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Sample records for immunodominant viral epitopes

  1. Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

    PubMed

    Palacios-Rodríguez, Yadira; Gazarian, Tatiana; Rowley, Merrill; Majluf-Cruz, Abraham; Gazarian, Karlen

    2007-02-01

    Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.

  2. Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes.

    PubMed

    Kelly, Christabel; Swadling, Leo; Brown, Anthony; Capone, Stefania; Folgori, Antonella; Salio, Mariolina; Klenerman, Paul; Barnes, Eleanor

    2015-01-01

    Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS3(1073) and NS3(1446), and multiple variants for epitope NS3(1406) that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross-reactivity of vaccine-induced T cells was determined using variant peptides in IFN-γ ELISPOT assays. Vaccine-induced T cells targeted approximately 90% of NS3(1073) genotype 1 sequences and 50% of NS3(1446) genotype 1 and 3 sequences. For NS3(1406), 62% of subtype-1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS3(1406) that was maximally cross-reactive. In vitro priming assays showed that of those tested the NS3(1406) vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross-reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross-reactive T cells at immunodominant epitopes. The degree of cross-reactivity varies between epitopes and may be HCV-subtype specific.

  3. Cross‐reactivity of hepatitis C virus specific vaccine‐induced T cells at immunodominant epitopes

    PubMed Central

    Kelly, Christabel; Swadling, Leo; Brown, Anthony; Capone, Stefania; Folgori, Antonella; Salio, Mariolina; Klenerman, Paul

    2014-01-01

    Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS31073 and NS31446, and multiple variants for epitope NS31406 that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross‐reactivity of vaccine‐induced T cells was determined using variant peptides in IFN‐γ ELISPOT assays. Vaccine‐induced T cells targeted approximately 90% of NS31073 genotype 1 sequences and 50% of NS31446 genotype 1 and 3 sequences. For NS31406, 62% of subtype‐1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS31406 that was maximally cross‐reactive. In vitro priming assays showed that of those tested the NS31406 vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross‐reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross‐reactive T cells at immunodominant epitopes. The degree of cross‐reactivity varies between epitopes and may be HCV‐subtype specific. PMID:25263407

  4. Localization of immunodominant epitopes within the "a" determinant of hepatitis B surface antigen using monoclonal antibodies.

    PubMed

    Golsaz-Shirazi, Forough; Mohammadi, Hamed; Amiri, Mohammad Mehdi; Khoshnoodi, Jalal; Kardar, Gholam Ali; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2016-10-01

    The common "a" determinant is the major immunodominant region of hepatitis B surface antigen (HBsAg) shared by all serotypes and genotypes of hepatitis B virus (HBV). Antibodies against this region are thought to confer protection against HBV and are essential for viral clearance. Mutations within the "a" determinant may lead to conformational changes in this region, which can affect the binding of neutralizing antibodies. There is an increasing concern about identification and control of mutant viruses which is possible by comprehensive structural investigation of the epitopes located within this region. Anti-HBs monoclonal antibodies (mAbs) against different epitopes of HBsAg are a promising tool to meet this goal. In the present study, 19 anti-HBs mAbs were employed to map epitopes localized within the "a" determinant, using a panel of recombinant mutant HBsAgs. The topology of the epitopes was analyzed by competitive enzyme-linked immunosorbent assay (ELISA). Our results indicate that all of the mAbs seem to recognize epitopes within or in the vicinity of the "a" determinant of HBsAg. Different patterns of binding with mutant forms were observed with different mAbs. Amino acid substitutions at positions 123, 126, 129, 144, and 145 dramatically reduced the reactivity of antibodies with HBsAg. The T123N mutation had the largest impact on antibody binding to HBsAg. The reactivity pattern of our panel of mAbs with mutant forms of HBsAg could have important clinical implications for immunoscreening, diagnosis of HBV infection, design of a new generation of recombinant HB vaccines, and immunoprophylaxis of HBV infection as an alternative to therapy with hepatitis B immune globulin (HBIG).

  5. Minute Time Scale Prolyl Isomerization Governs Antibody Recognition of an Intrinsically Disordered Immunodominant Epitope*

    PubMed Central

    Fassolari, Marisol; Chemes, Lucia B.; Gallo, Mariana; Smal, Clara; Sánchez, Ignacio E.; de Prat-Gay, Gonzalo

    2013-01-01

    Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a “hinge” region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t½ ∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 107 m−1 s−1, a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with KD = 1.2 × 10−7 m is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be “locked” in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation. PMID:23504368

  6. Vertical T cell immunodominance and epitope entropy determine HIV-1 escape

    PubMed Central

    Liu, Michael K.P.; Hawkins, Natalie; Ritchie, Adam J.; Ganusov, Vitaly V.; Whale, Victoria; Brackenridge, Simon; Li, Hui; Pavlicek, Jeffrey W.; Cai, Fangping; Rose-Abrahams, Melissa; Treurnicht, Florette; Hraber, Peter; Riou, Catherine; Gray, Clive; Ferrari, Guido; Tanner, Rachel; Ping, Li-Hua; Anderson, Jeffrey A.; Swanstrom, Ronald; B, CHAVI Core; Cohen, Myron; Karim, Salim S. Abdool; Haynes, Barton; Borrow, Persephone; Perelson, Alan S.; Shaw, George M.; Hahn, Beatrice H.; Williamson, Carolyn; Korber, Bette T.; Gao, Feng; Self, Steve; McMichael, Andrew; Goonetilleke, Nilu

    2012-01-01

    HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell–mediated in vivo control of HIV-1. Primary HIV-1–specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or “vertical” immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance. PMID:23221345

  7. Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.

    PubMed Central

    Eichinger, D J; Arnot, D E; Tam, J P; Nussenzweig, V; Enea, V

    1986-01-01

    The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes. Images PMID:2432395

  8. Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

    PubMed Central

    Lesénéchal, Mylène; Becquart, Laurence; Lacoux, Xavier; Ladavière, Laurent; Baida, Renata C. P.; Paranhos-Baccalà, Glaucia; da Silveira, José Franco

    2005-01-01

    Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules. PMID:15699429

  9. Mapping of the immunodominant T cell epitopes of the protein topoisomerase I

    PubMed Central

    Veeraraghavan, S; Renzoni, E; Jeal, H; Jones, M; Hammer, J; Wells, A; Black, C; Welsh, K; du Bois, R M

    2004-01-01

    Objectives: To identify the immunodominant T cell epitopes of the topoisomerase I protein in patients with systemic sclerosis (SSc) and control subjects, using computational analysis software (TEPITOPE) and T cell proliferation assays. Methods: Six oligopeptides, predicted by TEPITOPE software as potential topoisomerase protein epitopes, were used to perform T cell proliferation assays in 21 patients with SSc and 15 healthy controls. Results: A positive response to at least one of the peptides was seen in 10/21 patients and 7/15 healthy controls. Among responders, the proliferative response was limited to a single peptide in 6/7 healthy controls, whereas 5/10 patients responded to more than one peptide. In responding patients a significant correlation was found between disease duration and number of peptides inducing a response (p = 0.007). Conclusions: Several T cell epitopes of the topoisomerase I protein have been identified and evidence has been found to suggest epitope spreading in patients with SSc. PMID:15249326

  10. Cancer immunotherapy using a potent immunodominant CTL epitope.

    PubMed

    Song, Liwen; Yang, Ming-Chieh; Knoff, Jayne; Sun, Zu-Yue; Wu, T-C; Hung, Chien-Fu

    2014-10-21

    Immunotherapy has emerged as a promising approach that can be used in conjunction with conventional chemotherapy and radiotherapy to further improve the survival rate of patients with advanced cancer. We have recently shown in previous studies that chemotherapy and radiation therapy can alter the tumor microenvironment and allow intratumoral vaccination to prime the adaptive immune system leading to the generation of antigen-specific cell-mediated immune responses. Here, we investigated whether intratumoral injection of a foreign immunodominant peptide (GP33) and the adjuvant CpG into tumors following cisplatin chemotherapy could lead to potent antitumor effects and antigen-specific cell-mediated immune responses. We observed that treatment with all three agents produced the most potent antitumor effects compared to pairwise combinations. Moreover, treatment with cisplatin, CpG and GP33 was able to control tumors at a distant site, indicating that our approach is able to induce cross-presentation of the tumor antigen. Treatment with cisplatin, CpG and GP33 also enhanced the generation of GP33-specific and E7-specific CD8+ T cells and decreased the number of MDSCs in tumor loci, a process found to be mediated by the Fas-FasL apoptosis pathway. The treatment regimen presented here represents a universal approach to cancer control.

  11. T-Cell Proliferation Assay: Determination of Immunodominant T-Cell Epitopes of Food Allergens.

    PubMed

    Masilamani, Madhan; Pascal, Mariona; Sampson, Hugh A

    2017-01-01

    Characterization of allergen-specific T cells is critical to understand their contribution to disease pathogenesis. The identification of immunodominant T-cell epitopes is crucial for development of T-cell-based vaccines. Peptide-specific T-cell proliferation studies are usually performed in a library of short synthetic peptides (15mer or 20mer) with 3 or 5 offset spanning the entire length of the allergen. T-cell peptide epitopes lack the primary and tertiary structure of the native protein to cross-link IgE, but retain the ability to stimulate T cells. The peptides sequences can also be obtained either by in silico approaches and in vitro binding assays. The efficacy of T-cell epitope-based peptide immunotherapy has been proven in certain allergies. The present methodology describes T-cell proliferation assays using whole blood sample from allergic subjects.

  12. Immunodominant viral peptides as determinants of cross-reactivity in the immune system--Can we develop wide spectrum viral vaccines?

    PubMed

    Vieira, G F; Chies, J A B

    2005-01-01

    When we look back to Edward Jenner vaccination of a young man in 1796, we cannot help thinking that he was both lucky and crazy. Crazy because he decided to test in a human being a hypothesis based mainly in the traditional belief that people who had acquired cowpox from the udders of a cow were thereafter resistant to smallpox, a quite devastating disease, and lucky because (even considering that he did not know this at that time) he succeeded to induce protection against a pathogen through the induction of an immune response directed against a different agent. Not only was he able to protect the young man but he took the first step towards the development of a vast new field, vaccination. It is acceptable to say that Jenner was lucky because he succeeded in promoting protection against smallpox using a cowpox virus and this induction of protection in a cross-reactive way is believed to be quite rare. Nevertheless, more and more examples of cross-reactive immune responses are being described and we are beginning to admit that cross-reactivity is far more common and important than we used to think. Here we review cross-reactivity in the immune system and the plasticity of T cell recognition. Based on the existence of T cell receptor promiscuous recognition and cross-recognition of conserved viral immunodominant epitopes, we propose two approaches to develop wide spectrum viral vaccines. The first one is based on the identification, characterization, and cloning of immunodominant viral epitopes able to stimulate responses against different viruses. The produced peptides could then be purified and serve as a basis for vaccine therapies. A second strategy is based on the identification of conserved patterns in immunodominant viral peptides and the production of synthetic peptides containing the amino acid residues necessary for MHC anchoring and TCR contact. Although we are still far from a complete knowledge of the cross-reactivity phenomenon in the immune system

  13. Protective Effect of Human Leukocyte Antigen B27 in Hepatitis C Virus Infection Requires the Presence of a Genotype-Specific Immunodominant CD8+ T-Cell Epitope

    PubMed Central

    Kersting, Nadine; Fitzmaurice, Karen; Oniangue-Ndza, Cesar; Kemper, Michael N.; Humphreys, Isla; McKiernan, Susan; Kelleher, Dermot; Lohmann, Volker; Bowness, Paul; Huzly, Daniela; Rosen, Hugo R.; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Barnes, Eleanor; Roggendorf, Michael; Blum, Hubert E.; Thimme, Robert

    2015-01-01

    Human leukocyte antigen B27 (HLA-B27) is associated with protection in human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infection. This protective role is linked to single immunodominant HLA-B27-restricted CD8+ T-cell epitopes in both infections. In order to define the relative contribution of a specific HLA-B27-restricted epitope to the natural course of HCV infection, we compared the biological impact of the highly conserved HCV genotype 1 epitope, for which the protective role has been described, with the corresponding region in genotype 3 that differs in its sequence by three amino acid residues. The genotype 3a peptide was not recognized by CD8+ T cells specific for the genotype 1 peptide. Furthermore, patients with acute or chronic infection with HCV genotype 3a did not mount T-cell responses to this epitope region, and their autologous viral sequences showed no evidence of T-cell pressure. Finally, we found a significantly higher frequency of HLA-B27 positivity in patients with chronic HCV genotype 3a infection compared to genotype 1 infection, indicating that there is no protection by HLA-B27 in HCV genotype 3 infection. Conclusion Our data indicate that the protective effect of HLA-B27 is limited to HCV genotype 1 infection and does not expand to other genotypes such as genotype 3a. This can most likely be explained by intergenotype sequence diversity leading to the loss of the immunodominant HLA-B27 epitope in viral strains other than genotype 1. Our results underline the central role of a single HLA-B27-restricted epitope-specific CD8+ T-cell response in mediating protection in HCV genotype 1 infection. PMID:20034048

  14. Searching immunodominant epitopes prior to epidemic: HLA class II-restricted SARS-CoV spike protein epitopes in unexposed individuals.

