Planchot, V.; Monsan, P.

    1999-01-01

    The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the α-amylase family predicted a (β/α)8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in α-amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase–amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75. PMID:9882648

  8. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    SciTech Connect

    Dalton, J.P.; Tom, T.D.; Strand, M.

    1987-06-01

    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  9. Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice.

    PubMed

    Meng, Min; He, Shenyi; Zhao, Guanghui; Bai, Yang; Zhou, Huaiyu; Cong, Hua; Lu, Gang; Zhao, Qunli; Zhu, Xing-Quan

    2012-11-26

    Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly. In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×10(4) tachyzoites of T. gondii and the survival time of mice was observed and recorded every day. Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×10(4) tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05). The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the

  10. Granulocyte/macrophage-colony stimulating factor produced by recombinant avian poxviruses enriches the regional lymph nodes with antigen-presenting cells and acts as an immunoadjuvant.

    PubMed

    Kass, E; Panicali, D L; Mazzara, G; Schlom, J; Greiner, J W

    2001-01-01

    Recombinant avian poxviruses [fowlpox and canarypox (ALVAC)], restricted for replication in nonavian cell substrates and expressing granulocyte/macrophage-colony stimulating factor (avipox-GM-CSF), were evaluated for their ability to enrich an immunization site with antigen-presenting cells (APCs) and, in turn, function as biological vaccine adjuvants. Avipox-GM-CSF administered as a single s.c. injection significantly enhanced the percentage and absolute number of APCs in the regional lymph nodes that drain the injection site. Both the magnitude and duration of the cellular and phenotypic increases within the lymph nodes induced by the avipox-GM-CSF viruses were significantly (P < 0.05) greater than those measured in mice treated with four daily injections of recombinant GM-CSF protein. Temporal studies revealed that the APC enrichment of regional lymph nodes was sustained for 21-28 days after injection of the recombinant avipox virus expressing GM-CSF and, moreover, three injections of the recombinant virus could be given without any appreciable loss of in vivo bioactivity. Mice expressing human carcinoembryonic antigen (CEA) as a transgene (CEA.Tg) developed CEA-specific humoral and cell-mediated immunity after being immunized with avipox-CEA. The coadministration of recombinant avipox viruses expressing CEA and GM-CSF significantly enhanced CEA-specific host immunity with an accompanying immunotherapeutic response in tumor-bearing CEA.Tg mice. The optimal use of avipox-GM-CSF, in terms of dose and dose schedule, especially when used with different immunogens, remains to be determined. Nonetheless, the present findings demonstrate: (a) the effective delivery of GM-CSF to an immunization site using a recombinant avian poxvirus; (b) the compatibility of delivering an antigen and GM-CSF in replication-defective viruses to enhance antigen-specific immunity; and (c) the combined use of recombinant avipox viruses expressing CEA and GM-CSF to generate antitumor

  11. Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses.

    PubMed

    Sullender, W M; Britt, W J

    1996-04-01

    The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell-produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A-specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/- 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses.

  12. Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis

    PubMed Central

    Coler, Rhea N.; Bhatia, Ajay; Maisonneuve, Jean-Francois; Probst, Peter; Barth, Brenda; Ovendale, Pamela; Fang, Hang; Alderson, Mark; Lobet, Yves; Cohen, Joe; Mettens, Pascal; Reed, Steven G.

    2009-01-01

    Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous MHC population. Herein we describe the identification by CD4+ and CD8+ T cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome, of novel C. trachomatis antigens. These antigens elicited human CD4+ T cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in GSK proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis. Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells. PMID:19281568

  13. Identification and characterization of novel recombinant vaccine antigens for immunization against genital Chlamydia trachomatis.

    PubMed

    Coler, Rhea N; Bhatia, Ajay; Maisonneuve, Jean-Francois; Probst, Peter; Barth, Brenda; Ovendale, Pamela; Fang, Hang; Alderson, Mark; Lobet, Yves; Cohen, Joe; Mettens, Pascal; Reed, Steven G

    2009-03-01

    Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide, with over 91 million cases estimated annually. An effective subunit vaccine against Chlamydia may require a multivalent subunit cocktail of antigens in a single formulation for broad coverage of a heterogeneous major histocompatibility complex population. Herein, we describe the identification of novel C. trachomatis antigens by CD4+ and CD8+ T-cell expression cloning, serological expression cloning, and an in silico analysis of the C. trachomatis genome. These antigens elicited human CD4+ T-cell responses, and a subset proved to be immunogenic and protective when administered as immunoprophylactic vaccines against C. trachomatis challenge. Candidate vaccines consisting of the prioritized C. trachomatis antigens adjuvanted in a GlaxoSmithKline proprietary AS01B adjuvant were prioritized based on induction of solid protection against challenge in C57BL/6 and BALB/c mice with C. trachomatis. Some of the vaccines prevented bacterial shedding and colonization of the upper genital tract to varying degrees by mechanisms that may include CD4+ T cells.

  14. Oral immunization with attenuated Salmonella enterica serovar Typhimurium encoding Cryptosporidium parvum Cp23 and Cp40 antigens induces a specific immune response in mice.

    PubMed

    Benitez, Alvaro J; McNair, Nina; Mead, Jan R

    2009-09-01

    Attenuated Salmonella enterica serovar Typhimurium vaccine strain SL3261 was used as an antigen delivery system for the oral immunization of mice against two Cryptosporidium parvum antigens, Cp23 and Cp40. Each antigen was subcloned into the pTECH1 vector system, which allows them to be expressed as fusion proteins with highly immunogenic fragment C of tetanus toxin under the control of the anaerobically inducible nirB promoter. The recombinant vector was introduced into Salmonella Typhimurium vaccine strain SL3261, and the stable soluble expression of the chimeric protein was evaluated and confirmed by Western blotting with polyclonal C. parvum antisera. Mice were inoculated orally with a single dose of SL3261/pTECH-Cp23 or Cp40, respectively, and plasmid stability was demonstrated both in vitro and in vivo. Specific serum immunoglobulin G (IgG) antibodies against the Cp23 or Cp40 antigen were detected by enzyme-linked immunosorbent assay 35 days after immunization. Also, serum IgA and mucosal (feces) IgA antibodies were detected in 30% of the mice immunized with Cp23. In addition, prime-boosting with Cp23 and Cp40 DNA vaccine vectors followed by Salmonella immunization significantly increased antibody responses to both antigens. Our data show that a single oral inoculation with recombinant S. Typhimurium SL3261 can induce specific antibody responses to the Cp23 or Cp40 antigen from C. parvum in mice, suggesting that recombinant Salmonella is a feasible delivery system for a vaccine against C. parvum infection.

  15. Tandem repeat recombinant proteins as potential antigens for the sero-diagnosis of Schistosoma mansoni infection.

    PubMed

    Kalenda, Yombo Dan Justin; Kato, Kentaro; Goto, Yasuyuki; Fujii, Yoshito; Hamano, Shinjiro

    2015-12-01

    The diagnosis of schistosome infection, followed by effective treatment and/or mass drug administration, is crucial to reduce the disease burden. Suitable diagnostic tests and field-applicable tools are required to sustain schistosomiasis control programs. We therefore assessed the potential of tandem repeat (TR) proteins for sero-diagnosis of Schistosoma mansoni infection using an experimental mouse model. TR genes in the genome of S. mansoni were searched in silico and 7 candidates, named SmTR1, 3, 8, 9, 10, 11 and 15, were selected. Total RNA was extracted from S. mansoni adult worms and eggs. Target TR genes were amplified, cloned, and the proteins were expressed in Escherichia coli competent cells. Female BALB/c mice were infected with 100 S. mansoni cercariae and sera were collected each week post-infection for 18 weeks. The levels of IgG antibodies to SmTR antigens were compared to those to soluble egg antigen (SEA) and to soluble worm antigen preparation (SWAP). Sera of infected mice reacted to all the antigens whereas those of naïve mice did not. IgG responses to SmTR1, 3, 9 and 10 were detected at the early stage of infection. Interestingly, antibodies reacting to SmTR3, 9, 10 and 15 dramatically decreased 4 weeks after treatment with praziquantel, while those against SEA and SWAP remained elevated. Our study suggests that TR proteins, especially SmTR10, may be suitable antigens for sero-diagnosis of infection by S. mansoni and are potential markers for monitoring and surveillance of schistosomiasis, including re-infection after treatment with praziquantel.

  16. Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

    PubMed

    Boulila, Moncef

    2010-06-01

    To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

  17. Human parvovirus B19 serology and avidity using a combination of recombinant antigens enables a differentiated picture of the current state of infection.

    PubMed

    Pfrepper, K-I; Enders, M; Motz, M

    2005-01-01

    In order to improve serodiagnostic methods for the determination of the state of human parovirus B19 infection, a new test system, recomLine Parvovirus, based on the use of recombinant antigens, has been developed and evaluated. The test system combines the advantages of enzyme-linked immunosorbent assay (ELISA) methods with those of the Western blot technique. For the recombinant line assay, five antigens of human parvovirus B19 that were recombinantly produced in Escherichia coli were applied directly on nitrocellulose membranes: VP2, the aminoterminal and the carboxyterminal domain of VP1 (VP-N and VP-C), VP-1S another fragment of VP-N and NS1. In addition, empty virus particles isolated from eukaryotic cell cultures were also applied. The recombinant-line assay was used to detect human IgG and IgM antibodies directed against human parvovirus B19. In addition, the avidity of the IgG antibodies was investigated. The recombinant line assay was evaluated using 87 human serum samples of patients recently infected with human parvovirus B19 including 10 samples of three infection time courses and 100 serum samples of healthy blood donors. All results were compared with commercially available ELISAs. In the case of discrepancies, Western blot analysis was performed. The data revealed the recombinant line assay to be highly sensitive and specific. The individual determination of the human immune response against several recombinant antigens covering the structural proteins of human parvovirus B19 gives a deeper insight into the actual status of infection. In addition, the determination of IgG avidity against these individual recombinant antigens enables a more precise and differentiated picture of the infection event.

  18. Kaposi's sarcoma herpesvirus-encoded latency-associated nuclear antigen stabilizes intracellular activated Notch by targeting the Sel10 protein.

    PubMed

    Lan, Ke; Verma, Subhash C; Murakami, Masanao; Bajaj, Bharat; Kaul, Rajeev; Robertson, Erle S

    2007-10-09

    Deregulation of the evolutionarily conserved Notch signaling is highly correlated with oncogenesis. Intracellular activated Notch (ICN) is a protooncogene linked to the transcription activation of a number of cellular genes involved in cell cycle regulation, differentiation, and proliferation. Stability of ICN is tightly regulated by the Sel10-mediated ubiquitin-proteasome pathway. Sel10 can function as a negative regulator of Notch and exhibits activities of a tumor-suppressor protein. This article shows that the Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) directly interacts with Sel10 and forms a complex in KSHV-infected cells. This results in suppression of ICN ubiquitination and degradation. The carboxyl terminus of LANA interacts with the F-box and WD40 domains of Sel10 and competes with ICN for binding to Sel10. This elevated level of ICN is also critical for maintaining the enhanced proliferation of KSHV-infected tumor cells. These findings describe a mechanism by which the KSHV-encoded LANA protein regulates ubiquitination of ICN mediated by the F-box component of the E3 ligase Sel10, leading to proliferation of the virus-infected cells.

  19. Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

    PubMed

    Sawada, R; Ohashi, K; Anaguchi, H; Okazaki, H; Hattori, M; Minato, N; Naruto, M

    1990-04-01

    To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.

  20. Pro-recombination Role of Srs2 Protein Requires SUMO (Small Ubiquitin-like Modifier) but Is Independent of PCNA (Proliferating Cell Nuclear Antigen) Interaction.

    PubMed

    Kolesar, Peter; Altmannova, Veronika; Silva, Sonia; Lisby, Michael; Krejci, Lumir

    2016-04-01

    Srs2 plays many roles in DNA repair, the proper regulation and coordination of which is essential. Post-translational modification by small ubiquitin-like modifier (SUMO) is one such possible mechanism. Here, we investigate the role of SUMO in Srs2 regulation and show that the SUMO-interacting motif (SIM) of Srs2 is important for the interaction with several recombination factors. Lack of SIM, but not proliferating cell nuclear antigen (PCNA)-interacting motif (PIM), leads to increased cell death under circumstances requiring homologous recombination for DNA repair. Simultaneous mutation of SIM in asrs2ΔPIMstrain leads to a decrease in recombination, indicating a pro-recombination role of SUMO. Thus SIM has an ambivalent function in Srs2 regulation; it not only mediates interaction with SUMO-PCNA to promote the anti-recombination function but it also plays a PCNA-independent pro-recombination role, probably by stimulating the formation of recombination complexes. The fact that deletion of PIM suppresses the phenotypes of Srs2 lacking SIM suggests that proper balance between the anti-recombination PCNA-bound and pro-recombination pools of Srs2 is crucial. Notably, sumoylation of Srs2 itself specifically stimulates recombination at the rDNA locus.

  1. Expression of the ’Bacillus anthracis’ Protective Antigen Gene by Baculovirus and Vaccinia Virus Recombinants

    DTIC Science & Technology

    1990-02-01

    ANTIIRACIS PROTI-CTIVE ANTIGEN 367 in medium containing 10% fetal bovine serum. Vaccinia nitrocellulose filters . All cell lysate samples subjected...mM glycine. and 20% methanol at 4°C for 12 to 16 h. by CaCl. precipitation. A. cali irni’, nuclear polyhedrosis Nitrocellulose filters were washed in...and monoclonal antibodies used for these pl[XTJSCRilrr(Stratagcnc. La Jolla. Calif.I. pI3LSCRPPA studies. Filters were washed and incubated with

  2. Recombinant proteinase 3 (Wegener's antigen) expressed in Pichia pastoris is functionally active and is recognized by patient sera.

