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Sample records for fra recombinant antigens

  1. 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

  2. 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.

  3. Expression of co-stimulatory molecules CD80 and CD86 is altered in CD14 + HLA-DR + monocytes from patients with Chagas disease following induction by Trypanosoma cruzi recombinant antigens.

    PubMed

    Soares, Ana Karine de Araújo; Neves, Patrícia Areias Feitosa; Cavalcanti, Maria da Glória Aureliano de Melo; Marinho, Sílvia Martins; Oliveira, Wilson de; Souza, Joelma Rodrigues de; Lorena, Virginia Maria Barros de; Gomes, Yara de Miranda

    2016-01-01

    The relationships between monocytes and lymphocytes through MHC class II molecules and costimulatory, are of utmost importance for the production of an efficient immune response. In this work, we assessed the expression of surface molecules CD80 and CD86 on CD14+HLA-DR+ monocytes from patients with Chagas disease. The study population consisted of 31 patients with chronic clinical forms of Chagas disease. Patient blood samples were cultured in the presence of recombinant cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA). We found considerable differences in the expression profile of surface molecules involved in antigen presentation. CRA and FRA may contribute to host immune response evasion by Trypanozoma cruzi.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. Chagas disease-specific antigens: characterization of epitopes in CRA/FRA by synthetic peptide mapping and evaluation by ELISA-peptide assay

    PubMed Central

    2013-01-01

    Background The identification of epitopes in proteins recognized by medically relevant antibodies is useful for the development of peptide-based diagnostics and vaccines. In this study, epitopes in the cytoplasmic repetitive antigen (CRA) and flagellar repetitive antigen (FRA) proteins from Trypanosoma cruzi were identified using synthetic peptide techniques and pooled sera from Chagasic patients. The epitopes were further assayed with an ELISA assay based on synthetic peptides. Methods Twenty-two overlapping synthetic peptides representing the coding sequence of the T. cruzi CRA and FRA proteins were assessed by a Spot-synthesis array analysis using sera donated by patients with Chagas disease. Shorter peptides were selected that represented the determined epitopes and synthesized by solid phase synthesis to evaluate the patterns of cross-reactivities and discrimination through an ELISA-diagnostic assay. Results The peptide Spot-synthesis array successfully identified two IgG antigenic determinants in the CRA protein and four in FRA. Bioinformatics suggested that the CRA antigens were unique to T. cruzi while the FRA antigen showed similarity with sequences present within various proteins from Leishmania sp. Subsequently, shorter peptides representing the CRA-1, CRA-2 and FRA-1 epitopes were synthesized by solid phase synthesis and assayed by an ELISA-diagnostic assay. The CRA antigens gave a high discrimination between Chagasic, Leishmaniasis and T. cruzi-uninfected serum. A sensitivity and specificity of 100% was calculated for CRA. While the FRA antigen showed a slightly lower sensitivity (91.6%), its specificity was only 60%. Conclusions The epitopes recognized by human anti-T. cruzi antibodies have been precisely located in two biomarkers of T. cruzi, CRA and FRA. The results from screening a panel of patient sera through an ELISA assay based on peptides representing these epitopes strongly suggest that the sequences from CRA would be useful for the

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. [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.

  6. 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

  7. 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

  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. 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.

  10. 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.

  11. 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.

  12. [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.").

  13. 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

  14. 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.

  15. 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

  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. 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

  18. 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.

  19. 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.

  20. 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

  1. [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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  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. 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

  8. 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.

  9. 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).

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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...

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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).

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. 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.

  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. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  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. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. [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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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%.

  3. 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

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. [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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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

  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. 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.

  19. 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

  20. 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.

  1. 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

  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. 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.

  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. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Performance of different Trypanosoma cruzi antigens in the diagnosis of Chagas disease in patients with American cutaneous leishmaniasis from a co-endemic region in Argentina.

    PubMed

    Vega Benedetti, A F; Cimino, Rubén O; Cajal, Pamela S; Juarez, Marisa Del Valle; Villalpando, Carlos A; Gil, José F; Marcipar, Iván S; Krolewiecki, Alejandro J; Nasser, Julio R

    2013-09-01

    To determine the ability of recombinant antigens to detect cases of infection with Trypanosoma cruzi among cases of infection with Leishmania spp. by serological methods. Sera from 41 patients infected with Leishmania spp. were evaluated with ELISA using single (FRA, CP1 and TSSAVI) or pooled (commercial Rec-ELISA) recombinant proteins or homogenate antigens (commercial H-ELISA). As there is no gold standard antigen to discriminate Chagas disease from leishmaniasis, the correlation of results between defined antigens and the homogenate was made with Kappa Index (KI), the level of correlation considered being used as a criterion of specificity. Single recombinant antigens and Rec-ELISA showed good correlation (KI > 0.8). A low correlation (KI < 0.66) was observed between the results from single recombinant antigens or the commercial recombinant kit and H-ELISA. The highly correlated results between T. cruzi single or pooled recombinant proteins are indicative of the usefulness of recombinant antigens for Chagas diagnosis. Our results also indicate that in the city of Oran in Argentina, between 12% and 17% of patients with leishmaniasis are also infected with Chagas disease. The high KI values between TSSAVI and the other recombinant proteins suggest that in these patients, the infection may be caused by T. cruzi II and/or V and/or VI lineages. © 2013 John Wiley & Sons Ltd.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. [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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. [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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  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. 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.

  10. 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

  11. 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.

  12. A novel adjuvant-free H fusion system for the production of recombinant immunogens in Escherichia coli: Its application to a 12 kDa antigen from Cryptosporidium parvum.

    PubMed

    Costa, Sofia J; Silva, Pedro; Almeida, André; Conceição, Antónia; Domingues, Lucília; Castro, António

    2013-01-01

    The production of recombinant antigens in Escherichia coli and specific polyclonal antibodies for diagnosis and therapy is still a challenge for world-wide researchers. Several different strategies have been explored to improve both antigen and antibody production, all of them depending on a successful expression and immunogenicity of the antigen. Gene fusion technology attempted to address these challenges: fusion partners have been applied to optimize recombinant antigen production in E. coli, and to increase protein immunogenicity. Taking a 12-kDa surface adhesion antigen from Cryptosporidium parvum (CP12) by example, the novel H fusion partner was presented in this work as an attractive option for the development of recombinant immunogens and its adjuvant-free immunization. The H tag (of only 1 kDa) efficiently triggered a CP12-specific immune response, and it also improved the immunization procedure without requiring co-administration of adjuvants. Moreover, polyclonal antibodies raised against the HCP12 fusion antigen detected native antigen structures displayed on the surface of C. parvum oocysts. The H tag proved to be an advanced strategy and promising technology for the diagnosis and therapy of C. parvum infections in animals and humans, allowing a rapid and simple recombinant production of the CP12 antigen.

  13. Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays.

    PubMed

    Kumada, Yoichi

    2014-11-01

    The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

  14. Dendritic cell secretion of IL-15 is induced by recombinant huCD40LT and augments the stimulation of antigen-specific cytolytic T cells.

    PubMed

    Kuniyoshi, J S; Kuniyoshi, C J; Lim, A M; Wang, F Y; Bade, E R; Lau, R; Thomas, E K; Weber, J S

    1999-04-10

    Dendritic cells (DC) are professional antigen-presenting cells which stimulate strong proliferative and cytolytic T cell responses. Stimulation of CD40 on dendritic cells by its ligands and anti-CD40 antibodies induces maturation and enhances DC stimulatory ability. In order to understand the mechanism by which ligand:CD40 interactions augment DC function, we assessed the role of T cell stimulatory cytokines IL-12 and IL-15 in the function of DC stimulated with soluble trimeric CD40L, a recombinant fusion protein incorporating three covalently linked extracellular CD40L domains (huCD40LT). Peripheral blood derived DC treated with huCD40LT and/or IFN-gamma were used to stimulate T cell responses in vitro to specific antigens. DC treated with huCD40LT or IFN-gamma/huCD40LT stimulated enhanced T cell proliferation to CASTA, a soluble protein from C. albicans, induced T cells with augmented antigen-specific lysis, and increased the yield of antigen-specific IFN-gamma-producing T cells. IL-15 production by DC was enhanced in cultures treated with huCD40LT and correlated with expansion of antigen-specific cytolytic T cells. Addition of a neutralizing anti-IL-15 monoclonal antibody inhibited the expansion of viral and tumor antigen-specific T cells stimulated by IFN-gamma and huCD40LT-treated DC. In contrast, this enhanced stimulatory ability of DC did not appear to depend on synthesis of IL-12 since huCD40LT treatment stimulated the generation of antigen-specific cytokine producing and cytolytic T cells without increased IL-12 production. Addition of anti-IL-12 monoclonal antibody did not inhibit expansion of these cells. These data suggest that production of IL-15 but not IL-12 is an important factor in the enhanced immunostimulatory ability of huCD40LT-treated DC.

  15. Evaluation of an IgE ELISA with Culicoides spp. extracts and recombinant salivary antigens for diagnosis of insect bite hypersensitivity in Warmblood horses.

    PubMed

    Peeters, L M; Janssens, S; Goddeeris, B M; De Keyser, K; Wilson, A D; Kaufmann, C; Schaffartzik, A; Marti, E; Buys, N

    2013-10-01

    Insect bite hypersensitivity (IBH) in horses represents an immunoglobulin E (IgE)-mediated hypersensitivity to salivary antigens from biting midges (Culicoides spp.). The aim of this study was to evaluate and compare the performances of IgE ELISAs using recombinant Culicoides spp. Obsoletus group salivary gland antigens or crude whole body extracts ('ObsWBE'), C. nubeculosus recombinant proteins (Culn1, 3, 4, 5, 7, 8 and 10) and Obsoletus group recombinant proteins (Culo1 and 2). IgE levels were measured in plasma of 343 Warmblood horses classified as IBH-affected (n=167) and IBH-unaffected (n=176) according to the owners' descriptions. IBH-affected horses were subdivided based on the severity of their clinical signs at sampling and whether or not their IBH history was considered to be classical. The accuracies of the tests increased when clinical signs at sampling were more pronounced or when the IBH history could be considered as classical. A combination of IgE levels against the three best performing Culicoides spp. recombinant proteins (Culn4, Culo1 and Culo2) and ObsWBE resulted in the best performing test. When IBH-affected horses showing a classical history of the disease and severe clinical signs were compared with IBH-unaffected horses, the Youden's index at the optimal cut-off for the three tests in combination was 0.67. This optimal cut-off had a sensitivity of 70%, a specificity of 97% and a total accuracy of 92%. The performance of the IgE ELISA was affected by the severity of IBH clinical signs at sampling and was improved when IgE levels against several recombinant proteins were combined. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. 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.

  17. Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

    PubMed

    Liang, Jinping; Teng, Xindong; Yuan, Xuefeng; Zhang, Ying; Shi, Chunwei; Yue, Tingting; Zhou, Lei; Li, Jianrong; Fan, Xionglin

    2015-08-01

    Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

  18. Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China

    PubMed Central

    Wang, Wenling; Wang, Huijuan; Deng, Yao; Song, Tie; Lan, Jiaming; Wu, Guizhen; Ke, Changwen; Tan, Wenjie

    2016-01-01

    The first imported case of Middle East respiratory syndrome (MERS) in China recently occurred, allowing for the characterization of antibody titers in a series of the patient's sera using the following methods based on recombinant viral structural antigens: inactivated MERS coronavirus (MERS-CoV) enzyme-linked immunosorbent assay (ELISA), recombinant MERS-CoV spike (S, or fragments of S) ELISA, nucleoprotein (NP) ELISA and MERS S pseudovirus particle-based neutralization test (ppNT). A longitudinal profile of the infection showed that seroconversion detected by ELISAs based on the recombinant extracellular domain, S, S1 and receptor-binding domain (RBD) antigens occurred as early as neutralizing antibodies were detected by the ppNT and earlier than antibodies were detected by the inactivated MERS-CoV and N-terminal domain (NTD) ELISAs. Antibodies detected by the NP ELISA occurred last. Strong correlations were found between the S1, RBD and NP ELISAs and the inactivated MERS-CoV ELISA. The S and RBD ELISAs were highly correlated with the commercial S1 ELISA. The S ELISA strongly correlated with the ppNT, although the MERS-CoV, S1, NTD and RBD ELISAs were also significantly correlated with the ppNT (P<0.001). PMID:27826140

  19. Vector-primed mice display hypo-responsiveness to foreign antigen presented by recombinant Salmonella regardless of the route of delivery.

    PubMed

    Attridge, Stephen R; Vindurampulle, Christofer J

    2005-01-01

    Our previous studies have shown that mice which have been orally primed with an attenuated Salmonella vector [S. enterica serovar Stanley] are hypo-responsive to foreign antigens later delivered orally by the same vector strain, responding with significantly impaired serum and intestinal antibody responses compared with those seen in unprimed controls. Initial vector priming of the gut-associated lymphoid tissue (GALT) is likely to result in impaired persistence of recombinant Salmonella later administered orally. Delivery of recombinant bacteria by the intra-peritoneal or intra-nasal route, to avoid exposure to a primed GALT, did not allow vector-primed recipients to mount normal antibody responses to the foreign pilus protein K88. The negative impact of vector priming could be largely overcome, however, if mice were exposed to the foreign protein just prior to priming with the vector strain. Using this strategy, vector-primed mice displayed normal gut IgA and intermediate serum IgG responses to K88 following oral administration of recombinant Salmonella. Our findings are compatible with the concept of epitopic suppression, in which failure to respond to the foreign vaccine antigen reflects the clonal dominance of B cells specific for epitopes associated with the vector strain.

  20. Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China.

    PubMed

    Wang, Wenling; Wang, Huijuan; Deng, Yao; Song, Tie; Lan, Jiaming; Wu, Guizhen; Ke, Changwen; Tan, Wenjie

    2016-11-09

    The first imported case of Middle East respiratory syndrome (MERS) in China recently occurred, allowing for the characterization of antibody titers in a series of the patient's sera using the following methods based on recombinant viral structural antigens: inactivated MERS coronavirus (MERS-CoV) enzyme-linked immunosorbent assay (ELISA), recombinant MERS-CoV spike (S, or fragments of S) ELISA, nucleoprotein (NP) ELISA and MERS S pseudovirus particle-based neutralization test (ppNT). A longitudinal profile of the infection showed that seroconversion detected by ELISAs based on the recombinant extracellular domain, S, S1 and receptor-binding domain (RBD) antigens occurred as early as neutralizing antibodies were detected by the ppNT and earlier than antibodies were detected by the inactivated MERS-CoV and N-terminal domain (NTD) ELISAs. Antibodies detected by the NP ELISA occurred last. Strong correlations were found between the S1, RBD and NP ELISAs and the inactivated MERS-CoV ELISA. The S and RBD ELISAs were highly correlated with the commercial S1 ELISA. The S ELISA strongly correlated with the ppNT, although the MERS-CoV, S1, NTD and RBD ELISAs were also significantly correlated with the ppNT (P<0.001).

  1. Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis.

    PubMed

    Rastawicki, Waldemar; Smietafiska, Karolina; Chrost, Anna; Wolkowicz, Tomasz; Rokosz-Chudziak, Natalia

    Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections-The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, non- specific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. Recombinant YopD, YopB, YopE and V-Ag proteins of Y enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage ofpositive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA, IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to

  2. Evaluation of immune response to recombinant potential protective antigens of Mycoplasma hyopneumoniae delivered as cocktail DNA and/or recombinant protein vaccines in mice.

    PubMed

    Chen, Austen Y; Fry, Scott R; Daggard, Grant E; Mukkur, Trilochan K S

    2008-08-12

    Intramuscular immunization of mice with DNA cocktail vaccines, comprising potential protective antigens P36, P46, NrdF, and P97or P97R1 of Mycoplasma hyopneumoniae, induced strong Th1-polarized immune responses against each antigen, with only P46 eliciting a serum IgG response. Subcutaneous immunization with protein cocktail vaccines, surprisingly, induced both Th1-polarized immune response as well as antibody response whereas mice immunized with DNA cocktail vaccines followed by boosting with protein cocktail vaccines generated strong Th1-polarized and humoral immune responses. P97 was not recognized by serum antibodies from commercial bacterin-immunized mice indicating potential lack of expression of this important antigen in inactivated whole-cell vaccines.

  3. Involvement of Fra-1 in Retinal Ganglion Cell Apoptosis in Rat Light-Induced Retina Damage Model.

    PubMed

    Liu, Xiaojuan; Yang, Xiaowei; Zhu, Rongrong; Dai, Ming; Zhu, Manhui; Shen, Yuntian; Fang, Hongda; Sang, Aimin; Chen, Hui

    2017-01-01

    Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.

  4. Evaluation of an enzyme immunoassay for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen as an aid in the diagnosis of blastomycosis in dogs.

    PubMed

    Mourning, Alyssa C; Patterson, Edward E; Kirsch, Emily J; Renschler, Janelle S; Wolf, Linda A; Paris, Jasmin K; Durkin, Michelle M; Wheat, Lawrence J

    2015-11-15

    To evaluate the sensitivity and specificity of an enzyme immunoassay (EIA) for antibodies to a recombinant Blastomyces adhesin-1 repeat antigen (rBAD-1) to aid in the diagnosis of blastomycosis in dogs and compare the findings with results from other tests used for this purpose. Prospective analytic study. Serum and urine from 70 dogs with and without blastomycosis. Serum and urine samples were collected from dogs with blastomycosis (n = 21), histoplasmosis (8), or nonfungal pulmonary disease (21) and from healthy control dogs living in a blastomycosis-endemic area (20). Serum was tested for antibodies against Blastomyces dermatitidis with the rBAD-1 antibody EIA and an A-antigen antibody agar gel immunodiffusion (AGID) assay. Serum and urine were tested for B dermatitidis antigen with a quantitative EIA. Sensitivity of the quantitative antigen EIA was 100% in serum and urine samples from dogs with blastomycosis, with specificity of 95% in urine samples from dogs with nonfungal pulmonary disease and 100% in urine samples from healthy dogs. Sensitivity of the rBAD-1 antibody EIA (95%) was significantly greater than that of the A-antigen antibody AGID assay (65%). Specificity of the antibody EIA was 88% in dogs with histoplasmosis, 95% in healthy dogs, and 100% in dogs with nonfungal pulmonary disease. The rBAD-1 antibody EIA had greater sensitivity than the A-antigen antibody AGID assay in dogs with blastomycosis. This antibody EIA may assist in distinguishing histoplasmosis from blastomycosis. Further evaluation in a larger prospective study is needed to verify these results.