    PubMed

    Yang, Junbao; James, Eddie; Roti, Michelle; Huston, Laurie; Gebe, John A; Kwok, William W

    2009-01-01

    Identification of dominant T cell epitopes within newly emerging and re-emerging infectious organisms is valuable in understanding pathogenic immune responses and potential vaccine designs. However, difficulties in obtaining samples from patients or convalescent subjects have hampered research in this direction. We demonstrated a strategy, tetramer-guided epitope mapping, that specific CD4+ T cell epitopes can be identified by using PBMC from subjects that have not been exposed to the infectious organism. Sixteen HLA-DR0401- and 14 HLA-DR0701-restricted epitopes within spike protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) were identified. Among these, spike protein residues 159-171, 166-178, 449-461 and 1083-1097 were identified to contain naturally processed immunodominant epitopes based on strong in vitro T cell responses of PBMC (as assayed by tetramer staining) to intact spike protein stimulation. These immunodominant epitopes were confirmed in vivo in HLA-DR0401 transgenic mice by immunizing with spike protein. Furthermore, the epitope-specific T cells from naive donors secreted IFN-gamma and IL-13 upon re-stimulation with corresponding tetramers. Our study demonstrates a strategy to determine potential immunodominant epitopes for emerging infectious pathogens prior to their epidemic circulation.

  15. Searching immunodominant epitopes prior to epidemic: HLA class II-restricted SARS-CoV spike protein epitopes in unexposed individuals

    PubMed Central

    Yang, Junbao; James, Eddie; Roti, Michelle; Huston, Laurie; Gebe, John A.

    2009-01-01

    Identification of dominant T cell epitopes within newly emerging and re-emerging infectious organisms is valuable in understanding pathogenic immune responses and potential vaccine designs. However, difficulties in obtaining samples from patients or convalescent subjects have hampered research in this direction. We demonstrated a strategy, tetramer-guided epitope mapping, that specific CD4+ T cell epitopes can be identified by using PBMC from subjects that have not been exposed to the infectious organism. Sixteen HLA-DR0401- and 14 HLA-DR0701-restricted epitopes within spike protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) were identified. Among these, spike protein residues 159–171, 166–178, 449–461 and 1083–1097 were identified to contain naturally processed immunodominant epitopes based on strong in vitro T cell responses of PBMC (as assayed by tetramer staining) to intact spike protein stimulation. These immunodominant epitopes were confirmed in vivo in HLA-DR0401 transgenic mice by immunizing with spike protein. Furthermore, the epitope-specific T cells from naive donors secreted IFN-γ and IL-13 upon re-stimulation with corresponding tetramers. Our study demonstrates a strategy to determine potential immunodominant epitopes for emerging infectious pathogens prior to their epidemic circulation. PMID:19050106

  16. A human monoclonal antibody against HPV16 recognizes an immunodominant and neutralizing epitope partially overlapping with that of H16.V5

    PubMed Central

    Xia, Lin; Xian, Yangfei; Wang, Daning; Chen, Yuanzhi; Huang, Xiaofen; Bi, Xingjian; Yu, Hai; Fu, Zheng; Liu, Xinlin; Li, Shaowei; An, Zhiqiang; Luo, Wenxin; Zhao, Qinjian; Xia, Ningshao

    2016-01-01

    The presence of neutralizing epitopes in human papillomavirus (HPV) L1 virus-like particles (VLPs) is the structural basis of prophylactic vaccines. An anti-HPV16 neutralizing monoclonal antibody (N-mAb) 26D1 was isolated from a memory B cell of a human vaccinee. The pre-binding of heparan sulfate to VLPs inhibited the binding of both N-mAbs to the antigen, indicating that the epitopes are critical for viral cell attachment/entry. Hybrid VLP binding with surface loop swapping between types indicated the essential roles of the DE and FG loops for both 26D1 (DEa in particular) and H16.V5 binding. Specifically, Tyr135 and Val141 on the DEa loop were shown to be critical residues for 26D1 binding via site-directed mutagenesis. Partially overlap between the epitopes between 26D1 and H16.V5 was shown using pairwise epitope mapping, and their binding difference is demonstrated to be predominantly in DE loop region. In addition, 26D1 epitope is immunodominant epitope recognized by both antibodies elicited by the authentic virus from infected individuals and polyclonal antibodies from vaccinees. Overall, a partially overlapping but distinct neutralizing epitope from that of H16.V5 was identified using a human N-mAb, shedding lights to the antibody arrays as part of human immune response to vaccination and infection. PMID:26750243

  17. Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits.

    PubMed

    Acero, G; Manoutcharian, K; Vasilevko, V; Munguia, M E; Govezensky, T; Coronas, G; Luz-Madrigal, A; Cribbs, D H; Gevorkian, G

    2009-08-18

    N-truncated and N-modified forms of amyloid beta (Abeta) peptide are found in diffused and dense core plaques in Alzheimer's disease (AD) and Down's syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Abeta is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant N-truncated/modified Abeta peptide bearing amino-terminal pyroglutamate at position 3 (AbetaN3(pE)). We demonstrated that AbetaN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AbetaN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AbetaN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AbetaN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Abeta, which is absent in N-amino truncated peptides.

  18. IMMUNODOMINANT EPITOPE AND PROPERTIES OF PYROGLUTAMATE-MODIFIED Aβ-SPECIFIC ANTIBODIES PRODUCED IN RABBITS

    PubMed Central

    Acero, G.; Manoutcharian, K.; Vasilevko, V.; Munguia, M.E.; Govezensky, T.; Coronas, G.; Luz-Madrigal, A.; Cribbs, DH.; Gevorkian, G.

    2009-01-01

    N-truncated and N-modified forms of amyloid beta (Aß) peptide are found in diffused and dense core plaques in Alzheimer’s disease (AD) and Down’s syndrome patients as well as transgenic mouse models of AD. Although the pathological significance of these shortened forms Aβ is not completely understood, previous studies have demonstrated that these peptides are significantly more resistant to degradation, aggregate more rapidly in vitro and exhibit similar or, in some cases, increased toxicity in hippocampal neuronal cultures compared to the full-length peptides. In the present study we further investigated the mechanisms of toxicity of one of the most abundant Ntruncated/modified Aβ peptide bearing amino-terminal pyroglutamate at position 3 (AβN3(pE)). We demonstrated that AβN3(pE) oligomers induce phosphatidyl serine externalization and membrane damage in SH-SY5Y cells. Also, we produced AβN3(pE)-specific polyclonal antibodies in rabbit and identified an immunodominant epitope recognized by anti-AβN3(pE) antibodies. Our results are important for developing new immunotherapeutic compounds specifically targeting AβN3(pE) aggregates since the most commonly used immunogens in the majority of vaccines for AD have been shown to induce antibodies that recognize the N-terminal immunodominant epitope (EFRH) of the full length Aβ, which is absent in N-amino truncated peptides. PMID:19545911

  19. Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

    PubMed

    Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

    2015-05-01

    Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

  20. Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat.

    PubMed

    Tarp, Mads A; Sørensen, Anne Louise; Mandel, Ulla; Paulsen, Hans; Burchell, Joy; Taylor-Papadimitriou, Joyce; Clausen, Henrik

    2007-02-01

    The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, and cancer-associated MUC1 glycoforms represent potential targets for immunodiagnostic and therapeutic measures. We have recently shown that MUC1 with GalNAcalpha1-O-Ser/Thr (Tn) and NeuAcalpha2-6GalNAcalpha1-O-Ser/Thr (STn) O-glycosylation is a cancer-specific glycoform, and that Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance in human MUC1 transgenic mice and induce humoral immunity with high specificity for MUC1 cancer-specific glycoforms (Sorensen AL, Reis CA, Tarp MA, Mandel U, Ramachandran K, Sankaranarayanan V, Schwientek T, Graham R, Taylor-Papadimitriou J, Hollingsworth MA, et al. 2006. Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 16:96-107). In order to further characterize the immune response to Tn/STn-MUC1 glycoforms, we generated monoclonal antibodies with specificity similar to the polyclonal antibody response found in transgenic mice. In the present study, we define the immunodominant epitope on Tn/STn-MUC1 glycopeptides to the region including the amino acids GSTA of the MUC1 20-amino acid tandem repeat (HGVTSAPDTRPAPGSTAPPA). Most other MUC1 antibodies are directed to the PDTR region, although patients with antibodies to the GSTA region have been identified. A panel of other MUC1 glycoform-specific monoclonal antibodies was included for comparison. The study demonstrates that the GSTA region of the MUC1 tandem repeat contains a highly immunodominant epitope when presented with immature short O-glycans. The cancer-specific expression of this glycopeptide epitope makes it a prime candidate for immunodiagnostic and therapeutic measures.

  1. Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients

    PubMed Central

    Aw-Yong, Kam Leng; Sam, I-Ching; Koh, Mia Tuang

    2016-01-01

    Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates. PMID:27806091

  2. Subdominant CD8 T-cell epitopes account for protection against cytomegalovirus independent of immunodomination.

    PubMed

    Holtappels, Rafaela; Simon, Christian O; Munks, Michael W; Thomas, Doris; Deegen, Petra; Kühnapfel, Birgit; Däubner, Torsten; Emde, Simone F; Podlech, Jürgen; Grzimek, Natascha K A; Oehrlein-Karpi, Silke A; Hill, Ann B; Reddehase, Matthias J

    2008-06-01

    Cytomegalovirus (CMV) infection continues to be a complication in recipients of hematopoietic stem cell transplantation (HSCT). Preexisting donor immunity is recognized as a favorable prognostic factor for the reconstitution of protective antiviral immunity mediated primarily by CD8 T cells. Furthermore, adoptive transfer of CMV-specific memory CD8 T (CD8-T(M)) cells is a therapeutic option for preventing CMV disease in HSCT recipients. Given the different CMV infection histories of donor and recipient, a problem may arise from an antigenic mismatch between the CMV variant that has primed donor immunity and the CMV variant acquired by the recipient. Here, we have used the BALB/c mouse model of CMV infection in the immunocompromised host to evaluate the importance of donor-recipient CMV matching in immundominant epitopes (IDEs). For this, we generated the murine CMV (mCMV) recombinant virus mCMV-DeltaIDE, in which the two memory repertoire IDEs, the IE1-derived peptide 168-YPHFMPTNL-176 presented by the major histocompatibility complex class I (MHC-I) molecule L(d) and the m164-derived peptide 257-AGPPRYSRI-265 presented by the MHC-I molecule D(d), are both functionally deleted. Upon adoptive transfer, polyclonal donor CD8-T(M) cells primed by mCMV-DeltaIDE and the corresponding revertant virus mCMV-revDeltaIDE controlled infection of immunocompromised recipients with comparable efficacy and regardless of whether or not IDEs were presented in the recipients. Importantly, CD8-T(M) cells primed under conditions of immunodomination by IDEs protected recipients in which IDEs were absent. This shows that protection does not depend on compensatory expansion of non-IDE-specific CD8-T(M) cells liberated from immunodomination by the deletion of IDEs. We conclude that protection is, rather, based on the collective antiviral potential of non-IDEs independent of the presence or absence of IDE-mediated immunodomination.

  3. Definition of a discontinuous immunodominant epitope at the NH2 terminus of the La/SS-B ribonucleoprotein autoantigen.