    PubMed

    Harmsen, M C; Heeringa, P; van der Geld, Y M; Huitema, M G; Klimp, A; Tiran, A; Kallenberg, C G

    1997-11-01

    The open reading frame of human proteinase 3 (PR3) without the prepro-peptide was cloned and expressed in Escherichia coli (rcPR3) and in Pichia pastoris (rpPR3). The 6-histidine tagged rpPR3 was efficiently secreted into culture supernatant from which it could be purified by immobilized metal chelate chromatography. Purified rpPR3 migrated as a single 32-kD band on SDS-PAGE and harboured protease activity that could be inhibited with inhibitors specific for serine-proteases. By indirect antigen-capture ELISA using rpPR3, 60% of sera from patients with Wegener's granulomatosis bound to the recombinant product, although it was not recognized in ELISA with directly coated rpPR3.

  3. Poly-ε-caprolactone/Chitosan and Chitosan Particles: Two Recombinant Antigen Delivery Systems for Intranasal Vaccination.

    PubMed

    Jesus, Sandra; Soares, Edna; Borges, Olga

    2016-01-01

    Several evidences converge on the idea that among the mucosal administration routes, the nasal mucosa is the most attractive site for the delivery of vaccines. Mucoadhesive particulate adjuvants should be able to increase the residence time of antigens in nasal cavity in order to increase their probability of being taken up by nasopharynx-associated lymphoid tissue (NALT) cells and subsequently to initiate the innate and adaptive immune response. Focusing on chitosan, a mucoadhesive biopolymer, we describe in this chapter a method to prepare antigen loaded chitosan nanoparticles and a second method to prepare antigen loaded poly-ε-caprolactone/chitosan nanoparticles. Additionally the methodology for the assessment of mucoadhesivity of the delivery system is also described. The two critical procedures in mice intranasal immunization experiments include challenges in the intranasal administration itself due to the small mouse nose, and the other is related with the collection of mucosal secretions to assess the sIgA. The techniques are difficult to perform without advanced training. Therefore, protocols followed in our laboratory, as well as some tips, are described in this chapter.

  4. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    PubMed

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

  5. The low prevalence Rh antigen STEM (RH49) is encoded by two different RHCE*ce818T alleles that are often in cis to RHD*DOL

    PubMed Central

    Reid, Marion E.; Hipsky, Christine Halter; Hue-Roye, Kim; Coghlan, Gail; Olsen, Coral; Lomas-Francis, Christine

    2012-01-01

    Background STEM (RH49) is a low prevalence antigen in the Rh blood group system. A scarcity of anti-STEM has precluded extensive study of this antigen. We report that two alleles with a RHCE*ce818C>T change encode a partial e, and a hrS−, hrB+, STEM+ phenotype and that both alleles are frequently in cis to RHD*DOL1 or RHD*DOL2. Materials and methods Blood samples were from donors and patients in our collections. Hemagglutination, DNA and RNA testing was performed by standard techniques. Results Fourteen STEM+ samples were heterozygous RHCE*ce818C/T: six had RHCE*ceBI and eight had a novel allele, RHCE*ceSM. Eleven were heterozygous for RHD*DOL1 or RHD*DOL2. Eleven samples, previously typed STEM−, had RHCE*ce818C/C (consensus nucleotide). RBCs from informative STEM+ samples were e+/− hrS− hrB+. One person who was heterozygous RHCE*ceBI and RHCE*cE had an anti-e-like antibody in her plasma, and one person, who was hemizygous for RHD*DOL2 had anti-D in her plasma. Conclusions We show that two alleles with a RHCE*ce818C>T change (RHCE*ceBI and RHCE*ceSM) encode a hrS− hrB+ STEM+ phenotype. In addition, both alleles are frequently in cis to RHD*DOL1 or RHD*DOL2 and RHCE*ceBI encodes a partial e antigen. In the small cohort of samples tested, RHD*DOL invariably traveled with RHCE*ce818T. Our study also confirmed the presumption that RHD*DOL2, like RHD*DOL1, encodes a partial D antigen and the low prevalence antigen DAK. PMID:22738288

  6. Transduction of human dendritic cells with a recombinant modified vaccinia Ankara virus encoding MUC1 and IL-2.

    PubMed

    Trevor, K T; Hersh, E M; Brailey, J; Balloul, J M; Acres, B

    2001-10-01

    The epithelial mucin MUC1 is considered an opportune target antigen for cancer immunotherapy, as it is over-expressed and exhibits aberrant glycosylation in malignant cells. Because dendritic cells (DC) are powerful initiators of immune responses, efforts have focused on tumor antigen-bearing DC as potent cancer vaccines. In this study we have characterized the transduction of monocyte-derived DC with a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] encoding human MUC1 and the immunostimulatory cytokine IL-2. Analysis of transduced DC cultures generated from a number of donors revealed MUC1 expression in the range of 27-54% of the cells and a co-regulated secretion of bioactive IL-2. As shown by FACS analysis with MUCI-specific antibodies, the MVA-MUC1/IL-2-transduced DC predominantly expressed the fully processed glycoform of MUC1, typical of that displayed by normal epithelia. Over a 3-day period after transduction, transgene expression declined concurrent with an increase in MVA-induced cytopathic effects. The transduced DC stimulated allogeneic lymphocyte proliferation, indicating that DC immunostimulatory function is not impaired by vector transduction. In the presence of IL-2, MVA-transduced DC were able to enhance autologous lymphocyte proliferation. Also, vector expression was analyzed in DC cultures treated with TNF-alpha, a known DC maturation factor. As indicated by the up-regulation of several DC maturation markers, neither virus infection nor transgene expression influenced the maturation capacity of the cells. The MVA-MUC1/IL-2 vector effectively transduced both immature and TNF-alpha-matured DC. Overall, our results are encouraging for the clinical application of MVA-MUC1/IL-2-transduced DC.

  7. Alphavirus Replicon DNA Expressing HIV Antigens Is an Excellent Prime for Boosting with Recombinant Modified Vaccinia Ankara (MVA) or with HIV gp140 Protein Antigen

    PubMed Central

    Knudsen, Maria L.; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose. PMID:25643354

  8. Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA) or with HIV gp140 protein antigen.

    PubMed

    Knudsen, Maria L; Ljungberg, Karl; Tatoud, Roger; Weber, Jonathan; Esteban, Mariano; Liljeström, Peter

    2015-01-01

    Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP) vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA) and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF) adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

  9. New skin test for detection of bovine tuberculosis on the basis of antigen-displaying polyester inclusions produced by recombinant Escherichia coli.

    PubMed

    Chen, Shuxiong; Parlane, Natalie A; Lee, Jason; Wedlock, D Neil; Buddle, Bryce M; Rehm, Bernd H A

    2014-04-01

    The tuberculin skin test for diagnosing tuberculosis (TB) in cattle lacks specificity if animals are sensitized to environmental mycobacteria, as some antigens in purified protein derivative (PPD) prepared from Mycobacterium bovis are present in nonpathogenic mycobacteria. Three immunodominant TB antigens, ESAT6, CFP10, and Rv3615c, are present in members of the pathogenic Mycobacterium tuberculosis complex but absent from the majority of environmental mycobacteria. These TB antigens have the potential to enhance skin test specificity. To increase their immunogenicity, these antigens were displayed on polyester beads by translationally fusing them to a polyhydroxyalkanoate (PHA) synthase which mediated formation of antigen-displaying inclusions in recombinant Escherichia coli. The most common form of these inclusions is poly(3-hydroxybutyric acid) (PHB). The respective fusion proteins displayed on these PHB inclusions (beads) were identified using tryptic peptide fingerprinting analysis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The surface exposure and accessibility of antigens were assessed by enzyme-linked immunosorbent assay (ELISA). Polyester beads displaying all three TB antigens showed greater reactivity with TB antigen-specific antibody than did beads displaying only one TB antigen. This was neither due to cross-reactivity of antibodies with the other two antigens nor due to differences in protein expression levels between beads displaying single or three TB antigens. The triple-antigen-displaying polyester beads were used for skin testing of cattle and detected all cattle experimentally infected with M. bovis with no false-positive reactions observed in those sensitized to environmental mycobacteria. The results suggested applicability of TB antigen-displaying polyester inclusions as diagnostic reagents for distinguishing TB-infected from noninfected animals.

  10. Diagnostic value of the recombinant tandem repeat antigen TeGM6-4r for surra in water buffaloes.

    PubMed

    Nguyen, Thu-Thuy; Zhou, Mo; Ruttayaporn, Ngasaman; Nguyen, Quoc Doanh; Nguyen, Viet Khong; Goto, Yasuyuki; Suzuki, Yasuhiko; Kawazu, Shin-ichiro; Inoue, Noboru

    2014-03-17

    Trypanosoma evansi infection, or surra, is currently affecting various species of animals, especially water buffaloes. Since diagnosis is an important aspect of surra control, development of novel diagnostic antigens is of interest to implement and improve the currently utilized methods. Our study evaluated the tandem repeat antigen TeGM6-4r in T. evansi antibody detection in water buffaloes. TeGM6-4r-based ELISA was performed with 20 positive and 8 negative controls and 484 field samples from water buffaloes in Northern Vietnam. To examine cross-reactivity, sera from Japanese cattle that had been experimentally infected with Theileria orientalis (n=10), Babesia bovis (n=3), Babesia bigemina (n=7) and Trypanosoma theileri (n=59) were included in the study. The sensitivity of the test was 80%. TeGM6-4r did not react with Theileria or Babesia infected sera, however it showed cross reactivity with 11/59 T. theileri infected samples. The reference test, CATT/T. evansi also reacted with 3/59 T. theileri infected sera. The lysate antigen-based ELISA reacted with 4/59 T. theileri, 9/10 Theileria and 3/10 Babesia infected sera. In contrast, TeGM6-4r-based ELISA was 86.3% sensitive and 58.3% specific in the screening of field samples. The average seroprevalence of T. evansi infection among water buffaloes in Northern Vietnam was 27.1% by CATT/T. evansi and 53.7% by TeGM6-4r. Seroprevalence in the five surveyed provinces ranged from 17.4% to 39.8% in the reference test, and 47.3% to 67.3% in the recombinant antigen based test. The finding indicated that the disease is still widely endemic in the area and that surveillance programs need to be carried out regularly to better control surra. We proposed TeGM6-4r as a useful serodiagnostic antigen for the detection and epidemiological surveillance of T. evansi infection among water buffaloes. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi as an antigen for delayed-type hypersensitivity assays and serodiagnosis of canine visceral leishmaniasis.

    PubMed

    Pinheiro, Paulo Henrique da Costa; Pinheiro, Adriana Nunes; Ferreira, Josie Haydée Lima; Costa, Francisco Assis Lima; Katz, Simone; Barbiéri, Clara Lúcia

    2009-05-26

    A recombinant protein, rLdccys1, produced by expression of the gene encoding a 30kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used to detect specific antibodies in serum by enzyme-linked immunosorbent assays and to test for reactivity in delayed-type hypersensitivity (DTH) responses of dogs from an endemic region of visceral leishmaniasis (VL), Teresina, Piauí State, Brazil. Amastigote or promastigote extracts were also assayed for comparison. The sensitivity for detection of specific antibodies to L. (L.) chagasi using rLdccys1, lysates from L. (L.) chagasi promastigotes and amastigotes was 96%, 68%, and 69%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and little reactivity was found with sera from dogs with babesiosis and ehrlichiosis. Among 106 sera from symptomatic dogs and 22 from non-infected controls, no false negatives and only two false positive sera were found for rLdccys1. In contrast, amastigote lysates yielded 11 false positives and 13 false negatives, whereas the corresponding numbers for promastigote lysates were 17 and 16. DTH responses were determined after intradermal injection of rLdccys1 or amastigote extract and the induration area was measured at 24, 48 and 72h after injection. All asymptomatic dogs showed a positive intradermal response to rLdccys1 (>10mm) which peaked at 48h, whereas no significant reactivity to the recombinant antigen was found in the symptomatic group. Histological analysis of the intradermal induration showed a predominance of necrotic and hemorrhagic areas in sections from asymptomatic dogs injected with L. (L.) chagasi amastigote extract, whereas a typical granulomatous reaction mediated by mononuclear cells was observed in sections from asymptomatic animals injected with rLdccys1. Grouping data from ELISA and DTH assays with rLdccys1 and L. (L.) chagasi amastigote extracts showed that humoral and cellular responses were inversely correlated during the

  12. Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

    PubMed

    Su, Qiudong; Guo, Minzhuo; Jia, Zhiyuan; Qiu, Feng; Lu, Xuexin; Gao, Yan; Meng, Qingling; Tian, Ruiguang; Bi, Shengli; Yi, Yao

    2016-07-01

    Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

    PubMed

    Peretti, Leandro E; Gonzalez, Verónica D G; Marcipar, Iván S; Gugliotta, Luis M

    2014-08-01

    The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

  14. Mechanism of a decrease in potency for the recombinant influenza A virus hemagglutinin H3 antigen during storage.

    PubMed

    Hickey, John M; Holtz, Kathleen M; Manikwar, Prakash; Joshi, Sangeeta B; McPherson, Clifton E; Buckland, Barry; Srivastava, Indresh K; Middaugh, C Russell; Volkin, David B

    2014-03-01

    The recombinant hemagglutinin (rHA)-based influenza vaccine Flublok® has recently been approved in the United States as an alternative to the traditional egg-derived flu vaccines. Flublok is a purified vaccine with a hemagglutinin content that is threefold higher than standard inactivated influenza vaccines. When rHA derived from an H3N2 influenza virus was expressed, purified, and stored for 1 month, a rapid loss of in vitro potency (∼50%) was observed as measured by the single radial immunodiffusion (SRID) assay. A comprehensive characterization of the rHA protein antigen was pursued to identify the potential causes and mechanisms of this potency loss. In addition, the biophysical and chemical stability of the rHA in different formulations and storage conditions was evaluated over time. Results demonstrate that the potency loss over time did not correlate with trends in changes to the higher order structure or hydrodynamic size of the rHA. The most likely mechanism for the early loss of potency was disulfide-mediated cross-linking of rHA, as the formation of non-native disulfide-linked multimers over time correlated well with the observed potency loss. Furthermore, a loss of free thiol content, particularly in specific cysteine residues in the antigen's C-terminus, was correlated with potency loss measured by SRID.