  5. Combined Use of Paracoccidioides brasiliensis Recombinant 27-Kilodalton and Purified 87-Kilodalton Antigens in an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Paracoccidioidomycosis

    PubMed Central

    Díez, Soraya; Gómez, Beatriz L.; McEwen, Juan G.; Restrepo, Angela; Hay, Rod J.; Hamilton, Andrew J.

    2003-01-01

    The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM. PMID:12682142

  6. Swine adipose stromal cells loaded with recombinant bovine herpesvirus 4 virions expressing a foreign antigen induce potent humoral immune responses in pigs.

    PubMed

    Donofrio, Gaetano; Taddei, Simone; Franceschi, Valentina; Capocefalo, Antonio; Cavirani, Sandro; Martinelli, Nicola; Ottonello, Simone; Ferrari, Maura

    2011-01-29

    Increasingly effective vaccination strategies are needed to counteract the high incidence of contagious diseases associated with intensive swine breeding. Recombinant viral vaccines are a promising new avenue in this direction. Key features of viral vectors suitable for immunoprophylaxis are safety, ease of manipulation and the ability to replicate in a variety of hosts. Most of the above requirements are met by bovine herpesvirus 4 (BoHV-4), a non-pathogenic dsDNA virus capable of infecting a broad range of cell types in vitro. Here we report the results of an exploratory study using an engineered BoHV-4 virus (eBoHV-4) expressing two unrelated glycoprotein antigens from bovine viral diarrhea virus (BVDV) and bovine herpesvirus 1 (BoHV-1), to assess the potential of recombinant BoHV-4 as a self-adjuvanted immunogen in pigs. Free eBoHV-4 virions and virions preloaded into homologous swine adipose-derived stromal cells (SADSC) were tested. Neither virus formulation elicited neutralizing anti-BoHV-4 antibodies, nor any disease symptom, yet both induced specific immune responses against the heterologous antigens. However, a much earlier (18 vs 28 days post-infection) and more robust neutralizing response against BVDV and BoHV-1 viruses was elicited by eBoHV-4-preinfected SADSCs compared to free virions. The data validate BoHV-4 as a safe and effective heterologous antigen carrier/producer and identify SADSCs as helpful tools for the formulation of increasingly efficacious recombinant immunogens for pig vaccination.

  7. Oral immunization with recombinant hepatitis E virus antigen displayed on the Lactococcus lactis surface enhances ORF2-specific mucosal and systemic immune responses in mice.

    PubMed

    Gao, Shenyang; Li, Dandan; Liu, Ying; Zha, Enhui; Zhou, Tiezhong; Yue, Xiqing

    2015-01-01

    Hepatitis E virus (HEV) as a recognized zoonotic pathogen has posed global burden on public health, which is exacerbated by lack of efficient vaccine. In this study, we constructed a recombinant (inaQ-ORF2 gene fusion) Lactococcus lactis (L. lactis) strain NZ3900 that expresses and displays the hepatitis E virus antigen ORF2 utilizing an ice uncleation protein-based anchor system. After oral vaccination of BALB/c mice, significantly higher levels of ORF2-specific mucosal IgA and serum IgG were detected and cellular immunity was also induced. These findings further support that L. lactis-based HEV antigen vaccines could be used for human and animal protection against infection.

  8. Mucosal immunogenicity of a recombinant Salmonella typhimurium-cloned heterologous antigen in the absence or presence of coexpressed cholera toxin A2 and B subunits.

    PubMed Central

    Harokopakis, E; Hajishengallis, G; Greenway, T E; Russell, M W; Michalek, S M

    1997-01-01

    An avirulent Salmonella typhimurium vaccine strain expressing a streptococcal protein adhesin and a similar clone which produces the same streptococcal antigen linked to the cholera toxin (CT) A2 and B subunits (CTA2/B) were compared for the ability to induce antibody responses to the expressed heterologous antigen after oral or intranasal immunization of mice. Expression of cloned immunogens in these systems is temperature regulated, being optimal at 37 degrees C, and the two clones under comparison were shown to produce similar levels of the streptococcal antigen. Both clones were found to stimulate high levels of serum immunoglobulin G (IgG) and mucosal IgA antibodies to the cloned immunogen. A consistent trend was observed toward higher mucosal IgA but lower serum IgG responses in the case of the S. typhimurium vector that coexpressed CTA2/B, a potential mucosal adjuvant, regardless of the route of administration. Also noteworthy was the capacity of these antigen delivery systems to induce anamnestic systemic and secretory responses to the cloned immunogen 15 weeks after the primary immunization, despite preexisting immunity to the Salmonella vectors. These antibody responses were sustained for at least 7 months following the booster immunization, at which time the secretory IgA antibody levels were significantly higher in mice given the Salmonella clone that coexpressed CTA2/B. Although the serum IgG response against the Salmonella vector was characterized by a high IgG2a/IgG1 ratio (indicative of the T helper type 1 [Th1]/Th2 profile), a mixed IgG1 and IgG2a pattern was observed for the carried heterologous antigen, which displayed a dominant IgG1 response when administered as a purified immunogen. Our findings indicate that the recombinant streptococcal antigen and CTA2/B are strong immunogens when expressed by the antigen delivery system used in this study and suggest that CTA2/B may have an additional immunoenhancing activity in the mucosal compartment

  9. Recombinant hexon antigen based single serum dilution ELISA for rapid serological profiling against fowl adenovirus-4 causing hydropericardium syndrome in chickens.

    PubMed

    R, Rajasekhar; Roy, Parimal

    2014-10-01

    A recombinant hexon antigen based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Fowl adenovirus-4 (FAdV) causing Hydropericardium syndrome (HPS). An immunodominant partial hexon gene of 737bp was cloned into pRSET vector and expressed in Escherichia coli strain BL21 DE3 pLys S. Expression was tested by Western Blot test. The purified recombinant protein antigen was used in coating ELISA plate for FAdV-4 serology. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to Q-AGID test. Recombinant antigen was also used in Dot ELISA. In an experimental vaccination of broiler chicken at 10 days old age, the geometric mean (GM) antibody titres as measured by ELISA ranged from 5.006±0.11log10 to 4.526±0.04log10 and by Dot ELISA titre were from 2.240±0.08log10 to 0.180±0.04log10 during 5th-8th weeks of age, results were compared with Q-AGID results. Field samples were collected randomly from breeder flocks, found to have antibody titre by both ELISA and Dot ELISA at 10th and only 75% samples were positive at 14th weeks of age. After revaccination at 16th weeks of age, all sera samples were found have considerably high antibody titre on 24th week but all samples were negative at 32nd weeks. Advantages of recombinant hexon antigen based ELISA and Dot ELISA in FAdV-4 serology have been discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. 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

  11. Efficient in vivo priming by vaccination with recombinant NY-ESO-1 protein and CpG in antigen naive prostate cancer patients.

    PubMed

    Karbach, Julia; Neumann, Antje; Atmaca, Akin; Wahle, Claudia; Brand, Kathrin; von Boehmer, Lotta; Knuth, Alexander; Bender, Armin; Ritter, Gerd; Old, Lloyd J; Jäger, Elke

    2011-02-15

    NY-ESO-1, one of the most immunogenic tumor antigens, is expressed in 15% to 25% of metastatic prostate cancers. The immunological and clinical effects of vaccination with recombinant NY-ESO-1 protein combined with CpG as adjuvant were evaluated. In a phase I clinical study, patients with advanced prostate cancer were vaccinated with recombinant NY-ESO-1 protein (100 μg) mixed with CpG 7909 (2.5 mg) every 3 weeks intradermally for 4 doses. Objectives of the study were the safety of the vaccine and changes of specific humoral and cellular immunological responses to NY-ESO-1 in relation to detectable NY-ESO-1 expression in the individual tumor. All 12 baseline sero-negative patients developed high-titer NY-ESO-1 antibody responses. B-cell epitope mapping identified NY-ESO-1 p91-110 to be recognized most frequently by vaccine-induced antibodies. Two patients developed significant antibody titers against the adjuvant CpG. NY-ESO-1-specific CD4+ and/or CD8+ T-cell responses were induced in 9 patients (69%). Five of these 9 patients did not express NY-ESO-1 in the autologous tumor. Postvaccine CD8+ T-cell clones recognized and lyzed HLA-matched tumor cell lines in an antigen-specific manner. Our data provide clear evidence for the capacity of NY-ESO-1 protein/CpG vaccine to induce integrated antigen-specific immune responses in vivo and to efficiently prime CD8+ T-cell responses in NY-ESO-1 antigen-negative patients. Our results may also support further clinical vaccination protocols with NY-ESO-1 protein not only focused on the treatment of existing cancer, but also to prevent further development of NY-ESO-1 positive cancers in vivo. ©2010 AACR.

  12. Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats.

    PubMed

    Liu, Wen-xing; Hu, Sen; Qiao, Zu-jian; Chen, Wei-ye; Liu, Lin-tao; Wang, Fang-kun; Hua, Rong-hong; Bu, Zhi-gao; Li, Xiang-rui

    2011-01-01

    Antibodies produced in animals vaccinated using live attenuated vaccines against Brucella spp. are indistinguishable using current conventional serological tests from those produced in infected animals. One potential approach is to develop marker vaccines in which specific genes have been deleted from parental vaccine strains that show good immunogenicity and vaccine efficacy. Corresponding methods of detection for antibodies raised by the marker vaccine should also be developed. A specific fragment of the bp26 gene of Brucella melitensis M5-90 was cloned into vector pQE32 to construct the recombinant plasmid (pQE32-rΔbp26). It was used to transform Escherichia coli M15 (pREP4) host cells, which expressed the rΔbp26 protein. Subsequently, the recombinant protein was purified by immobilized metal affinity chromatography and size-exclusion chromatography. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified rΔbp26 protein was represented by only one band, with a molecular weight of 14 kDa, and it showed good antigenic specificity on western blot and enzyme-linked immunosorbent assay (ELISA). The purified rΔbp26 protein was intended to be used as an antigen to develop a novel ELISA to differentiate animals vaccinated with bp26 mutants of Brucella spp. from those infected naturally and those vaccinated with the parental vaccine strains.

  13. Complete genome sequencing of a recombinant strain between human astrovirus antigen types 2 and 8 isolated from South Korea.

    PubMed

    Ha, Hyun-Ju; Lee, Sung-Geun; Cho, Han-Gil; Jin, Ji-Young; Lee, Jae Woong; Paik, Soon-Yong

    2016-04-01

    Human astroviruses (HAstVs) occur worldwide and are known to the causative agents of diarrhea in infants and elderly patients with immune dysfunction. This study aimed to identify recombinant HAstV strains and characterize rare genotypes. The full-length genome of a recombinant HAstV strain isolated from the stool sample of a patient with acute gastroenteritis from South Korea was amplified using three pairs of previously designed primers and seven newly designed primers. The recombinant HAstV was 6757-bp long and contained three sequential open reading frames (ORFs), designated as ORF1a (2781 bp), ORF1b (1548 bp), and ORF2 (2349 bp). Our findings suggested that a recombination event had occurred between ORF1b and ORF2 of the isolated strain, with a recombination breakpoint at 4081 bp. To our knowledge, this is the first study to reveal the complete nucleotide sequence of a recombinant HAstV strain from South Korea. Our study findings might be useful for identifying other recombinant HAstV strains and for developing vaccines against this pathogenic virus. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

    PubMed

    Higuchi, Akira; Toriniwa, Hiroko; Komiya, Tomoyoshi; Nakayama, Tetsuo

    2016-01-01

    An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

  15. Immunoenhancing effects of MontanideTM ISA oil-based adjuvants on recombinant coccidia antigen vaccination against Eimeria acervulina infection

    USDA-ARS?s Scientific Manuscript database

    The current study was conducted to investigate the immunoenhancing effects of Montanide' adjuvants on protein subunit vaccination against avian coccidiosis. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with one ...

  16. Vaccine Properties of a Novel Marker Gene-Free Recombinant Modified Vaccinia Ankara Expressing Immunodominant CMV Antigens pp65 and IE1

    PubMed Central

    Wang, Zhongde; La Rosa, Corinna; Li, Zhongqi; Ly, Heang; Krishnan, Aparna; Martinez, Joy; Britt, William J.; Diamond, Don

    2007-01-01

    CMV tegument protein pp65 and CMV immediate early gene product IE1 are both considered immunodominant targets of cell-mediated immunity (CMI) and potentially capable of controlling CMV infection. To better assess their role in host defense, we have constructed a novel MVA transfer vector named pZWIIA and generated a recombinant MVA (rMVA) expressing both full-length pp65 and exon4 of IE1 (pp65-IE1-MVA) at high levels, followed by the genetic removal of the bacterial marker gene used to distinguish recombinant forms. Immunogenicity evaluation indicates that pp65-IE1-MVA not only can induce robust primary CMI to both antigens in HLA A2.1 Tg mice, but also can stimulate vigorous expansion of memory T lymphocyte responses to pp65 and IE1 in PBMC of CMV-positive donors. These properties make the MVA-based vaccine ideal for the dual role of priming and boosting CMV-specific T cell immunity as a means to control CMV disease in recipients of hematopoietic cell or solid organ transplantation (HCT or SOT). pZWIIA alone or in combination with other MVA transfer vectors can be used to generate MVA based multiple-antigen vaccine which have application in vaccine development for a wide spectrum of infectious diseases and cancer. PMID:17049414

  17. Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines.

    PubMed

    Galli, V; Simionatto, S; Marchioro, S B; Fisch, A; Gomes, C K; Conceição, F R; Dellagostin, O A

    2012-12-17

    Porcine enzootic pneumonia (PEP), which is caused by the fastidious bacterium Mycoplasma hyopneumoniae, is one of the most economically important diseases in the pig industry worldwide. Commercial bacterins provide only partial protection; therefore, the development of more efficient vaccines against PEP is necessary. In this study, the cellular and humoral immune responses elicited by DNA and recombinant subunit vaccines based on the P37, P42, P46 and P95 antigens of M. hyopneumoniae were evaluated after the intramuscular inoculation of BALB/c mice. The expression of the cytokines INFγ, TNFα and IL1 was evaluated by real-time RT-PCR in splenocytes from vaccinated mice. All antigens delivered as subunit vaccines, especially P42 and P95, and the pcDNA3/P46 DNA vaccine were able to elicit strong immune responses. These vaccines induced cellular immune responses and the production of antibodies able to react with native M. hyopneumoniae proteins. Because both cellular and humoral immune responses were induced, P42 and P95 are promising candidates for a recombinant subunit vaccine and P46 is a promising candidate for a DNA vaccine against PEP.

  18. Generation of a baculovirus recombinant prostate-specific membrane antigen and its use in the development of a novel protein biochip quantitative immunoassay.

    PubMed

    Xiao, Z; Jiang, X; Beckett, M L; Wright, G L

    2000-06-01

    Prostate-specific membrane antigen (PSMA) is a 100-kDa transmembrane glycoprotein identified by the monoclonal antibody 7E11-C5.3 from the human prostate tumor cell line LNCaP. Because of its significant upregulation in androgen refractory and metastatic prostate cancers, PSMA may be a useful prognostic biomarker and a target for developing novel therapeutic strategies. However, the lack of abundant pure PSMA protein and the low efficacy in immunoaffinity isolation from LNCaP cells have hampered the development of clinical assays. In order to obtain a renewable and reliable source of pure antigen, we used the baculovirus/insect cell system to express and purify a recombinant PSMA. A recombinant baculovirus containing a 6x histidine-tagged PSMA gene was generated, from which rPSMA was expressed and purified using cobalt-chelating affinity chromatography. The purity and correct molecular size of rPSMA were demonstrated by gel electrophoresis and mass spectrometry. Glycosidase digestions showed that the oligosaccharides on rPSMA are primarily N-linked high-mannose type. Although the glycosylation is different from the native PSMA, it did not affect the immunoreactivity of rPSMA to antibodies specific for either the intra- or the extracellular domains of PSMA. Finally, the purified rPSMA was successfully used to develop a quantitative PSMA immunoassay using the novel ProteinChip surface-enhanced laser desorption/ionization mass spectrometry technology. Copyright 2000 Academic Press.

  19. Enzyme-linked immunosorbent assay using recombinant SAG1 antigen to detect Toxoplasma gondii-specific immunoglobulin G antibodies in human sera and saliva.