    PubMed Central

    McNeilage, L J; Umapathysivam, K; Macmillan, E; Guidolin, A; Whittingham, S; Gordon, T

    1992-01-01

    High-titer IgG autoantibodies to the La/SS-B ribonucleoprotein (RNP) are a hallmark of patients with primary Sjogren's syndrome. Anti-La/SS-B-positive human sera bind to multiple epitopes on recombinant La/SS-B, although the initial response is against an immunodominant epitope within the first 107 NH2-terminal amino acids (aa). Sequence analysis has identified a striking homology between aa 88-101 in this NH2-terminal region of La/SS-B and a feline retroviral gag polypeptide suggesting the anti-La/SS-B response may be initiated by cross-reactivity with an exogenous agent. In the present study, detailed mapping of this NH2-terminal epitope, using recombinant La/SS-B purified from the expression of overlapping DNA fragments spanning aa 1-107, has shown that this immunodominant epitope is a complex conformational or discontinuous epitope dependent upon both aa 12-28 and 82-99 for expression, even though these regions share no homology with each other. This requirement questions the significance of the homology between La/SS-B and a retroviral gag polypeptide in the generation of the B cell response to La/SS-B and is in accord with the general concept that B cells recognize conformational epitopes on antigens rather than small linear peptide sequences. The finding also reinforces the notion that native autoantigen could be the initiator of the autoimmune response. Images PMID:1373741

  4. Immunisation With Immunodominant Linear B Cell Epitopes Vaccine of Manganese Transport Protein C Confers Protection against Staphylococcus aureus Infection

    PubMed Central

    Yang, Hui-Jie; Zhang, Jin-Yong; Wei, Chao; Yang, Liu-Yang; Zuo, Qian-Fei; Zhuang, Yuan; Feng, You-Jun; Srinivas, Swaminath; Zeng, Hao; Zou, Quan-Ming

    2016-01-01

    Vaccination strategies for Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA) infections have attracted much research attention. Recent efforts have been made to select manganese transport protein C, or manganese binding surface lipoprotein C (MntC), which is a metal ion associated with pathogen nutrition uptake, as potential candidates for an S. aureus vaccine. Although protective humoral immune responses to MntC are well-characterised, much less is known about detailed MntC-specific B cell epitope mapping and particularly epitope vaccines, which are less-time consuming and more convenient. In this study, we generated a recombinant protein rMntC which induced strong antibody response when used for immunisation with CFA/IFA adjuvant. On the basis of the results, linear B cell epitopes within MntC were finely mapped using a series of overlapping synthetic peptides. Further studies indicate that MntC113-136, MntC209-232, and MntC263-286 might be the original linear B-cell immune dominant epitope of MntC, furthermore, three-dimensional (3-d) crystal structure results indicate that the three immunodominant epitopes were displayed on the surface of the MntC antigen. On the basis of immunodominant MntC113-136, MntC209-232, and MntC263-286 peptides, the epitope vaccine for S. aureus induces a high antibody level which is biased to TH2 and provides effective immune protection and strong opsonophagocytic killing activity in vitro against MRSA infection. In summary, the study provides strong proof of the optimisation of MRSA B cell epitope vaccine designs and their use, which was based on the MntC antigen in the development of an MRSA vaccine. PMID:26895191

  5. Oral administration of an immunodominant T-cell epitope downregulates Th1/Th2 cytokines and prevents experimental myasthenia gravis

    PubMed Central

    Baggi, Fulvio; Andreetta, Francesca; Caspani, Elisabetta; Milani, Monica; Longhi, Renato; Mantegazza, Renato; Cornelio, Ferdinando; Antozzi, Carlo

    1999-01-01

    The mucosal administration of the native antigen or peptide fragments corresponding to immunodominant regions is effective in preventing or treating several T cell–dependent models of autoimmune disease. No data are yet available on oral tolerance with immunodominant T-cell peptides in experimental autoimmune myasthenia gravis (EAMG), an animal model of B cell–dependent disease. We report that oral administration of the T-cell epitope α146-162 of the Torpedo californica acetylcholine receptor (TAChR) α-subunit suppressed T-cell responses to AChR and ameliorated the disease in C57Bl/6 (B6) mice. Protection from EAMG was associated with reduced serum Ab’s to mouse AChR and reduced AChR loss in muscle. The effect of Tα146-162 feeding was specific; treatment with a control peptide did not affect EAMG manifestations. The protective effect induced by peptide Tα146-162 was mediated by reduced production of IFN-γ, IL-2, and IL-10 by TAChR-reactive cells, suggesting T-cell anergy. TGF-β–secreting Th3 cells did not seem to be involved in tolerance induction. We therefore demonstrate that feeding a single immunodominant epitope can prevent an Ab-mediated experimental model of autoimmune disease. PMID:10545527

  6. 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.

  7. Excavating the surface-associated and secretory proteome of Mycobacterium leprae for identifying vaccines and diagnostic markers relevant immunodominant epitopes.

    PubMed

    Rana, Aarti; Thakur, Shweta; Bhardwaj, Nupur; Kumar, Devender; Akhter, Yusuf

    2016-12-01

    For centuries, Mycobacterium leprae, etiological agent of leprosy, has been afflicting mankind regardless of extensive use of live-attenuated vaccines and antibiotics. Surface-associated and secretory proteins (SASPs) are attractive targets against bacteria. We have integrated biological knowledge with computational approaches and present a proteome-wide identification of SASPs. We also performed computational assignment of immunodominant epitopes as coordinates of prospective antigenic candidates in most important class of SASPs, the outer membrane proteins (OMPs). Exploiting the known protein sequence and structural characteristics shared by the SASPs from bacteria, 17 lipoproteins, 11 secretory and 19 novel OMPs (including 4 essential proteins) were identified in M. leprae As OMPs represent the most exposed antigens on the cell surface, their immunoinformatics analysis showed that the identified 19 OMPs harbor T-cell MHC class I epitopes and class II epitopes against HLA-DR alleles (54), while 15 OMPs present potential T-cell class II epitopes against HLA-DQ alleles (6) and 7 OMPs possess T-cell class II epitopes against HLA-DP alleles (5) of humans. Additionally, 11 M. leprae OMPs were found to have B-cell epitopes and these may be considered as prime candidates for the development of new immunotherapeutics against M. leprae.

  8. Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective.

    PubMed

    Guo, Hailong; Zhou, E M; Sun, Z F; Meng, X J

    2008-03-01

    Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

  9. Plant-based Production of Two Chimeric Monoclonal IgG Antibodies Directed against Immunodominant Epitopes of Vibrio cholerae Lipopolysaccharide

    PubMed Central

    Levinson, Kara J.; Giffen, Samantha R.; Pauly, Michael H.; Kim, Do H.; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J.; Zeitlin, Larry; Mantis, Nicholas J.

    2015-01-01

    We have produced and characterized two chimeric IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest. PMID:25865265

  10. Plant-based production of two chimeric monoclonal IgG antibodies directed against immunodominant epitopes of Vibrio cholerae lipopolysaccharide.

    PubMed

    Levinson, Kara J; Giffen, Samantha R; Pauly, Michael H; Kim, Do H; Bohorov, Ognian; Bohorova, Natasha; Whaley, Kevin J; Zeitlin, Larry; Mantis, Nicholas J

    2015-07-01

    We have produced and characterized two chimeric human IgG1 monoclonal antibodies that bind different immunodominant epitopes on Vibrio cholerae lipopolysaccharide (LPS). MAb 2D6 IgG1 recognizes Ogawa O-polysaccharide antigen, while mAb ZAC-3 IgG1 recognizes core/lipid A moiety of Ogawa and Inaba LPS. Both antibodies were expressed using a Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) and evaluated in vitro for activities associated with immunity to V. cholerae, including vibriocidal activity, bacterial agglutination and motility arrest.

  11. Recovery of known T-cell epitopes by computational scanning of a viral genome

    NASA Astrophysics Data System (ADS)

    Logean, Antoine; Rognan, Didier

    2002-04-01

    A new computational method (EpiDock) is proposed for predicting peptide binding to class I MHC proteins, from the amino acid sequence of any protein of immunological interest. Starting from the primary structure of the target protein, individual three-dimensional structures of all possible MHC-peptide (8-, 9- and 10-mers) complexes are obtained by homology modelling. A free energy scoring function (Fresno) is then used to predict the absolute binding free energy of all possible peptides to the class I MHC restriction protein. Assuming that immunodominant epitopes are usually found among the top MHC binders, the method can thus be applied to predict the location of immunogenic peptides on the sequence of the protein target. When applied to the prediction of HLA-A*0201-restricted T-cell epitopes from the Hepatitis B virus, EpiDock was able to recover 92% of known high affinity binders and 80% of known epitopes within a filtered subset of all possible nonapeptides corresponding to about one tenth of the full theoretical list. The proposed method is fully automated and fast enough to scan a viral genome in less than an hour on a parallel computing architecture. As it requires very few starting experimental data, EpiDock can be used: (i) to predict potential T-cell epitopes from viral genomes (ii) to roughly predict still unknown peptide binding motifs for novel class I MHC alleles.

  12. Diagnosis of AIDS Using Designed Amino Acid Peptides Representing Immunodominant Epitopes of HIV

    DTIC Science & Technology

    1990-07-03

    completed two main areas of research. First, we mapped the immune response during primary acute infection to the HIV-1 immunodominant domain GP41 12...Generation and analysis of monoclonal antibodies to the HIV-1 and HIV-2 immunodominant GP41 domains. ii. Analysis of monoclonal antibody(s) that see...both GP41 HIV-1 and HIV-2; study of sera from a subset of African patients •n which individuals show a positive serologic response to both specific

  13. Identification of Zika virus epitopes reveals immunodominant and protective roles for dengue virus cross-reactive CD8(+) T cells.

    PubMed

    Wen, Jinsheng; Tang, William Weihao; Sheets, Nicholas; Ellison, Julia; Sette, Alessandro; Kim, Kenneth; Shresta, Sujan

    2017-03-13

    CD8(+) T cells play an important role in controlling Flavivirus infection, including Zika virus (ZIKV). Here, we have identified 25 HLA-B*0702-restricted epitopes and 1 HLA-A*0101-restricted epitope using interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and intracellular cytokine staining (ICS) in ZIKV-infected IFN-α/β receptor-deficient HLA transgenic mice. The cross-reactivity of ZIKV epitopes to dengue virus (DENV) was tested using IFN-γ-ELISPOT and IFN-γ-ICS on CD8(+) T cells from DENV-infected mice, and five cross-reactive HLA-B*0702-binding peptides were identified by both assays. ZIKV/DENV cross-reactive CD8(+) T cells in DENV-immune mice expanded post ZIKV challenge and dominated in the subsequent CD8(+) T cell response. ZIKV challenge following immunization of mice with ZIKV-specific and ZIKV/DENV cross-reactive epitopes elicited CD8(+) T cell responses that reduced infectious ZIKV levels, and CD8(+) T cell depletions confirmed that CD8(+) T cells mediated this protection. These results identify ZIKV-specific and ZIKV/DENV cross-reactive epitopes and demonstrate both an altered immunodominance pattern in the DENV-immune setting relative to naive, as well as a protective role for epitope-specific CD8(+) T cells against ZIKV. These results have important implications for ZIKV vaccine development and provide a mouse model for evaluating anti-ZIKV CD8(+) T cell responses of human relevance.

  14. Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae

    PubMed Central

    De Paolis, Francesca; Beghetto, Elisa; Spadoni, Andrea; Montagnani, Francesca; Felici, Franco; Oggioni, Marco R; Gargano, Nicola

    2007-01-01

    Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA) of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery. PMID:18088426

  15. Characterization of the banana streak virus capsid protein and mapping of the immunodominant continuous B-cell epitopes to the surface-exposed N terminus.

    PubMed

    Vo, Jenny N; Campbell, Paul R; Mahfuz, Nur N; Ramli, Ras; Pagendam, Daniel; Barnard, Ross; Geering, Andrew D W

    2016-12-01

    This study identified the structural proteins of two badnavirus species, Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV), and mapped the distribution of continuous B-cell epitopes. Two different capsid protein (CP) isoforms of about 44 and 40 kDa (CP1 and CP2) and the virion-associated protein (VAP) were consistently associated with purified virions. For both viral species, the N terminus of CP2 was successfully sequenced by Edman degradation but that of CP1 was chemically blocked. De novo peptide sequencing of tryptic digests suggested that CP1 and CP2 derive from the same region of the P3 polyprotein but differ in the length of either the N or the C terminus. A three-dimensional model of the BSMYV-CP was constructed, which showed that the CP is a multi-domain structure, containing homologues of the retroviral capsid and nucleocapsid proteins, as well as a third, intrinsically disordered protein region at the N terminus, henceforth called the NID domain. Using the Pepscan approach, the immunodominant continuous epitopes were mapped to the NID domain for five different species of banana streak virus. Anti-peptide antibodies raised against these epitopes in BSMYV were successfully used for detection of native virions and denatured CPs in serological assays. Immunoelectron microscopy analysis of the virion surface using the anti-peptide antibodies confirmed that the NID domain is exposed on the surface of virions, and that the difference in mass of the two CP isoforms is due to variation in length of the NID domain.

  16. Limited effect on NS3-NS4A protein cleavage after alanine substitutions within the immunodominant HLA-A2-restricted epitope of the hepatitis C virus genotype 3a non-structural 3/4A protease.

    PubMed

    Ahlén, Gustaf; Chen, Antony; Roe, Barbara; Falkeborn, Tina; Frelin, Lars; Hall, William W; Sällberg, Matti; Söderholm, Jonas

    2012-08-01

    It has been well established that immunological escape mutations within the hepatitis C virus genotype (gt) 1a non-structural (NS) 3/4A protease are partly prevented by a reduction in viral protease fitness. Surprisingly little is known about whether similar mutations affect proteases from other genotypes. In the present study, we assessed both the HLA-A2-restricted CTL response and gt3a NS3/4A protease fitness. Similar to gt1, the 1073-1081 epitope was immunodominant within the gt3a-specific HLA-A2-restricted CTL response, despite sequence similarity of only 56 % between the gt1a and gt3a genes. However, unlike the gt1a NS3/4A protease, all residues within the gt3a 1073-1081 epitope could be replaced sequentially by alanine while retaining protease activity, at least in part.

  17. Tetramer-guided epitope mapping: rapid identification and characterization of immunodominant CD4+ T cell epitopes from complex antigens.