  15. Serologic Diagnosis of Human Taenia solium Cysticercosis by Using Recombinant and Synthetic Antigens in QuickELISA™

    PubMed Central

    Lee, Yeuk-Mui; Handali, Sukwan; Hancock, Kathy; Pattabhi, Sowmya; Kovalenko, Victor A.; Levin, Andrew; Rodriguez, Silvia; Lin, Sehching; Scheel, Christina M.; Gonzalez, Armando E.; Gilman, Robert H.; Garcia, Hector H.; Tsang, Victor C. W.

    2011-01-01

    Diagnosis of Taenia solium cysticercosis is an important component in the control and elimination of cysticercosis and taeniasis. New detection assays using recombinant and synthetic antigens originating from the lentil lectin-purified glycoproteins (LLGPs) of T. solium cysticerci were developed in a QuickELISA™ format. We analyzed a panel of 474 serum samples composed of 108 serum samples from donors with two or more viable cysts, 252 serum samples from persons with other parasitic infections, and 114 serum samples from persons with no documented illnesses. The sensitivities and specificities of T24H QuickELISA™, GP50 QuickELISA™, and Ts18var1 QuickELISA™ were 96.3% and 99.2%, 93.5% and 98.6%, and 89.8% and 96.4%, respectively, for detecting cases with multiple, viable cysts. T24H QuickELISA™ performs best among the three assays, and has sensitivity and specificity values comparable to those of the LLGP enzyme-linked immunosorbent blot. The QuickELISA™ are simple, rapid quantitative methods for detecting antibodies specific for T. solium cysticerci antigens. PMID:21460015

  16. Effect of single-point mutations on the stability and immunogenicity of a recombinant ricin A chain subunit vaccine antigen.

    PubMed

    Thomas, Justin C; O'Hara, Joanne M; Hu, Lei; Gao, Fei P; Joshi, Sangeeta B; Volkin, David B; Brey, Robert N; Fang, Jianwen; Karanicolas, John; Mantis, Nicholas J; Middaugh, C Russell

    2013-04-01

    There is great interest in the design and development of highly thermostable and immunogenic protein subunit vaccines for biodefense. In this study, we used two orthogonal and complementary computational protein design approaches to generate a series of single-point mutants of RiVax, an attenuated recombinant ricin A chain (RTA) protein subunit vaccine antigen. As assessed by differential scanning calorimetry, the conformational stabilities of the designed mutants ranged from 4°C less stable to 4.5°C more stable than RiVax, depending on solution pH. Two more thermostable (V18P, C171L) and two less thermostable (T13V, S89T) mutants that displayed native-like secondary and tertiary structures (as determined by circular dichroism and fluorescence spectral analysis, respectively) were tested for their capacity to elicit RTA-specific antibodies and toxin-neutralizing activity. Following a prime-boost regimen, we found qualitative differences with respect to specific antibody titers and toxin neutralizing antibody levels induced by the different mutants. Upon a second boost with the more thermostable mutant C171L, a statistically significant increase in RTA-specific antibody titers was observed when compared with RiVax-immunized mice. Notably, the results indicate that single residue changes can be made to the RiVax antigen that increase its thermal stability without adversely impacting the efficacy of the vaccine.

  17. Recombinant mid gut antigen (Bm95) as a vaccine against Indian Rhiphicephalus haemaphysaloides in Bos indicus cattle.

    PubMed

    Sugumar, Parthasarathy; Chandran, Dev; Sudha Rani, Gudavalli; Shahana, Pallichera Vijayan; Maske, Dasarath Keshavrao; Rangarajan, Pundi Narasimhan; Mangamoori, Lakshmi Narasu; Srinivasan, Villuppanoor Alwar

    2011-04-01

    In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of Rhipicephalus microplus strain A in controlling the tick infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography-mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. Immunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naïve Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling R. haemaphysaloides infestations in the field. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

    PubMed Central

    Alves, Rúbens Prince dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta

    2016-01-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. PMID:27030586

  19. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

    PubMed Central

    2012-01-01

    Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

  20. Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology.

    PubMed

    Tam, Yew Joon; Allaudin, Zeenathul Nazariah; Lila, Mohd Azmi Mohd; Bahaman, Abdul Rani; Tan, Joo Shun; Rezaei, Morvarid Akhavan

    2012-10-05

    Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

  1. Serum immunoglobulin G4 antibodies to the recombinant antigen, Ll-SXP-1, are highly specific for Loa loa infection.

    PubMed

    Klion, Amy D; Vijaykumar, Aarthi; Oei, Tamara; Martin, Brian; Nutman, Thomas B

    2003-01-01

    The clinical manifestations and geographic distribution of loiasis overlap with those of other human filarial parasites, presenting challenges in the specific diagnosis of loiasis that may lead to delays in appropriate therapy. A recombinant antigen (Ll-SXP-1), preferentially recognized by serum samples from experimentally infected rhesus monkeys, was identified from a Loa loa L3 cDNA library. IgG4 antibody reactivity to purified Ll-SXP-1 was assessed by means of ELISA, using serum samples from patients with loiasis, lymphatic filariasis, onchocerciasis, mansonellosis, or other helminthiases and healthy control subjects. The assay was 56% sensitive and 98% specific for loiasis. Antibody reactivity was detectable before microfilaremia in experimentally infected rhesus monkeys and declined (but did not disappear) after diethylcarbamazine therapy in infected patients. IgG4 antibodies to recombinant Ll-SXP-1 are a highly specific marker of L. loa infection and may be useful for the diagnostic evaluation of persons with filariasis of unclear etiology.

  2. Regeneration of Recombinant Antigen Microarrays for the Automated Monitoring of Antibodies against Zoonotic Pathogens in Swine Sera

    PubMed Central

    Meyer, Verena K.; Kober, Catharina; Niessner, Reinhard; Seidel, Michael

    2015-01-01

    The ability to regenerate immobilized proteins like recombinant antigens (rAgs) on surfaces is an unsolved problem for flow-based immunoassays on microarray analysis systems. The regeneration on microarray chip surfaces is achieved by changing the protein structures and desorption of antibodies. Afterwards, reactivation of immobilized protein antigens is necessary for reconstitution processes. Any backfolding should be managed in a way that antibodies are able to detect the protein antigens in the next measurement cycle. The regeneration of rAg microarrays was examined for the first time on the MCR3 flow-based chemiluminescence (CL) microarray analysis platform. The aim was to reuse rAg microarray chips in order to reduce the screening effort and costs. An antibody capturing format was used to detect antibodies against zoonotic pathogens in sera of slaughtered pigs. Different denaturation and reactivation buffers were tested. Acidic glycine-SDS buffer (pH 2.5) and 8 M guanidinium hydrochloride showed the best results in respect of denaturation efficiencies. The highest CL signals after regeneration were achieved with a carbonate buffer containing 10 mM DTT and 0.1% BSA for reactivation. Antibodies against Yersinia spp. and hepatitis E virus (HEV) were detected in swine sera on one immunochip over 4 days and 25 measurement cycles. Each cycle took 10 min for detection and regeneration. By using the rAg microarray chip, a fast and automated screening of antibodies against pathogens in sera of slaughtered pigs would be possible for zoonosis monitoring. PMID:25625908

  3. Analysis of the Cross-Reactivity of Various 56 kDa Recombinant Protein Antigens with Serum Samples Collected after Orientia tsutsugamushi Infection by ELISA

    DTIC Science & Technology

    2011-01-01

    has increased the yield of Orientia , 28 considerable difficulties still exist in mass production of pure Orientia and in retaining its stability...Rickettsia tsutsugamushi Boryong with partial 56-kilodalton recombinant antigen fused with the maltose - binding protein MBP-Bor56 . Infect Immun 65

  4. Live recombinant Lactococcus lactis vaccine expressing immobilization antigen (i-Ag) for protection against Ichthyophthirius multifiliis in goldfish.

    PubMed

    Yao, Jia-Yun; Yuan, Xue-Mei; Xu, Yang; Yin, Wen-Lin; Lin, Ling-Yun; Pan, Xiao-Yi; Yang, Gui-Lian; Wang, Chun-Feng; Shen, Jin-Yu

    2016-11-01

    The parasite Ichthyophthirius multifiliis (Ich) has been reported in various freshwater fishes worldwide and results in severe losses to both food and aquarium fish production. Lactobacillus strains have a number of properties that make them attractive candidates as delivery vehicles for the presentation to the mucosa of compounds with pharmaceutical interest, in particular vaccines. Here, the present study was conducted to evaluate a live recombinant Lactococcus lactis vaccine expressing immobilization antigen (IAG-52X) in protection against I. multifiliis. A 1266 bp gene fragment containing a potential antigenic epitope of the 48 kDa immobilization antigen of I. multifiliis was assembled from six synthetic ohgonucleotides and cloned into pSIP409 and electrotransformed into Lactobacillus plantarum NC8. The recombinant vaccine candidate was then orally fed into goldfish. The expression of immune-related genes: complement component 3 (C3), MHC I, IgM gene in blood from goldfish at different time points after immunization were evaluated. Immunized fish were than challenged with a lethal dose of infectious I. multifiliis. The cumulative mortality and relative percentage survival (RPS) were also determined. Our results showed that the antibody level in the blood and skin of the immunized fish was statistically significant (P < 0.05) in relation to the control groups. Goldfish orally immunized with NC8-pSIP409- IAG-52X had high serum antibody titers that ranged from 32 to 256 after 28d post immunization, while fish fed with NC8-pSIP409 or PBS had no detectable immobilizing antibody response. Expression of IgM, C3, MHC I genes in the group immunized with IAG-52X were significantly (P < 0.05) up regulated as compared with control group, indicating that different immune cells were actively involved in cellular immune response. The results showed that the average survival rate of fish orally immunized with 10(8) and 10(6)NC8-pSIP409-IAG-52X was 60% and 50

  5. Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen.

    PubMed

    Byrne, Guerard W; Du, Zeji; Stalboerger, Paul; Kogelberg, Heide; McGregor, Christopher G A

    2014-01-01

    Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in

  6. Cloning and expression of porcine β1,4 N-acetylgalactosaminyl transferase encoding a new xenoreactive antigen

    PubMed Central

    Byrne, Guerard W; Du, Zeji; Stalboerger, Paul; Kogelberg, Heide; McGregor, Christopher G A

    2014-01-01

    Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4

  7. Protective antibodies against Taenia taeniaeformis in rats infected with eggs or injected with non-viable oncospheres or recombinant antigens of oncospheres.

    PubMed

    Ito, A; Asano, K; Okamoto, K

    1994-09-01

    Antibody responses against Taenia taeniaeformis in rats infected with eggs or injected with non-viable oncospheres or recombinant antigens of oncospheres were analysed by passive transfer of serum and Western blotting. When recipient rats were injected with 1 ml serum from donors infected with eggs (infected serum), they all showed complete resistance to oral egg challenge, whereas those injected with 1 ml serum from donors injected with either oncospheres or recombinant antigens (vaccinated serum) showed no resistance. IgG and IgG subclass responses detected by Western blotting revealed that antibody responses to oncosphere antigens in infected serum thoroughly differed from those in vaccinated serum. It is suggested that IgG2 alpha responses in infected serum should be used for screening of epitopes for candidate vaccine.

  8. Evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching

    PubMed Central

    Allison, Andrew B.; Stallknecht, David E.; Holmes, Edward C.

    2014-01-01

    Western equine encephalitis virus (WEEV), Highlands J virus (HJV), and Fort Morgan virus (FMV) are the sole representatives of the WEE antigenic complex of the genus Alphavirus, family Togaviridae, that are endemic to North America. All three viruses have their ancestry in a recombination event involving eastern equine encephalitis virus (EEEV) and a Sindbis (SIN)-like virus that gave rise to a chimeric alphavirus that subsequently diversified into the present-day WEEV, HJV, and FMV. Here, we present a comparative analysis of the genetic, ecological, and evolutionary relationships among these recombinant-origin viruses, including the description of a nsP4 polymerase mutation in FMV that allows it to circumvent the host range barrier to Asian tiger mosquito cells, a vector species that is normally refractory to infection. Notably, we also provide evidence that the recombination event that gave rise to these three WEEV antigenic complex viruses may have occurred in North America. PMID:25463613

  9. Evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching.

    PubMed

    Allison, Andrew B; Stallknecht, David E; Holmes, Edward C

    2015-01-01

    Western equine encephalitis virus (WEEV), Highlands J virus (HJV), and Fort Morgan virus (FMV) are the sole representatives of the WEE antigenic complex of the genus Alphavirus, family Togaviridae, that are endemic to North America. All three viruses have their ancestry in a recombination event involving eastern equine encephalitis virus (EEEV) and a Sindbis (SIN)-like virus that gave rise to a chimeric alphavirus that subsequently diversified into the present-day WEEV, HJV, and FMV. Here, we present a comparative analysis of the genetic, ecological, and evolutionary relationships among these recombinant-origin viruses, including the description of a nsP4 polymerase mutation in FMV that allows it to circumvent the host range barrier to Asian tiger mosquito cells, a vector species that is normally refractory to infection. Notably, we also provide evidence that the recombination event that gave rise to these three WEEV antigenic complex viruses may have occurred in North America.

  10. Production of hepatitis B surface antigen in recombinant plant systems: an update.

    PubMed

    Kumar, G B Sunil; Ganapathi, T R; Bapat, V A

    2007-01-01

    There is a growing interest to develop oral vaccines for infectious diseases, as it is the most convenient and effective way to attain mucosal immunity. Hepatitis B continues to be a major infectious disease in many developing countries despite the availability of recombinant vaccine. On a global scenario, Hepatitis B Virus infection is probably the single most prevalent cause of persistent viraemia in humans. There are about 350 million chronic carriers of HBV, which is about 5% of the total world population. It is estimated that 75-100 million of them will die of liver cirrhosis and/or hepatocellular carcinoma. Progress in plant genetic engineering has enabled the transfer of useful genes for desirable traits. The recent trend is to use this technique to exploit plants as biofactories for the production of therapeutic proteins including vaccines. Rapid progress has been made in this area to develop plant-based vaccines for hepatitis B. This review describes the expression, characterization, and immunogenicity studies of hepatitis B vaccines produced in recombinant plant systems and their implications for developing a plant-based vaccine.