    PubMed

    Chahed Bel-Ochi, Nouha; Bouratbine, Aïda; Mousli, Mohamed

    2013-04-01

    Serologic detection of Toxoplasma gondii IgG antibodies is widely accepted as a means to determine immune status and susceptibility to Toxoplasma infection during pregnancy. However, current commercial kits present some drawbacks, such as a requirement for whole-parasite antigen preparation or interassay variability. To address these problems, the purpose of this study was to produce a whole sequence of the recombinant T. gondii SAG1 antigen (rSAG1) to assess its diagnostic performance in Toxoplasma IgG screening and to explore a saliva-based method as a noninvasive alternative to serum-based testing. rSAG1 was expressed in recombinant bacteria as inclusion bodies, purified through one-step affinity chromatography, and refolded in native form by dialysis. A large amount was obtained, and the specific antigen immunoreactivity was confirmed by immunoblotting. Two rSAG1-based enzyme-linked immunosorbent assays (ELISAs) applied to paired serum and saliva samples were designed. The rSAG1-based ELISA evaluation consisted of testing intrinsic sensitivity and specificity of 49 serum samples from patients immune to toxoplasmosis and 42 serum samples from nonimmune controls identified by routinely used kits. To assess agreement between serum-based and saliva-based tests, the positive percent agreement (PPA) and negative percent agreement (NPA) between the 2 tests were estimated. The rSAG1 serum-based ELISA detected specific IgG with 100% sensitivity and specificity. The PPA and NPA between the serum-based and saliva-based tests varied according to the selected optical density threshold in saliva. Thus, for a selected cutoff of 0.14, the PPA was 100% and the NPA was 88.1%, whereas for a selected cutoff of 0.29, the PPA was 67.3% and the NPA was 100%.

  20. Evaluation of recombinant LigB antigen-based indirect ELISA and latex agglutination test for the serodiagnosis of bovine leptospirosis in India.

    PubMed

    Deneke, Yosef; Sabarinath, T; Gogia, Neha; Lalsiamthara, Jonathan; Viswas, K N; Chaudhuri, Pallab

    2014-08-01

    Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the genus Leptospira, causing febrile infection characterized by multi-organ failure in humans and animals. Leptospiral Ig-like protein B (LigB) is a surface-expressed antigen that mediates host cell invasion or attachment. In this study, N-terminal conserved region of LigB protein (46 kDa) was evaluated for its diagnostic potential to detect anti-leptospiral antibodies in the sera of various animal species. Dot blot analysis revealed immunoreactivity of Leptospira-positive sera of cattle, buffalo, dog, sheep and goat to purified LigB protein. We have analyzed 1126 bovine serum samples, collected from Northern and Eastern part of India, by microscopic agglutination test (MAT) and recombinant LigB (rLigB) based ELISA and latex agglutination test (LAT). The sensitivity of rLigB based ELISA for 554 MAT positive sera was 96.9% and the specificity with 572 MAT negative sera was 91.08% whereas LAT showed sensitivity and specificity of 93.68% and 92.31%, respectively. Kappa values of 0.879 and 0.860 for recombinant antigen based ELISA and LAT indicate excellent agreement with the gold standard serological test, MAT, for the detection of anti-leptospiral antibodies in sera. Further, LAT based on rLigB antigen is a simple and rapid test, suitable for serodiagnosis of leptospirosis under field conditions, owing to its portability and longer shelf life. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Freeze-thaw stress of Alhydrogel ® alone is sufficient to reduce the immunogenicity of a recombinant hepatitis B vaccine containing native antigen.

    PubMed

    Clapp, Tanya; Munks, Michael W; Trivedi, Ruchit; Kompella, Uday B; Braun, LaToya Jones

    2014-06-24

    Preventing losses in vaccine potency due to accidental freezing has recently become a topic of interest for improving vaccines. All vaccines with aluminum-containing adjuvants are susceptible to such potency losses. Recent studies have described excipients that protect the antigen from freeze-induced inactivation, prevent adjuvant agglomeration and retain potency. Although these strategies have demonstrated success, they do not provide a mechanistic understanding of freeze-thaw (FT) induced potency losses. In the current study, we investigated how adjuvant frozen in the absence of antigen affects vaccine immunogenicity and whether preventing damage to the freeze-sensitive recombinant hepatitis B surface antigen (rHBsAg) was sufficient for maintaining vaccine potency. The final vaccine formulation or Alhydrogel(®) alone was subjected to three FT-cycles. The vaccines were characterized for antigen adsorption, rHBsAg tertiary structure, particle size and charge, adjuvant elemental content and in-vivo potency. Particle agglomeration of either vaccine particles or adjuvant was observed following FT-stress. In vivo studies demonstrated no statistical differences in IgG responses between vaccines with FT-stressed adjuvant and no adjuvant. Adsorption of rHBsAg was achieved; regardless of adjuvant treatment, suggesting that the similar responses were not due to soluble antigen in the frozen adjuvant-containing formulations. All vaccines with adjuvant, including the non-frozen controls, yielded similar, blue-shifted fluorescence emission spectra. Immune response differences could not be traced to differences in the tertiary structure of the antigen in the formulations. Zeta potential measurements and elemental content analyses suggest that FT-stress resulted in a significant chemical alteration of the adjuvant surface. This data provides evidence that protecting a freeze-labile antigen from subzero exposure is insufficient to maintain vaccine potency. Future studies should

  2. 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.

  3. A recombinant protein based on the Trypanosoma cruzi metacyclic trypomastigote 82-kilodalton antigen that induces and effective immune response to acute infection.

    PubMed Central

    Santori F, R; Paranhos-Bacalla, G S; Franco DA Silveira, J; Yamauchi, L M; Araya, J E; Yoshida, N

    1996-01-01

    To further investigate the immunological properties of the stage-specific 82-kDa glycoprotein (gp82) of Trypanosoma cruzi metacyclic trypomastigotes, previously shown to induce antigen-specific humoral and T-cell responses in mice, we performed a series of experiments with recombinant proteins containing sequences of gp82 fused to glutathione S-transferase. Of five fusion proteins tested, only J18b and J18b1, the carboxyproximal peptides containing amino acids 224 to 516 and 303 to 516, respectively, were recognized by monoclonal antibody 3F6 as well as by various anti-T. cruzi antisera and, when administered to mice, were capable of eliciting antibodies directed to the native gp82. The amino-terminal peptide and other carboxyterminal recombinant proteins lacking the central domain of gp82 (amino acids 224 to 356), which is exposed on the surface of live metacyclic forms, did not display any of these properties. Spleen cells derived from mice immunized with any of the five recombinant proteins proliferated in vitro in the presence of native gp82.J18b was the most stimulatory, whereas J18b3, the peptide containing amino acids 408 to 516, elicited the weakest response. When BALB/c mice immunized with J18b antigen plus A1(OH)3 as adjuvant were challenged 10 5 metacyclic trypomastigotes, 85% of them resisted acute infection, in comparison with control mice that received glutathione S-transferase plus adjuvant. Antibodies induced by J18b protein lacked agglutinating or complement-dependent lytic activity and failed to neutralize parasite infectivity. On the other hand, CD4+T cells from the spleens of J18b-immunized mice displayed an intense proliferative activity upon stimulation with 1.25 microgram of native gp82 per ml, which resulted in increased production of gamma interferon, a cytokine associated with resistance to T. cruzi infection. PMID:8606064

  4. Recombinant subunit ORF2.1 antigen and induction of antibody against immunodominant epitopes in the hepatitis E virus capsid protein.

    PubMed

    Li, F; Riddell, M A; Seow, H F; Takeda, N; Miyamura, T; Anderson, D A

    2000-04-01

    A recombinant subunit antigen (ORF2.1), representing the carboxy-terminal 267 amino acids of the 660-amino-acid hepatitis E virus (HEV) capsid protein, was expressed in Escherichia coli and used for the immunisation of rats. Purified antigen formulated with either Aluminium Hydroxide Gel Adjuvant (Alum) or Titermax gave high and equivalent levels of antibody after three doses. Responses to two doses of 15, 75, or 150 microg antigen, formulated with Alum and given at 0 and 4 weeks, were also equivalent by 17 weeks after immunisation. Rats initially developed antibody to a wide range of linear epitopes in the ORF2.1 region, but by 27 weeks the predominant response detected by Western immunoblotting was restricted to the conformational epitope unique to ORF2.1 [Li et al. (1997) Journal of Medical Virology 52:289-300], a pattern that was also observed when comparing acute-phase patient serum samples with serum samples from convalescing patients. Antibody from immunised rats blocked the majority of patients' serum reactivity in enzyme-linked immunosorbent assay against both ORF2.1 (57-92% inhibition) and virus-like particles of HEV produced using the baculovirus system (74-97% inhibition). Together, these results suggest that the ORF2.1 subunit vaccine induces an antibody response against immunodominant, conformational epitopes in the viral capsid, which largely mimics that seen in convalescent patients, who are presumed to be immune to HEV infection.

  5. Oral immunization with recombinant Lactococcus lactis delivering a multi-epitope antigen CTB-UE attenuates Helicobacter pylori infection in mice.

    PubMed

    Li, Xinyang; Xing, Yingying; Guo, Le; Lv, Xiaobo; Song, Hui; Xi, Tao

    2014-10-01

    Urease is an essential virulence factor and colonization factor for Helicobacter pylori (H. pylori) and is considered as an excellent vaccine candidate antigen. However, conventional technologies for preparing an injectable vaccine require purification of the antigenic protein and preparation of an adjuvant. Lactococcus lactis NZ9000 (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. In previous study, we constructed a multi-epitope vaccine, designated CTB-UE, which is composed of the mucosal adjuvant cholera toxin B subunit (CTB), three Th cell epitopes and two B-cell epitopes from urease subunits. To develop a novel type of oral vaccine against H. pylori, genetically modified L. lactis strains were established to secrete this epitope vaccine extracellularly in this study. Oral prophylactic immunization with recombinant L. lactis significantly elicited humoral anti-urease antibody responses (P < 0.001) and reduced the gastric colonization of H. pylori from 7.14 ± 0.95 to 4.68 ± 0.98 log10 CFU g(-1) stomach. This L. lactis oral vaccine offers a promising vaccine candidate for the control of H. pylori infection.

  6. ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera

    PubMed Central

    Ahn, Hye-Jin; Kim, Sera; Kim, Dae-Yong

    2003-01-01

    An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a 6 × His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals. PMID:12972732

  7. The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method

    PubMed Central

    Clargo, Alison M; Hudson, Ashley R; Ndlovu, Welcome; Wootton, Rebecca J; Cremin, Louise A; O'Dowd, Victoria L; Nowosad, Carla R; Starkie, Dale O; Shaw, Sophie P; Compson, Joanne E; White, Dominic P; MacKenzie, Brendon; Snowden, James R; Newnham, Laura E; Wright, Michael; Stephens, Paul E; Griffiths, Meryn R; Lawson, Alastair DG; Lightwood, Daniel J

    2014-01-01

    Single B cell technologies, which avoid traditional hybridoma fusion and combinatorial display, provide a means to interrogate the naturally-selected antibody repertoire of immunized animals. Many methods enable the sampling of memory B cell subsets, but few allow for the direct interrogation of the plasma cell repertoire, i.e., the subset of B cells responsible for producing immunoglobulin in serum. Here, we describe the use of a robust and simple fluorescence-based technique, called the fluorescent foci method, for the identification and isolation of antigen-specific IgG-secreting cells, such as plasma cells, from heterogeneous bone marrow preparations. Following micromanipulation of single cells, cognate pairs of heavy and light chain variable region genes were recovered by reverse transcription (RT)-polymerase chain reaction (PCR). During the PCR, variable regions were combined with a promoter fragment and a relevant constant region fragment to produce two separate transcriptionally-active PCR (TAP) fragments that were directly co-transfected into a HEK-293F cell line for recombinant antibody expression. The technique was successfully applied to the generation of a diverse panel of high-affinity, functional recombinant antibodies to human tumor necrosis factor (TNF) receptor 2 and TNF derived from the bone marrow of immunized rabbits and rats, respectively. Progression from a bone marrow sample to a panel of functional recombinant antibodies was possible within a 2-week timeframe. PMID:24423622

  8. Development of a Recombinant Cell-Based Indirect Immunofluorescence Assay for the Determination of Autoantibodies against Soluble Liver Antigen in Autoimmune Hepatitis

    PubMed Central

    Radzimski, Christiane; Probst, Christian; Teegen, Bianca; Rentzsch, Kristin; Blöcker, Inga Madeleine; Dähnrich, Cornelia; Schlumberger, Wolfgang; Stöcker, Winfried; Bogdanos, Dimitrios P.; Komorowski, Lars

    2013-01-01

    Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases. PMID:23401700

  9. Development of a recombinant cell-based indirect immunofluorescence assay for the determination of autoantibodies against soluble liver antigen in autoimmune hepatitis.

    PubMed

    Radzimski, Christiane; Probst, Christian; Teegen, Bianca; Rentzsch, Kristin; Blöcker, Inga Madeleine; Dähnrich, Cornelia; Schlumberger, Wolfgang; Stöcker, Winfried; Bogdanos, Dimitrios P; Komorowski, Lars

    2013-01-01

    Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

  10. Vaccination with a novel recombinant Leishmania antigen plus MPL provides partial protection against L. donovani challenge in experimental model of visceral leishmaniasis.

    PubMed

    Bhardwaj, Suvercha; Vasishta, R K; Arora, Sunil K

    2009-01-01

    The acquisition of immunity following subclinical or resolved infection with the intracellular parasite Leishmania donovani suggests that vaccination could prevent visceral leishmaniasis. The characteristics and in vitro stimulating capability of the recombinant proteins expressed by previously identified clones on the basis of their capacity to stimulate an indigenously established Leishmania-specific cell line leading to high level of IFN-gamma suggested these to be potential candidates for immunoprophylaxis against leishmaniasis. In this study, we investigated the protective efficacy of purified recombinant proteins from two of the identified cDNA clones along with the adjuvant MPL, in a hamster model of experimental leishmaniasis. We demonstrate here that the immunization of animals with one of the recombinant proteins (rF14) having 97% similarity to C1 clone of L. chagasi ribosomal protein gene P0 (rLiP0) along with MPL provided partial protection against the virulent challenge of L. donovani. The absence of antigen-specific lymphoproliferative responses in these immunized animals may be responsible for the lack of complete and long-lasting protection.

  11. 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.

  12. 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

  13. 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.

  14. The Immunomodulatory Role of Adjuvants in Vaccines Formulated with the Recombinant Antigens Ov-103 and Ov-RAL-2 against Onchocerca volvulus in Mice.

    PubMed

    Hess, Jessica A; Zhan, Bin; Torigian, April R; Patton, John B; Petrovsky, Nikolai; Zhan, Tingting; Bottazzi, Maria Elena; Hotez, Peter J; Klei, Thomas R; Lustigman, Sara; Abraham, David

    2016-07-01

    In some regions in Africa, elimination of onchocerciasis may be possible with mass drug administration, although there is concern based on several factors that onchocerciasis cannot be eliminated solely through this approach. A vaccine against Onchocerca volvulus would provide a critical tool for the ultimate elimination of this infection. Previous studies have demonstrated that immunization of mice with Ov-103 and Ov-RAL-2, when formulated with alum, induced protective immunity. It was hypothesized that the levels of protective immunity induced with the two recombinant antigens formulated with alum would be improved by formulation with other adjuvants known to enhance different types of antigen-specific immune responses. Immunizing mice with Ov-103 and Ov-RAL-2 in conjunction with alum, Advax 2 and MF59 induced significant levels of larval killing and host protection. The immune response was biased towards Th2 with all three of the adjuvants, with IgG1 the dominant antibody. Improved larval killing and host protection was observed in mice immunized with co-administered Ov-103 and Ov-RAL-2 in conjunction with each of the three adjuvants as compared to single immunizations. Antigen-specific antibody titers were significantly increased in mice immunized concurrently with the two antigens. Based on chemokine levels, it appears that neutrophils and eosinophils participate in the protective immune response induced by Ov-103, and macrophages and neutrophils participate in immunity induced by Ov-RAL-2. The mechanism of protective immunity induced by Ov-103 and Ov-RAL-2, with the adjuvants alum, Advax 2 and MF59, appears to be multifactorial with roles for cytokines, chemokines, antibody and specific effector cells. The vaccines developed in this study have the potential of reducing the morbidity associated with onchocerciasis in humans.

  15. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    PubMed

    Li, Wu; Deng, Guangcun; Li, Min; Zeng, Jin; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  16. 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.

  17. Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients

    PubMed Central

    Odunsi, Kunle; Matsuzaki, Junko; Karbach, Julia; Neumann, Antje; Mhawech-Fauceglia, Paulette; Miller, Austin; Beck, Amy; Morrison, Carl D.; Ritter, Gerd; Godoy, Heidi; Lele, Shashikant; duPont, Nefertiti; Edwards, Robert; Shrikant, Protul; Old, Lloyd J.; Gnjatic, Sacha; Jäger, Elke

    2012-01-01

    Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4+ and CD8+ T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0–84 mo) and the median OS was 48 mo (range, 3–106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16–29 mo), and median OS was 48 mo (CI, not estimable). CD8+ T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations. PMID:22454499

  18. Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients.

    PubMed

    Odunsi, Kunle; Matsuzaki, Junko; Karbach, Julia; Neumann, Antje; Mhawech-Fauceglia, Paulette; Miller, Austin; Beck, Amy; Morrison, Carl D; Ritter, Gerd; Godoy, Heidi; Lele, Shashikant; duPont, Nefertiti; Edwards, Robert; Shrikant, Protul; Old, Lloyd J; Gnjatic, Sacha; Jäger, Elke

    2012-04-10

    Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4(+) and CD8(+) T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0-84 mo) and the median OS was 48 mo (range, 3-106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16-29 mo), and median OS was 48 mo (CI, not estimable). CD8(+) T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

  19. Western blot assay using recombinant p26 antigen for detection of equine infectious anemia virus-specific antibodies.

    PubMed

    Alvarez, I; Gutierrez, G; Ostlund, E; Barrandeguy, M; Trono, K

    2007-12-01

    We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.