    PubMed

    Novak, E J; Liu, A W; Gebe, J A; Falk, B A; Nepom, G T; Koelle, D M; Kwok, W W

    2001-06-01

    T cell responses to Ags involve recognition of selected peptide epitopes contained within the antigenic protein. In this report, we describe a new approach for direct identification of CD4+ T cell epitopes of complex Ags that uses human class II tetramers to identify reactive cells. With a panel of 60 overlapping peptides covering the entire sequence of the VP16 protein, a major Ag for HSV-2, we generated a panel of class II MHC tetramers loaded with peptide pools that were used to stain peripheral lymphocytes of an HSV-2 infected individual. With this approach, we identified four new DRA1*0101/DRB1*0401- and two DRA1*0101/DRB1*0404-restricted, VP16-specific epitopes. By using tetramers to sort individual cells, we easily obtained a large number of clones specific to these epitopes. Although DRA1*0101/DRB1*0401 and DRA1*0101/DRB1*0404 are structurally very similar, nonoverlapping VP16 epitopes were identified, illustrating high selectivity of individual allele polymorphisms within common MHC variants. This rapid approach to detecting CD4+ T cell epitopes from complex Ags can be applied to any known Ag that gives a T cell response.

  18. Immunodominant SARS Coronavirus Epitopes in Humans Elicited both Enhancing and Neutralizing Effects on Infection in Non-human Primates.

    PubMed

    Wang, Qidi; Zhang, Lianfeng; Kuwahara, Kazuhiko; Li, Li; Liu, Zijie; Li, Taisheng; Zhu, Hua; Liu, Jiangning; Xu, Yanfeng; Xie, Jing; Morioka, Hiroshi; Sakaguchi, Nobuo; Qin, Chuan; Liu, Gang

    2016-05-13

    Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection.

  19. Cytolytic T lymphocytes (CTLs) from HIV-1 subtype C-infected Indian patients recognize CTL epitopes from a conserved immunodominant region of HIV-1 Gag and Nef.

    PubMed

    Thakar, Madhuri R; Bhonge, Leena S; Lakhashe, Samir K; Shankarkumar, U; Sane, Suvarna S; Kulkarni, Smita S; Mahajan, Bharati A; Paranjape, Ramesh S

    2005-09-01

    Analysis of the human immunodeficiency virus type 1 (HIV-1) cytolytic T lymphocyte (CTL) epitopes recognized by the targeted population is critical for HIV-1 vaccine design. Peripheral blood mononuclear cells from 47 Indian subjects at different stages of HIV-1 infection were tested for HIV-1 Gag-, Nef-, and Env-specific T cell responses by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay, using pools of overlapping peptides. The Gag and Nef antigens were targeted by 83% and 36% of responders. Five immunodominant regions, 4 in Gag and 1 in Nef, were identified in the study; these regions are conserved across clades, including the African subtype C clade. Three antigenic regions were also found to be recognized by CTLs of the study participants. These regions were not identified as immunodominant regions in studies performed in Africa, which highlights the importance of differential clustering of responses within HIV-1 subtype C. Twenty-six putative epitopes--15 Gag (10 in p24 and 5 in p17), 10 Nef, and 1 Env (gp 41)--were predicted using a combination of peptide matrix ELISPOT assay and CTL epitope-prediction software. Ninety percent of the predicted epitopes were clustered in the conserved immunodominant regions of the Gag and Nef antigens. Of 26 predicted epitopes, 8 were promiscuous, 3 of which were highly conserved across clades. Three Gag and 4 Nef epitopes were novel. The identification of conserved epitopes will be important in the planning of an HIV-1 vaccine strategy for subtype C-affected regions.

  20. Extensive HLA class I allele promiscuity among viral CTL epitopes

    PubMed Central

    Frahm, Nicole; Yusim, Karina; Suscovich, Todd J.; Adams, Sharon; Sidney, John; Hraber, Peter; Hewitt, Hannah S.; Linde, Caitlyn H.; Kavanagh, Daniel G.; Woodberry, Tonia; Henry, Leah M.; Faircloth, Kellie; Listgarten, Jennifer; Kadie, Carl; Jojic, Nebojsa; Sango, Kaori; Brown, Nancy V.; Pae, Eunice; Zaman, M. Tauheed; Bihl, Florian; Khatri, Ashok; John, Mina; Mallal, Simon; Marincola, Francesco M.; Walker, Bruce D.; Sette, Alessandro; Heckerman, David; Korber, Bette T.; Brander, Christian

    2008-01-01

    Summary Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I restricted antigen presentation and vaccine development. PMID:17705138

  1. Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

    PubMed

    Bhullar, S S; Chandak, N H; Baheti, N N; Purohit, H J; Taori, G M; Daginawala, H F; Kashyap, R S

    2014-01-01

    Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

  2. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance

    PubMed Central

    van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H. J.; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines. PMID:27333291

  3. Chemical Modification of Influenza CD8+ T-Cell Epitopes Enhances Their Immunogenicity Regardless of Immunodominance.

    PubMed

    Rosendahl Huber, Sietske K; Luimstra, Jolien J; van Beek, Josine; Hoppes, Rieuwert; Jacobi, Ronald H J; Hendriks, Marion; Kapteijn, Kim; Ouwerkerk, Casper; Rodenko, Boris; Ovaa, Huib; de Jonge, Jørgen

    2016-01-01

    T cells are essential players in the defense against infection. By targeting the MHC class I antigen-presenting pathway with peptide-based vaccines, antigen-specific T cells can be induced. However, low immunogenicity of peptides poses a challenge. Here, we set out to increase immunogenicity of influenza-specific CD8+ T cell epitopes. By substituting amino acids in wild type sequences with non-proteogenic amino acids, affinity for MHC can be increased, which may ultimately enhance cytotoxic CD8+ T cell responses. Since preventive vaccines against viruses should induce a broad immune response, we used this method to optimize influenza-specific epitopes of varying dominance. For this purpose, HLA-A*0201 epitopes GILGFVFTL, FMYSDFHFI and NMLSTVLGV were selected in order of decreasing MHC-affinity and dominance. For all epitopes, we designed chemically enhanced altered peptide ligands (CPLs) that exhibited greater binding affinity than their WT counterparts; even binding scores of the high affinity GILGFVFTL epitope could be improved. When HLA-A*0201 transgenic mice were vaccinated with selected CPLs, at least 2 out of 4 CPLs of each epitope showed an increase in IFN-γ responses of splenocytes. Moreover, modification of the low affinity epitope NMLSTVLGV led to an increase in the number of mice that responded. By optimizing three additional influenza epitopes specific for HLA-A*0301, we show that this strategy can be extended to other alleles. Thus, enhancing binding affinity of peptides provides a valuable tool to improve the immunogenicity and range of preventive T cell-targeted peptide vaccines.

  4. Selective Pressure to Increase Charge in Immunodominant Epitopes of the H3 Hemagglutinin Influenza Protein

    PubMed Central

    Pan, Keyao; Long, Jinxue; Sun, Haoxin; Tobin, Gregory J.; Nara, Peter L.

    2010-01-01

    The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein–protein interactions, including antibody–antigen binding and ligand–receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R2 > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies. Electronic supplementary material The online version of this article (doi:10.1007/s00239-010-9405-4) contains supplementary material, which is available to authorized users. PMID:21086120

  5. Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in spike 1 domain and membrane protein of feline infectious peritonitis virus.

    PubMed

    Takano, Tomomi; Morioka, Hiroyuki; Gomi, Kohji; Tomizawa, Keisuke; Doki, Tomoyoshi; Hohdatsu, Tsutomu

    2014-04-01

    Feline infectious peritonitis virus (FIP virus: FIPV) causes a fatal disease in wild and domestic cats. The development of an FIP-preventive vaccine requires an antigen that does not induce antibody-dependent enhancement, and T helper (Th)1 activity plays an important role in protect against FIPV infection. In the present study, we identified synthetic peptides including Th1 and a linear immunodominant antibody-binding epitope in the S1 domain and M protein of FIPV. We also identified peptides that strongly induce Th1 activity from those derived from the structural proteins (S, M, and N proteins) of FIPV based on this and previous studies (Satoh et al. [19]). No Th1 epitope-containing peptide was identified in the peptides derived from the S1 domain of type I FIPV. In contrast, 7 Th1 epitope-containing peptides were identified in the S1 domain of type II FIPV, and no linear immunodominant antibody-binding epitope was contained in any of these peptides. Eleven Th1 epitope-containing peptides common to each serotype were identified in the M protein-derived peptides, and 2 peptides (M-11 and M-12) contained the linear immunodominant antibody-binding epitope. Of the peptides derived from the S, M, and N proteins of FIPV, those that induced significantly stronger Th1 activity than that of the FIPV antigen were rescreened, and 4 peptides were identified. When 3 of these peptides (M-9, I-S2-15, and II-S1-24) were selected and administered with CpG-ODNs to SPF cats, M-9 and II-S1-24 induced Th1 activity. Our results may provide important information for the development of a peptide-based vaccine against FIPV infection.

  6. Screening and identification of T helper 1 and linear immunodominant antibody-binding epitopes in the spike 2 domain and the nucleocapsid protein of feline infectious peritonitis virus.

    PubMed

    Satoh, Ryoichi; Furukawa, Tomoko; Kotake, Masako; Takano, Tomomi; Motokawa, Kenji; Gemma, Tsuyoshi; Watanabe, Rie; Arai, Setsuo; Hohdatsu, Tsutomu

    2011-02-17

    The antibody-dependent enhancement (ADE) of feline infectious peritonitis virus (FIPV) infection has been recognized in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of feline infectious peritonitis (FIP). In the present study, we synthesized eighty-one kinds of peptides derived from the spike (S)2 domain of type I FIPV KU-2 strain, the S2 domain of type II FIPV 79-1146 strain, and the nucleocapcid (N) protein of FIPV KU-2 strain. To detect the T helper (Th)1 epitope, peripheral blood mononuclear cells (PBMCs) obtained from FIPV-infected cats were cultured with each peptide, and Th1-type immune responses were measured using feline interferon (fIFN)-γ production as an index. To detect the linear immunodominant antibody-binding epitope, we investigated the reactivity of plasma collected from FIPV-infected cats against each peptide by ELISA. Four and 2 peptides containing Th1 epitopes were identified in the heptad repeat (HR)1 and inter-helical (IH) regions of the S2 domain of type I FIPV, respectively, and these were located on the N-terminal side of the regions. In the S2 domain of type II FIPV, 2, 3, and 2 peptides containing Th1 epitopes were identified in the HR1, IH, and HR2 regions, respectively, and these were mainly located on the C-terminal side of the regions. In the S2 domain of type I FIPV, 3 and 7 peptides containing linear immunodominant antibody-binding epitopes were identified in the IH and HR2 regions, respectively. In the S2 domain of type II FIPV, 4 peptides containing linear immunodominant antibody-binding epitopes were identified in the HR2 region. The Th1 epitopes in the S2 domain of type I and II FIPV were located in different regions, but the linear immunodominant antibody-binding epitopes were mostly located in the HR2 region. Eight peptides containing Th1 epitopes were identified in N protein, and 3 peptides derived from residues 81 to 100 and 137 to 164 showed strong

  7. Mapping of immunodominant B-cell epitopes and the human serum albumin-binding site in natural hepatitis B virus surface antigen of defined genosubtype.

    PubMed

    Sobotta, D; Sominskaya, I; Jansons, J; Meisel, H; Schmitt, S; Heermann, K H; Kaluza, G; Pumpens, P; Gerlich, W H

    2000-02-01

    Twelve MAbs were generated by immunization of BALB/c mice with plasma-derived hepatitis B virus surface spherical antigen particles subtype ayw2 (HBsAg/ayw2 genotype D). Their epitopes were mapped by analysis of reactivity with plasma-derived HBsAg/ayw2 and HBsAg/adw2 (genotype A) in enzyme immunoassays and blots. Mapping was supported by nested sets of truncated preS2 proteins and preS2 peptides. Five antibodies were S domain-specific, seven were preS2-specific and 11 had a preference for genotype D. According to our data, group I of the three known epitope groups of preS2 has to be divided into IA and IB. Three preS2-specific MAbs forming the new group IA reacted with genotype D residues 3-15 which have not yet been described as an epitope region. IA antibodies strongly inhibited the binding of polymerized human serum albumin. Two antibodies (group II) reacted with the glycosylated N-terminal region of preS2 in plasma-derived HBsAg, but not with a preparation from transfected murine cells. One group III antibody was subtype-specific and reacted with the highly variable preS2 sequence 38-48. Only one antibody (group IB) mapped to the region (old group I) which was believed to be immunodominant and genotype-independent. Geno(sub)type-specific epitopes of preS2 are obviously the immunodominant components of natural HBsAg in BALB/c mice, but these epitopes may be masked by serum albumins in humans. The data may explain why it is difficult to detect anti-preS2 antibodies in human recipients of preS2-containing vaccines, in spite of the preS2 immunodominance in mice.