  11. Complete nucleotide sequence of a gene encoding a functional human class I histocompatibility antigen (HLA-CW3).

    PubMed Central

    Sodoyer, R; Damotte, M; Delovitch, T L; Trucy, J; Jordan, B R; Strachan, T

    1984-01-01

    The HLA-CW3 gene contained in a cosmid clone identified by transfection expression experiments has been completely sequenced. This provides, for the first time, data on the structure of HLA-C locus products and constitutes, together with that of the gene coding for HLA-A3, the first complete nucleotide sequences of genes coding for serologically defined class I HLA molecules. In contrast to the organisation of the two class I HLA pseudogenes whose sequences have previously been determined, the sequence of the HLA-CW3 gene reveals an additional cytoplasmic encoding domain, making the organisation of this gene very similar to that of known H-2 class I genes and also the HLA-A3 gene. The deduced amino acid sequences of HLA-CW3 and HLA-A3 now allow a systematic comparison of such sequences of HLA class I molecules from the three classical transplantation antigen loci A, B, C. The compared sequences include the previously determined partial amino acid sequences of HLA-B7, HLA-B40, HLA-A2 and HLA-A28. The comparisons confirm the extreme polymorphism of HLA classical class I molecules, and permit a study of the level of diversity and the location of sequence differences. The distribution of differences is not uniform, most of them being located in the first and second extracellular domains, the third extracellular domain is extremely conserved, and the cytoplasmic domain is also a variable region. Although it is difficult to determine locus-specific regions, we have identified several candidate positions which may be C locus-specific. PMID:6609813

  12. Immunotherapeutic effects of recombinant adenovirus encoding granulocyte–macrophage colony-stimulating factor in experimental pulmonary tuberculosis

    PubMed Central

    Francisco-Cruz, A.; Mata-Espinosa, D.; Estrada-Parra, S.; Xing, Z.; Hernández-Pando, R.

    2013-01-01

    Summary BALB/c mice with pulmonary tuberculosis (TB) develop a T helper cell type 1 that temporarily controls bacterial growth. Bacterial proliferation increases, accompanied by decreasing expression of interferon (IFN)-γ, tumour necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS). Activation of dendritic cells (DCs) is delayed. Intratracheal administration of only one dose of recombinant adenoviruses encoding granulocyte–macrophage colony-stimulating factor (AdGM-CSF) 1 day before Mycobacterium tuberculosis (Mtb) infection produced a significant decrease of pulmonary bacterial loads, higher activated DCs and increased expression of TNF-α, IFN-γ and iNOS. When AdGM-CSF was given in female mice B6D2F1 (C57BL/6J X DBA/2J) infected with a low Mtb dose to induce chronic infection similar to latent infection and corticosterone was used to induce reactivation, a very low bacilli burden in lungs was detected, and the same effect was observed in healthy mice co-housed with mice infected with mild and highly virulent bacteria in a model of transmissibility. Thus, GM-CSF is a significant cytokine in the immune protection against Mtb and gene therapy with AdGM-CSF increased protective immunity when administered in a single dose 1 day before Mtb infection in a model of progressive disease, and when used to prevent reactivation of latent infection or transmission. PMID:23379435

  13. Systemic Administration of a Recombinant AAV1 Vector Encoding IGF-1 Improves Disease Manifestations in SMA Mice

    PubMed Central

    Tsai, Li-Kai; Chen, Chien-Lin; Ting, Chen-Hung; Lin-Chao, Sue; Hwu, Wuh-Liang; Dodge, James C; Passini, Marco A; Cheng, Seng H

    2014-01-01

    Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron. PMID:24814151

  14. Multiplex PCR assay for detection of recombinant genes encoding fatty acid desaturases fused with lichenase reporter protein in GM plants.

    PubMed

    Berdichevets, Iryna N; Shimshilashvili, Hristina R; Gerasymenko, Iryna M; Sindarovska, Yana R; Sheludko, Yuriy V; Goldenkova-Pavlova, Irina V

    2010-07-01

    Thermostable lichenase encoded by licB gene of Clostridium thermocellum can be used as a reporter protein in plant, bacterial, yeast, and mammalian cells. It has important advantages of high sensitivity and specificity in qualitative and quantitative assays. Deletion variants of LicB (e.g., LicBM3) retain its enzymatic activity and thermostability and can be expressed in translational fusion with target proteins without compromising with their properties. Fusion with the lichenase reporter is especially convenient for the heterologous expression of proteins whose analysis is difficult or compromised by host enzyme activities, as it is in case of fatty acid desaturases occurring in all groups of organisms. Recombinant desaturase-lichenase genes can be used for creating genetically modified (GM) plants with improved chill tolerance. Development of an analytical method for detection of fused desaturase-lichenase transgenes is necessary both for production of GM plants and for their certification. Here, we report a multiplex polymerase chain reaction method for detection of desA and desC desaturase genes of cyanobacteria Synechocystis sp. PCC6803 and Synechococcus vulcanus, respectively, fused to licBM3 reporter in GM plants.

  15. Dose of incorporated immunodominant antigen in recombinant BCG impacts modestly on Th1 immune response and protective efficiency against Mycobacterium tuberculosis in mice.

    PubMed

    Ma, Hui; Wu, Kang; Liu, Fang; Yang, Hua; Kang, Han; Chen, Ning-Ning; Yuan, Qin; Zhou, Wen-Jiang; Fan, Xiao-Yong

    2014-01-01

    One approach for improving BCG efficacy is to utilize BCG as vehicle to develop recombinant BCG (rBCG) strains overexpressing Mycobacterium tuberculosis (M. tb) antigens. Also expression level of a candidate antigen should impact the final T cell responses conferred by rBCG. In this study, based on our previously constructed differential expression system, we developed two rBCG strains overexpressing M. tb chimeric antigen Ag856A2 (coding a recombinant ag85a with 2 copies of esat-6 inserted at Acc I site of ag85a) at differential levels under the control of the subtly modified furA promoters. These two rBCG strains were used to vaccinate C57BL/6 mice and exploit dose of incorporated antigen in rBCG to optimize immune response and protective efficiency against M. tb challenge in mouse model. The results showed that rBCG strains overexpressing Ag856A2 at differential levels induced different antigen-specific IFN-γ production and comparable number of M. tb-specific CD4 T cells expressing IL-2. M. tb challenge experiment showed that rBCG strains afforded enhanced but comparable immune protection characterized by reduced bacillary load, lung pathology, and inflammation. These results suggested that the dose of antigens incorporated in rBCG can impact T cell immune responses but imposed no significantly differential protective efficacies.

  16. Design, engineering, and production of human recombinant t cell receptor ligands derived from human leukocyte antigen DR2.

    PubMed

    Chang, J W; Mechling, D E; Bächinger, H P; Burrows, G G

    2001-06-29

    Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers on the surface of antigen-presenting cells that bind the T cell receptor, initiating a cascade of interactions that results in antigen-specific activation of clonal populations of T cells. Susceptibility to multiple sclerosis is associated with certain MHC class II haplotypes, including human leukocyte antigen (HLA) DR2. Two DRB chains, DRB5*0101 and DRB1*1501, are co-expressed in the HLA-DR2 haplotype, resulting in the formation of two functional cell surface heterodimers, HLA-DR2a (DRA*0101, DRB5*0101) and HLA-DR2b (DRA*0101, DRB1*1501). Both isotypes can present an immunodominant peptide of myelin basic protein (MBP-(84-102)) to MBP-specific T cells from multiple sclerosis patients. We have previously demonstrated that the peptide binding/T cell recognition domains of rat MHC class II (alpha1 and beta1 domains) could be expressed as a single exon for structural and functional characterization; Burrows, G. G., Chang, J. W., Bächinger, H.-P., Bourdette, D. N., Wegmann, K. W., Offner, H., and Vandenbark A. A. (1999) Protein Eng. 12, 771-778; Burrows, G. G., Adlard, K. L., Bebo, B. F., Jr., Chang, J. W., Tenditnyy, K., Vandenbark, A. A., and Offner, H. (2000) J. Immunol. 164, 6366-6371). Single-chain human recombinant T cell receptor ligands (RTLs) of approximately 200 amino acid residues derived from HLA-DR2b were designed using the same principles and have been produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides. Structural characterization using circular dichroism predicted that these molecules retained the antiparallel beta-sheet platform and antiparallel alpha-helices observed in the native HLA-DR2 heterodimer. The proteins exhibited a cooperative two-state thermal unfolding transition, and DR2-derived RTLs with a covalently linked MBP peptide (MBP-(85-99)) showed increased stability to thermal unfolding relative to the

  17. Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi.

    PubMed

    Weatherly, D Brent; Peng, Duo; Tarleton, Rick L

    2016-09-13

    The protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, depends upon a cell surface-expressed trans-sialidase (ts) to avoid activation of complement-mediated lysis and to enhance intracellular invasion. However these functions alone fail to account for the size of this gene family in T. cruzi, especially considering that most of these genes encode proteins lacking ts enzyme activity. Previous whole genome sequencing of the CL Brener clone of T. cruzi identified ~1400 ts variants, but left many partially assembled sequences unannotated. In the current study we reevaluated the trans-sialidase-like sequences in this reference strain, identifying an additional 1779 full-length and partial ts genes with their important features annotated, and confirming the expression of previously annotated "pseudogenes" and newly annotated ts family members. Multiple EM for Motif Elicitation (MEME) analysis allowed us to generate a model T. cruzi ts (TcTS) based upon the most conserved motif patterns and demonstrated that a common motif order is highly conserved among ts family members. Using a newly developed pipeline for the analysis of recombination within large gene families, we further demonstrate that TcTS family members are undergoing frequent recombination, generating new variants from the thousands of functional and non-functional ts gene segments but retaining the overall structure of the core TcTS family members. The number and variety as well as high recombination frequency of TcTS family members supports strong evolutionary pressure, probably exerted by immune selection, for continued variation in ts sequences in T. cruzi, and thus for a unique immune evasion mechanism for the large ts gene family.

  18. Multi-isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen.

    PubMed

    Ong, Eugene Boon Beng; Ignatius, Joshua; Anthony, Amy Amilda; Aziah, Ismail; Ismail, Asma; Lim, Theam Soon

    2015-01-01

    The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

  19. Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species

    PubMed Central

    Sato, Camila Massae; Sanchez, Maria Carmen Arroyo; Celeste, Beatriz Julieta; Duthie, Malcolm S.; Guderian, Jeffrey; Reed, Steven G.; de Brito, Maria Edileuza Felinto; Campos, Marliane Batista; de Souza Encarnação, Helia Valeria; Guerra, Jorge; de Mesquita, Tirza Gabrielle Ramos; Pinheiro, Suzana Kanawati; Ramasawmy, Rajendranath; Silveira, Fernando Tobias; de Assis Souza, Marina

    2016-01-01

    ABSTRACT American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania. The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L. (Viannia) braziliensis, and L. (V.) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL. PMID:27927927

  20. Use of Recombinant Antigens for Sensitive Serodiagnosis of American Tegumentary Leishmaniasis Caused by Different Leishmania Species.

    PubMed

    Sato, Camila Massae; Sanchez, Maria Carmen Arroyo; Celeste, Beatriz Julieta; Duthie, Malcolm S; Guderian, Jeffrey; Reed, Steven G; de Brito, Maria Edileuza Felinto; Campos, Marliane Batista; de Souza Encarnação, Helia Valeria; Guerra, Jorge; de Mesquita, Tirza Gabrielle Ramos; Pinheiro, Suzana Kanawati; Ramasawmy, Rajendranath; Silveira, Fernando Tobias; de Assis Souza, Marina; Goto, Hiro

    2017-02-01

    American tegumentary leishmaniasis (ATL) (also known as cutaneous leishmaniasis [CL]) is caused by various species of protozoa of the genus Leishmania The diagnosis is achieved on a clinical, epidemiological, and pathological basis, supported by positive parasitological exams and demonstration of leishmanin delayed-type hypersensitivity. Serological assays are not routinely used in the diagnosis because many are considered to have low sensitivity and the particular Leishmania species causing the disease can lead to variable performance. In the present study, we generated recombinant versions of two highly conserved Leishmania proteins, Leishmania (Viannia) braziliensis-derived Lb8E and Lb6H, and evaluated both in enzyme-linked immunosorbent assays (ELISA). Recombinant Lb6H (rLb6H) had better performance and reacted with 100.0% of the ATL and 89.4% of the VL samples. These reactions with rLb6H were highly specific (98.5%) when compared against those for samples from healthy control individuals. We then assessed rLb6H against sera from ATL patients infected with different species of Leishmania prevalent in Brazil [Leishmania (Leishmania) amazonensis, L (Viannia) braziliensis, and L (V) guyanensis] and samples from patients with other infectious diseases. In analyses of 500 sera, ELISA using rLb6H detected all 219 ATL samples (sensitivity of 100.0%) with an overall specificity of 93.9% (considering healthy individuals and other infectious diseases patients). Only a minority of samples from Chagas disease patients possessed antibodies against rLb6H, and all of these responses were low (with a highest reactivity index of 2.2). Taken together, our data support further evaluation of rLb6H and the potential for its routine use in the serological diagnosis of ATL. Copyright © 2017 Sato et al.

  1. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease.

    PubMed

    Weiner, Zachary P; Crew, Rebecca M; Brandt, Kevin S; Ullmann, Amy J; Schriefer, Martin E; Molins, Claudia R; Gilmore, Robert D

    2015-11-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Evaluation of Selected Borrelia burgdorferi lp54 Plasmid-Encoded Gene Products Expressed during Mammalian Infection as Antigens To Improve Serodiagnostic Testing for Early Lyme Disease

    PubMed Central

    Weiner, Zachary P.; Crew, Rebecca M.; Brandt, Kevin S.; Ullmann, Amy J.; Schriefer, Martin E.; Molins, Claudia R.