  20. Brucella abortus Omp19 recombinant protein subcutaneously co-delivered with an antigen enhances antigen-specific T helper 1 memory responses and induces protection against parasite challenge.

    PubMed

    Coria, Lorena M; Ibañez, Andrés E; Pasquevich, Karina A; Cobiello, Paula L González; Frank, Fernanda M; Giambartolomei, Guillermo H; Cassataro, Juliana

    2016-01-20

    The discovery of effective adjuvants for many vaccines especially those with limited commercial appeal, such as vaccines to poverty-related diseases, is required. In this work, we demonstrated that subcutaneous co-administration of mice with the outer membrane protein U-Omp19 from Brucella spp. plus OVA as antigen (Ag) increases Ag-specific T cell proliferation and T helper (Th) 1 immune responses in vitro and in vivo. U-Omp19 treated dendritic cells promote IFN-γ production by specific CD4(+) T cells and increases T cell proliferation. U-Omp19 co-administration induces the production of Ag specific effector memory T cell populations (CD4(+) CD44(high) CD62L(low) T cells). Finally, subcutaneous co-administration of U-Omp19 with Trypanosoma cruzi Ags confers protection against virulent parasite challenge, reducing parasitemia and weight loss while increasing mice survival. These results indicate that the bacterial protein U-Omp19 when delivered subcutaneously could be a suitable component of vaccine formulations against infectious diseases requiring Th1 immune responses.

  1. Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

    PubMed Central

    Sedlik, C.; Saron, M.-F.; Sarraseca, J.; Casal, I.; Leclerc, C.

    1997-01-01

    To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells. PMID:9207121

  2. Comparative Vaccine Efficacy of Different Isoforms of Recombinant Protective Antigen Against Bacillus anthracis Spore Challenge in Rabbits

    DTIC Science & Technology

    2006-02-06

    bovine serum, 4 mM glutamine and 00 U of Penicillin G and 100 g of streptomycin per ml D-MEM complete) supplemented with 25 mM HEPES. ne -hundred...contains small, but varying quantities of other bacterial com- ponents. A second-generation vaccine currently undergoing clinical trials is a purified...Friedlander AM. Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb Pathog 1988;5(2):127–39

  3. Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

    PubMed Central

    Halpern, Micah D.; Molins, Claudia R.; Schriefer, Martin

    2014-01-01

    A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. PMID:24899074

  4. Simple objective detection of human lyme disease infection using immuno-PCR and a single recombinant hybrid antigen.

    PubMed

    Halpern, Micah D; Molins, Claudia R; Schriefer, Martin; Jewett, Mollie W

    2014-08-01

    A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections.

    PubMed

    Rosati, S; Profiti, M; Lorenzetti, R; Bandecchi, P; Mannelli, A; Ortoffi, M; Tolari, F; Ciabatti, I M

    2004-10-01

    Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period. Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses. In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections. Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA. The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test.

  6. Immunization against Lamb Haemonchosis with a Recombinant Somatic Antigen of Haemonchus contortus (rHcp26/23)

    PubMed Central

    García-Coiradas, Leticia; Angulo-Cubillán, Francisco; Valladares, Basilio; Martínez, Enrique; de la Fuente, Concepción; Alunda, José María; Cuquerella, Montserrat

    2010-01-01

    Haemonchosis, caused by the abomasal nematode Haemonchus contortus, is a common parasitic disease of sheep. Our previous results showed that a soluble fraction from adult stages of the nematode (p26/23) induced partial protection against challenge. Recombinant DNA technology was applied to obtain a synthetic protein (rHcp26/23). Immunological assays (ELISA, Western blotting, and immunolocalization), using sera from lambs immunized with p26/23, confirmed the identity of the recombinant protein and demonstrated that the synthetic protein is equivalent to the purified protein employed in the previous immunoprophylaxis studies. Vaccination of lambs with 300 μg of rHcp26/23 and Freund's adjuvant elicited a notable specific antibody response. Immunization did not induce any significant protection after challenge with 16000 infective larvae of H. contortus, and comparable values for parasite faecal egg output, packed cell volume, and abomasal parasite burdens were found in vaccinated and control animals. PMID:20631899

  7. 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.

  8. Western Blot Assay Using Recombinant p26 Antigen for Detection of Equine Infectious Anemia Virus-Specific Antibodies▿

    PubMed Central

    Alvarez, I.; Gutierrez, G.; Ostlund, E.; Barrandeguy, M.; Trono, K.

    2007-01-01

    We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results. PMID:17959820

  9. Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

    PubMed

    Anderson, John P; Rascoe, Lisa N; Levert, Keith; Chastain, Holly M; Reed, Matthew S; Rivera, Hilda N; McAuliffe, Isabel; Zhan, Bin; Wiegand, Ryan E; Hotez, Peter J; Wilkins, Patricia P; Pohl, Jan; Handali, Sukwan

    2015-01-01

    The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.

  10. 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.

  11. 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

  12. Immunogenic targeting of recombinant peptide vaccines to human antigen-presenting cells by chimeric anti-HLA-DR and anti-surface immunoglobulin D antibody Fab fragments in vitro.

    PubMed Central

    Baier, G; Baier-Bitterlich, G; Looney, D J; Altman, A

    1995-01-01

    To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system. PMID:7533857

  13. Seroprevalence of Cryptosporidium parvum infection of dairy cows in three northern provinces of Thailand determined by enzyme-linked immunosorbent assay using recombinant antigen CpP23.

    PubMed

    Inpankaew, T; Jittapalapong, S; Phasuk, J; Pinyopanuwut, N; Chimnoi, W; Kengradomkit, C; Sunanta, C; Zhang, G; Aboge, G O; Nishikawa, Y; Igarashi, I; Xuan, X

    2009-06-01

    Cryptosporidium parvum is the most frequent parasitic agent that causes diarrhoea in AIDS patients in Thailand. Cryptosporidiosis outbreaks in humans may be attributed to contamination of their drinking water from infected dairy pastures. A 23-kDa glycoprotein of C. parvum (CpP23) is a sporozoite surface protein that is geographically conserved among C. parvum isolates. This glycoprotein is a potentially useful candidate antigen for the diagnosis of cryptosporidiosis by enzyme-linked immunosorbent assay. Therefore, we investigated the seroprevalence of C. parvum infection in dairy cows in northern Thailand using an ELISA based on recombinant CpP23 antigen. Sera were randomly collected from 642 dairy cows of 42 small-holder farmers, which had the top three highest number of the dairy cows' population in Northern Thailand, that included Chiang Mai, Chiang Rai and Lumpang provinces. The overall seroprevalence of the infection was 4.4%, and the seropositive rates for the three provinces were 3.3% in Chiang Mai, 5.1% in Chiang Rai and 3% in Lumpang. These results suggest that cattle could play a role in zoonotic cryptosporidiosis in Thailand.

  14. 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.

  15. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection.

    PubMed

    Reed, Matthew D; Wilder, Julie A; Mega, William M; Hutt, Julie A; Kuehl, Philip J; Valderas, Michelle W; Chew, Lawrence L; Liang, Bertrand C; Squires, Charles H

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg.

  16. The use of hybrid phage displaying antigen epitope and recombinant protein in the diagnosis of systemic Candida albicans infection in rabbits and cancer patients.

    PubMed

    Quanping, Su; Yanyan, Huai; Yicun, Wang; Zhigang, Ju; Yuling, Geng; Li, Wang

    2010-12-01

    Hsp90 and Sap2 are 2 immunodominant antigens of Candida albicans. Both of them can induce the production of antibody. In this article, systemically infected rabbits were used to study the Hsp90 and Sap2 antibody production. Also, pET28a-Hsp90 protein, pET28a-Sap2 protein, hybrid phage displaying LKVIRK epitope, and hybrid phage displaying VKYTS epitope were used for diagnosis of the antibody in cancer patients. The results showed that the Sap2 antibody appeared earlier than Hsp90 antibody in systemically infected rabbits. Meanwhile, both of the antibodies can perform protection in rabbits. The conclusion is that Sap2 antibody, which appears at early stage in systemic candidiasis, may be better than Hsp90 antibody for the diagnosis of invasive candidiasis. For 141 sera of cancer patients, 52 sera were detected Sap2 antibody and 57 sera were detected Hsp90 antibody. Only 14 sera contained both the 2 antibodies. Although recombinant protein was slightly more sensitive than hybrid phage, there was no significant difference between them. For its easy preparation, less expensive hybrid phage displaying antigen epitope may be a better agent for diagnosis of candidiasis.

  17. Macrophage Immune Response Suppression by Recombinant Mycobacterium tuberculosis Antigens, the ESAT-6, CFP-10, and ESAT-6/CFP-10 Fusion Proteins

    PubMed Central

    Seghatoleslam, Atefeh; Hemmati, Mina; Ebadat, Saeedeh; Movahedi, Bahram; Mostafavi-Pour, Zohreh

    2016-01-01

    Background: Macrophage immune responses are affected by the secretory proteins of Mycobacterium tuberculosis (Mtb). This study aimed to examine the immune responses of macrophages to Mtb secretory antigens, namely ESAT-6, CFP-10, and ESAT-6/CFP-10. Methods: THP-1 cells (a human monocytic cell line) were cultured and differentiated to macrophages by phorbol 12-myristate 13-acetate. The cytotoxicity of the recombinant Mtb proteins was assessed using the MTT assay. Two important immune responses of macrophages, namely NO and ROS production, were measured in response to the ESAT-6, CFP-10, and ESAT-6/CFP-10 antigens. The data were analyzed using one-way ANOVA with SPSS, version 16, and considered significant at P<0.05. Results: The results showed that the ESAT-6, CFP-10, and ESAT-6/CFP-10 proteins markedly reduced macrophage immune response. The treatment of the THP-1-differentiated cells with ESAT-6, CFP-10, and ESAT-6/CFP-10 reduced NO and ROS production. The treated THP-1-differentiated cells exhibited less inducible NO synthase activity than did the untreated cells. No toxic effect on macrophage viability was observed for the applied proteins at the different concentrations. Conclusion: It seems that the decline in macrophage immune response is due to the suppression of NO and ROS production pathways without any effect on cell viability. PMID:27365551

  18. Poly (lactide-co-glycolide) microspheres in respirable sizes enhance an in vitro T cell response to recombinant Mycobacterium tuberculosis antigen 85B.

    PubMed

    Lu, Dongmei; Garcia-Contreras, Lucila; Xu, Ding; Kurtz, Sherry L; Liu, Jian; Braunstein, Miriam; McMurray, David N; Hickey, Anthony J

    2007-10-01

    To investigate the use of poly (lactide-co-glycolide) (PLGA) microparticles in respirable sizes as carriers for Antigen 85B (Ag85B), a secreted protein of Mycobacterium tuberculosis, with the ultimate goal of employing them in pulmonary delivery of tuberculosis vaccine. Recombinant Ag85B was expressed from two Escherichia coli strains and encapsulated by spray-drying in PLGA microspheres with/without adjuvants. These microspheres containing rAg85B were assessed for their ability to deliver antigen to macrophages for subsequent processing and presentation to the specific CD4 T-hybridoma cells DB-1. DB-1 cells recognize the Ag85B(97-112) epitope presented in the context of MHC class II and secrete IL-2 as the cytokine marker. Microspheres suitable for aerosol delivery to the lungs (3.4-4.3 microm median diameter) and targeting alveolar macrophages were manufactured. THP-1 macrophage-like cells exposed with PLGA-rAg85B microspheres induced the DB-1 cells to produce IL-2 at a level that was two orders of magnitude larger than the response elicited by soluble rAg85B. This formulation demonstrated extended epitope presentation. PLGA microspheres in respirable sizes were effective in delivering rAg85B in an immunologically relevant manner to macrophages. These results are a foundation for further investigation into the potential use of PLGA particles for delivery of vaccines to prevent M. tuberculosis infection.

  19. Immunization with a Recombinant, Pseudomonas fluorescens-Expressed, Mutant Form of Bacillus anthracis-Derived Protective Antigen Protects Rabbits from Anthrax Infection

    PubMed Central

    Reed, Matthew D.; Wilder, Julie A.; Mega, William M.; Hutt, Julie A.; Kuehl, Philip J.; Valderas, Michelle W.; Chew, Lawrence L.; Liang, Bertrand C.; Squires, Charles H.

    2015-01-01

    Protective antigen (PA), one of the components of the anthrax toxin, is the major component of human anthrax vaccine (Biothrax). Human anthrax vaccines approved in the United States and Europe consist of an alum-adsorbed or precipitated (respectively) supernatant material derived from cultures of toxigenic, non-encapsulated strains of Bacillus anthracis. Approved vaccination schedules in humans with either of these vaccines requires several booster shots and occasionally causes adverse injection site reactions. Mutant derivatives of the protective antigen that will not form the anthrax toxins have been described. We have cloned and expressed both mutant (PA SNKE167-ΔFF-315-E308D) and native PA molecules recombinantly and purified them. In this study, both the mutant and native PA molecules, formulated with alum (Alhydrogel), elicited high titers of anthrax toxin neutralizing anti-PA antibodies in New Zealand White rabbits. Both mutant and native PA vaccine preparations protected rabbits from lethal, aerosolized, B. anthracis spore challenge subsequent to two immunizations at doses of less than 1 μg. PMID:26207820

  20. Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

    PubMed Central

    Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

    2013-01-01

    Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

  1. Immunogenicity of recombinant Mycobacterium bovis bacille Calmette–Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens

    PubMed Central

    2013-01-01

    Recombinant Mycobacterium bovis bacille Calmette–Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4+ and CD8+ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB. PMID:23458635

  2. The Immunomodulatory Role of Adjuvants in Vaccines Formulated with the Recombinant Antigens Ov-103 and Ov-RAL-2 against Onchocerca volvulus in Mice

    PubMed Central

    Hess, Jessica A.; Zhan, Bin; Torigian, April R.; Patton, John B.; Petrovsky, Nikolai; Zhan, Tingting; Bottazzi, Maria Elena; Hotez, Peter J.; Klei, Thomas R.; Lustigman, Sara; Abraham, David

    2016-01-01

    Background In some regions in Africa, elimination of onchocerciasis may be possible with mass drug administration, although there is concern based on several factors that onchocerciasis cannot be eliminated solely through this approach. A vaccine against Onchocerca volvulus would provide a critical tool for the ultimate elimination of this infection. Previous studies have demonstrated that immunization of mice with Ov-103 and Ov-RAL-2, when formulated with alum, induced protective immunity. It was hypothesized that the levels of protective immunity induced with the two recombinant antigens formulated with alum would be improved by formulation with other adjuvants known to enhance different types of antigen-specific immune responses. Methodology/ Principal Findings Immunizing mice with Ov-103 and Ov-RAL-2 in conjunction with alum, Advax 2 and MF59 induced significant levels of larval killing and host protection. The immune response was biased towards Th2 with all three of the adjuvants, with IgG1 the dominant antibody. Improved larval killing and host protection was observed in mice immunized with co-administered Ov-103 and Ov-RAL-2 in conjunction with each of the three adjuvants as compared to single immunizations. Antigen–specific antibody titers were significantly increased in mice immunized concurrently with the two antigens. Based on chemokine levels, it appears that neutrophils and eosinophils participate in the protective immune response induced by Ov-103, and macrophages and neutrophils participate in immunity induced by Ov-RAL-2. Conclusions/Significance The mechanism of protective immunity induced by Ov-103 and Ov-RAL-2, with the adjuvants alum, Advax 2 and MF59, appears to be multifactorial with roles for cytokines, chemokines, antibody and specific effector cells. The vaccines developed in this study have the potential of reducing the morbidity associated with onchocerciasis in humans. PMID:27387453

  3. Analysis of antibody responses to Mycobacterium leprae phenolic glycolipid I, lipoarabinomannan, and recombinant proteins to define disease subtype-specific antigenic profiles in leprosy.

    PubMed

    Spencer, John S; Kim, Hee Jin; Wheat, William H; Chatterjee, Delphi; Balagon, Marivic V; Cellona, Roland V; Tan, Esterlina V; Gelber, Robert; Saunderson, Paul; Duthie, Malcolm S; Reece, Stephen T; Burman, William; Belknap, Robert; Mac Kenzie, William R; Geluk, Annemieke; Oskam, Linda; Dockrell, Hazel M; Brennan, Patrick J

    2011-02-01

    A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.

  4. A Comprehensive Preclinical Model Evaluating the Recombinant PRAME Antigen Combined With the AS15 Immunostimulant to Fight Against PRAME-expressing Tumors

    PubMed Central

    Baudson, Nathalie; Ory, Thierry; Segal, Lawrence; Louahed, Jamila

    2015-01-01

    The PRAME tumor antigen is a potential target for immunotherapy. We assessed the immunogenicity, the antitumor activity, and the safety and the tolerability of a recombinant PRAME protein (recPRAME) combined with the AS15 immunostimulant (recPRAME+AS15) in preclinical studies in mice and Cynomolgus monkeys. Four groups of 12 CB6F1 mice received 4 injections of phosphate-buffered saline (PBS), recPRAME, AS15, or recPRAME+AS15. Immunized mice were injected with tumor cells expressing PRAME (CT26-PRAME) 2 weeks or 2 months after the last injection. The mean tumor surface was measured twice a week. Two groups of 10 monkeys received 7 injections of saline or recPRAME+AS15. T-cell responses were measured by flow cytometry using intracellular cytokine staining (ICS). In CB6F1 mice, repeated injections of recPRAME+AS15 induced high PRAME-specific antibody titers and mostly CD4+ T cells producing cytokines. This immune response was long-lasting in these animals and was associated with protection against a challenge with PRAME-expressing tumor cells (CT26-PRAME) applied either 2 weeks or 2 months after the last injection; these data indicate the induction of an immune memory. In HLA-A02.01/HLA-DR1 transgenic mice, recPRAME+AS15 induced both CD4+ and CD8+ T-cell responses, indicating that this antigen can be processed by the human leukocyte antigen and is potentially immunogenic in humans. In addition, a repeated-dose toxicity study in monkeys showed that 7 biweekly injections of recPRAME+AS15 were well tolerated, and induced PRAME-specific antibodies and T cells. In conclusion, these preclinical data indicate that repeated injections of the PRAME cancer immunotherapeutic are immunogenic and have an acceptable safety profile. PMID:26325375

  5. A Comprehensive Preclinical Model Evaluating the Recombinant PRAME Antigen Combined With the AS15 Immunostimulant to Fight Against PRAME-expressing Tumors.