  8. Characterization of an Immunodominant Antigenic Epitope from Trypanosoma cruzi as a Biomarker of Chronic Chagas' Disease Pathology

    PubMed Central

    Thomas, M. Carmen; Fernández-Villegas, Ana; Carrilero, Bartolomé; Marañón, Concepción; Saura, Daniel; Noya, Oscar; Segovia, Manuel; Alarcón de Noya, Belkisyolé; Alonso, Carlos

    2012-01-01

    Nowadays, the techniques available for chronic Chagas' disease diagnosis are very sensitive; however, they do not allow discrimination of the patient's clinical stages of the disease. The present paper describes that three out of the five different repeats contained in the Trypanosoma cruzi TcCA-2 membrane protein (3972-FGQAAAGDKPPP, 6303-FGQAAAGDKPAP, and 3973-FGQAAAGDKPSL) are recognized with high sensitivity (>90%) by sera from chronic Chagas' disease patients and that they are not recognized by sera from patients in the acute phase of the disease. A total of 133 serum samples from chagasic patients and 50 serum samples from healthy donors were tested. In addition, sera from 15 patients with different autoimmune diseases, 43 serum samples from patients suffering an infectious disease other than Chagas' disease, and 38 serum samples from patients with nonchagasic cardiac disorders were also included in this study. The residue 3973 peptide shows a specificity of >98%, as it is not recognized by individuals with autoimmune and inflammatory processes or by patients with a nonchagasic cardiomyopathy. Remarkably, the levels of antibody against the 3973 epitope detected by the sera from Chagas' disease patients in the symptomatic chronic phase, involving cardiac or digestive alterations, are higher than those detected by the sera from Chagas' disease patients in the indeterminate phase of the disease. It is suggested that the diagnostic technique described could also be used to indicate the degree of pathology. The amino acids F, Q, and DKP located in the peptide at positions 1, 3, and 8 to 10, respectively, are essential to conform to the immunodominant antigenic epitope. PMID:22155766

  9. Amyloid-β-Anti-Amyloid-β Complex Structure Reveals an Extended Conformation in the Immunodominant B-Cell Epitope

    SciTech Connect

    Miles, Luke A; Wun, Kwok S; Crespi, Gabriela A.N.; Fodero-Tavoletti, Michelle T; Galatis, Denise; Bagley, Christopher J; Beyreuther, Konrad; Masters, Colin L; Cappai, Roberto; McKinstry, William J; Barnham, Kevin J; Parker, Michael W

    2008-04-29

    Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.

  10. Amyloid-β-Anti-Amyloid-β Complex Structure Reveals an Extended Conformation in the Immunodominant B-Cell Epitope

    SciTech Connect

    Miles, Luke A; Wun, Kwok S; Crespi, Gabriela A.N.; Fodero-Tavoletti, Michelle T; Galatis, Denise; Bagley, Christopher J; Beyreuther, Konrad; Masters, Colin L; Cappai, Roberto; McKinstry, William J; Barnham, Kevin J; Parker, Michael W

    2012-04-17

    Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.

  11. HLA-DQ tetramers identify epitope-specific T cells in peripheral blood of herpes simplex virus type 2-infected individuals: direct detection of immunodominant antigen-responsive cells.

    PubMed

    Kwok, W W; Liu, A W; Novak, E J; Gebe, J A; Ettinger, R A; Nepom, G T; Reymond, S N; Koelle, D M

    2000-04-15

    Ag-specific CD4+ T cells are present in peripheral blood in low frequency, where they undergo recruitment and expansion during immune responses and in the pathogenesis of numerous autoimmune diseases. MHC tetramers, which constitute a labeled MHC-peptide ligand suitable for binding to the Ag-specific receptor on T cells, provide a novel approach for the detection and characterization of such rare cells. In this study, we utilized this technology to identify HLA DQ-restricted Ag-specific T cells in the peripheral blood of human subjects and to identify immunodominant epitopes associated with viral infection. Peptides representing potential epitope regions of the VP16 protein from HSV-2 were loaded onto recombinant DQ0602 molecules to generate a panel of Ag-specific DQ0602 tetramers. VP16 Ag-specific DQ-restricted T cells were identified and expanded from the peripheral blood of HSV-2-infected individuals, representing two predominant epitope specificities. Although the VP16 369-380 peptide has a lower binding affinity for DQ0602 molecules than the VP16 33-52 peptide, T cells that recognized the VP16 369-380 peptide occurred at a much higher frequency than those that were specific for the VP16 33-52 peptide.

  12. HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T-cell epitope

    PubMed Central

    Nitschke, Katja; Barriga, Alejandro; Schmidt, Julia; Timm, Jörg; Viazov, Sergei; Kuntzen, Thomas; Kim, Arthur Y.; Lauer, Georg M.; Allen, Todd M.; Gaudieri, Silvana; Rauch, Andri; Lange, Christian M.; Sarrazin, Christoph; Eiermann, Thomas; Sidney, John; Sette, Alessandro; Thimme, Robert; López, Daniel; Neumann-Haefelin, Christoph

    2013-01-01

    Background & Aims HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T-cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Methods Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T-cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. Results The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. Conclusions The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines. PMID:23978718

  13. An immunodominant epitope of myelin basic protein is an amphipathic alpha-helix.

    PubMed

    Bates, Ian R; Feix, Jimmy B; Boggs, Joan M; Harauz, George

    2004-02-13

    Myelin basic protein is a candidate autoantigen in multiple sclerosis. One of its dominant antigenic epitopes is segment Pro85 to Pro96 (human sequence numbering, corresponding to Pro82 to Pro93 in the mouse). There have been several, contradictory predictions of secondary structure in this region; either beta-sheet, alpha-helix, random coil, or combinations thereof have all been proposed. In this paper, molecular dynamics and site-directed spin labeling in aqueous solution indicate that this segment forms a transient alpha-helix, which is stabilized in 30% trifluoroethanol. When bound to a myelin-like membrane surface, this antigenic segment exhibits a depth profile that is characteristic of an amphipathic alpha-helix, penetrating up to 12 A into the bilayer. The alpha-helix is tilted approximately 9 degrees, and the central lysine is in an ideal snorkeling position for side-chain interaction with the negatively charged phospholipid head groups.

  14. The value of HIV protective epitope research for informed vaccine design against diverse viral pathogens.

    PubMed

    Kramer, Victor G; Byrareddy, Siddappa N

    2014-08-01

    The success of vaccine regimens against viral pathogens hinges on the elicitation of protective responses. Hypervariable pathogens such as HIV avoid neutralization by masking protective epitopes with more immunogenic decoys. The identification of protective, conserved epitopes is crucial for future vaccine candidate design. The strategies employed for identification of HIV protective epitopes will also aid towards rational vaccine design for other viral pathogens.

  15. Identification of a Novel Immunodominant HLA-B*07: 02-restricted Adenoviral Peptide Epitope and Its Potential in Adoptive Transfer Immunotherapy.

    PubMed

    Günther, Patrick S; Peper, Janet K; Faist, Benjamin; Kayser, Simone; Hartl, Lena; Feuchtinger, Tobias; Jahn, Gerhard; Neuenhahn, Michael; Busch, Dirk H; Stevanović, Stefan; Dennehy, Kevin M

    2015-09-01

    Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

  16. Fine Epitope Mapping of the Central Immunodominant Region of Nucleoprotein from Crimean-Congo Hemorrhagic Fever Virus (CCHFV)

    PubMed Central

    Liu, Dongliang; Li, Yang; Zhao, Jing; Deng, Fei; Duan, Xiaomei; Kou, Chun; Wu, Ting; Li, Yijie; Wang, Yongxing; Ma, Ji; Yang, Jianhua; Hu, Zhihong; Zhang, Fuchun; Zhang, Yujiang; Sun, Surong

    2014-01-01

    Crimean-Congo hemorrhagic fever (CCHF), a severe viral disease known to have occurred in over 30 countries and distinct regions, is caused by the tick-borne CCHF virus (CCHFV). Nucleocapsid protein (NP), which is encoded by the S gene, is the primary antigen detectable in infected cells. The goal of the present study was to map the minimal motifs of B-cell epitopes (BCEs) on NP. Five precise BCEs (E1, 247FDEAKK252; E2a, 254VEAL257; E2b, 258NGYLNKH264; E3, 267EVDKA271; and E4, 274DSMITN279) identified through the use of rabbit antiserum, and one BCE (E5, 258NGYL261) recognized using a mouse monoclonal antibody, were confirmed to be within the central region of NP and were partially represented among the predicted epitopes. Notably, the five BCEs identified using the rabbit sera were able to react with positive serum mixtures from five sheep which had been infected naturally with CCHFV. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. Interestingly, the identified BCEs with only one residue variation can apparently be recognized by the positive sera of sheep naturally infected with CCHFV. Computer-generated three-dimensional structural models indicated that all the antigenic motifs are located on the surface of the NP stalk domain. This report represents the first identification and mapping of the minimal BCEs of CCHFV-NP along with an analysis of their primary and structural properties. Our identification of the minimal linear BCEs of CCHFV-NP may provide fundamental data for developing rapid diagnostic reagents and illuminating the pathogenic mechanism of CCHFV. PMID:25365026

  17. Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

    PubMed

    Becker, Pablo D; Nörder, Miriam; Weissmann, Sebastian; Ljapoci, Ronny; Erfle, Volker; Drexler, Ingo; Guzmán, Carlos A

    2014-07-22

    Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

  18. Exclusion of two major areas on thyroid peroxidase from the immunodominant region containing the conformational epitopes recognized by human autoantibodies.

    PubMed

    Nishikawa, T; Rapoport, B; McLachlan, S M

    1994-12-01

    We have used a chimeric molecule between thyroid peroxidase (TPO) and myeloperoxidase (MPO) as well as new information on the three-dimensional structure of MPO to refine further our understanding of the location of the TPO-immunodominant region recognized by TPO autoantibodies in patients' sera. In TPO-MPO chimera A, the amino-terminal 146 amino acids of MPO substitute for the amino-terminal 121 amino acids of TPO. We performed fluorescence-activated cell sorter analysis of Chinese hamster ovary cells expressing TPO-MPO-A on their surface using four monoclonal human autoantibody F(ab) (WR1.7, TR1.8, TR1.9, and SP1.4) that define the immunodominant region. All four F(ab) recognized the TPO-MPO-A chimeric molecule to the same extent. In a second approach to refine the location on the TPO-immunodominant region, we compared the ability of the TPO autoantibody F(ab) to inhibit the binding of serum autoantibodies to the monomeric and dimeric forms of human TPO. The F(ab) inhibited equally (approximately 80%) the binding to the TPO monomer and dimer by autoantibodies in the sera of six individual patients. The present observations exclude two major regions of TPO from the autoantibody-immunodominant region, namely the amino-terminal 121 amino acids of the TPO extracellular domain and the contact region between the two TPO monomers. These findings together with previous data on the Mab47/C21 region of TPO and the recently elucidated 3-dimensional structure of highly homologous MPO, narrow, by a process of exclusion, the site on TPO comprising the immunodominant region. The data provide further support for the thesis, still controversial, that the majority of TPO autoantibodies recognize the native molecule.

  19. Age-Dependent Loss of Tolerance to an Immunodominant Epitope of Glutamic Acid Decarboxylase in Diabetic prone RIP-B7/DR4 Mice

    PubMed Central

    Gebe, John A.; Unrath, Kellee A; Falk, Ben A.; Ito, Kouichi; Wen, Li; Daniels, Terri L.; Lernmark, Åke; Nepom, Gerald T.

    2007-01-01

    We have identified for the first time an age-dependent spontaneous loss of tolerance to two self-antigenic epitopes derived from putative diabetes associated antigens glutamic acid decarboxylase (GAD65) and glial fibrillary acidic protein (GFAP) in RIP-B7/DRB1*0404 HLA transgenic mice. Diabetic and older non-diabetic mice exhibited a proliferative response to an immunodominant epitope from GAD65 (555-567) and also from GFAP (240-252) but not from an immunogenic epitope from diabetes associated islet-specific glucose-6-phosphatase catalytic subunit-related protein. The response to both of these self-antigens is not observed in young mice but is observed in older non-diabetic mice, and is accompanied by histological evidence of insulitis in the absence of overt diabetes. Islet infiltrates in older non-diabetic mice and diabetic mice contain CD4+/FoxP3+ cells and suggest the presence of a regulatory mechanism prior and during diabetic disease. Diabetes penetrance in RIP-B7/DR0404 mice is 23% with a mean onset age of 40 weeks and is similar to that reported for RIP-B7/DR0401 mice. A gender preference is observed in that 38% of female mice become diabetic compared to 8% of male mice. PMID:16979383

  20. Age-dependent loss of tolerance to an immunodominant epitope of glutamic acid decarboxylase in diabetic-prone RIP-B7/DR4 mice.

    PubMed

    Gebe, John A; Unrath, Kellee A; Falk, Ben A; Ito, Kouichi; Wen, Li; Daniels, Terri L; Lernmark, Ake; Nepom, Gerald T

    2006-12-01

    We have identified for the first time an age-dependent spontaneous loss of tolerance to two self-antigenic epitopes derived from putative diabetes-associated antigens glutamic acid decarboxylase (GAD65) and glial fibrillary acidic protein (GFAP) in RIP-B7/DRB1*0404 HLA transgenic mice. Diabetic and older non-diabetic mice exhibited a proliferative response to an immunodominant epitope from GAD65 (555-567) and also from GFAP (240-252) but not from an immunogenic epitope from diabetes-associated islet-specific glucose-6-phosphatase catalytic subunit-related protein. The response to both of these self-antigens is not observed in young mice but is observed in older non-diabetic mice and is accompanied by histological evidence of insulitis in the absence of overt diabetes. Islet infiltrates in older non-diabetic mice and diabetic mice contain CD4(+)/FoxP3(+) cells and suggest the presence of a regulatory mechanism prior and during diabetic disease. Diabetes penetrance in RIP-B7/DR0404 mice is 23% with a mean onset age of 40 weeks and is similar to that reported for RIP-B7/DR0401 mice. A gender preference is observed in that 38% of female mice become diabetic compared to 8% of male mice.