    2015-01-01

    Laboratory testing for the diagnosis of Lyme disease is performed primarily by serologic assays and is accurate for detection beyond the acute stage of the infection. Serodiagnostic assays to detect the early stages of infection, however, are limited in their sensitivity, and improvement is warranted. We analyzed a series of Borrelia burgdorferi proteins known to be induced within feeding ticks and/or during mammalian infection for their utility as serodiagnostic markers against a comprehensive panel of Lyme disease patient serum samples. The antigens were assayed for IgM and IgG reactivity in line immunoblots and separately by enzyme-linked immunosorbent assay (ELISA), with a focus on reactivity against early Lyme disease with erythema migrans (EM), early disseminated Lyme neuroborreliosis, and early Lyme carditis patient serum samples. By IgM immunoblotting, we found that recombinant proteins BBA65, BBA70, and BBA73 reacted with early Lyme EM samples at levels comparable to those of the OspC antigen used in the current IgM blotting criteria. Additionally, these proteins reacted with serum samples from patients with early neuroborreliosis and early carditis, suggesting value in detecting early stages of this disease progression. We also found serological reactivity against recombinant proteins BBA69 and BBA73 with early-Lyme-disease samples using IgG immunoblotting and ELISA. Significantly, some samples that had been scored negative by the Centers for Disease Control and Prevention-recommended 2-tiered testing algorithm demonstrated positive reactivity to one or more of the antigens by IgM/IgG immunoblot and ELISA. These results suggest that incorporating additional in vivo-expressed antigens into the current IgM/IgG immunoblotting tier in a recombinant protein platform assay may improve the performance of early-Lyme-disease serologic testing. PMID:26376927

  3. Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein.

    PubMed

    Sun, Lina; Chen, Zhe; Yu, Li; Wei, Jingshuang; Li, Chuan; Jin, Jing; Shen, Xinxin; Lv, Xinjun; Tang, Qing; Li, Dexin; Liang, Mifang

    2012-10-01

    The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.

  4. Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc).

    PubMed

    Niccheri, Francesca; Real-Fernàndez, Feliciana; Ramazzotti, Matteo; Lolli, Francesco; Rossi, Giada; Rovero, Paolo; Degl'Innocenti, Donatella

    2014-10-01

    Multiple sclerosis (MS) is a chronic auto-immune disease characterized by a damage to the myelin component of the central nervous system. Self-antigens created by aberrant glycosylation have been described to be a key component in the formation of auto-antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS-specific auto-antibodies, and it is actively used in diagnostics and research to monitor and quantify MS-associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His-tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide-clone interaction were calculated obtaining a value of KD in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies.

  5. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity.

    PubMed

    Maynard, Jennifer A; Maassen, Catharina B M; Leppla, Stephen H; Brasky, Kathleen; Patterson, Jean L; Iverson, Brent L; Georgiou, George

    2002-06-01

    The tripartite toxin produced by Bacillus anthracis is the key determinant in the etiology of anthrax. We have engineered a panel of toxin-neutralizing antibodies, including single-chain variable fragments (scFvs) and scFvs fused to a human constant kappa domain (scAbs), that bind to the protective antigen subunit of the toxin with equilibrium dissociation constants (K(d)) between 63 nM and 0.25 nM. The entire antibody panel showed high serum, thermal, and denaturant stability. In vitro, post-challenge protection of macrophages from the action of the holotoxin correlated with the K(d) of the scFv variants. Strong correlations among antibody construct affinity, serum half-life, and protection were also observed in a rat model of toxin challenge. High-affinity toxin-neutralizing antibodies may be of therapeutic value for alleviating the symptoms of anthrax toxin in infected individuals and for medium-term prophylaxis to infection.

  6. [Antigenic analysis of the recombinant capsid protein of porcine circovirus type 2].

    PubMed

    Shang, Shao-bin; Zhou, Ji-yong; Wu, Jian-xiang; Chen, Qing-xin; Gong, Hui

    2005-06-01

    The nuclear localization signal (NLS)-defected capsid protein gene (dCap) of porcine circovirus type 2 (PCV2) was expressed firstly in Escherichia coli as a fusion protein with glutathione S-transferase (rGST-dCap protein). The purified rGST-dCap protein and NLS-defected Cap protein of PCV2 (rdCap protein) from the purified rGST-dCap protein reacted specifically with swine antiserum to PCV2. Furthermore, the obtained monoclonal antibodies (mAbs) to rdCap protein were shown to bind to PCV2 particles replicated in PK15 cell. MAbs to rdCap protein also revealed the neutralizing ability to PCV2 particles. These results demonstrate that rGST-dCap protein expressed in E. coli is folded correctly or at least partly, and all mAbs to rdCap protein possess the binding epitopes of PCV2 particle whereas mAbs 4C4,3F6 and 2G7 to rdCap protein keep the neutralization epitopes of PCV2 particle, showing a potential of rGST-dCap protein as a vaccine antigen or serodiagnostic reagent.

  7. Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

    PubMed

    López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

    2007-11-01

    An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

  8. Recombinant Adenovirus Delivery of Calreticulin–ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

    PubMed Central

    Esparza-González, S. C.; Troy, A.; Troudt, J.; Loera-Arias, M. J.; Villatoro-Hernández, J.; Torres-López, E.; Ancer-Rodríguez, J.; Gutiérrez-Puente, Y.; Muñoz-Maldonado, G.; Saucedo-Cárdenas, O.; Montes-de-Oca-Luna, R.; Izzo, A.

    2015-01-01

    Bacillus Calmette–Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT–ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT–ESAT-6–CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis. PMID:22010821

  9. Immune response and functional role of antibodies raised in heifers against a Staphylococcus aureus CP5 lysate and recombinant antigens vaccine formulated with Iscom Matrix adjuvant.

    PubMed

    Camussone, C M; Pujato, N; Renna, M S; Veaute, C M; Morein, B; Marcipar, I S; Calvinho, L F

    2014-12-15

    Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or inactivated whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 lysate vaccine adjuvanted with Iscom Matrix to generate a longer lasting specific antibody response in blood and milk, with improved opsonic capacity, compared with a S. aureus CP5 whole-cell formulation. The aim of the present study was to obtain an experimental immunogen composed of lysed cells of a CP5 S. aureus strain supplemented with recombinant clumping factor A, fibronectin binding protein A and β-toxin formulated with Iscom Matrix, characterize the immune response generated when immunizing pregnant heifers and assess the functional role of antibodies raised against this immunogen in experimental models. Both a lysate vaccine and a lysate+recombinant antigens vaccine elicited antibodies that promoted neutrophil phagocytosis and inhibited internalization into mammary epithelial cells, in vitro. Incorporation of defined antigenic molecules to the lysate formulation elicited a strong specific humoral immune response against both lysate and recombinant antigens and was associated with higher expression of regulatory and pro-inflammatory cytokines. In addition, antibodies were efficient for blocking S. aureus binding to bovine fibrinogen and fibronectin, and neutralizing β-toxin effect in vitro, placing these antigens as candidates to be included in a formulation directed to prevent staphylococcal bovine mastitis. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Cysteine mutagenesis improves the production without abrogating antigenicity of a recombinant protein vaccine candidate for human chagas disease.

    PubMed

    Seid, Christopher A; Jones, Kathryn M; Pollet, Jeroen; Keegan, Brian; Hudspeth, Elissa; Hammond, Molly; Wei, Junfei; McAtee, C Patrick; Versteeg, Leroy; Gutierrez, Amanda; Liu, Zhuyun; Zhan, Bin; Respress, Jonathan L; Strych, Ulrich; Bottazzi, Maria Elena; Hotez, Peter J

    2017-03-04

    A therapeutic vaccine for human Chagas disease is under development by the Sabin Vaccine Institute Product Development Partnership. The aim of the vaccine is to significantly reduce the parasite burden of Trypanosoma cruzi in humans, either as a standalone product or in combination with conventional chemotherapy. Vaccination of mice with Tc24 formulated with monophosphoryl-lipid A (MPLA) adjuvant results in a Th1 skewed immune response with elevated IgG2a and IFNγ levels and a statistically significant decrease in parasitemia following T. cruzi challenge. Tc24 was therefore selected for scale-up and further evaluation. During scale up and downstream process development, significant protein aggregation was observed due to intermolecular disulfide bond formation. To prevent protein aggregation, cysteine codons were replaced with serine codons which resulted in the production of a non-aggregated and soluble recombinant protein, Tc24-C4. No changes to the secondary structure of the modified molecule were detected by circular dichroism. Immunization of mice with wild-type Tc24 or Tc24-C4, formulated with E6020 (TLR4 agonist analog to MPLA) emulsified in a squalene-oil-in-water emulsion, resulted in IgG2a and antigen specific IFNγ production levels from splenocytes that were not significantly different, indicating that eliminating putative intermolecular disulfide bonds had no significant impact on the immunogenicity of the molecule. In addition, vaccination with either formulated wild type Tc24 or Tc24-C4 antigen also significantly increased survival and reduced cardiac parasite burden in mice. Investigations are now underway to examine the efficacy of Tc24-C4 formulated with other adjuvants to reduce parasite burden and increase survival in pre-clinical studies.

  11. M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes.

    PubMed

    Shukla, Anshuman; Katare, O P; Singh, Bhupinder; Vyas, Suresh P

    2010-01-29

    The present study aims to improve upon our earlier findings with bilosomes as potential delivery vehicle through oral route for recombinant hepatitis B surface antigen (HBsAg). The work entails the conjugation of bilosomal system with cholera toxin B subunit (CTB) to increase transmucosal uptake via M-cell specific delivery approach. The study encompasses the development and characterization of HBsAg-loaded CTB-conjugated system for percent antigen entrapment, size, shape, and stability in SGF (USP, pH 1.2), SIF (USP, pH 7.5) and in bile salt solutions. Biological activity of CTB, subsequent to conjugation, was verified by hemagglutination test. Anti-HBsAg IgG response in serum and anti-HBsAg sIgA in various body secretions were estimated using ELISA, following oral immunization with 10 microg dose-loaded CTB-conjugated bilosomes (CTB2) and 20 microg dose-loaded CTB-conjugated bilosomes (CTB1) in BALB/c mice. The results showed that CTB1 produced anti-HBsAg IgG antibody titre response comparable to that of the intramuscular (i.m.) injection of 10 microg of alum-adsorbed HBsAg. Moreover, all the bilosomal preparations elicited measurable sIgA vis-à-vis negligible response with i.m. administered HBsAg. Thus, HBsAg-loaded CTB-conjugated bilosomes provide a promising potential for targeted oral immunization against hepatitis B. 2009 Elsevier B.V. All rights reserved.

  12. Evaluation of a New Immunochromatographic Test Using Recombinant Antigen B8/1 for Diagnosis of Cystic Echinococcosis

    PubMed Central

    Rodriguez, Mary L.; Rodriguez, Silvia; Sako, Yashuito; Nkouawa, Agathe; Kobayashi, Yukuharu; Sotomayor, Alfredo L.; Peralta, Julio E.; Valcarcel, Maria; Gonzalez, Armando E.; Garcia, Hector H.; Ito, Akira

    2015-01-01

    Diagnosis of cystic echinococcosis (CE) is based on the identification of the cyst(s) by imaging, using immunodiagnostic tests mainly as complementary tools in clinical settings. Among the antigens used for immunodiagnosis, previous studies described a good performance of the recombinant antigen B8/1 (rAgB) in an enzyme-linked immunosorbent assay (ELISA) format; however, in remote parts of areas where the disease is endemic, the implementation of an ELISA is difficult, so a more simple, rapid, and reliable method such as the immunochromatographic test (ICT) is required. In this study, using a set of 50 serum samples from patients with surgically confirmed CE, we compared the performance of an ICT and that of an ELISA using the rAgB. The overall sensitivities of ICT and ELISA were not statistically different (78% versus 72%; P = 0.36). The overall agreement between both tests was moderate (κ = 0.41; P < 0.01). Concordance between ICT and ELISA was substantial or almost perfect for patients with liver involvement (κ = 0.65; P < 0.001) and patients with more than one hydatid cyst (κ = 0.82; P < 0.001), respectively. Moreover, specificity analysis using a total of 88 serum samples from healthy individuals (n = 20) and patients (n = 68) with other parasitic infections revealed that ICT had a specificity of 89.8%. ICT and ELISA had similar performance for the detection of specific antibodies to E. granulosus, and ICT had a high specificity, opening the possibility of using ICT as a screening tool in rural settings. PMID:26447116

  13. Evaluation of a New Immunochromatographic Test Using Recombinant Antigen B8/1 for Diagnosis of Cystic Echinococcosis.

    PubMed

    Santivañez, Saul J; Rodriguez, Mary L; Rodriguez, Silvia; Sako, Yashuito; Nkouawa, Agathe; Kobayashi, Yukuharu; Sotomayor, Alfredo L; Peralta, Julio E; Valcarcel, Maria; Gonzalez, Armando E; Garcia, Hector H; Ito, Akira

    2015-12-01

    Diagnosis of cystic echinococcosis (CE) is based on the identification of the cyst(s) by imaging, using immunodiagnostic tests mainly as complementary tools in clinical settings. Among the antigens used for immunodiagnosis, previous studies described a good performance of the recombinant antigen B8/1 (rAgB) in an enzyme-linked immunosorbent assay (ELISA) format; however, in remote parts of areas where the disease is endemic, the implementation of an ELISA is difficult, so a more simple, rapid, and reliable method such as the immunochromatographic test (ICT) is required. In this study, using a set of 50 serum samples from patients with surgically confirmed CE, we compared the performance of an ICT and that of an ELISA using the rAgB. The overall sensitivities of ICT and ELISA were not statistically different (78% versus 72%; P = 0.36). The overall agreement between both tests was moderate (κ = 0.41; P < 0.01). Concordance between ICT and ELISA was substantial or almost perfect for patients with liver involvement (κ = 0.65; P < 0.001) and patients with more than one hydatid cyst (κ = 0.82; P < 0.001), respectively. Moreover, specificity analysis using a total of 88 serum samples from healthy individuals (n = 20) and patients (n = 68) with other parasitic infections revealed that ICT had a specificity of 89.8%. ICT and ELISA had similar performance for the detection of specific antibodies to E. granulosus, and ICT had a high specificity, opening the possibility of using ICT as a screening tool in rural settings.

  14. Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China.