    PubMed

    Gérard, Catherine; Baudson, Nathalie; Ory, Thierry; Segal, Lawrence; Louahed, Jamila

    2015-10-01

    The PRAME tumor antigen is a potential target for immunotherapy. We assessed the immunogenicity, the antitumor activity, and the safety and the tolerability of a recombinant PRAME protein (recPRAME) combined with the AS15 immunostimulant (recPRAME+ AS15) in preclinical studies in mice and Cynomolgus monkeys. Four groups of 12 CB6F1 mice received 4 injections of phosphate-buffered saline (PBS), recPRAME, AS15, or recPRAME+AS15. Immunized mice were injected with tumor cells expressing PRAME (CT26-PRAME) 2 weeks or 2 months after the last injection. The mean tumor surface was measured twice a week. Two groups of 10 monkeys received 7 injections of saline or recPRAME+ AS15. T-cell responses were measured by flow cytometry using intracellular cytokine staining (ICS). In CB6F1 mice, repeated injections of recPRAME+ AS15 induced high PRAME-specific antibody titers and mostly CD4+ T cells producing cytokines. This immune response was long-lasting in these animals and was associated with protection against a challenge with PRAME-expressing tumor cells (CT26-PRAME) applied either 2 weeks or 2 months after the last injection; these data indicate the induction of an immune memory. In HLA-A02.01/HLA-DR1 transgenic mice, recPRAME+ AS15 induced both CD4+ and CD8+ T-cell responses, indicating that this antigen can be processed by the human leukocyte antigen and is potentially immunogenic in humans. In addition, a repeated-dose toxicity study in monkeys showed that 7 biweekly injections of recPRAME+ AS15 were well tolerated, and induced PRAME-specific antibodies and T cells. In conclusion, these preclinical data indicate that repeated injections of the PRAME cancer immunotherapeutic are immunogenic and have an acceptable safety profile.

  6. Immunogenicity of recombinant Mycobacterium bovis bacille Calmette-Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens.

    PubMed

    Mohamud, Rohimah; Azlan, Maryam; Yero, Daniel; Alvarez, Nadine; Sarmiento, Maria E; Acosta, Armando; Norazmi, Mohd-Nor

    2013-01-01

    Recombinant Mycobacterium bovis bacille Calmette-Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.

  7. Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

    USDA-ARS?s Scientific Manuscript database

    This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

  8. 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.

  9. Recombinant Lactobacillus plantarum induces immune responses to cancer testis antigen NY-ESO-1 and maturation of dendritic cells.

    PubMed

    Mobergslien, Anne; Vasovic, Vlada; Mathiesen, Geir; Fredriksen, Lasse; Westby, Phuong; Eijsink, Vincent G H; Peng, Qian; Sioud, Mouldy

    2015-01-01

    Given their safe use in humans and inherent adjuvanticity, Lactic Acid Bacteria may offer several advantages over other mucosal delivery strategies for cancer vaccines. The objective of this study is to evaluate the immune responses in mice after oral immunization with Lactobacillus (L) plantarum WCFS1 expressing a cell-wall anchored tumor antigen NY-ESO-1. And to investigate the immunostimulatory potency of this new candidate vaccine on human dendritic cells (DCs). L. plantarum displaying NY-ESO-1 induced NY-ESO-1 specific antibodies and T-cell responses in mice. By contrast, L. plantarum displaying conserved proteins such as heat shock protein-27 and galectin-1, did not induce immunity, suggesting that immune tolerance to self-proteins cannot be broken by oral administration of L. plantarum. With respect to immunomodulation, immature DCs incubated with wild type or L. plantarum-NY-ESO-1 upregulated the expression of co-stimulatory molecules and secreted a large amount of interleukin (IL)-12, TNF-α, but not IL-4. Moreover, they upregulated the expression of immunosuppressive factors such as IL-10 and indoleamine 2,3-dioxygenase. Although L. plantarum-matured DCs expressed inhibitory molecules, they stimulated allogeneic T cells in-vitro. Collectively, the data indicate that L. plantarum-NY-ESO-1 can evoke antigen-specific immunity upon oral administration and induce DC maturation, raising the potential of its use in cancer immunotherapies.

  10. Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen.

    PubMed

    Ng, Michelle Y T; Tan, Wen Siang; Abdullah, Norhafizah; Ling, Tau Chuan; Tey, Beng Ti

    2006-10-01

    Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.

  11. Recombinant DNA technology for melanoma immunotherapy: anti-Id DNA vaccines targeting high molecular weight melanoma-associated antigen.

    PubMed

    Barucca, A; Capitani, M; Cesca, M; Tomassoni, D; Kazmi, U; Concetti, F; Vincenzetti, L; Concetti, A; Venanzi, F M

    2014-11-01

    Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V H) and light (V L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V H/V L, and V L/V H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.

  12. Dysregulation of Fra1 expression by Wnt/β-catenin signalling promotes glioma aggressiveness through epithelial-mesenchymal transition.

    PubMed

    Zhang, Li; Liu, Huaijun; Mu, Xiaodan; Cui, Jianling; Peng, Zhigang

    2017-04-28

    Aberrant expression of Fos-related antigen-1 (Fra1) is commonly elevated in various malignant cancers and is strongly implicated in invasion and metastasis. However, the molecular mechanisms underlying its dysregulation in human glioma remain poorly understood. In the present study, we demonstrate that up-regulation of Fra1 plays a crucial role in the glioma aggressiveness and epithelial-mesenchymal transition (EMT) activated by Wnt/β-catenin signal pathway. In glioma cells, activation of Wnt/β-catenin signalling by Wnt3a administration obviously induced EMT and directly activated the transcription of Fra1. Phenotype experiments revealed that up-regulation of Fra1 induced by Wnt/β-catenin signalling drove the EMT of glioma cells. Furthermore, it was found that the cisplatin resistance acquired by Wnt/β-catenin signalling activation depended on increased expression of Fra1. Analysis of clinical specimens verified a positive correlation between Fra1 and β-catenin as well as a poor prognosis in glioma patients with double-high expressions of them. These findings indicate that an aberrant Wnt/β-catenin signalling leads to the EMT and drug resistance of glioma via Fra1 induction, which suggests novel therapeutic strategies for the malignant disease.

  13. Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

    PubMed Central

    Law, John; Wong, Jason Alexander Ji-Xhin; Hockman, Darren; Landi, Amir; Chen, Chao; Crawford, Kevin; Kundu, Juthika; Baldwin, Lesley; Johnson, Janelle; Dahiya, Anita; LaChance, Gerald; Marcotrigiano, Joseph; Law, Mansun; Foung, Steven; Tyrrell, Lorne

    2016-01-01

    ABSTRACT A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine

  14. Protection against aerosolized Yersinia pestis challenge following homologous and heterologous prime-boost with recombinant plague antigens.

    PubMed

    Glynn, Audrey; Roy, Chad J; Powell, Bradford S; Adamovicz, Jeffrey J; Freytag, Lucy C; Clements, John D

    2005-08-01

    A Yersinia pestis-derived fusion protein (F1-V) has shown great promise as a protective antigen against aerosol challenge with Y. pestis in murine studies. In the current study, we examined different prime-boost regimens with F1-V and demonstrate that (i) boosting by a route other than the route used for the priming dose (heterologous boosting) protects mice as well as homologous boosting against aerosol challenge with Y. pestis, (ii) parenteral immunization is not required to protect mice against aerosolized plague challenge, (iii) the route of immunization and choice of adjuvant influence the magnitude of the antibody response as well as the immunoglobulin G1 (IgG1)/IgG2a ratio, and (iv) inclusion of an appropriate adjuvant is critical for nonparenteral immunization.

  15. Comparison of T24H-his, GST-T24H and GST-Ts8B2 recombinant antigens in western blot, ELISA and multiplex bead-based assay for diagnosis of neurocysticercosis.

    PubMed

    Hernández-González, Ana; Noh, John; Perteguer, María Jesús; Gárate, Teresa; Handali, Sukwan

    2017-05-15

    Currently, the reference standard assay for the serodiagnosis of neurocysticercosis (NCC) is the lentil lectin-bound glycoproteins/enzyme-linked immunoelectrotransfer blot (LLGP-EITB). The main disadvantage of this technique is the complexity of obtaining and purifying the LLGP extract. This could be solved by replacement with highly specific recombinant antigens from Taenia solium. Based on previous studies, we selected and produced the recombinant Ts8B2 and T24H proteins and applied them to three diagnostic techniques: western blot (WB), enzyme-linked immunosorbent assay (ELISA) and the multiplex bead-based assay (MBA). The Ts8B2 and T24H cDNA sequences were expressed in a prokaryotic system and the corresponding expression products purified; three recombinant proteins were further characterized: T24H-his, GST-T24H and GST-Ts8B2. The proteins on WB, ELISA and MBA were tested against 149 sera from patients with NCC confirmed by brain imaging, 40 sera from patients with other parasitic diseases, and 131 sera from US. individuals without evidence of neurocysticercosis (clinical/serological/brain imaging). The sensitivity and specificity of each antigen by WB were calculated by counting the number of true positive, false positive, true negative and false negative results. Using the receiver operating characteristic (ROC) curves, the cut-off values for the ELISA and MBA were established as well as the sensitivity and specificity of each assay. All three antigens showed a high sensitivity on WB in active NCC cases with two or more viable cysts and low sensitivity for cases with single viable cyst or calcified lesions and inactive NCC. WB showed the highest specificity and sensitivity out of the three diagnostic techniques. The recombinant T24H-his was the best diagnostic reagent in WB (100% sensitivity, 99.4% specificity), exhibiting similar results to the LLGP-EITB, against the same panel of NCC sera. The GST-T24H antigen worked better than the others in ELISA and MBA

  16. Evaluation of an ELISA using recombinant Ssλ20ΔB3 antigen for the serological diagnosis of Sarcoptes scabiei infestation in domestic and wild rabbits.

    PubMed

    Casais, Rosa; Millán, Javier; Rosell, Joan Maria; Dalton, Kevin P; Prieto, José Miguel

    2015-12-15

    An ELISA, based on the Sarcoptes scabiei Ssλ20ΔB3 inmunodominant antigen, was evaluated for the detection of antibodies to S. scabiei in experimentally infested (n=10), farm (n=109), and wild (n=78) rabbit sera. The S. scabiei antigen Ssλ20ΔB3, a major structural protein present over the entire mite's body, was produced as a recombinant protein in Escherichia coli and purified for its use in the ELISA. The resulting ELISA showed, in experimentally infested domestic rabbits, detectable specific antibody responses (IgG) above the cut off level from week three post-infestation indicating that the assay is able to detect positive rabbits very early during the course of the infestation. The ELISA was validated on a panel of 109 domestic breeding rabbit sera collected from 26 Spanish farms, of which 41 were obtained from rabbits with skin lesions compatible with sarcoptic mange, 26 with skin lesions compatible with psoroptic mange, and 42 from unexposed individuals from mange-free farms. The ELISA in this group was characterized by 95% sensitivity, 97% specificity, and a high degree of repeatability. In the psoroptic mange compatible lesions group, included in the study as control group for cross-reactivity with the closely related mite Psoroptes cuniculi, cross-reacting antibodies to Ssλ20ΔB3 S. scabiei antigen were detected in 42.30% of the rabbit sera. However, mean% OD values of the sarcoptic-mange group (55.61 ± 39.20%) were significantly higher (p<0.001) than OD values of the psoroptic-mange (3.64% ± 5.4%) and also of the free-mange (0.21% ± 0.67%) groups. In addition, the ELISA was also evaluated in serum samples obtained from both naturally infested and non-infested wild rabbits from Mallorca Island. The sensitivity of the assay for this group was 100% (4 out of the 4 rabbits with sarcoptic mange compatible lesions and presence of S. scabiei mites were seropositive) and the specificity was 90% (67 out of 74 wild rabbits without detectable mange lesions

  17. Recombinant Lipoprotein Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand that Induces Macrophages Apoptosis and Inhibits MHC II Antigen Processing.

    PubMed

    Su, Haibo; Zhu, Shenglin; Zhu, Lin; Huang, Wei; Wang, Honghai; Zhang, Zhi; Xu, Ying

    2016-01-01

    TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2(-/-) mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4(+) T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

  18. Recombinant Lipoprotein Rv1016c Derived from Mycobacterium tuberculosis Is a TLR-2 Ligand that Induces Macrophages Apoptosis and Inhibits MHC II Antigen Processing

    PubMed Central

    Su, Haibo; Zhu, Shenglin; Zhu, Lin; Huang, Wei; Wang, Honghai; Zhang, Zhi; Xu, Ying

    2016-01-01

    TLR2-dependent cellular signaling in Mycobacterium tuberculosis-infected macrophages causes apoptosis and inhibits class II major histocompatibility complex (MHC-II) molecules antigen processing, leading to evasion of surveillance. Mycobacterium tuberculosis (MTB) lipoproteins are an important class of Toll-like receptor (TLR) ligand, and identified as specific components that mediate these effects. In this study, we identified and characterized MTB lipoprotein Rv1016c (lpqT) as a cell wall associated-protein that was exposed on the cell surface and enhanced the survival of recombinants M. smegmatis_Rv1016c under stress conditions. We found that Rv1016c lipoprotein was a novel TLR2 ligand and able to induce macrophage apoptosis in a both dose- and time-dependent manner. Additionally, apoptosis induced by Rv1016c was reserved in THP-1 cells blocked with anti-TLR-2 Abs or in TLR2−/− mouse macrophages, indicating that Rv1016c-induced apoptosis is dependent on TLR2. Moreover, we demonstrated that Rv1016c lipoprotein inhibited IFN-γ-induced MHC-II expression and processing of soluble antigens in a TLR2 dependent manner. Class II transactivator (CIITA) regulates MHC II expression. In this context, Rv1016c lipoprotein diminished IFN-γ-induced expression of CIITA IV through TLR2 and MAPK Signaling. TLR2-dependent apoptosis and inhibition of MHC-II Ag processing induced by Rv1016c during mycobacteria infection may promote the release of residual bacilli from apoptotic cells and decrease recognition by CD4+ T cells. These mechanisms may allow intracellular MTB to evade immune surveillance and maintain chronic infection. PMID:27917375

  19. Assessing drivers of the IgG4 antibody reactivity to recombinant antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa.

    PubMed

    Damgaard, Johanne; Meyrowitsch, Dan W; Rwegoshora, Rwehumbiza T; Magesa, Stephen M; Mukoko, Dunstan A; Simonsen, Paul E

    2016-09-01

    A high proportion of the human population in lymphatic filariasis (LF) endemic areas is positive for filarial specific IgG4 antibodies, including many individuals without microfilariae (mf; circulating larvae in the human blood) or circulating filarial antigens (CFA; marker of adult worm infection). The antibodies are commonly regarded as markers of infection and/or exposure to filarial larvae, but a direct association between the antibodies and these indices has not been well documented. The present study assessed the role and relative effect of potential drivers of the human IgG4 antibody reactivity to the recombinant filarial antigen Bm14 in Wuchereria bancrofti endemic populations in East Africa. Sera collected during previous studies from 395 well characterized individuals with regard to age, sex, mf, CFA, household vector biting and household exposure to infective filarial larvae were tested for IgG4 antibodies to Bm14, and associations between antibody reactivity and the different variables were statistically analyzed. IgG4 reactivity to Bm14 was highly positively associated with CFA, and to a lesser extent with age. However, an expected association with household exposure to infective filarial larvae was not found. Bm14 antibody reactivity thus appeared mainly to reflect actual infection of individuals with adult filarial worms rather than ongoing exposure to transmission. The analyses moreover suggested that many of the CFA negative but Bm14 positive individuals had early or low level infections where antibodies had been induced but where CFA was not (yet?) measurable. Although the study indicated that IgG4 reactivity to Bm14 is a marker of filarial infection, assessment of this reactivity, especially in children, will still be useful for indirect monitoring of changes in transmission intensity, including break of transmission and post-elimination surveillance, in LF control.

  20. Simulation of control strategies for the cattle tick Boophilus microplus employing vaccination with a recombinant Bm86 antigen preparation.

    PubMed

    Labarta, V; Rodríguez, M; Penichet, M; Lleonart, R; Luaces, L L; de la Fuente, J

    1996-05-01

    Current strategies for the control of the cattle tick Boophilus microplus include the use of chemicals as the principal control method. These methods, however, have met with partially successful results. The recent development of immunological methods for the control of the cattle tick has opened new possibilities for the design of control strategies. Employing the results obtained by us in experiments testing the effect of vaccination with the recombinant vaccine, Gavac (Heber Biotec S.A.), on tick populations, we have developed a model to evaluate, through a computer program, the efficacy of the vaccine as a control method. The action of the vaccine on the control of tick populations was simulated and the specific serum antibody titers required to decrease the tick population in the field were calculated. The specific serum antibody titer required to decrease the tick population in the field after the first vaccination scheme was found to be > or = 57,200 and the antibody titer required to maintain this effect when the vaccine is already acting and after successive revaccinations was found to be > or = 27,500. Considerations about revaccination schemes and combination between vaccination and acaricide treatments as possible control strategies are discussed.