  1. Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation

    PubMed Central

    Goulder, P.J.R.; Sewell, A.K.; Lalloo, D.G.; Price, D.A.; Whelan, J.A.; Evans, J.; Taylor, G.P.; Luzzi, G.; Giangrande, P.; Phillips, R.E.; McMichael, A.J.

    1997-01-01

    Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201–restricted SLYNTVATL and HLA-A3–restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201–restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response. PMID:9126923

  2. Vaccine Targeting of Subdominant CD8+ T Cell Epitopes Increases the Breadth of the T Cell Response upon Viral Challenge, but May Impair Immediate Virus Control.

    PubMed

    Steffensen, Maria A; Pedersen, Louise H; Jahn, Marie L; Nielsen, Karen N; Christensen, Jan P; Thomsen, Allan R

    2016-03-15

    As a result of the difficulties in making efficient vaccines against genetically unstable viruses such as HIV, it has been suggested that future vaccines should preferentially target subdominant epitopes, the idea being that this should allow a greater breadth of the induced T cell response and, hence, a greater efficiency in controlling escape variants. However, to our knowledge the evidence supporting this concept is limited at best. To improve upon this, we used the murine lymphocytic choriomeningitis virus model and adenoviral vectors to compare a vaccine expressing unmodified Ag to a vaccine expressing the same Ag without its immunodominant epitope. We found that removal of the dominant epitope allowed the induction of CD8(+) T cell responses targeting at least two otherwise subdominant epitopes. Importantly, the overall magnitude of the induced T cell responses was similar, allowing us to directly compare the efficiency of these vaccines. Doing this, we observed that mice vaccinated with the vaccine expressing unmodified Ag more efficiently controlled an acute viral challenge. In the course of a more chronic viral infection, mice vaccinated using the vaccine targeting subdominant epitopes caught up with the conventionally vaccinated mice, and analysis of the breadth of the CD8(+) T cell response revealed that this was notably greater in the former mice. However, under the conditions of our studies, we never saw any functional advantage of this. This may represent a limitation of our model, but clearly our findings underscore the importance of carefully weighing the pros and cons of changes in epitope targeting before any implementation.

  3. Comprehensive Mapping of Common Immunodominant Epitopes in the Eastern Equine Encephalitis Virus E2 Protein Recognized by Avian Antibody Responses

    PubMed Central

    Sun, EnCheng; Zhao, Jing; Sun, Liang; Xu, QingYuan; Yang, Tao; Qin, YongLi; Wang, WenShi; Wei, Peng; Sun, Jing; Wu, DongLai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211–226 and 331–352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11–26, 30–45 and 151–166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein. PMID:23922704

  4. Comprehensive mapping of common immunodominant epitopes in the eastern equine encephalitis virus E2 protein recognized by avian antibody responses.

    PubMed

    Sun, Encheng; Zhao, Jing; Sun, Liang; Xu, Qingyuan; Yang, Tao; Qin, Yongli; Wang, Wenshi; Wei, Peng; Sun, Jing; Wu, Donglai

    2013-01-01

    Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that can cause both human and equine encephalitis with high case fatality rates. EEEV can also be widespread among birds, including pheasants, ostriches, emu, turkeys, whooping cranes and chickens. The E2 protein of EEEV and other Alphaviruses is an important immunogenic protein that elicits antibodies of diagnostic value. While many therapeutic and diagnostic applications of E2 protein-specific antibodies have been reported, the specific epitopes on E2 protein recognized by the antibody responses of different susceptible hosts, including avian species, remain poorly defined. In the present study, the avian E2-reactive polyclonal antibody (PAb) response was mapped to linear peptide epitopes using PAbs elicited in chickens and ducks following immunization with recombinant EEEV E2 protein and a series of 42 partially overlapping peptides covering the entire EEEV E2 protein. We identified 12 and 13 peptides recognized by the chicken and duck PAb response, respectively. Six of these linear peptides were commonly recognized by PAbs elicited in both avian species. Among them five epitopes recognized by both avian, the epitopes located at amino acids 211-226 and 331-352 were conserved among the EEEV antigenic complex, but not other associated alphaviruses, whereas the epitopes at amino acids 11-26, 30-45 and 151-166 were specific to EEEV subtype I. The five common peptide epitopes were not recognized by avian PAbs against Avian Influenza Virus (AIV) and Duck Plague Virus (DPV). The identification and characterization of EEEV E2 antibody epitopes may be aid the development of diagnostic tools and facilitate the design of epitope-based vaccines for EEEV. These results also offer information with which to study the structure of EEEV E2 protein.

  5. Structures of HLA-A*1101 complexed with immunodominant nonamer and decamer HIV-1 epitopes clearly reveal the presence of a middle, secondary anchor residue.

    PubMed

    Li, Lenong; Bouvier, Marlene

    2004-05-15

    HLA-A*1101 is one of the most common human class I alleles worldwide. An increased frequency of HLA-A*1101 has been observed in cohorts of female sex workers from Northern Thailand who are highly exposed to HIV-1 and yet have remained persistently seronegative. In view of this apparent association of HLA-A*1101 with resistance to acquisition of HIV-1 infection, and given the importance of eliciting strong CTL responses to control and eliminate HIV-1, we have determined the crystal structure of HLA-A*1101 complexed with two immunodominant HIV-1 CTL epitopes: the nonamer reverse transcriptase(313-321) (AIFQSSMTK) and decamer Nef(73-82) (QVPLRPMTYK) peptides. The structures confirm the presence of primary anchor residues P2-Ile/-Val and P9-/P10-Lys, and also clearly reveal the presence of secondary anchor residues P6-Ser for reverse transcriptase and P7-Met for Nef. The overall backbone conformation of both peptides is defined as two bulges that are separated by a more buried middle residue. In this study, we discuss how this topology may offer functional advantages in the selection and presentation of HIV-1 CTL epitopes by HLA-A*1101. Overall, this structural analysis permits a more accurate definition of the peptide-binding motif of HLA-A*1101, the characterization of its antigenic surface, and the correlation of molecular determinants with resistance to HIV-1 infection. These studies are relevant for the rational design of HLA-A*1101-restricted CTL epitopes with improved binding and immunological properties for the development of HIV-1 vaccines.

  6. Cross-genotype-reactivity of the immunodominant HCV CD8 T-cell epitope NS3-1073.

    PubMed

    Fytili, P; Dalekos, G N; Schlaphoff, V; Suneetha, P V; Sarrazin, C; Zauner, W; Zachou, K; Berg, T; Manns, M P; Klade, C S; Cornberg, M; Wedemeyer, H

    2008-07-23

    The HCV-specific HLA-A2-restricted NS3(1073) epitope is one of the most frequently recognized epitopes in hepatitis C. NS3(1073)-specific T-cell responses are associated with clearance of acute HCV-infection. Therefore this epitope is an interesting candidate for a HCV-peptide vaccine. However, heterogeneity between genotypes and mutations in the epitope has to be considered as an obstacle. We here identified 34 naturally occurring NS3(1073)-variants, as compared with the wild type genotype-1 variants (CVNGVCWTV/CINGVCWTV) by sequencing sera of 251 Greek and German patients and searching for published HCV-genomes. The frequency of variants among genotype-1 patients was 10%. Importantly, HLA-A2 binding was reduced only in 3 genotype 1 mutants while all non-genotype 1 variants showed strong HLA-A2-binding. By screening 28 variants in ELISPOT assays from T cell lines we could demonstrate that HCV-NS3(1073)-wild-type-specific T-cells displayed cross-genotype-reactivity, in particular against genotypes 4-6 variants. However, single aa changes within the TCR-binding domain completely abolished recognition even in case of conservative aa exchanges within genotype-1. NS3(1073)-specific T-cell lines from recovered, chronically infected, and HCV-negative individuals showed no major difference in the pattern of cross-recognition although the proliferation of NS3(1073)-specific T-cells differed significantly between the groups. Importantly, the recognition pattern against the 28 variants was also identical directly ex vivo in a patient with acute HCV infection and a healthy volunteer vaccinated with the peptide vaccine IC41 containing the NS3(1073)-wild-type peptide. Thus, partial cross-genotype recognition of HCV NS3(1073)-specific CD8 T cells is possible; however, even single aa exchanges can significantly limit the potential efficacy of vaccines containing the NS3(1073)-wild-type peptide.

  7. Restricted V gene usage and VH/VL pairing of mouse humoral response against the N-terminal immunodominant epitope of the amyloid β peptide

    PubMed Central

    Robert, Remy; Lefranc, Marie-Paule; Ghochikyan, Anahit; Agadjanyan, Michael G.; Cribbs, David H.; Van Nostrand, William E.; Wark, Kim L.; Dolezal, Olan

    2011-01-01

    Over the last decade, the potential of antibodies as therapeutic strategies to treat Alzheimer’s disease (AD) has been growing, based on successful experimental and clinical trials in transgenic mice. Despite, undesirable side effects in humans using an active immunization approach, immunotherapy still remains one of the most promising treatments for AD. In this study, we analyzed the V genes of twelve independently isolated monoclonal antibodies raised against the N-terminal immunodominant epitope of the amyloid β peptide (Aβ or A beta). Surprisingly, we found a high and unusual level of restriction in the VH/VL pairing of these antibodies. Moreover, these antibodies mostly differ in their heavy chain complementary determining region 3 (HCDR3) and the residues in the antibodies which contact Aβ are already present in the germline V-genes. Based on these observations and or co-crystal structures of antibodies with Aβ, the aim of the current study was to better understand the role of antibody V-domains, HCDR3 regions, key contact residue (H58) and germline encoded residues in Aβ recognition. For that purpose, we designed and produced a range of recombinant Fab constructs. All the Fabs were tested and compared by surface plasmon resonance on Aβ1–16, Aβ1–42 high molecular weight and Aβ1–42 low molecular weight soluble oligomers. Although all the Fabs recognized the Aβ1–16 peptide and the Aβ1–42 high molecular weight soluble oligomers, they did not bind the Aβ1–42 low molecular weight soluble oligomers. Furthermore, we demonstrated that: (1) an aromatic residue at position H58 in the antibody is essential in the recognition of Aβ and (2) Fabs based on germline V-genes bind to Aβ monomers with a low affinity. These findings may have important implications in designing more effective therapeutic antibodies against Aβ. PMID:20970857

  8. Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening.

    PubMed

    Han, Jin-Hee; Li, Jian; Wang, Bo; Lee, Seong-Kyun; Nyunt, Myat Htut; Na, Sunghun; Park, Jeong-Hyun; Han, Eun-Taek

    2015-08-01

    Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

  9. Reconstitution of CD8 T Cells Protective against Cytomegalovirus in a Mouse Model of Hematopoietic Cell Transplantation: Dynamics and Inessentiality of Epitope Immunodominance

    PubMed Central

    Holtappels, Rafaela; Lemmermann, Niels A. W.; Podlech, Jürgen; Ebert, Stefan; Reddehase, Matthias J.

    2016-01-01

    Successful reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells by hematopoietic cell transplantation (HCT) gives a favorable prognosis for the control of CMV reactivation and prevention of CMV disease after hematoablative therapy of hematopoietic malignancies. In the transient immunocompromised state after HCT, pre-emptive cytoimmunotherapy with viral epitope-specific effector or memory CD8+ T cells is a promising option to speed up antiviral control. Despite high-coding capacity of CMVs and a broad CD8+ T-cell response on the population level, which reflects polymorphism in major histocompatibility complex class-I (MHC-I) glycoproteins, the response in terms of quantity of CD8+ T cells in any individual is directed against a limited set of CMV-encoded epitopes selected for presentation by the private repertoire of MHC-I molecules. Such epitopes are known as “immunodominant” epitopes (IDEs). Besides host immunogenetics, genetic variance in CMV strains harbored as latent viruses by an individual HCT recipient can also determine the set of IDEs, which complicates a “personalized immunotherapy.” It is, therefore, an important question if IDE-specific CD8+ T-cell reconstitution after HCT is critical or dispensable for antiviral control. As viruses with targeted mutations of IDEs cannot be experimentally tested in HCT patients, we employed the well-established mouse model of HCT. Notably, control of murine CMV (mCMV) after HCT was comparably efficient for IDE-deletion mutant mCMV-Δ4IDE and the corresponding IDE-expressing revertant virus mCMV-Δ4IDE-rev. Thus, antigenicity-loss mutations in IDEs do not result in loss-of-function of a polyclonal CD8+ T-cell population. Although IDE deletion was not associated with global changes in the response to non-IDE epitopes, the collective of non-IDE-specific CD8+ T-cells infiltrates infected tissue and confines infection within nodular inflammatory foci. We conclude from the model, and predict also for human

  10. New Viral Vector for Superproduction of Epitopes of Vaccine Proteins in Plants

    PubMed Central

    Tyulkina, L.G.; Skurat, E.V.; Frolova, O.Yu.; Komarova, T.V.; Karger, E.M.; Atabekov, I.G.