    PubMed

    Lu, Shuai; Chen, Yingzhu; Qin, Kun; Zhou, Jianfang; Lou, Yongliang; Tan, Wenjie

    2016-06-01

    To characterize the antigenicity of nucleocapsid proteins (NP) derived from canine coronavirus (CCoV) and canine respiratory coronavirus (CRCoV) in China, the N genes of CCoV (CCoV-BJ70) and CRCoV (CRCoV-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs (rNPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCoV-BJ70 and CRCoV-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that rNPs of CCoV and CRCoV were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the rNP from CRCoV could cross-react with mice antisera against human coronavirus (HCoV-229E, NL63, OC43, HKU1), while rNP of CCoV had cross-reactivity with only anti-sera against viruses belonging to the same group (HCoV-229E and NL63). In summary, CCoV and CRCoV rNPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.

  15. Enzyme-linked immunosorbent assay employing a recombinant antigen for detection of protective antibody against swine erysipelas.

    PubMed

    Imada, Yumiko; Mori, Yasuyuki; Daizoh, Masaji; Kudoh, Kazuma; Sakano, Tetsuya

    2003-11-01

    The specificities and sensitivities of five recombinant proteins of the surface protective antigen (SpaA) of Erysipelothrix rhusiopathiae were examined by indirect enzyme-linked immunosorbent assay (ELISA) with the aim of developing a reliable serological test for the detection of protective antibody against E. rhusiopathiae. Fully mature protein and the N-terminal 416 amino acids (SpaA416) showed sufficient antigenicities, and further examination was done with SpaA416 because of its higher yield. The antibody titers of pigs experimentally immunized with commercial live vaccine and two types of inactivated vaccines clearly increased after immunization, and all pigs were completely protected against challenge with virulent strains. On the other hand, the antibody titers of nonimmunized control pigs remained very low until they were challenged, and all showed severe symptoms or subsequently died. Interference with the production of antibody against live vaccine by maternal antibody or porcine respiratory and reproductive syndrome virus infection 1 week after vaccination was also clearly detected. Because the ELISA titer correlated well with the protection results, the specificity and sensitivity of the ELISA were further evaluated with sera collected from pigs reared on 1 farm on which animals had acute septicemia, 2 farms on which the animals were infected or free from infection, and 10 farms on which the animals were vaccinated with live vaccine, among others. The ELISA titers clearly revealed the conditions of the herds. These results indicate that the SpaA416 ELISA is an effective method not only for evaluating pigs for the presence of protective antibody levels resulting from vaccination or maternal antibody but also for detecting antibody produced by natural infection. This test has important potential for the effective control of swine erysipelas.

  16. Identification of antigenic differences of recombinant and pituitary bovine growth hormone using monoclonal antibodies.

    PubMed

    Erhard, M H; Kellner, J; Schmidhuber, S; Schams, D; Lösch, U

    1994-02-01

    For characterization and determination of recombinant bovine GH (rbGH) eight monoclonal antibodies (MAb) were produced against rbGH from Monsanto. The various MAb showed different affinities to rbGH, pituitary bovine GH (pbGH), and pituitary ovine GH (poGH). With epitope analysis several MAb were shown to recognize different epitopes of rbGH. The MAb MUC-rbGH-3A11 and MUC-rbGH-1E5 were used to develop a Sandwich ELISA. By checking the specificity of the assay no cross reactivity was found with pituitary porcine GH, pituitary human GH, bovine or ovine prolactin and little cross reactivity with poGH could be found. The Sandwich ELISA detected various rbGH (Monsanto, Elanco, Cyanamid) with different N-terminal amino acids and discriminated between rbGH and pituitary bovine GH by an affinity factor of 2.0. The detection level was 2 ng rbGH per ml PBS buffer. The recovery was about 86% in bovine serum. It might therefore be possible to detect rbGH-treated cows using a Sandwich ELISA, but this would need a field study.

  17. Development of a Highly Specific Recombinant Toxocara canis Second-Stage Larva Excretory-Secretory Antigen for Immunodiagnosis of Human Toxocariasis

    PubMed Central

    Yamasaki, Hiroshi; Araki, Kunioki; Lim, Patricia Kim Chooi; Zasmy, Ngah; Mak, Joon Wah; Taib, Radzan; Aoki, Takashi

    2000-01-01

    The specificity of the recombinant Toxocara canis antigen developed for the immunodiagnosis of human toxocariasis was compared with that of the excretory-secretory antigen from T. canis second-stage larvae (TES) by enzyme-linked immunosorbent assay. A total of 153 human serum samples from patients infected with 20 different helminths, including 11 cases of toxocariasis, were examined. No false-negative reactions were observed for the toxocariasis cases. When the TES was used at concentrations of 0.5 and 0.125 μg/ml, cross-reactions were observed in 79 (55.6%) and 61 (43.0%) of 142 cases, respectively. In contrast, when the recombinant antigen was tested at a concentration of 0.5 μg/ml, cross-reactions were observed in 19 (13.4%) of 142 cases. At a concentration of 0.125 μg/ml, however, the cross-reaction rate decreased sharply to only 2.1%, corresponding to 3 of 142 cases. The cross-reactions occurred with one case each of gnathostomiasis, paragonimiasis with Paragonimus miyazakii, and spirometriasis, in which high antibody titers were detected. In addition, the recombinant antigen showed negative reactions with serum samples from patients infected with Ascaris and hookworms, which are the most common parasites in the world. These findings are also supported by experiments with animals infected with Ascaris and hookworm. From these results, the recombinant antigen is highly specific for toxocariasis and may provide more reliable diagnostic results than other methods. PMID:10747116

  18. Live Recombinant Salmonella Typhi Vaccines Constructed to Investigate the Role of rpoS in Eliciting Immunity to a Heterologous Antigen

    PubMed Central

    Brenneman, Karen E.; Wanda, Soo-Young; Wang, Shifeng; Senechal, Patti; Sun, Wei; Roland, Kenneth L.; Curtiss, Roy

    2010-01-01

    We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (χ9639 and χ9640) were derived from the rpoS mutant strain Ty2 and one (χ9633) from the RpoS+ strain ISP1820. In χ9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS+ vaccines induced a balanced Th1/Th2 immune response while the RpoS− strain χ9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS+ strain χ9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, χ9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human

  19. Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA.

    PubMed

    Oem, Jae-Ku; Park, Jong-Hyeon; Lee, Kwang-Nyeong; Kim, Yong-Joo; Kye, Soo-Jeong; Park, Jee-Yong; Song, Hee-Jong

    2007-05-16

    Non-infectious recombinant pentamer-like structures of the foot-and-mouth disease virus (FMDV) were expressed by baculovirus, and the antigenicity and immunogenicity of the proteins were analyzed in a blocking ELISA for the detection of FMDV antibodies. The recombinant pentamer-like structures were produced in insect (Sf9) cells that were inoculated with recombinant baculoviruses that expressed, simultaneously, the genes for the P1 and 3C proteins of FMDV from individual promoters. The FMDV pentamer-like structures were processed by viral 3C protease, as shown in Western blots, and were antigenic, as revealed by their reactivities in an indirect ELISA. Analysis by CsCl gradient centrifugation showed that the pentamer-like structures were similar to authentic pentameric subunits from FMDV in terms of sedimentation velocity. Furthermore, the pentamer-like structures induced high levels of FMDV-specific antibodies in mice following immunization. Observations made under the electron microscope revealed that the pentamer-like structures expressed by insect cells self-assembled to form pentameric subunits of 7-8 nm in diameter, which resemble the authentic FMDV (23+/-2 nm in diameter). The results indicate that these pentamer-like structures are as antigenic and immunogenic as authentic FMDV, although the former are smaller in size. Based on these results, a blocking ELISA was developed using the recombinant pentamer-like structure. The ELISA showed specificity of 99.5% and sensitivity of 98.5% when tested with FMDV antibody-negative and -positive sera, respectively. This blocking ELISA is highly specific and offers many advantages over the current ELISAs that use inactivated FMDV antigen. This is the first report of the production and diagnostic application of recombinant pentameric subunits of FMDV.

  20. Recombinant chimeric vaccine composed of PRRSV antigens and truncated Pseudomonas exotoxin A (PE-K13).

    PubMed

    Yang, Hsin-Ping; Wang, Tsan-Chih; Wang, Shiou-Jen; Chen, Shih-Ping; Wu, Eva; Lai, Shao-Qun; Chang, Hsueh-Wei; Liao, Chao-Wei

    2013-10-01

    A Pseudomonas exotoxin (PE-KDEL)-based chimeric subunit vaccine system was recently developed using a reverse vaccinology technique. In this study, the plasmids containing PE-PRRS chimeric subunits were constructed that composed of porcine reproductive and respiratory syndrome virus (PRRSV) antigen moieties, a ligand moiety and a Pseudomonas exotoxin A deleted domain III (PE (ΔIII)), and a carboxyl terminal moiety that includes a polypeptide with amino acid sequence KDEL (K3). The PE-PRRS combination vaccine can effectively induce not only PRRSV-specific INF-γ cellular immunity but also a slow-reacting and complement-requiring type serum neutralizing antibody in pigs. In a specific pathogen free (SPF) pig challenge model, body temperature (colonic temperature), occurrence of PRRSV viremia, nasal excretions, gross and histopathological appearances of pneumonia, and serum antibody activity (IFA and SN) titers significantly differed between the immunized group and the control group. The survey showed that a 0.3mg/dose PE-PRRS vaccine formula conferred protection against PRRSV. A field trial of PE-PRRS vaccine was performed to study the immune response of pregnant sows after vaccination in a PRRSV persist farm. The RT-PCR analysis of viremia and serological titers showed that the PE-PRRS vaccine not only increased sow reproductive performance and evoked its immune response to PRRS viremia, it also activated maternal immune protections to prevent piglets from inflicting viremia. In conclusion, we developed a novel and effective PRRS cytotoxic T-cells (CTLs)-based vaccine containing Pseudomonas exotoxin (PE-KDEL) carrier in combination with PRRSV conserved epitopes against PRRS virus.

  1. Comparative evaluation of recombinant LigB protein and heat-killed antigen-based latex agglutination test with microscopic agglutination test for diagnosis of bovine leptospirosis.

    PubMed

    Nagalingam, Mohandoss; Thirumalesh, Sushma Rahim Assadi; Kalleshamurthy, Triveni; Niharika, Nakkala; Balamurugan, Vinayagamurthy; Shome, Rajeswari; Sengupta, Pinaki Prasad; Shome, Bibek Ranjan; Prabhudas, Krishnamsetty; Rahman, Habibur

    2015-10-01

    This study aimed to develop latex agglutination test (LAT) using recombinant leptospiral immunoglobulin-like protein (LigB) (rLigB) antigen and compare its diagnostic efficacy with LAT using conventional heat-killed leptospiral antigen and microscopic agglutination test (MAT) in diagnosing bovine leptospirosis. The PCR-amplified 1053-bp ligB gene sequences from Leptospira borgpetersenii Hardjo serovar were cloned in pET 32 (a) vector at EcoRI and NotI sites and expressed in BL21 E. coli cells as fusion protein with thioredoxin (-57 kDa) and characterized by SDS-PAGE and immunoblot. Out of 390 serum samples [cattle (n = 214), buffaloes (n = 176)] subjected to MAT, 115 samples showed reciprocal titre≥100 up to 1600 against one or more serovars. For recombinant LigB protein/antigen-based LAT, agglutination was observed in the positive sample, while no agglutination was observed in the negative sample. Similarly, heat-killed leptospiral antigen was prepared from and used in LAT for comparison with MAT. A two-sided contingency table was used for analysis of LAT using both the antigens separately against MAT for 390 serum samples. The sensitivity, specificity and positive and negative predictive values of recombinant LigB LAT were found to be 75.65, 91.27, 78.38 and 89.96 %, respectively, and that of heat-killed antigen-based LAT were 72.17, 89.82, 74.77 and 88.53 %, respectively, in comparison with MAT. This developed test will be an alternative/complementary to the existing battery of diagnostic assays/tests for specific detection of pathogenic Leptospira infection in bovine population.

  2. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

    PubMed Central

    Guo, D; Bowden, M G; Pershad, R; Kaplan, H B

    1996-01-01

    A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID:8626291

  3. Immunization of Bos taurus steers with Babesia bovis recombinant antigens MSA-1, MSA-2c and 12D3.

    PubMed

    Antonio Alvarez, J; Lopez, U; Rojas, C; Borgonio, V M; Sanchez, V; Castañeda, R; Vargas, P; Figueroa, J V

    2010-04-01

    The purpose of this research was to evaluate the recombinant proteins MSA-1, MSA-2c and 12D3 as a combined immunogen for cattle. Fifteen steers were randomly assigned into three groups of five animals each (I, II and III). On day 0, cattle in group I were injected with 50 microg each of rMSA-1, rMSA-2c and r12D3 with the adjuvant Montanide 75; cattle in Group II received adjuvant-PBS, and Group III were untreated controls. On day 14, cattle in Group I received a second injection of the three recombinant proteins in adjuvant and cattle in Group II again received adjuvant alone. On day 28, all groups of cattle were challenged with a field strain of Babesia bovis. After challenge, the experimental cattle were clinically and serologically monitored. Three of the five steers immunized with the combined recombinant B. bovis proteins seroconverted on day 14 post-immunization (P.I.) and the maximum titre was 1 : 1600. All five immunized steers presented strong seropositivity to B. bovis antigens at day 21 P.I. The prepatent periods of vaccinated cattle were delayed until day 10 post-challenge exposure versus 8 and 7 days in Groups II and III, respectively. Cattle in all groups had fever above 41 degrees C; the reduction in packed cell volume was not significantly different (P > 0.05) in vaccinated group I compared with Groups II and III (29% versus 26% and 31%, respectively). Treatment was required for one steer in the control group. During the period of the study, the weight of cattle in Groups I and II increased an average of 9 and 7 kg, whereas the weight of the control cattle was reduced on average 4 kg. Immunization with rMSA-1-rMSA-2c-r12D3 proteins was not sufficient to prevent clinical symptoms against challenge, but the immunologic response was sufficient to protect steers against a mild virulent strain of B. bovis.

  4. Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations.