  1. Purification and characterization of pre-S-containing hepatitis B surface antigens produced in recombinant mammalian cell culture.

    PubMed

    Youn, B W; Samanta, H

    1989-02-01

    Heterogeneous, pre-S-rich HBsAg particles were expressed in recombinant mammalian cell culture and purified to near homogeneity. The purification process comprises: concentration of cell culture medium, protein precipitation by poly(ethylene glycol), gel filtration column chromatography, isopycnic ultracentrifugation by KBr and sucrose density gradient ultracentrifugation. The resulting HBsAg product was greater than 98% pure, and contained much of pre-S1 and pre-S2 components. Scanning densitometry analysis of the silver-stained HBsAg product showed approximately 70-80% S protein, approximately 10-20% pre-S2 protein, and approximately 5-15% pre-S1 protein. It was estimated that the amount of HBV-specific DNA present the final product was less than 7 pg mg-1 HBsAg. Further biochemical analysis has demonstrated that the HBsAg particles are very heterogeneous in charge and density. Charge heterogeneity was quite random among the particles, but density heterogeneity could be related to the different amounts of pre-S2 component in the particles.

  2. Design and optimization of a recombinant system for large-scale production of the MPT64 antigen from Mycobacterium tuberculosis.

    PubMed

    Geisbrecht, Brian V; Nikonenko, Boris; Samala, Rowena; Nakamura, Reiko; Nacy, Carol A; Sacksteder, Katherine A

    2006-03-01

    Early clinical trials of a potential new tuberculosis (TB) diagnostic, the Patch Test for Active TB (PTAT), used MPB64 protein that was purified from the spent medium of Bacillus Calmette-Guérin (BCG) Tokyo 172 vaccine production. The yield was poor, 0.05 mg/L, and the process for purification of the protein was complex, requiring four chromatographic steps. The combination of yield and purification complexity compromised the ability to produce the PTAT diagnostic in quantities sufficient for larger clinical trials and commercialization. We report here a highly efficient method for the overexpression and purification of recombinant MPT64 from Escherichia coli (rMPT64) based upon a mild insolubility of rMPT64 following induction, and scalable anion-exchange and gel filtration chromatographies. Yields of protein were improved substantially to approximately 250 mg/L, and resulted in a preparation greater than 98% pure. Quantitative release assays were developed and used with MALDI-TOF mass spectrometry to confirm the identity of rMPT64. Using a guinea pig model of active TB, we found that rMPT64 elicited a specific immune response indistinguishable from that of MPB64 purified from BCG Tokyo culture filtrates. These results describe the first efficient and scalable protocol for production of rMPT64, demonstrate its activity in an animal model of active TB, and lay the foundation of ongoing and future use of the PTAT in clinical settings.

  3. Immune protection conferred by recombinant MRLC (myosin regulatory light chain) antigen in TiterMax Gold® adjuvant against experimental fasciolosis in rats.

    PubMed

    Henker, Luan C; Schwertz, Claiton I; Lucca, Neuber J; Piva, Manoela M; Prior, Keila C; Baska, Piotr; Norbury, Luke; Januszkiewicz, Kamil; Dezen, Diogenes; Duarte, Marta M M F; Moresco, Rafael N; Bertagnolli da Rosa, Liana; Mendes, Ricardo E

    2017-01-23

    Protection against experimental fasciolosis in rats immunized with recombinant myosin regulatory light chain (MRLC) in TiterMax Gold® adjuvant was assessed. The experimental trial consisted of four groups of 15 animals; group 1 was unimmunized and infected, group 2 was immunized with MRLC in adjuvant and infected, group 3 was infected and immunized with adjuvant only and group 4 was unimmunized and uninfected. Immunization with MRLC in TiterMax Gold® adjuvant (group 2) induced a reduction in fluke burdens of 51.0% (p<0.001) when compared with the adjuvant control group, and 61.5% (p<0.001) when compared with the unimmunized infected controls. There was a reduction in fecal egg output in group 2 of 44.8% and 37.3% compared with group 1 and group 3, respectively; although this difference was not statistically significant. Measurement of cytokine levels revealed higher levels of TNF-alpha and IL-2 as well as lower levels of IL-4 in group 2 during the chronic stage of infection (p<0.05), along with higher levels of IFN-gamma during early stages of infection (p<0.05). These results suggest a mixed Th1/Th2 phenotype immune response; however predominance of Th1 cytokines was observed. Levels of anti-MRLC serum IgG in group 2 were significantly higher than controls at the time of euthanasia (p<0.05). This is the first report of immunization with recombinant MRLC in rats, demonstrating that this antigen significantly reduces fluke burdens, increases the Th1 immune response and encourages further studies to improve the vaccine's efficacy.

  4. Whole recombinant Pichia pastoris expressing HPV16 L1 antigen is superior in inducing protection against tumor growth as compared to killed transgenic Leishmania

    PubMed Central

    Bolhassani, Azam; Muller, Martin; Roohvand, Farzin; Motevalli, Fatemeh; Agi, Elnaz; Shokri, Mehdi; Rad, Mahdieh Motamedi; Hosseinzadeh, Sahar

    2015-01-01

    The development of an efficient vaccine against high-risk HPV types can reduce the incidence rates of cervical cancer by generating anti-tumor protective responses. Traditionally, the majority of prophylactic viral vaccines are composed of live, attenuated or inactivated viruses. Among them, the design of an effective and low-cost vaccine is critical. Inactivated vaccines especially heat-killed yeast cells have emerged as a promising approach for generating antigen-specific immunotherapy. Recent studies have indicated that yeast cell wall components possess adjuvant activities. Moreover, a non-pathogenic protozoan, Leishmania tarentolae (L.tar) has attracted a great attention as a live candidate vaccine. In current study, immunological and protective efficacy of whole recombinant killed Pichia pastoris and Leishmania tarentolae expressing HPV16 L1 capsid protein was evaluated in tumor mice model. We found that Pichia-L1, L.tar-L1 and Gardasil groups increase the IgG2a/IgG1 ratio, indicating a relative preference for the induction of Th1 immune responses. Furthermore, subcutaneous injection of killed Pichia-L1 generated the significant L1-specific IFN-γ immune response as well as the best protective effects in vaccinated mice as compared to killed L.tar-L1, killed Pichia pastoris, killed L.tar and PBS groups. Indeed, whole recombinant Leishmania tarentolae could not protect mice against C3 tumor mice model. These data suggest that Pichia-L1 may be a candidate for the control of HPV infections. PMID:25668661

  5. Enhancing recovery of recombinant hepatitis B surface antigen in lab-scale and large-scale anion-exchange chromatography by optimizing the conductivity of buffers.

    PubMed

    Mojarrad Moghanloo, Gol Mohammad; Khatami, Maryam; Javidanbardan, Amin; Hosseini, Seyed Nezamedin

    2018-01-01

    In biopharmaceutical science, ion-exchange chromatography (IEC) is a well-known purification technique to separate the impurities such as host cell proteins from recombinant proteins. However, IEC is one of the limiting steps in the purification process of recombinant hepatitis B surface antigen (rHBsAg), due to its low recovery rate (<50%). In the current study, we hypothesized that ionic strengths of IEC buffers are easy-to-control parameters which can play a major role in optimizing the process and increasing the recovery. Thus, we investigated the effects of ionic strengths of buffers on rHBsAg recovery via adjusting Tris-HCl and NaCl concentrations. Increasing the conductivity of equilibration (Eq.), washing (Wash.) and elution (Elut.) buffers from their initial values of 1.6 mS/cm, 1.6 mS/cm, and 7.0 mS/cm to 1.6 mS/cm, 7 mS/cm and 50 mS/cm, respectively yielded an average recovery rate of 82% in both lab-scale and large-scale weak anion-exchange chromatography without any harsh effect on the purity percentage of rHBsAg. The recovery enhancement via increasing the conductivity of Eq. and Wash. buffers can be explained by their roles in reducing the binding strength and aggregation of retained particles in the column. Moreover, further increase in the salt concentration of Elut. Buffer could substantially promote the ion exchange process and the elution of retained rHBsAg. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen

    PubMed Central

    Gurramkonda, Chandrasekhar; Adnan, Ahmad; Gäbel, Thomas; Lünsdorf, Heinrich; Ross, Anton; Nemani, Satish Kumar; Swaminathan, Sathyamangalam; Khanna, Navin; Rinas, Ursula

    2009-01-01

    Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries. PMID:19208244

  7. Recombinant interferon gamma up-regulates in vivo and down-regulates in vitro monocyte CD14 antigen expression in cancer patients.

    PubMed

    Landmann, R; Wesp, M; Ludwig, C; Obrist, R; Knüsli, C; Obrecht, J P

    1990-01-01

    The expression of the monocyte membrane glycoprotein CD14 was measured and related to the serum interferon gamma (IFN gamma) concentration in thirteen patients with disseminated cancer during treatment with human recombinant interferon gamma (rIFN gamma). The drug was administered by continuous subcutaneous infusion using an escalating dose schedule, starting at 50 micrograms/day or 100 micrograms/day and increasing weekly up to 600 micrograms/day, if tolerated. Treatment was continued at a mean maximal tolerated dose of 200 micrograms/day for a median duration of 43 days. Serum IFN gamma concentration and monocyte CD14 antigen expression (immunofluorescence with the monoclonal antibody LeuM3 and fluorescence-activated cell sorting analysis) were determined weekly. The serum IFN gamma concentration was positively correlated with the rIFN gamma dose (P less than 0.05). Therapy induced a dose-dependent enhancement of CD14 antigen expression. The increase in mean CD14 fluorescence intensity was on average 60% after 3 weeks of treatment at a mean dose of 220 micrograms rIFN gamma/day and was reversed after withdrawal of therapy. Patients with a rapidly rising serum IFN gamma concentration (starting dose 100 micrograms/day) showed a smaller increment in CD14 fluorescence intensity than those with slowly rising serum IFN gamma levels (starting dose 50 micrograms/day). Since rIFN gamma is known to down-regulate CD14 antigen expression in vitro, monocytes from patients off therapy and from healthy volunteers were cultured with this cytokine. A similar decrease of CD14 fluorescence was observed in both groups. In patients several factors, such as IFN gamma concentration, duration of drug effect and type of serum, were evaluated and could not explain the discrepant in vivo and in vitro findings. In conclusion, the monocyte marker CD14 was found to be differentially regulated by rIFN gamma in vivo and in vitro. In vivo, secondary mediators, induced by rIFN gamma and acting on

  8. Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid.

    PubMed Central

    Mayer, L W; Aasted, B; Garon, C F; Bloom, M E

    1983-01-01

    Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization. Images PMID:6313959

  9. Recombinant antigens based on toxins A and B of Clostridium difficile that evoke a potent toxin-neutralising immune response.

    PubMed

    Maynard-Smith, Michael; Ahern, Helen; McGlashan, Joanna; Nugent, Philip; Ling, Roger; Denton, Harriet; Coxon, Ruth; Landon, John; Roberts, April; Shone, Clifford

    2014-02-03

    Infection with the bacterium Clostridium difficile causes symptoms ranging from mild to severe diarrhoea with life-threatening complications and remains a significant burden to healthcare systems throughout the developed world. Two potent cytotoxins, TcdA and TcdB are the prime mediators of the syndrome and rapid neutralisation of these would afford significant benefits in disease management. In the present study, a broad range of non-toxic, recombinant fragments derived from TcdA and TcdB were designed for soluble expression in E. coli and assessed for their capacity to generate a potent toxin-neutralising immune response as assessed by cell-based assays. Significant differences between the efficacies of isolated TcdA and TcdB regions with respect to inducing a neutralising immune response were observed. While the C-terminal repeat regions played the principal role in generating neutralising antibodies to TcdA, in the case of TcdB, the central region domains dominated the neutralising immune response. For both TcdA and TcdB, fragments which comprised domains from both the central and C-terminal repeat region of the toxins were found to induce the most potent neutralising immune responses. Generated antibodies neutralised toxins produced by a range of C. difficile isolates including ribotype 027 and 078 strains. Passive immunisation of hamsters with a combination of antibodies to TcdA and TcdB fragments afforded complete protection from severe CDI induced by a challenge of bacterial spores. The results of the study are discussed with respect to the development of a cost effective immunotherapeutic approach for the management of C. difficile infection.

  10. Intrarectal vaccination with recombinant vaccinia virus expressing carcinoembronic antigen induces mucosal and systemic immunity and prevents progression of colorectal cancer.

    PubMed

    Kim-Schulze, Seunghee; Kim, Hong Sung; Wainstein, Alberto; Kim, Dae Won; Yang, Wein Cui; Moroziewicz, Dorota; Mong, Phyllus Y; Bereta, Michal; Taback, Bret; Wang, Qin; Kaufman, Howard L

    2008-12-01

    The gastrointestinal mucosa contains an intact immune system that protects the host from pathogens and communicates with the systemic immune system. Absorptive epithelial cells in the mucosa give rise to malignant tumors although the interaction between tumor cells and the mucosal immune system is not well defined. The pathophysiology of colorectal cancer has been elucidated through studies of hereditary syndromes, such as familial adenomatous polyposis, a cancer predisposition syndrome caused by germline mutations in the adenomatous polyposis coli tumor suppressor gene. Patients with FAP develop adenomas and inevitably progress to invasive carcinomas by the age of 40. To better delineate the role of mucosal immunity in colorectal cancer, we evaluated the efficacy of intrarectal recombinant vaccinia virus expressing the human carcinoembryonic Ag (CEA) in a murine FAP model in which mice are predisposed to colorectal cancer and also express human CEA in the gut. Mucosal vaccination reduced the incidence of spontaneous adenomas and completely prevented progression to invasive carcinoma. The therapeutic effects were associated with induction of mucosal CEA-specific IgA Ab titers and CD8(+) CTLs. Mucosal vaccination was also associated with an increase in systemic CEA-specific IgG Ab titers, CD4(+) and CD8(+) T cell responses and resulted in growth inhibition of s.c. implanted CEA-expressing tumors suggesting communication between mucosal and systemic immune compartments. Thus, intrarectal vaccination induces mucosal and systemic antitumor immunity and prevents progression of spontaneous colorectal cancer. These results have implications for the prevention of colorectal cancer in high-risk individuals.

  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. Production, characterization, and application of monoclonal antibodies specific to recombinant (E2) structural protein in antigen-capture ELISA for clinical diagnosis of Chikungunya virus.

    PubMed

    Kumar, Jyoti S; Khan, Mohsin; Gupta, Garima; Bhoopati, Manna; Lakshmana Rao, P V; Parida, Manmohan

    2012-04-01

    The resurgence of Chikungunya (CHIK) virus in the form of an explosive, unprecedented epidemic with high virulence and unusual numbers of fatalities has created an immense public health concern in recent years. In the absence of an effective vaccine and specific antiviral therapy, early accurate diagnosis is essential for the best patient management. The present study describes the production and characterization of high-affinity and selective monoclonal antibodies (Mabs) against recombinant E2 protein (rE2) of the CHIK virus. The reactivity of Mabs for rE2 protein was demonstrated using ELISA. The specificity of the generated Mabs for rE2 was demonstrated by Western blot and indirect immunofluorescence. The application of this CHIK virus E2-specific monoclonal antibody in early clinical diagnosis was demonstrated by various analytical methods, such as immunoblotting, indirect immunofluorescence assay (IFA), and antigen-capture ELISA (AC-ELISA), for the detection as well as the identification of the novel ECSA genotypes of CHIK virus. These findings suggest that the high-affinity E2-specific monoclonal antibodies reported in this study will be useful for early clinical diagnosis and epidemiological studies of CHIK virus in developing countries.

  13. Usefulness of Four Different Echinococcus granulosus Recombinant Antigens for Serodiagnosis of Unilocular Hydatid Disease (UHD) and Postsurgical Follow-Up of Patients Treated for UHD▿

    PubMed Central

    Hernández-González, Ana; Muro, Antonio; Barrera, Inmaculada; Ramos, Guillermo; Orduña, Antonio; Siles-Lucas, Mar

    2008-01-01

    Four different recombinant antigens derived from Echinococcus granulosus, designated B1t, B2t, E14t, and C317, were tested with enzyme-linked immunosorbent assays (ELISAs) for the detection of specific immunoglobulin G (IgG) in patients with unilocular hydatid disease (UHD). The results were compared to those obtained with hydatid fluid and were subjected to receiver operator characteristic analysis. The diagnostic performance of the above-listed proteins was defined with respect to their specificity, sensitivity, and predictive values (PV); the influence of cyst location; and usefulness in the follow-up of surgical treatment for UHD and in the determination of whether or not patients have been surgically cured of UHD. The best diagnostic results were obtained with the anti-B2t IgG ELISA, with 91.2% sensitivity, 93% specificity, and high positive and negative PV (89.4 and 94.2, respectively). In addition, this diagnostic tool proved to be useful for the follow-up of surgically treated UHD patients. The anti-B2t IgG ELISA may find an application in the serodiagnosis of UHD in clinical laboratories. PMID:17989342

  14. Development of EMA-2 recombinant antigen based enzyme-linked immunosorbent assay for seroprevalence studies of Theileria equi infection in Indian equine population.