    2011-01-01

    The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome andAlternantheramosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVX-CP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines. PMID:22649706

  11. Definition of MHC and T cell receptor contacts in the HLA-DR4restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice

    PubMed Central

    Andersson, Ellen Christina; Hansen, Bjarke Endel; Jacobsen, Helle; Madsen, Lars S.; Andersen, Claus B.; Engberg, Jan; Rothbard, Jonathan B.; McDevitt, Grete Sønderstrup; Malmström, Vivianne; Holmdahl, Rikard; Svejgaard, Arne; Fugger, Lars

    1998-01-01

    Rheumatoid arthritis (RA) is an autoimmune disease associated with the HLA-DR4 and DR1 alleles. The target autoantigen(s) in RA is unknown, but type II collagen (CII) is a candidate, and the DR4- and DR1-restricted immunodominant T cell epitope in this protein corresponds to amino acids 261–273 (CII 261–273). We have defined MHC and T cell receptor contacts in CII 261–273 and provide strong evidence that this peptide corresponds to the peptide binding specificity previously found for RA-associated DR molecules. Moreover, we demonstrate that HLA-DR4 and human CD4 transgenic mice homozygous for the I-Abβ0 mutation are highly susceptible to collagen-induced arthritis and describe the clinical course and histopathological changes in the affected joints. PMID:9636191

  12. Identification of immunodominant VP1 linear epitope of enterovirus 71 (EV71) using synthetic peptides for detecting human anti-EV71 IgG antibodies in Western blots.

    PubMed

    Foo, D G W; Ang, R X; Alonso, S; Chow, V T K; Quak, S H; Poh, C L

    2008-03-01

    A major IgG-specific immunodominant VP1 linear epitope of enterovirus 71 (EV71) strain 41 (5865/SIN/00009), defined by the core sequence LEGTTNPNG, was identified by Pepscan analysis. Oligonucleotides corresponding to the amino-acid sequence of synthetic peptide SP32 were cloned and over-expressed in Escherichia coli as a recombinant glutathione-S-transferase (GST)-SP32 fusion protein. In ELISAs, this protein did not react with human anti-EV71 IgG antibodies, but there was significant immunoreactivity according to western blot analysis. The amino-acid sequence of SP32 was highly specific for detecting EV71 strains in western blot analysis, and showed no immunoreactivity with monoclonal antibodies raised against other enteroviruses, e.g., CA9 and Echo 6.

  13. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

    PubMed

    Shorter, Shayla K; Schnell, Frederick J; McMaster, Sean R; Pinelli, David F; Andargachew, Rakieb; Evavold, Brian D

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

  14. Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

    PubMed Central

    Shorter, Shayla K.; Schnell, Frederick J.; McMaster, Sean R.; Pinelli, David F.; Andargachew, Rakieb; Evavold, Brian D.

    2016-01-01

    T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant. PMID:26915099

  15. The universal epitope of influenza A viral neuraminidase fundamentally contributes to enzyme activity and viral replication.

    PubMed

    Doyle, Tracey M; Jaentschke, Bozena; Van Domselaar, Gary; Hashem, Anwar M; Farnsworth, Aaron; Forbes, Nicole E; Li, Changgui; Wang, Junzhi; He, Runtao; Brown, Earl G; Li, Xuguang

    2013-06-21

    The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

  16. An immunodominant epitope in a functional domain near the N-terminus of human granulocyte-macrophage colony-stimulating factor identified by cross-reaction of synthetic peptides with neutralizing anti-protein and anti-peptide antibodies.

    PubMed

    Beffy, P; Rovero, P; Di Bartolo, V; Laricchia Robbio, L; Dané, A; Pegoraro, S; Bertolero, F; Revoltella, R P

    1994-12-01

    We produced polyclonal and monoclonal antibodies (MAbs) against recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and performed studies of epitope mapping by ELISA, using five synthetic peptides corresponding to sequences along this molecule. Additionally, anti-peptide MAbs were generated. The antibody ability to inhibit rhGM-CSF activity was determined using as bioassay the MO7e cell line, which is dependent on hGM-CSF for growth in vitro. An immunodominant epitope able to induce the highest neutralization antibody titers was identified near the N terminus of hGM-CSF. A synthetic peptide 14-24, homologous to a sequence including part of the first alpha-helix of the molecule, was recognized by neutralizing anti-protein antibodies. Similarly, MAbs anti- 14-24 cross-reacted with rhGM-CSF and specifically blocked its function. Replacement of Val16 or Asn17 with alanine greatly reduced the antibody-binding capacity to peptide 14-24, whereas substitution of Gln20 or Glu21 was less critical. Monoclonal antibodies generated against residues 30-41 (corresponding to an intrahelical loop) and 79-91 (homologous to a sequence including part of the third alpha-helix) or its analog [Ala88](79-91)beta Ala-Cys, were conformation dependent and nonneutralizing: they failed to react or bound poorly to rhGM-CSF in ELISA, but readily recognized the homologous sequence in the denatured protein, by Western blotting.

  17. Novel immunodominant peptide presentation strategy: a featured HLA-A*2402-restricted cytotoxic T-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein.

    PubMed

    Liu, Jun; Wu, Peng; Gao, Feng; Qi, Jianxun; Kawana-Tachikawa, Ai; Xie, Jing; Vavricka, Christopher J; Iwamoto, Aikichi; Li, Taisheng; Gao, George F

    2010-11-01

    Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are presented by major histocompatibility complex (MHC; or human leukocyte antigen [HLA] in humans) molecules, and the peptide selection and presentation strategy of the host has been studied to guide our understanding of cellular immunity and vaccine development. Here, a severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein-derived CTL epitope, N1 (QFKDNVILL), restricted by HLA-A*2402 was identified by a series of in vitro studies, including a computer-assisted algorithm for prediction, stabilization of the peptide by co-refolding with HLA-A*2402 heavy chain and β(2)-microglobulin (β(2)m), and T2-A24 cell binding. Consequently, the antigenicity of the peptide was confirmed by enzyme-linked immunospot (ELISPOT), proliferation assays, and HLA-peptide complex tetramer staining using peripheral blood mononuclear cells (PBMCs) from donors who had recovered from SARS donors. Furthermore, the crystal structure of HLA-A*2402 complexed with peptide N1 was determined, and the featured peptide was characterized with two unexpected intrachain hydrogen bonds which augment the central residues to bulge out of the binding groove. This may contribute to the T-cell receptor (TCR) interaction, showing a host immunodominant peptide presentation strategy. Meanwhile, a rapid and efficient strategy is presented for the determination of naturally presented CTL epitopes in the context of given HLA alleles of interest from long immunogenic overlapping peptides.

  18. Human serum antibodies to a major defined epitope of human herpesvirus 8 small viral capsid antigen.

    PubMed

    Tedeschi, R; De Paoli, P; Schulz, T F; Dillner, J

    1999-04-01

    The major antibody-reactive epitope of the small viral capsid antigen (sVCA) of human herpesvirus 8 (HHV-8) was defined by use of overlapping peptides. Strong IgG reactivity was found among approximately 50% of 44 human immunodeficiency virus-positive or -negative patients with Kaposi's sarcoma and 13 subjects who were seropositive by immunofluorescence assay (IFA) for the latent HHV-8 nuclear antigen. Only 1 of 106 subjects seronegative for both lytic and latent HHV-8 antigens and 10 of 81 subjects IFA-seropositive only for the lytic HHV-8 antigen had strong IgG reactivity to this epitope. Among 534 healthy Swedish women, only 1.3% were strongly seropositive. Comparison of the peptide-based and purified sVCA protein-based ELISAs found 55% sensitivity and 98% specificity. However, only 1 of 452 serum samples from healthy women was positive in both tests. In conclusion, the defined sVCA epitope was a specific, but not very sensitive, serologic marker of active HHV-8 infection. Such infection appears to be rare among Swedish women, even with sexual risk-taking behavior.

  19. Shifting immunodominance pattern of two cytotoxic T-lymphocyte epitopes in the F glycoprotein of the Long strain of respiratory syncytial virus.

    PubMed

    Johnstone, Carolina; de León, Patricia; Medina, Francisco; Melero, José A; García-Barreno, Blanca; Del Val, Margarita

    2004-11-01

    Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2K(d)-restricted CTL epitopes (F85-93 and F92-106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85-93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249-258, which is presented by K(d) as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous K(d) ligand. The CD8(+) T-lymphocyte responses to epitopes F85-93 and F249-258 present in the F protein of RSV Long were found to be strongly skewed to F85-93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8(+) T-lymphocyte responses to F85-93 and F249-258 epitopes was observed in vivo during a primary response.

  20. Fine specificity analysis of an HLA-A2.1-restricted immunodominant T cell epitope derived from human alpha-fetoprotein.

    PubMed

    Meng, W S; Butterfield, L H; Ribas, A; Heller, J B; Dissette, V B; Glaspy, J A; McBride, W H; Economou, J S

    2000-11-01

    Human alpha-fetoprotein (AFP) is a potentially important target for the immunotherapy of hepatocellular carcinoma (HCC). AFP(542-550) (GVALQTMKQ) is one of several HLA-A2.1-restricted immunodominant AFP peptides that consistently generate AFP-specific T cell responses in human T cell cultures and in HLA-A2.1/K(b) transgenic (A2.1 tg) mice. We performed a fine specificity analysis of this nonamer to determine which amino acid side chains were critical for T cell priming and recognition. Using peptide-pulsed dendritic cells (DC) as an immunization strategy, we characterized the effects of AFP(542-550) amino acid substitutions on priming and recognition in A2.1 tg mice. Replacing the glutamine at anchor position 9 with a leucine enhanced MHC binding and AFP-specific T cell responses. Substitution of leucine at non-anchor position 4 with an alanine did not alter binding but greatly diminished T cell recognition. Computer-generated three-dimensional models provided the structural rationale for these observed effects in MHC binding and T cell responses resulted from the modifications in the AFP(542-550) sequence.

  1. H5N1 strain-specific hemagglutinin CD4+ T cell epitopes restricted by HLA DR4.

    PubMed

    Yang, Junbao; Gebe, John A; Huston, Laurie; James, Eddie; Tan, Venus; Yue, Betty B; Nepom, Gerald T; Kwok, William W

    2009-06-12

    CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain-specific HLA class II restricted epitopes is essential for monitoring cellular strain-specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA(441-460) and H5HA(57-76) were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA(57-76) epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA(57-76) response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA(57-76) immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide.

  2. H5N1 Strain-Specific Hemagglutinin CD4+ T cell Epitopes Restricted by HLA DR4

    PubMed Central

    Yang, Junbao; Gebe, John A.; Huston, Laurie; James, Eddie; Tan, Venus; Yue, Betty B.; Nepom, Gerald T.; Kwok, William W.

    2009-01-01

    CD4+ T cells play a pivotal role in the viral immunity, and as such identification of unique strain specific HLA class II restricted epitopes is essential for monitoring cellular strain specific viral immunity. Using Tetramer-Guided Epitope Mapping technique, we identified HLA-DR0401 restricted HA epitopes that are strain-specific to H5N1 virion. Two immunodominant epitopes H5HA441-460 and H5HA57-76 were identified from in vitro stimulated human PBMC. Both epitopes elicit strong cellular immune responses when HLA-DR0401 transgenic mice are immunized with H5N1 subvirion indicating in vivo naturally processed immunodominant epitopes. The H5HA57-76 epitope is unique for the H5N1 strain but conserved among all H5N1 clades recommended for vaccine development by World Health Organization. The unique H5HA57-76 response was uncommon in unexposed individuals and only observed in the naïve T cell subset. Thus, H5N1 strain-specific H5HA57-76 immunogenic epitope represents a unique marker for monitoring the efficacy of vaccination or as a candidate vaccine peptide. PMID:19446935

  3. Crystal Structure of West Nile Virus Envelope Glycoprotein Reveals Viral Surface Epitopes

    SciTech Connect

    Kanai,R.; Kar, K.; Anthony, K.; Gould, L.; Ledizet, M.; Fikrig, E.; Marasco, W.; Koski, R.; Modis, Y.

    2006-01-01

    West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

  4. TCR Affinity Associated with Functional Differences between Dominant and Subdominant SIV Epitope-Specific CD8+ T Cells in Mamu-A*01+ Rhesus Monkeys

    PubMed Central

    Osuna, Christa E.; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S. Munir; Letvin, Norman L.

    2014-01-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design. PMID:24743648

  5. TCR affinity associated with functional differences between dominant and subdominant SIV epitope-specific CD8+ T cells in Mamu-A*01+ rhesus monkeys.