    PubMed Central

    Green, K Y; Lew, J F; Jiang, X; Kapikian, A Z; Estes, M K

    1993-01-01

    Since the discovery of the Norwalk virus (NV) by immune electron microscopy (IEM) in 1972, serologic studies with this virus have relied on particle-positive fecal material from infected volunteers as the source of antigen because it has not been possible to propagate this virus in cell culture. However, the recent cloning of the NV (strain 8FIIa) genome and expression of the capsid protein in a baculovirus system to form "virus-like particles" has provided a consistent source of antigen (designated rNV). The purpose of the present study was to compare the antigenicities of these rNV particles with those of native NV antigen derived from human fecal material by using well-characterized sera obtained from earlier studies. In IEM studies, the rNV antigen reacted with NV-specific antibodies in a manner similar to that observed previously when particle-positive fecal material was used as antigen. In addition, a direct enzyme-linked immunosorbent assay, in which the rNV antigen was used as antigen, proved efficient and specific for the detection of serologic responses to NV compared with the previously established techniques of IEM and blocking antibody immunoassays in which particle-positive fecal material was used as the antigen. The availability of an unlimited source of antigen will enable serologic studies that will greatly increase our understanding of the epidemiology of NV and its role in human enteric illness. Images PMID:8396590

  5. Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

    PubMed

    Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

    2016-12-01

    Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

  6. Adaptation of the Endogenous Salmonella enterica Serovar Typhi clyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity of Anthrax Protective Antigen Domain 4 Expressed by the Attenuated Live-Vector Vaccine Strain CVD 908-htrA

    PubMed Central

    Galen, James E.; Zhao, Licheng; Chinchilla, Magaly; Wang, Jin Yuan; Pasetti, Marcela F.; Green, Jeffrey; Levine, Myron M.

    2004-01-01

    Bacterial live-vector vaccines aim to deliver foreign antigens to the immune system and induce protective immune responses, and surface-expressed or secreted antigens are generally more immunogenic than cytoplasmic constructs. We hypothesize that an optimum expression system will use an endogenous export system to avoid the need for large amounts of heterologous DNA encoding additional proteins. Here we describe the cryptic chromosomally encoded 34-kDa cytolysin A hemolysin of Salmonella enterica serovar Typhi (ClyA) as a novel export system for the expression of heterologous antigens in the supernatant of attenuated Salmonella serovar Typhi live-vector vaccine strains. We constructed a genetic fusion of ClyA to the reporter green fluorescent protein and showed that in Salmonella serovar Typhi CVD 908-htrA, the fusion protein retains biological activity in both domains and is exported into the supernatant of an exponentially growing live vector in the absence of detectable bacterial lysis. The utility of ClyA for enhancing the immunogenicity of an otherwise problematic antigen was demonstrated by engineering ClyA fused to the domain 4 (D4) moiety of Bacillus anthracis protective antigen (PA). A total of 11 of 15 mice immunized intranasally with Salmonella serovar Typhi exporting the protein fusion manifested fourfold or greater rises in serum anti-PA immunoglobulin G, compared with only 1 of 16 mice immunized with the live vector expressing cytoplasmic D4 (P = 0.0002). In addition, the induction of PA-specific gamma interferon and interleukin 5 responses was observed in splenocytes. This technology offers exceptional versatility for enhancing the immunogenicity of bacterial live-vector vaccines. PMID:15557633

  7. Gamma delta T cells recognize a microbial encoded B Cell antigen to initiate a rapid antigen-specific Interleukin-17 response

    USDA-ARS?s Scientific Manuscript database

    Gamma delta T cells contribute uniquely to host immune defense, but the way in which they do so remains an enigma. Here we show that an algae protein, phycoerythrin (PE) is recognized by gamma delta T cells from mice, bovine and humans and binds directly to specific gamma delta T cell antigen recept...

  8. Optimal attenuation of a PR8-derived mouse pathogenic H5N1 recombinant virus for testing antigenicity and protective efficacy in mice.

    PubMed

    Kim, Il-Hwan; Kwon, Hyuk-Joon; Park, Jae-Keun; Song, Chang-Seon; Kim, Jae-Hong

    2015-11-17

    The PR8-based reverse genetics vector system is widely used to generate commercial vaccine strains, but the pathogenicity of PR8-derived recombinant viruses in mice hinders further immunological studies. In the present study, we generated PR8-derived H5N1 recombinant viruses, in which haemagglutinin (HA) and neuraminidase (NA) originated from a mouse-pathogenic H5N1 low pathogenic avian influenza virus (LPAIV), and the non-structural proteins (NS) and polymerase basic protein 2 (PB2) originated from different H9N2 LPAIVs. In contrast to the control H5N1 recombinant virus, harboring six internal genes from PR8, the NS and PB2 recombinant viruses did not cause body weight loss in mice. However, the NS recombinant virus replicated in the lungs of mice. It was more immunogenic than the PB2 recombinant virus to protect efficiently against a lethal challenge of a H5N1 highly pathogenic AIV with 89 and 88% amino acid identity in HA and NA, respectively. Therefore, the NS gene may be useful for generating nonpathogenic and immunogenic PR8-derived recombinant viruses for studies of antigenicity and protective efficacy in mice.

  9. Advax-Adjuvanted Recombinant Protective Antigen Provides Protection against Inhalational Anthrax That Is Further Enhanced by Addition of Murabutide Adjuvant

    PubMed Central

    Feinen, Brandon; Petrovsky, Nikolai; Verma, Anita

    2014-01-01

    Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy. PMID:24554695

  10. Structural characterization of the N-glycans of a recombinant hepatitis B surface antigen derived from yeast

    SciTech Connect

    Ip, C.C.Y.; Miller, W.J.; Kubek, D.J. ); Strang, A.M.; van Halbeek, H. ); Piesecki, S.J.; Alhadeff, J.A. )

    1992-01-14

    The N-glycans of purified recombinant middle surface protein (preS2+S) from hepatitis B virus, a candidate vaccine antigen expressed in a mnn9 mutant strain of Saccharomyces cerevisiae, have been characterized structurally. The glycans were released by N-glycanase treatment, isolated by size-exclusion chromatography on Sephadex G-50 and Bio-Gel P-4 columns, and analyzed by 500-MHz {sup 1}H NMR spectroscopy and fast atom bombardment mass spectrometry. The mixture of oligosaccharides was fractionated by HPLC, the major subfractions were isolated, and their carbohydrate compositions were determined by high-pH anion-exchange chromatography with pulsed amperometric detection. The combined results suggest that high-mannose oligosaccharides account for all the N-glycans released from preS2+S: structures include Man{sub 7}GlcNAc{sub 2}, Man{sub 8}GlcNAc{sub 2} isomers in the ratios of 3:6:1. Approximately 80% of the oligosaccharides contain the C2, C6-branched trimannosyl structural element typical of yeast high-mannose oligosaccharides but not usually found in high-mannose oligosaccharides in animal glycoproteins.

  11. Fasciola hepatica - the pilot study of in vitro assessing immune response against native and recombinant antigens of the fluke.

    PubMed

    Bąska, Piotr; Zawistowska-Deniziak, Anna; Zdziarska, Anna M; Wasyl, Katarzyna; Wiśniewski, Marcin; Cywińska, Anna; Klockiewicz, Maciej; Januszkiewicz, Kamil; Wędrychowicz, Halina

    2013-12-01

    Fasciola hepatica is a liver fluke that infects 2.4 million of people and causes great economical loss in animal production. To date a 100% effective vaccine has not been developed and the disease is controlled by drug therapy. Great efforts are put into development of effective vaccine against parasite what is difficult since Fasciola spp. (like other helmints) during evolutionary process has developed sophisticated and efficient methods to evade immune response. During preliminary experiments it is convenient to use cell lines which are relatively cheap and allow for reproducible comparison of results between laboratories. We stimulated BOMA (bovine monocyte/macrophage cell line) and BOMAC (bovine macrophage cell line) with native or recombinant antigens of Fasciola hepatica and assessed IFN-γ, IL-4 and TNF-α level upon stimulation. We observed diminished secretion of proinflammatory TNF-α in LPS activated BOMA cells stimulated with Excretory/Secretory products of adult fluke (Fh-ES). We also observed greater changes in gene expression in LPS activated BOMA cells than in non activated BOMA cells upon stimulation using Fh-ES. The results show possibility of using cell lines for in vitro research of bovine immune response against liver fluke, although this model still requires validation and further characterization.

  12. Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis.

    PubMed

    Rodríguez, Islay; Alvarez, Elvio L; Fernández, Carmen; Miranda, Alina

    2002-04-01

    A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.

  13. Advax-adjuvanted recombinant protective antigen provides protection against inhalational anthrax that is further enhanced by addition of murabutide adjuvant.

    PubMed

    Feinen, Brandon; Petrovsky, Nikolai; Verma, Anita; Merkel, Tod J

    2014-04-01

    Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy.

  14. [Development of a new hydrophobic interaction chromatography absorbent and its application to the purification of recombinant hepatitis B surface antigen].

    PubMed

    Wang, Yang-Mu; Bi, Jing-Xiu; Zhao, Lan; Zhou, Wei-Bin; Li, Yan; Huang, Yong-Dong; Zhang, Yan; Lin, Hai; Su, Zhi-Guo

    2006-03-01

    A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.

  15. Immune memory response induced in vitro by recombinant hepatitis B surface antigen challenge 13-18 years after primary vaccination.

    PubMed

    do Livramento, Andréa; Schultz, Júnia; Batista, Keila Zaniboni Siqueira; Treitinger, Arício; de Cordova, Caio Maurício Mendes; Spada, Celso

    2014-10-01

    The question of whether booster doses are required to maintain long-term protection against hepatitis B virus (HBV) after primary vaccination remains to be determined. Thus, the aim of this study was to evaluate the immune memory responses to hepatitis B surface antigen (HBsAg) challenge in vaccinated individuals through an in vitro-specific stimulation assay. Peripheral blood mononuclear cells (4 × 10(6)  cells/ml) were stimulated with 50 ng/ml of recombinant HBsAg. In vitro anamnestic antibody responses, as shown by detection of high avidity antibody in culture supernatants, were found 13-18 years after primary vaccination and were not correlated with serum antibodies (r = -0.177; P = 0.377). In addition, the findings from this study indicate that immune memory against hepatitis B was well preserved in 40.0% and 60.0% of vaccinees with anti-HBs levels less than 10 IU/L or lacking serum antibodies altogether, respectively. In conclusion, the data suggest the presence of immunological memory in vaccinated individuals, including those who showed anti-HBs <10 IU/L or undetectable antibody. © 2014 Wiley Periodicals, Inc.

  16. Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar canicola in ELISA for serodiagnosis of bovine leptospirosis.

    PubMed

    Mariya, R; Chaudhary, Pallab; Kumar, A A; Thangapandian, E; Amutha, R; Srivastava, S K

    2006-11-01

    The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis.

  17. 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

  18. Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

    PubMed

    Virginio, Veridiana Gomes; Gonchoroski, Taylor; Paes, Jéssica Andrade; Schuck, Desirée Cigaran; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2014-10-07

    Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP.

  19. Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis.

    PubMed

    Cheng, Weiqiang; Curti, Elena; Rezende, Wanderson C; Kwityn, Clifford; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan; Joshi, Sangeeta B; Volkin, David B; Hotez, Peter J; Middaugh, C Russell; Bottazzi, Maria Elena

    2013-11-01

    A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0-8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine.

  20. Biophysical and formulation studies of the Schistosoma mansoni TSP-2 extracellular domain recombinant protein, a lead vaccine candidate antigen for intestinal schistosomiasis

    PubMed Central

    Cheng, Weiqiang; Curti, Elena; Rezende, Wanderson C; Kwityn, Clifford; Zhan, Bin; Gillespie, Portia; Plieskatt, Jordan; Joshi, Sangeeta B.; Volkin, David B.; Hotez, Peter J; Middaugh, C Russell; Bottazzi, Maria Elena

    2013-01-01

    A candidate vaccine to prevent human schistosomiasis is under development. The vaccine is comprised of a recombinant 9 kDa antigen protein corresponding to the large extracellular domain of a tetraspanin surface antigen protein of Schistosoma mansoni, Sm-TSP-2. Here, we describe the biophysical profile of the purified, recombinant Sm-TSP-2 produced in the yeast PichiaPink™, which in preclinical studies in mice was shown to be an effective vaccine against intestinal schistosomiasis. Biophysical techniques including circular dichroism, intrinsic and extrinsic fluorescence and light scattering were employed to generate an empirical phase diagram, a color based map of the physical stability of the vaccine antigen over a wide range of temperatures and pH. From these studies a pH range of 6.0–8.0 was determined to be optimal for maintaining the stability and conformation of the protein at temperatures up to 25 °C. Sorbitol, sucrose and trehalose were selected as excipients that prevented physical degradation during storage. The studies described here provide guidance for maximizing the stability of soluble recombinant Sm-TSP-2 in preparation of its further development as a vaccine. PMID:23880663

  1. TMV recombinants encoding fused foreign transmembrane domains to the CP subunit caused local necrotic response on susceptible tobacco.

    PubMed

    Li, Qiaoli; Li, Mangmang; Jiang, Lubin; Zhang, Qingqi; Song, Rentao; Xu, Zhengkai

    2006-05-10

    With regard to the effects of various foreign peptides fused to the coat protein subunits on the infectivity of corresponding TMV recombinants, some of TMV recombinants were found to induce necrotic local lesions on the inoculated leaves of susceptible tobacco. This paper reported that there existed a group of TMV recombinants in which the fused foreign peptides contained a transmembrane domain according to the predictions by three programs of SOSUI, TMpred and DAS. Further studies showed for the first time that a foreign transmembrane domain in a fused peptide of the corresponding TMV recombinant would result in the local lesions on the susceptible tobacco leaves. In addition, it was concluded that none of the TMV recombinants that systematically infected susceptible tobacco contained a transmembrane domain in the coat protein subunits.

  2. The use of halloysite clay and carboxyl-functionalised multi-walled carbon nanotubes for recombinant LipL32 antigen delivery enhanced the IgG response.