    PubMed

    Kumar, Sanjay; Kumar, Rajender; Gupta, Ashok K; Yadav, Suresh C; Goyal, Sachin K; Khurana, Sandip K; Singh, Raj K

    2013-11-15

    Equine piroplasmosis is a tick-transmitted protozoan disease caused by Theileria equi and/or Babesia caballi. In the present study, we expressed a 53kDa protein from the truncated EMA-2 gene of T. equi (Indian strain) and developed EMA-2ELISA using this expressed protein. This ELISA is able to detect T. equi-specific antibodies in experimentally infected animals as early as 9 days post-infection. The assay developed was validated with the OIE recommended competitive ELISA (cELISA) on 120 serum samples and significant agreement (kappa=0.93) was observed between results of both the ELISAs which indicates suitability of EMA-2ELISA for use in sero-diagnosis. Diagnostic sensitivity and specificity of EMA-2ELISA - as compared with cELISA - were 0.97 and 0.96, respectively. Analysis of 5651 equine serum samples - collected during 2007-2012 from 12 states of India representing eight agro-climatic zones - by EMA-2ELISA revealed 32.65% seroprevalence of T. equi in India. In conclusion, the EMA-2ELISA developed using the T. equi EMA-2 recombinant protein as antigen for detecting T. equi-specific antibodies has good diagnostic potential for sero-epidemiological surveys.

  15. Development of an enzyme-linked immunoelectrotransfer blot (EITB) assay using two baculovirus expressed recombinant antigens for diagnosis of Taenia solium taeniasis.

    PubMed

    Levine, Min Z; Lewis, Melissa M; Rodriquez, Silvia; Jimenez, Juan A; Khan, Azra; Lin, Sehching; Garcia, Hector H; Gonzales, Armando E; Gilman, Robert H; Tsang, Victor C W

    2007-04-01

    Taeniasis diagnosis is an important step in the control and elimination of both cysticercosis and taeniasis. We report the development of 2 serological taeniasis diagnostic tests using recombinant antigens rES33 and rES38 expressed by baculovirus in insect cells in an EITB format. In laboratory testing with defined sera from nonendemic areas, rES33 has a sensitivity of 98% (n = 167) and a specificity of 99% (n = 310) (J index: 0.97); rES38 has a sensitivity of 99% (n = 146) and a specificity of 97% (n = 275) (J index: 0.96). Independent field testing in Peru showed 97% (n = 203) of the taeniasis sera were positive with rES33, and 100% of the nontaeniasis sera (n = 272) were negative with rES33; 98% (n = 198) of taeniasis sera were positive with rES38, and 91% (n = 274) of the nontaeniasis sera were negative with rES38. Among the Peruvian sera tested, 17 of 26 Peruvian Taenia saginata sera were false positive with rES38 test. Both tests were also examined with cysticercosis sera, with a positive rate ranging from 21% to 46%. rES33 and rES38 tests offer sensitive and specific diagnosis of taeniasis and easy sample collection through finger sticks that can be used in large-scale studies. They are currently being used in cysticercosis elimination programs in Peru.

  16. Serodiagnosis of equine infectious anemia by agar gel immunodiffusion and ELISA using a recombinant p26 viral protein expressed in Escherichia coli as antigen.

    PubMed

    Piza, Adriana S Toledo; Pereira, Alessandra Rael; Terreran, Maria Thereza; Mozzer, Otto; Tanuri, Amílcar; Brandão, Paulo Eduardo; Richtzenhain, Leonardo José

    2007-03-17

    We used a p26 recombinant protein (p26r) from equine infectious-anemia virus (EIAV) expressed in Escherichia coli as antigen to standardize an agar-gel immunodiffusion (AGIDp26r) test and an indirect ELISA (ELISAp26r) for the detection of antibodies against EIAV in 720 equine sera from Brazil. We evaluated the tests's relative diagnostic sensitivities (relSe) and relative diagnostic specificities (relSp) against a commercial AGID kit (Idexx, USA). We used three sera panels: panel A--196 AGID-negative sera from an AIE non-endemic controlled area; panel B--194 AGID-negative sera from an AIE endemic area and panel C--330 AGID-positive sera from an AIE endemic area. ELISAp26r cut-off value was defined with TG-ROC using sera from panels A and C. AGIDp26r showed an agreement of 100% with the commercial kit. When applied to sera from panels A and C, ELISAp26r showed an agreement of 100% with the kit, but, although relSe was 100% for panel C, the ELISAp26r had relSp of 93.3%.

  17. Standardization and validation of an agar gel immunodiffusion test for the diagnosis of equine infectious anemia using a recombinant p26 antigen.

    PubMed

    Alvarez, I; Gutierrez, G; Vissani, A; Rodriguez, S; Barrandeguy, M; Trono, K

    2007-04-15

    We developed and validated an agar gel immunodiffusion test (AGID) test for the diagnosis of equine infectious anemia (EIA) using as antigen the p26 protein of equine infectious anemia virus (EIAV) produced in the Escherichia coli expression system. The developed rp26-AGID test showed an excellent diagnostic relative sensitivity (100%) and specificity (100%) compared to a commercial AGID assay when 1855 field serum samples were analyzed. In addition, the rp26-AGID demonstrated to be a precise assay with excellent repeatability and reproducibility. In the analytical sensitivity trial, positive sera showed nearly the same endpoint dilutions for both compared tests. No positive-reactions were observed with 35 serum samples with antibodies related to other endemic agents and also with severely hemolysed samples, demonstrating that the rp26-AGID has an excellent analytical specificity. Complete concordance with blind previous results from five proficiency test panels confirmed the capability of the assay of accurate detection of EIAV antibodies. This is the first time that a recombinant AGID assay able to identify EIAV infections has been standardized and validated in Argentina according to international guidelines. Taking into account the results obtained, the p26-AGID could be adopted as an official test method for the diagnosis and control of EIA in this country.

  18. Production of recombinant antigens and antibodies in Nicotiana benthamiana using 'magnifection' technology: GMP-compliant facilities for small- and large-scale manufacturing.

    PubMed

    Klimyuk, Victor; Pogue, Gregory; Herz, Stefan; Butler, John; Haydon, Hugh

    2014-01-01

    This review describes the adaptation of the plant virus-based transient expression system, magnICON(®) for the at-scale manufacturing of pharmaceutical proteins. The system utilizes so-called "deconstructed" viral vectors that rely on Agrobacterium-mediated systemic delivery into the plant cells for recombinant protein production. The system is also suitable for production of hetero-oligomeric proteins like immunoglobulins. By taking advantage of well established R&D tools for optimizing the expression of protein of interest using this system, product concepts can reach the manufacturing stage in highly competitive time periods. At the manufacturing stage, the system offers many remarkable features including rapid production cycles, high product yield, virtually unlimited scale-up potential, and flexibility for different manufacturing schemes. The magnICON system has been successfully adaptated to very different logistical manufacturing formats: (1) speedy production of multiple small batches of individualized pharmaceuticals proteins (e.g. antigens comprising individualized vaccines to treat NonHodgkin's Lymphoma patients) and (2) large-scale production of other pharmaceutical proteins such as therapeutic antibodies. General descriptions of the prototype GMP-compliant manufacturing processes and facilities for the product formats that are in preclinical and clinical testing are provided.

  19. A novel ELISA using a recombinant outer membrane protein, rTp0663, as the antigen for serological diagnosis of syphilis.

    PubMed

    Xu, Man; Xie, Yafeng; Jiang, Chuanhao; Xiao, Yongjian; Kuang, Xingxing; Zhao, Feijun; Zeng, Tiebing; Liu, Shuangquan; Liang, Mingxing; Li, Li; Wang, Chuan; Wu, Yimou

    2016-02-01

    The lack of Treponema pallidum-specific antigens with highly accurate diagnosis makes the diagnosis of syphilis challenging. A soluble recombinant version of a new diagnostic protein Tp0663 has been produced. The serodiagnostic potential of this protein was assessed by screening 3326 serum samples simultaneously evaluated by rapid plasma reagin and T. pallidum particle agglutination tests. Kappa (κ) coefficients were used to compare the concordance between clinical diagnosis and the Tp0663-based ELISA or the ARCHITECT Syphilis TP chemiluminescent immunoassay (Abbott GmbH and Co. KG). Using the results of clinical diagnosis as the gold standard, the sensitivity and specificity of Tp0663 were found to be 98.83% (95% confidence interval (CI) 96.61-99.60%) and 100% (95% CI 99.88-100%), respectively. In comparison, the ARCHITECT Syphilis TP assay was found to have a lower sensitivity (97.27%, 95% CI 94.46-98.67%) and specificity (99.61%, 95% CI 99.32-99.78%). In particular, the ARCHITECT Syphilis TP exhibited a false-positive rate of 0.39%. Moreover, the ELISA was in perfect agreement with the gold standard, with a κ value of 0.99, comparable to that of ARCHITECT Syphilis TP (0.96). These results identified Tp0663 as a novel serodiagnostic candidate with great potential for developing novel tests for the diagnosis of syphilis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. 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

  1. A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

    PubMed

    Drapała, Dorota; Holec-Gąsior, Lucyna; Kur, Józef; Ferra, Bartłomiej; Hiszczyńska-Sawicka, Elżbieta; Lautenbach, Dariusz

    2014-07-01

    The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection.

  2. Immunogenic Salivary Proteins of Triatoma infestans: Development of a Recombinant Antigen for the Detection of Low-Level Infestation of Triatomines

    PubMed Central

    Schwarz, Alexandra; Helling, Stefan; Collin, Nicolas; Teixeira, Clarissa R.; Medrano-Mercado, Nora; Hume, Jen C. C.; Assumpção, Teresa C.; Marcus, Katrin; Stephan, Christian; Meyer, Helmut E.; Ribeiro, José M. C.; Billingsley, Peter F.; Valenzuela, Jesus G.; Sternberg, Jeremy M.; Schaub, Günter A.

    2009-01-01

    Background Triatomines are vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The most effective vector, Triatoma infestans, has been controlled successfully in much of Latin America using insecticide spraying. Though rarely undertaken, surveillance programs are necessary in order to identify new infestations and estimate the intensity of triatomine bug infestations in domestic and peridomestic habitats. Since hosts exposed to triatomines develop immune responses to salivary antigens, these responses can be evaluated for their usefulness as epidemiological markers to detect infestations of T. infestans. Methodology/Principal Findings T. infestans salivary proteins were separated by 2D-gel electrophoresis and tested for their immunogenicity by Western blotting using sera from chickens and guinea pigs experimentally exposed to T. infestans. From five highly immunogenic protein spots, eight salivary proteins were identified by nano liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) and comparison to the protein sequences of the National Center for Biotechnology Information (NCBI) database and expressed sequence tags of a unidirectionally cloned salivary gland cDNA library from T. infestans combined with the NCBI yeast protein sub-database. The 14.6 kDa salivary protein [gi|149689094] was produced as recombinant protein (rTiSP14.6) in a mammalian cell expression system and recognized by all animal sera. The specificity of rTiSP14.6 was confirmed by the lack of reactivity to anti-mosquito and anti-sand fly saliva antibodies. However, rTiSP14.6 was recognized by sera from chickens exposed to four other triatomine species, Triatoma brasiliensis, T. sordida, Rhodnius prolixus, and Panstrongylus megistus and by sera of chickens from an endemic area of T. infestans and Chagas disease in Bolivia. Conclusions/Significance The recombinant rTiSP14.6 is a suitable and promising epidemiological marker for

  3. Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications.

    PubMed

    Lai, Guan-Hua; Lin, Ming-Kuem; Lien, Yi-Yang; Fu, Jiun-Hau; Chen, Hsi-Jien; Huang, Chi-Hung; Tzen, Jason T C; Lee, Meng-Shiou

    2013-08-13

    Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other subunit proteins

  4. A new ELISA kit which uses a combination of Plasmodium falciparum extract and recombinant Plasmodium vivax antigens as an alternative to IFAT for detection of malaria antibodies

    PubMed Central

    Doderer, Cecile; Heschung, Aurelie; Guntz, Phillippe; Cazenave, Jean-Pierre; Hansmann, Yves; Senegas, Alexandre; Pfaff, Alexander W; Abdelrahman, Tamer; Candolfi, Ermanno

    2007-01-01

    Background The methods most commonly used to measure malarial antibody titres are the Indirect Fluorescence Antibody Test (IFAT), regarded as the gold standard, and the Enzyme-Linked ImmunoSorbent Assay (ELISA). The objective here was to assess the diagnostic performance, i.e. the sensitivity and specificity, of a new malaria antibody ELISA kit in comparison to IFAT. This new ELISA kit, the ELISA malaria antibody test (DiaMed), uses a combination of crude soluble Plasmodium falciparum extract and recombinant Plasmodium vivax antigens. Methods Two groups were used: 95 samples from malaria patients to assess the clinical sensitivity and 2,152 samples from blood donors, who had not been exposed to malaria, to assess the clinical specificity. Results The DiaMed ELISA test kit had a clinical sensitivity of 84.2% and a clinical specificity of 99.6% as compared with 70.5% and 99.6% respectively, using the IFAT method. The ELISA method was more sensitive than the IFAT method for P. vivax infections (75% vs. 25%). However, in 923 malaria risk donors the analytical sensitivity of the ELISA test was 40% and its specificity 98.3%, performances impaired by large numbers of equivocal results non-concordant between ELISA and IFAT. When the overall analytical performances of ELISA was compared to IFAT, the ELISA efficiency J index was 0.84 versus 0.71 for IFAT. Overall analytical sensitivity was 93.1% and the analytical specificity 96.7%. Overall agreement between the two methods reached 0.97 with a reliability k index of 0.64. Conclusion The DiaMed ELISA test kit shows a good correlation with IFAT for analytical and clinical parameters. It may be an interesting method to replace the IFAT especially in blood banks, but further extensive investigations are needed to examine the analytical performance of the assay, especially in a blood bank setting. PMID:17313669

  5. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata).

    PubMed

    SongLin, Guo; PanPan, Lu; JianJun, Feng; JinPing, Zhao; Peng, Lin; LiHua, Duan

    2015-04-01

    The immogenicity of a novel vaccine antigen was evaluated after immunized American eels (Anguilla rostrata) with a recombinant bivalent expressed outer membrane protein (OMP) of Vibrio vulnificus and Aeromonas hydrophila. Three groups of eels were intraperitoneal (i.p) injected with phosphate-buffered saline (PBS group), formaline-killed-whole-cell (FKC) of A. hydrophila and V. vulnificus (FKC group) or the bivalent OMP (OMP group). On 14, 21, 28 and 42 days post-vaccination respectively, proliferation of the whole blood cells, titers of specific antibody and lysozyme activities of experimental eels were detected. On 28 day post-vaccination, eels from three groups were challenged by i.p injection of live A. hydrophila or V. vulnificus. The results showed that, compared with the PBS group, proliferation of whole blood cells in OMP group was significant enhanced on 28 days, and the serum titers of anti-A.hydrophila and anti-V. vulnificus antibody in eels of FKC and OMP group were significant increased on 14, 21 and 28d. Lysozyme Activities in serum, skin mucus, liver and kidney were significant changed between the three groups. Relative Percent Survival (RPS) after challenged A. hydrophila in KFC vs. PBS group and OMP vs. PBS group were 62.5% and 50% respectively, and the RPS challenged V. vulnificus in FKC and OMP vs. PBS group were 37.5% and 50% respectively. These results suggest that American eels immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect against infection by the two pathogens in fresh water farming. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. IFN-γ and IL-2 Responses to Recombinant AlaDH against ESAT-6/CFP-10 Fusion Antigens in the Diagnosis of Latent versus Active Tuberculosis Infection

    PubMed Central

    Movahedi, Bahram; Mokarram, Pooneh; Hemmati, Mina; Mosavari, Nader; Zare, Razie; Ardekani, Leila Safaee; Mostafavi-Pour, Zohreh

    2017-01-01

    Background: Discriminating latent tuberculosis infection (LTBI) from active TBI may be challenging. The objective of this study was to produce the recombinant L-alanine dehydrogenase (AlaDH) antigen and evaluate individuals with LTBI, those with active TBI, and uninfected individuals by enzyme-linked immunospot assay (ELISPOT) in order to distinguish LTBI from active TBI. Methods: This exploratory study was performed in the Iranian city of Shiraz from 2014 to 2015. The study population (N=99) was divided into 3 groups: individuals with newly diagnosed active TBI (n=33), their household contacts (n=33), and controls (n=33). AlaDH was produced through PCR and cloning methods. The diagnostic characteristics of AlaDH vs. ESAT-6/CFP-10 were evaluated in responses to interferon-γ (IFN-γ) and interleukin-2 (IL-2) with ELISPOT. Differences between the groups were assessed with the Kruskal–Wallis and Mann–Whitney tests for nonparametric data analysis. The statistical analyses were performed with SPSS, version 16. Results: IFN-γ responses to both ESAT-6/CFP-10 (P=0.81) and AlaDH (P=0.18) revealed that there were no significant differences between the individuals with LTBI and those with active TBI. The same results were determined for IL-2 responses to ESAT-6/CFP-10 between the 2 groups, while significantly higher IL-2 responses to AlaDH were observed in LTBI than in active TBI. According to the ROC curve analysis, a cutoff value of 275 SFC showed sensitivity of 75.8% and specificity of 78.8% for distinguishing LTBI from active TBI by IL-2 responses to AlaDH. Conclusion: The current study suggests that it may be possible to discriminate LTBI from active TBI by IL-2 responses to AlaDH. PMID:28533576

  7. The Yeast Iron Regulatory Proteins Grx3/4 and Fra2 Form Heterodimeric Complexes Containing a [2Fe-2S] Cluster with Cysteinyl and Histidyl Ligation†

    PubMed Central

    Li, Haoran; Mapolelo, Daphne T.; Dingra, Nin N.; Naik, Sunil G.; Lees, Nicolas S.; Hoffman, Brian M.; Riggs-Gelasco, Pamela J.; Huynh, Boi Hanh; Johnson, Michael K.; Outten, Caryn E.