    PubMed

    Hartwig, Daiane D; Bacelo, Kátia L; Oliveira, Thaís L; Schuch, Rodrigo; Seixas, Fabiana K; Collares, Tiago; Rodrigues, Oscar; Hartleben, Cláudia P; Dellagostin, Odir A

    2015-02-01

    We studied the feasibility of using halloysite clay nanotubes (HNTs) and carboxyl-functionalised multi-walled carbon nanotubes (COOH-MWCNTs) as antigen carriers to improve immune responses against a recombinant LipL32 protein (rLipL32). Immunisation using the HNTs or COOH-MWCNTs significantly increased the rLipL32-specific IgG antibody titres (p < 0.05) of Golden Syrian hamsters. None of the vaccines tested conferred protection against a challenge using a virulent Leptospira interrogans strain. These results demonstrated that nanotubes can be used as antigen carriers for delivery in hosts and the induction of a humoral immune response against purified leptospiral antigens used in subunit vaccine preparations.

  3. Expression of recombinant West Nile virus prM protein fused to an affinity tag for use as a diagnostic antigen.

    PubMed

    Setoh, Y X; Hobson-Peters, J; Prow, N A; Young, P R; Hall, R A

    2011-07-01

    Previous studies have concluded that the Flavivirus prM protein is a suitable viral antigen to distinguish serologically between infections with closely related Flaviviruses (Cardosa et al., 2002). To express the recombinant West Nile virus (WNV) prM antigen fused to a suitable affinity tag for purification, a series of prM-His-tag and prM-V5-tag fusion proteins were generated. Analysis of the prM-His-tag fusion proteins revealed that either prM epitopes were disrupted or the His-tag was not presented properly depending on the location of the His tag and the presence of the prM transmembrane domains in these constructs. This identified domains critical for proper folding of prM, and arrangements that allowed the correct presentation of the His-tag. However, the inclusion of the V5 epitope tag fused to the C terminus of prM allowed formation of the authentic antigenic structure of prM and the proper presentation of the V5 epitope. Capture of tagged recombinant WNV(NY99) prM antigen to the solid phase with anti-V5 antibody in ELISA enabled the detection of prM-specific antibodies in WNV(NY99)-immune horse serum, confirming its potential as a useful diagnostic reagent.

  4. Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

    PubMed

    Peron, G; Fernandes, F F; Landgraf, T N; Martinez, R; Panunto-Castelo, A

    2017-04-03

    Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

  5. Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity.

    PubMed

    Santana, Vinicius C; Diniz, Mariana O; Cariri, Francisco A M O; Ventura, Armando M; Cunha-Neto, Edécio; Almeida, Rafael R; Campos, Marco A; Lima, Graciela K; Ferreira, Luís C S

    2013-01-01

    Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

  6. BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems.

    PubMed

    Patnaik, Santosh Kumar; Helmberg, Wolfgang; Blumenfeld, Olga O

    2012-01-01

    Analogous to human leukocyte antigens, blood group antigens are surface markers on the erythrocyte cell membrane whose structures differ among individuals and which can be serologically identified. The Blood Group Antigen Gene Mutation Database (BGMUT) is an online repository of allelic variations in genes that determine the antigens of various human blood group systems. The database is manually curated with allelic information collated from scientific literature and from direct submissions from research laboratories. Currently, the database documents sequence variations of a total of 1251 alleles of all 40 gene loci that together are known to affect antigens of 30 human blood group systems. When available, information on the geographic or ethnic prevalence of an allele is also provided. The BGMUT website also has general information on the human blood group systems and the genes responsible for them. BGMUT is a part of the dbRBC resource of the National Center for Biotechnology Information, USA, and is available online at http://www.ncbi.nlm.nih.gov/projects/gv/rbc/xslcgi.fcgi?cmd=bgmut. The database should be of use to members of the transfusion medicine community, those interested in studies of genetic variation and related topics such as human migrations, and students as well as members of the general public.

  7. Characterization of a cDNA clone encoding the carboxy-terminal domain of a 90-kilodalton surface antigen of Trypanosoma cruzi metacyclic trypomastigotes.

    PubMed Central

    Franco, F R; Paranhos-Bacallà, G S; Yamauchi, L M; Yoshida, N; da Silveira, J F

    1993-01-01

    We have cloned and sequenced a cDNA for a metacyclic trypomastigote-specific glycoprotein with a molecular mass of 90 kDa, termed MTS-gp90. By immunoblotting, antibodies to the MTS-gp90 recombinant protein reacted exclusively with a 90-kDa antigen of metacyclic trypomastigotes. The insert of the MTS-gp90 cDNA clone strongly hybridized with a single 3.0-kb mRNA of metacyclic forms, whereas the hybridization signal with epimastigote mRNA was weak and those with RNAs from other developmental stages were negative, indicating that transcription of the MTS-gp90 gene is developmentally regulated. A series of experiments showed that the MTS-gp90 gene is present in multiple copies in the Trypanosoma cruzi genome, arranged in a nontandem manner, and that there are at least 40 copies of the gene per haploid genome. Sequence analysis of recombinant MTS-gp90 revealed 40 to 60% identity at the amino acid level with members of a family of mammalian stage-specific, 85-kDa surface antigens of T. cruzi. However, there are considerable differences in the amino acid compositions outside the homology region. Images PMID:8406808

  8. Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery.

    PubMed

    La, Tom; Phillips, Nyree D; Hampson, David J

    2009-01-01

    Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera (n=1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.

  9. Molecular analysis of region 1 of the Escherichia coli K5 antigen gene cluster: a region encoding proteins involved in cell surface expression of capsular polysaccharide.

    PubMed Central

    Pazzani, C; Rosenow, C; Boulnois, G J; Bronner, D; Jann, K; Roberts, I S

    1993-01-01

    The nucleotide sequence of region 1 of the K5 antigen gene cluster of Escherichia coli was determined. This region is postulated to encode functions which, at least in part, participate in translocation of polysaccharide across the periplasmic space and onto the cell surface. Analysis of the nucleotide sequence revealed five genes that encode proteins with predicted molecular masses of 75.7, 60.5, 44, 43, and 27 kDa. The 27-kDa protein was 70.7% homologous to the CMP-2-keto-3-deoxyoctulosonic acid synthetase enzyme encoded by the E. coli kdsB gene, indicating the presence of a structural gene for a similar enzyme within the region 1 operon. The 43-kDa protein was homologous to both the Ctrb and BexC proteins encoded by the Neisseria meningitidis and Haemophilus influenzae capsule gene clusters, respectively, indicating common stages in the expression of capsules in these gram-negative bacteria. However, no homology was detected between the 75.7, 60.5-, and 44-kDa proteins and any of the proteins so far described for the H. influenzae and N. meningitidis capsule gene clusters. Images PMID:8397187

  10. Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family.

    PubMed

    Bierhuizen, M F; Mattei, M G; Fukuda, M

    1993-03-01

    The blood group i/I antigens were the first identified alloantigens that display a dramatic change during human development. The i and I antigens are determined by linear and branched poly-N-acetyllactosaminoglycans, respectively. In human erythrocytes during embryonic development, the fetal (i) antigen is replaced by the adult (I) antigen as a result of the appearance of a beta-1,6-N-acetylglucosaminyltransferase, the I-branching enzyme. Here, we report the cDNA cloning and expression of this branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans, thus introducing the I antigen in transfected cells. The cDNA sequence predicts a protein with type II membrane topology as has been found for all other mammalian glycosyltransferases cloned to date. The Chinese hamster ovary cells that stably express the isolated cDNA acquire I-branched structures as evidenced by the structural analysis of glycopeptides from these cells. Comparison of the amino acid sequence with those of other glycosyltransferases revealed that this I-branching enzyme and another beta-1,6-N-acetylglucosaminyltransferase that forms a branch in O-glycans are strongly homologous in the center of their putative catalytic domains. Moreover, the genes encoding these two beta-1,6-N-acetylglucosaminyltransferases were found to be located at the same locus on chromosome 9, band q21. These results indicate that the I-branching enzyme represents a member of a beta-1,6-N-acetylglucosaminyltransferase gene family of which expression is controlled by developmental programs.

  11. A recombinant multi-antigen vaccine formulation containing Babesia bovis merozoite surface antigens MSA-2a1, MSA-2b and MSA-2c elicits invasion-inhibitory antibodies and IFN-γ producing cells.

    PubMed

    Gimenez, Alba Marina; Françoso, Katia S; Ersching, Jonatan; Icimoto, Marcelo Y; Oliveira, Vitor; Rodriguez, Anabel E; Schnittger, Leonhard; Florin-Christensen, Monica; Rodrigues, Mauricio M; Soares, Irene S

    2016-11-14

    Babesia bovis is a tick-transmitted protozoan hemoparasite and the causative agent of bovine babesiosis, a potential risk to more than 500 million cattle worldwide. The vaccines currently available are based on attenuated parasites, which are difficult to produce, and are only recommended for use in bovines under one year of age. When used in older animals, these vaccines may cause life-threatening clinical symptoms and eventually death. The development of a multi-subunit recombinant vaccine against B. bovis would be attractive from an economic standpoint and, most importantly, could be recommended for animals of any age. In the present study, recombinant ectodomains of MSA-2a1, MSA-2b and MSA-2c antigens were expressed in Pichia pastoris yeast as secreted soluble peptides. The antigens were purified to homogeneity, and biochemically and immunologically characterized. A vaccine formulation was obtained by emulsifying a mixture of the three peptides with the adjuvant Montanide ISA 720, which elicited high IgG antibody titers against each of the above antigens. IgG antibodies generated against each MSA-antigen recognized merozoites and significantly inhibited the invasion of bovine erythrocytes. Cellular immune responses were also detected, which were characterized by splenic and lymph node CD4(+) T cells producing IFN-γ and TNF-α upon stimulation with the antigens MSA-2a1 or MSA-2c. These data strongly suggest the high protective potential of the presented formulation, and we propose that it could be tested in vaccination trials of bovines challenged with B. bovis.

  12. Tumor-specific immunity and antiangiogenesis generated by a DNA vaccine encoding calreticulin linked to a tumor antigen.

    PubMed

    Cheng, W F; Hung, C F; Chai, C Y; Hsu, K F; He, L; Ling, M; Wu, T C

    2001-09-01

    Antigen-specific cancer immunotherapy and antiangiogenesis have emerged as two attractive strategies for cancer treatment. An innovative approach that combines both mechanisms will likely generate the most potent antitumor effect. We tested this approach using calreticulin (CRT), which has demonstrated the ability to enhance MHC class I presentation and exhibit an antiangiogenic effect. We explored the linkage of CRT to a model tumor antigen, human papilloma virus type-16 (HPV-16) E7, for the development of a DNA vaccine. We found that C57BL/6 mice vaccinated intradermally with CRT/E7 DNA exhibited a dramatic increase in E7-specific CD8(+) T cell precursors and an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with wild-type E7 DNA or CRT DNA. Vaccination of CD4/CD8 double-depleted C57BL/6 mice and immunocompromised (BALB/c nu/nu) mice with CRT/E7 DNA or CRT DNA generated significant reduction of lung tumor nodules compared with wild-type E7 DNA, suggesting that antiangiogenesis may have contributed to the antitumor effect. Examination of microvessel density in lung tumor nodules and an in vivo angiogenesis assay further confirmed the antiangiogenic effect generated by CRT/E7 and CRT. Thus, cancer therapy using CRT linked to a tumor antigen holds promise for treating tumors by combining antigen-specific immunotherapy and antiangiogenesis.

  13. Development of a single-antigen magnetic bead assay (SAMBA) for the sensitive detection of HPA-1a alloantibodies using tag-engineered recombinant soluble β3 integrin.

    PubMed

    Skaik, Younis; Battermann, Anja; Hiller, Oliver; Meyer, Oliver; Figueiredo, Constanca; Salama, Abdulgabar; Blasczyk, Rainer

    2013-05-31

    Timely and accurate testing for human platelet antigen 1a (HPA-1a) alloantibodies is vital for clinical diagnosis of neonatal alloimmune thrombocytopenia (NAIT). Current antigen-specific assays used for the detection of HPA-1 alloantibodies are technically very complex and cumbersome for most diagnostic laboratories. Hence, we designed and applied recombinant soluble (rs) β3 integrins displaying HPA-1a or HPA-1b epitopes for the development of a single-antigen magnetic bead assay (SAMBA). Soluble HPA-1a and HPA-1b were produced recombinantly in human embryonic kidney 293 (HEK293) cells and differentially tagged. The recombinant soluble proteins were then immobilized onto paramagnetic beads and used for analysis of HPA-1 alloantibodies by enzyme-linked immunosorbent assay (ELISA). HPA-1a serum samples (n=7) from NAIT patients, inert sera and sera containing non-HPA-1a antibodies were used to evaluate the sensitivity and specificity of the SAMBA. Fusion of V5-His or GS-SBP-His tags to the rsβ3 integrins resulted in high-yield expression. SAMBA was able to detect all HPA-1a and -1b alloantibodies recognized by monoclonal antibody-specific immobilization of platelet antigens assay (MAIPA). No cross-reactions between the sera were observed. Two out of seven of the HPA-1a alloantibody-containing sera demonstrated weak to moderate reactivity in MAIPA but strong signals in SAMBA. SAMBA provides a very reliable method for the detection of HPA-1 antibodies with high specificity and sensitivity. This simple and rapid assay can be adapted for use in any routine laboratory and can be potentially adapted for use on automated systems.

  14. Constitutive expression of Epstein-Barr virus-encoded RNAs and nuclear antigen during latency and after induction of Epstein-Barr virus replication.

    PubMed Central

    Weigel, R; Fischer, D K; Heston, L; Miller, G

    1985-01-01

    We examined the fate of two major products of latency as Epstein-Barr virus was induced to replicate. We studied a superinducible clone of HR-1 cells in the presence and absence of induction by phorbol ester, and we analyzed the X50-7 line with and without superinfection by an HR-1 viral variant which disrupts latency. The two methods of induction yielded qualitatively similar results. After induction, there was abundant synthesis of viral transcripts, amplification of viral DNA, and the appearance of many new viral polypeptides. Nonetheless, there were no changes in the cytoplasmic abundance of Epstein-Barr virus-encoded RNAs and no alteration in the level of Epstein-Barr virus nuclear antigen mRNA or polypeptide. Thus, under conditions in which numerous other Epstein-Barr virus gene products are activated, the two major latent gene products are expressed at a constitutive level. Expression of Epstein-Barr virus-encoded RNAs and nuclear antigen must therefore be regulated in a manner completely different from expression of replicative functions. Images PMID:2981344

  15. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli: Its application to a 12 kDa antigen from