    2009-01-01

    The transcription of iron uptake and storage genes in S. cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in E. coli. We have shown that co-expression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable [2Fe-2S]2+ cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a [2Fe-2S] cluster on Grx3 or Grx4 without Fra2 produces a [2Fe-2S]-bridged homodimer. UV-visible absorption and CD, resonance Raman, EPR, ENDOR, Mössbauer, and EXAFS studies of [2Fe-2S] Grx3/4 homodimers and the [2Fe-2S] Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a [2Fe-2S] cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast. PMID:19715344

  8. Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems.

    PubMed

    Smith, Mark L; Keegan, Mark E; Mason, Hugh S; Shuler, Michael L

    2002-01-01

    The expression of vaccine antigens in edible plant material together with their delivery by the oral route constitutes a powerful paradigm, with the potential to dramatically reduce the cost of vaccine production and administration, in addition to improving distribution and patient compliance. These products will be subject to many of the same regulations applied to current injectable vaccines, so reliable methods to quantify antigen and ensure stability in crude plant extracts are required. As a model system the hepatitis B surface antigen (HBsAg) was expressed in soybean and tobacco cell cultures. This complex antigen consists of membrane-associated small surface antigen proteins (p24(s)), disulfide cross-linked to yield dimers and higher multimers. Although the total p24(s) extracted from plant cells was relatively unaffected by detergent concentration, the quantification of antigenically reactive product depended strongly on the ratio of detergent to cell concentration. Furthermore, 1-20% w/v sodium ascorbate improved the measured levels of monoclonal-reactive antigen 4- to 12-fold. Detergent also influenced antigen stability in cell lysates stored at 4 degrees C; under optimum conditions stability was maintained for at least 1 month, whereas excess detergent rendered the antigen susceptible to proteolytic degradation. This proteolysis could be counteracted by the addition of skim milk or its protein component, which stabilized antigenically reactive p24(s) for up to 2 months. The immunologically relevant epitopes of HBsAg are critically dependent on disulfide bonding. By altering the sodium ascorbate concentration or buffer pH the proportion of HBsAg displaying the monoclonal reactive epitopes was increased between 8- and 20-fold. In addition, under certain conditions the dimerized p24(s) could be converted to oligomeric aggregates, resembling the form of the serum-derived antigen. These simple in vitro manipulations, compatible with the goal of a minimally

  9. Immune responses in mice induced by prime-boost schemes of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1)-based DNA, protein and recombinant modified vaccinia Ankara vaccines.

    PubMed

    Miao, Jun; Li, Xun; Liu, Zhongxiang; Xue, Caifang; Bujard, Hermann; Cui, Liwang

    2006-09-11

    The apical membrane antigen 1 (AMA1) of malaria parasites is a leading vaccine candidate. Its expression in merozoites and sporozoites and its importance for erythrocyte and hepatocyte invasion underline the significance of both humoral and cellular immunities against this antigen in malaria protection. We have generated a DNA construct and a recombinant poxvirus (rMVA) for expressing the Plasmodium falciparum AMA1 ectodomain, produced recombinant AMA1 protein (rAMA1) and evaluated their antigenicity in mice using single and combinatory vaccine schemes. Our results showed that although vaccinations of mice by either DNA or rMVA alone did not yield high antibody responses, they had primed significant numbers of rAMA1-responsive splenocytes. Under heterologous prime-boost schemes, priming with DNA followed by boosting with rMVA or rAMA1 protein resulted in a significant increase in antibody titers. In addition, the antibody titers to AMA1 appeared to be correlated with the levels of inhibition of merozoite invasion of erythrocytes in vitro. Furthermore, different prime-boost schemes resulted in different AMA1-specific antibody isotype (IgG1/IgG2a) ratios, providing us with an indication about Th1 or Th2 responses the vaccination regimens have induced. This study has yielded useful information for further in vivo evaluation of the suitability and effectiveness of the heterologous prime-boost strategy in AMA1 vaccination.

  10. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant

    PubMed Central

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant. PMID:25750999

  11. Oral administration of recombinant Neisseria meningitidis PorA genetically fused to H. pylori HpaA antigen increases antibody levels in mouse serum, suggesting that PorA behaves as a putative adjuvant.

    PubMed

    Vasquez, Abel E; Manzo, Ricardo A; Soto, Daniel A; Barrientos, Magaly J; Maldonado, Aurora E; Mosqueira, Macarena; Avila, Anastasia; Touma, Jorge; Bruce, Elsa; Harris, Paul R; Venegas, Alejandro

    2015-01-01

    The Neisseria meningitidis outer membrane protein PorA from a Chilean strain was purified as a recombinant protein. PorA mixed with AbISCO induced bactericidal antibodies against N. meningitidis in mice. When PorA was fused to the Helicobacter pylori HpaA antigen gene, the specific response against H. pylori protein increased. Splenocytes from PorA-immunized mice were stimulated with PorA, and an increase in the secretion of IL-4 was observed compared with that of IFN-γ. Moreover, in an immunoglobulin sub-typing analysis, a substantially higher IgG1 level was found compared with IgG2a levels, suggesting a Th2-type immune response. This study revealed a peculiar behavior of the purified recombinant PorA protein per se in the absence of AbISCO as an adjuvant. Therefore, the resistance of PorA to proteolytic enzymes, such as those in the gastrointestinal tract, was analyzed, because this is an important feature for an oral protein adjuvant. Finally, we found that PorA fused to the H. pylori HpaA antigen, when expressed in Lactococcus lactis and administered orally, could enhance the antibody response against the HpaA antigen approximately 3 fold. These observations strongly suggest that PorA behaves as an effective oral adjuvant.

  12. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

    PubMed

    Rhee, Elizabeth G; Mendez, Susana; Shah, Javeed A; Wu, Chang-you; Kirman, Joanna R; Turon, Tara N; Davey, Dylan F; Davis, Heather; Klinman, Dennis M; Coler, Rhea N; Sacks, David L; Seder, Robert A

    2002-06-17

    CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

  13. Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

    PubMed

    Logan, Michael; Law, John; Wong, Jason Alexander Ji-Xhin; Hockman, Darren; Landi, Amir; Chen, Chao; Crawford, Kevin; Kundu, Juthika; Baldwin, Lesley; Johnson, Janelle; Dahiya, Anita; LaChance, Gerald; Marcotrigiano, Joseph; Law, Mansun; Foung, Steven; Tyrrell, Lorne; Houghton, Michael

    2017-01-01

    A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography.

  14. Estrogen reduces aldosterone, upregulates adrenal angiotensin II AT2 receptors and normalizes adrenomedullary Fra-2 in ovariectomized rats.

    PubMed

    Macova, Miroslava; Armando, Ines; Zhou, Jin; Baiardi, Gustavo; Tyurmin, Dmitri; Larrayoz-Roldan, Ignacio M; Saavedra, Juan M

    2008-01-01

    We studied the effect of ovariectomy and estrogen replacement on expression of adrenal angiotensin II AT1 and AT2 receptors, aldosterone content, catecholamine synthesis, and the transcription factor Fos-related antigen 2 (Fra-2). Ovariectomy increased AT1 receptor expression in the adrenal zona glomerulosa and medulla, and decreased adrenomedullary catecholamine content and Fra-2 expression when compared to intact female rats. In the zona glomerulosa, estrogen replacement normalized AT1 receptor expression, decreased AT1B receptor mRNA, and increased AT2 receptor expression and mRNA. Estrogen treatment decreased adrenal aldosterone content. In the adrenal medulla, the effects of estrogen replacement were: normalized AT1 receptor expression, increased AT2 receptor expression, AT2 receptor mRNA, and tyrosine hydroxylase mRNA, and normalized Fra-2 expression and catecholamine content. We demonstrate that the constitutive adrenal expression of AT1 receptors, catecholamine synthesis and Fra-2 expression are partially under the control of reproductive hormones. Our results suggest that estrogen treatment decreases aldosterone production through AT1 receptor downregulation and AT2 receptor upregulation. AT2 receptor upregulation and modulation of Fra-2 expression may participate in the estrogen-dependent normalization of adrenomedullary catecholamine synthesis in ovariectomized rats. The AT2 receptor upregulation and the decrease in AT1 receptor function and in the production of the fluid-retentive, pro-inflammatory hormone aldosterone partially explain the protective effects of estrogen therapy. Copyright 2008 S. Karger AG, Basel.

  15. Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

    PubMed

    Li, Wu; Li, Min; Deng, Guangcun; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2015-08-01

    Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection.

  16. Comparative evaluation of immunization with recombinant protein and plasmid DNA vaccines of fusion antigen ROP2 and SAG1 from Toxoplasma gondii in mice: cellular and humoral immune responses.

    PubMed

    Li, Wen-Shu; Chen, Qing-Xin; Ye, Ju-Xiu; Xie, Zi-Xin; Chen, Jun; Zhang, Li-Fang

    2011-09-01

    The aim of this work was to evaluate immune responses in BALB/c mice vaccinated subcutaneously by recombinant protein, or intramuscularly by plasmid DNA with fusion antigen of rhoptry protein 2 (ROP2) and major surface protein 1 (SAG1) from Toxoplasma gondii (T. gondii). BALB/c mice were immunized with one of three different antigen formulations respectively, which were rROP2-SAG1, pcROP2-SAG1, and pcROP2-SAG1 boosted with rROP2-SAG1. The production of IgG, IgG subclasses, lymphoproliferation, and level of gamma interferon (IFN-γ) were detected after vaccination. The animals vaccinated with rROP2-SAG1 quickly developed specific anti-TLA (T. gondii lysate antigen) antibodies, which continued to rise after immunization. However, production of IgG against TLA in mice vaccinated with pcROP2-SAG1 was relatively slow and maintained a high level after reaching plateau. There are more vigorous specific lymphoproliferative responses observed in mice of group rROP2-SAG1 than in pcROP2-SAG1. Immune responses in mice of group pcROP2-SAG1 boosted with rROP2-SAG1 were similar to the protein immunization group. Three immunization procedures resulted in a similar level of IFN-γ production. Our results indicate that BALB/c mice vaccinated by three immunization procedures induce similar humoral and cellular immunity against infection of T. gondii. Mice immunized with recombinant protein rROP2-SAG1 produce more humoral immune responses than mice immunized with other antigen formulations.

  17. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    PubMed

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (P<0.05). The adjuvant rLTB enhanced the immune response in both fused and co-administered antigens; however, better results were obtained with the chimeric protein. This multi-antigen is a promising vaccine candidate that may help control M. hyopneumoniae infection.

  18. A Combinatory Antibody–Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries

    PubMed Central

    Jansson, Bo; Stuhr-Hansen, Nicolai; Kovács, András; Welinder, Charlotte

    2016-01-01

    We have developed a combinatory antibody–antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a “spot-on-spot” print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation. PMID:28002485

  19. 49 CFR 230.19 - Posting of FRA Form No. 1 and FRA Form No. 3.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION STEAM LOCOMOTIVE INSPECTION AND MAINTENANCE STANDARDS.... 1. The steam locomotive owner and/or operator shall place a copy of the 31 and 92 service day... the cab of the steam locomotive before the inspected boiler is put into service. This FRA Form No. 1...

  20. 49 CFR 230.19 - Posting of FRA Form No. 1 and FRA Form No. 3.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION STEAM LOCOMOTIVE INSPECTION AND MAINTENANCE STANDARDS.... 1. The steam locomotive owner and/or operator shall place a copy of the 31 and 92 service day... the cab of the steam locomotive before the inspected boiler is put into service. This FRA Form No. 1...

  1. 49 CFR 230.19 - Posting of FRA Form No. 1 and FRA Form No. 3.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION STEAM LOCOMOTIVE INSPECTION AND MAINTENANCE STANDARDS.... 1. The steam locomotive owner and/or operator shall place a copy of the 31 and 92 service day... the cab of the steam locomotive before the inspected boiler is put into service. This FRA Form No. 1...

  2. 49 CFR 230.19 - Posting of FRA Form No. 1 and FRA Form No. 3.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION STEAM LOCOMOTIVE INSPECTION AND MAINTENANCE STANDARDS.... 1. The steam locomotive owner and/or operator shall place a copy of the 31 and 92 service day... the cab of the steam locomotive before the inspected boiler is put into service. This FRA Form No. 1...

  3. 49 CFR 230.19 - Posting of FRA Form No. 1 and FRA Form No. 3.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... RAILROAD ADMINISTRATION, DEPARTMENT OF TRANSPORTATION STEAM LOCOMOTIVE INSPECTION AND MAINTENANCE STANDARDS.... 1. The steam locomotive owner and/or operator shall place a copy of the 31 and 92 service day... the cab of the steam locomotive before the inspected boiler is put into service. This FRA Form No. 1...

  4. 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.

  5. Recombinant Ov-ASP-1, a Th1-biased protein adjuvant derived from the helminth Onchocerca volvulus, can directly bind and activate antigen-presenting cells.

    PubMed

    He, Yuxian; Barker, Sophie J; MacDonald, Angus J; Yu, Yu; Cao, Long; Li, Jingjing; Parhar, Ranjit; Heck, Susanne; Hartmann, Susanne; Golenbock, Douglas T; Jiang, Shibo; Libri, Nathan A; Semper, Amanda E; Rosenberg, William M; Lustigman, Sara

    2009-04-01

    We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant.

  6. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    PubMed

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex.

    PubMed

    Lala, Puloma; Higashiyama, Tadayoshi; Erman, Mary; Griswold, Jennifer; Wagner, Traci; Osawa, Yoshio; Ghosh, Debashis

    2004-03-01

    Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2). The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

  8. Heteropentameric Cholera Toxin B Subunit Chimeric Molecules Genetically Fused to a Vaccine Antigen Induce Systemic and Mucosal Immune Responses: a Potential New Strategy To Target Recombinant Vaccine Antigens to Mucosal Immune Systems

    PubMed Central

    Harakuni, Tetsuya; Sugawa, Hideki; Komesu, Ai; Tadano, Masayuki; Arakawa, Takeshi

    2005-01-01

    Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB “molecular buffers” into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design. PMID:16113283

  9. Characterization of a double-CRD-mutated Gal-8 recombinant protein that retains co-stimulatory activity on antigen-specific T-cell response.

    PubMed

    Schroeder, Matías Nicolás; Tribulatti, María Virginia; Carabelli, Julieta; André-Leroux, Gwenaëlle; Caramelo, Julio Javier; Cattaneo, Valentina; Campetella, Oscar

    2016-04-01

    Galectins (Gals) constitute a family of mammalian lectins with affinity for β-galactosides, characterized by the presence of conserved CRDs (carbohydrate-recognition domains). We have found previously that Gal-8, from the tandem-repeat group with two linked CRDs, exerts two separate actions on CD4(+)T-cells: antigen-independent proliferation and, at lower concentration, antigen-specific co-stimulation. Whereas proliferation can be ascribed to the pro-inflammatory role of Gal-8, the co-stimulatory activity of borderline T-cell-specific responses allows the proposal of Gal-8 as an adjuvant in vaccination. To study the relevance of glycan-lectin interaction to these T-cell activities, we generated a double-mutated protein (Gal-8mut) by replacing canonical arginine residues on each CRD, so as to abolish sugar-binding capacity. As expected, Gal-8mut was unable to bind to lactosyl-Sepharose, confirming that lactose recognition was precluded; however, preservation of lectin activity was still evident since Gal-8mut displayed haemoagglutinatory effects and binding capacity to the T-cell surface. To search for glycan affinity, a glycan microarray analysis was conducted which revealed that Gal-8mut lost most low- and intermediate-, but retained high-, affinity interactions, mainly to polylactosamines and blood group antigens. These findings were supported further by molecular modelling. Regarding biological activity, Gal-8mut was unable to induce T-cell proliferation, but efficiently co-stimulated antigen-specific responses, bothin vitroandin vivo.Therefore Gal-8mut represents a useful tool to dissect the specificities of lectin-glycan interactions underlying distinctive Gal-8 activities on T-cell biology. Moreover, given its distinguishing properties, Gal-8mut could be used to enhance borderline immune responses without the non-specific pro-inflammatory activity or other potential adverse effects.

  10. Dogs as alternative intermediate hosts of Taenia solium in Papua (Irian Jaya), Indonesia confirmed by highly specific ELISA and immunoblot using native and recombinant antigens and mitochondrial DNA analysis.

    PubMed

    Ito, A; Putra, M I; Subahar, R; Sato, M O; Okamoto, M; Sako, Y; Nakao, M; Yamasaki, H; Nakaya, K; Craig, P S; Margono, S S

    2002-12-01

    Serology (ELISA and immunoblot) using native glycoproteins, affinity purified glycoproteins, and a recombinant antigen is known to be highly specific to Taenia solium cysticercosis in humans and pigs. These techniques were applied for dogs in the highly endemic area of cysticercosis in Papua (Irian Jaya), Indonesia. Analysis of dog sera by both ELISA and immunoblot revealed 7 of 64 dogs were highly positive. Examination of two sero-positive dogs revealed cysticerci of T. solium in the brain and heart of these dogs. Mitochondrial DNA analysis confirmed that they were the same as T. solium previously confirmed from pigs and biopsies from local people from Irian Jaya. It is suggested that the life cycle of T. solium may be completed not only between humans and pigs but also between humans and dogs.

  11. The Use of Recombinant Pseudotype Virus-Like Particles Harbouring Inserted Target Antigen to Generate Antibodies against Cellular Marker p16INK4A

    PubMed Central

    Lasickienė, Rita; Gedvilaite, Alma; Norkiene, Milda; Simanaviciene, Vaida; Sezaite, Indre; Dekaminaviciute, Dovile; Shikova, Evelina; Zvirbliene, Aurelija

    2012-01-01

    Protein engineering provides an opportunity to generate new immunogens with desired features. Previously,