    PubMed

    Osuna, Christa E; Gonzalez, Ana Maria; Chang, Hsun-Hsien; Hung, Amy Shi; Ehlinger, Elizabeth; Anasti, Kara; Alam, S Munir; Letvin, Norman L

    2014-04-01

    Many of the factors that contribute to CD8+ T cell immunodominance hierarchies during viral infection are known. However, the functional differences that exist between dominant and subdominant epitope-specific CD8+ T cells remain poorly understood. In this study, we characterized the phenotypic and functional differences between dominant and subdominant simian immunodeficiency virus (SIV) epitope-specific CD8+ T cells restricted by the major histocompatibility complex (MHC) class I allele Mamu-A*01 during acute and chronic SIV infection. Whole genome expression analyses during acute infection revealed that dominant SIV epitope-specific CD8+ T cells had a gene expression profile consistent with greater maturity and higher cytotoxic potential than subdominant epitope-specific CD8+ T cells. Flow-cytometric measurements of protein expression and anti-viral functionality during chronic infection confirmed these phenotypic and functional differences. Expression analyses of exhaustion-associated genes indicated that LAG-3 and CTLA-4 were more highly expressed in the dominant epitope-specific cells during acute SIV infection. Interestingly, only LAG-3 expression remained high during chronic infection in dominant epitope-specific cells. We also explored the binding interaction between peptide:MHC (pMHC) complexes and their cognate TCRs to determine their role in the establishment of immunodominance hierarchies. We found that epitope dominance was associated with higher TCR:pMHC affinity. These studies demonstrate that significant functional differences exist between dominant and subdominant epitope-specific CD8+ T cells within MHC-restricted immunodominance hierarchies and suggest that TCR:pMHC affinity may play an important role in determining the frequency and functionality of these cell populations. These findings advance our understanding of the regulation of T cell immunodominance and will aid HIV vaccine design.

  6. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

    NASA Astrophysics Data System (ADS)

    Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

    1994-06-01

    IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

  7. Kinetics of Antigen Expression and Epitope Presentation during Virus Infection

    PubMed Central

    Croft, Nathan P.; Smith, Stewart A.; Wong, Yik Chun; Tan, Chor Teck; Dudek, Nadine L.; Flesch, Inge E. A.; Lin, Leon C. W.; Tscharke, David C.; Purcell, Anthony W.

    2013-01-01

    Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity. PMID:23382674

  8. Towards peptide vaccines against Zika virus: Immunoinformatics combined with molecular dynamics simulations to predict antigenic epitopes of Zika viral proteins

    PubMed Central

    Usman Mirza, Muhammad; Rafique, Shazia; Ali, Amjad; Munir, Mobeen; Ikram, Nazia; Manan, Abdul; Salo-Ahen, Outi M. H.; Idrees, Muhammad

    2016-01-01

    The recent outbreak of Zika virus (ZIKV) infection in Brazil has developed to a global health concern due to its likely association with birth defects (primary microcephaly) and neurological complications. Consequently, there is an urgent need to develop a vaccine to prevent or a medicine to treat the infection. In this study, immunoinformatics approach was employed to predict antigenic epitopes of Zika viral proteins to aid in development of a peptide vaccine against ZIKV. Both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted for ZIKV Envelope (E), NS3 and NS5 proteins. We further investigated the binding interactions of altogether 15 antigenic CTL epitopes with three class I major histocompatibility complex (MHC I) proteins after docking the peptides to the binding groove of the MHC I proteins. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlight the limits of rigid-body docking methods. Some of the antigenic epitopes predicted and analyzed in this work might present a preliminary set of peptides for future vaccine development against ZIKV. PMID:27934901

  9. Generation of T-cell receptors targeting a genetically stable and immunodominant cytotoxic T-lymphocyte epitope within hepatitis C virus non-structural protein 3.

    PubMed

    Pasetto, Anna; Frelin, Lars; Brass, Anette; Yasmeen, Anila; Koh, Sarene; Lohmann, Volker; Bartenschlager, Ralf; Magalhaes, Isabelle; Maeurer, Markus; Sällberg, Matti; Chen, Margaret

    2012-02-01

    Hepatitis C virus (HCV) is a major cause of severe liver disease, and one major contributing factor is thought to involve a dysfunction of virus-specific T-cells. T-cell receptor (TCR) gene therapy with HCV-specific TCRs would increase the number of effector T-cells to promote virus clearance. We therefore took advantage of HLA-A2 transgenic mice to generate multiple TCR candidates against HCV using DNA vaccination followed by generation of stable T-cell-BW (T-BW) tumour hybrid cells. Using this approach, large numbers of non-structural protein 3 (NS3)-specific functional T-BW hybrids can be generated efficiently. These predominantly target the genetically stable HCV genotype 1 NS3(1073-1081) CTL epitope, frequently associated with clearance of HCV in humans. These T-BW hybrid clones recognized the NS3(1073) peptide with a high avidity. The hybridoma effectively recognized virus variants and targeted cells with low HLA-A2 expression, which has not been reported previously. Importantly, high-avidity murine TCRs effectively redirected human non-HCV-specific T-lymphocytes to recognize human hepatoma cells with HCV RNA replication driven by a subgenomic HCV replicon. Taken together, TCR candidates with a range of functional avidities, which can be used to study immune recognition of HCV-positive targets, have been generated. This has implications for TCR-related immunotherapy against HCV.

  10. Detection of Aichi virus with antibody targeting of conserved viral protein 1 epitope.

    PubMed

    Chen, Yao-Shen; Chen, Bao-Chen; Lin, You-Sheng; Chang, Jenn-Tzong; Huang, Tsi-Shu; Chen, Jih-Jung; Chang, Tsung-Hsien

    2013-10-01

    Aichi virus (AiV) is an emerging single-stranded, positive-sense, non-enveloped RNA virus in the Picornaviridae that causes acute gastroenteritis in humans. The first case of AiV infection in Taiwan was diagnosed in a human neonate with enterovirus-associated symptoms; the virus was successfully isolated and propagated. To establish a method to detect AiV, we analyzed the antigen epitope and generated a polyclonal antibody against AiV viral protein 1 (VP1). This peptide-purified anti-AiV VP1 antibody showed high specificity against AiV VP1 without cross-reaction to nine other tested strains of Picornaviruses. The anti-AiV VP1 antibody was used in immunofluorescence analysis, immunoblotting, and enzyme-linked immunosorbent assay to elucidate the cell tropism and replication kinetics of AiV. Use of the anti-AiV VP1 antibody also revealed AiV infection restriction with interferon type I and polyI/C antiviral treatment. The AiV infection and detection system may provide an in vitro platform for AiV virology study.

  11. Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: biological validation of the proposed HLA A24 supertype.

    PubMed

    Burrows, Scott R; Elkington, Rebecca A; Miles, John J; Green, Katherine J; Walker, Susan; Haryana, Sofia M; Moss, Denis J; Dunckley, Heather; Burrows, Jacqueline M; Khanna, Rajiv

    2003-08-01

    Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.

  12. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies.

    PubMed

    Dong, Shiwei; Zhao, Qin; Lu, Mingzhe; Sun, Peiming; Qiu, Hongkai; Zhang, Lu; Lv, Junhua; Zhou, En-Min

    2011-02-01

    Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.

  13. An immunodominant HLA-A*1101-restricted CD8+ T-cell response targeting hepatitis B surface antigen in chronic hepatitis B patients.

    PubMed

    Chen, Xiaoling; Wang, Wenbo; Wang, Shufeng; Meng, Gang; Zhang, Mengjun; Ni, Bing; Wu, Yuzhang; Wang, Li

    2013-12-01

    Hepatitis B virus (HBV) infection is a worldwide public health problem. HBV-specific CD8(+) CTLs are vital for viral clearance. Identification of immunodominant CTL epitopes from HBV-associated antigens is necessary for therapeutic vaccine development. We showed that the HLA-A*1101 allele is one of the most common alleles in both healthy individuals and chronic hepatitis B (CHB) patients in the Chongqing area, China. However, less than 10% of epitopes of HBV-associated antigens have been identified in an HLA-A*1101 context. Here, we describe an immunodominant CD8(+) T-cell response targeting a hepatitis B surface antigen determinant (HBs(295-304)) restricted by HLA-A*1101 in both healthy individuals and CHB patients. Moreover, HBs(295-304) is more immunogenic for CTL induction than a known naturally HLA-A*1101-processed epitope from hepatitis B core antigen (HBc(88-96)). Therefore, the newly identified epitope, HBs(295-304), will benefit the development of immunotherapeutic approaches for HBV infection.

  14. The two-faced T cell epitope

    PubMed Central

    Moise, Leonard; Gutierrez, Andres H.; Bailey-Kellogg, Chris; Terry, Frances; Leng, Qibin; Abdel Hady, Karim M.; VerBerkmoes, Nathan C.; Sztein, Marcelo B.; Losikoff, Phyllis T.; Martin, William D.; Rothman, Alan L; De Groot, Anne S.

    2013-01-01

    Advances in the field of T cell immunology have contributed to the understanding that cross-reactivity is an intrinsic characteristic of the T cell receptor (TCR), and that each TCR can potentially interact with many different T cell epitopes. To better define the potential for TCR cross-reactivity between epitopes derived from the human genome, the human microbiome, and human pathogens, we developed a new immunoinformatics tool, JanusMatrix, that represents an extension of the validated T cell epitope mapping tool, EpiMatrix. Initial explorations, summarized in this synopsis, have uncovered what appear to be important differences in the TCR cross-reactivity of selected regulatory and effector T cell epitopes with other epitopes in the human genome, human microbiome, and selected human pathogens. In addition to exploring the T cell epitope relationships between human self, commensal and pathogen, JanusMatrix may also be useful to explore some aspects of heterologous immunity and to examine T cell epitope relatedness between pathogens to which humans are exposed (Dengue serotypes, or HCV and Influenza, for example). In Hand-Foot-Mouth disease (HFMD) for example, extensive enterovirus and human microbiome cross-reactivity (and limited cross-reactivity with the human genome) seemingly predicts immunodominance. In contrast, more extensive cross-reactivity with proteins contained in the human genome as compared to the human microbiome was observed for selected Treg epitopes. While it may be impossible to predict all immune response influences, the availability of sequence data from the human genome, the human microbiome, and an array of human pathogens and vaccines has made computationally–driven exploration of the effects of T cell epitope cross-reactivity now possible. This is the first description of JanusMatrix, an algorithm that assesses TCR cross-reactivity that may contribute to a means of predicting the phenotype of T cells responding to selected T cell

  15. Epitope-specific CD8+ T cell kinetics rather than viral variability determine the timing of immune escape in simian immunodeficiency virus infection.

    PubMed

    Martyushev, Alexey P; Petravic, Janka; Grimm, Andrew J; Alinejad-Rokny, Hamid; Gooneratne, Shayarana L; Reece, Jeanette C; Cromer, Deborah; Kent, Stephen J; Davenport, Miles P

    2015-05-01

    CD8(+) T cells are important for the control of chronic HIV infection. However, the virus rapidly acquires "escape mutations" that reduce CD8(+) T cell recognition and viral control. The timing of when immune escape occurs at a given epitope varies widely among patients and also among different epitopes within a patient. The strength of the CD8(+) T cell response, as well as mutation rates, patterns of particular amino acids undergoing escape, and growth rates of escape mutants, may affect when escape occurs. In this study, we analyze the epitope-specific CD8(+) T cells in 25 SIV-infected pigtail macaques responding to three SIV epitopes. Two epitopes showed a variable escape pattern and one had a highly monomorphic escape pattern. Despite very different patterns, immune escape occurs with a similar delay of on average 18 d after the epitope-specific CD8(+) T cells reach 0.5% of total CD8(+) T cells. We find that the most delayed escape occurs in one of the highly variable epitopes, and that this is associated with a delay in the epitope-specific CD8(+) T cells responding to this epitope. When we analyzed the kinetics of immune escape, we found that multiple escape mutants emerge simultaneously during the escape, implying that a diverse population of potential escape mutants is present during immune selection. Our results suggest that the conservation or variability of an epitope does not appear to affect the timing of immune escape in SIV. Instead, timing of escape is largely determined by the kinetics of epitope-specific CD8(+) T cells.

  16. A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.

    PubMed

    House, J A; Stott, J L; Blanchard, M T; LaRocco, M; Llewellyn, M E

    1996-07-23

    A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described.

  17. Atomic-level mapping of antibody epitopes on a GPCR.

    PubMed

    Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A; Rucker, Joseph B; Doranz, Benjamin J

    2009-05-27

    Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.

  18. CD8 epitope escape and reversion in acute HCV infection.

    PubMed

    Timm, Joerg; Lauer, Georg M; Kavanagh, Daniel G; Sheridan, Isabelle; Kim, Arthur Y; Lucas, Michaela; Pillay, Thillagavathie; Ouchi, Kei; Reyor, Laura L; Schulze zur Wiesch, Julian; Gandhi, Rajesh T; Chung, Raymond T; Bhardwaj, Nina; Klenerman, Paul; Walker, Bruce D; Allen, Todd M

    2004-12-20

    In the setting of acute hepatitis C virus (HCV) infection, robust HCV-specific CD8+ cytotoxic T lymphocyte (CTL) responses are associated with initial control of viremia. Despite these responses, 70-80% of individuals develop persistent infection. Although viral escape from CD8 responses has been illustrated in the chimpanzee model of HCV infection, the effect of CD8 selection pressure on viral evolution and containment in acute HCV infection in humans remains unclear. Here, we examined viral evolution in an immunodominant human histocompatibility leukocyte antigen (HLA)-B8-restricted NS3 epitope in subjects with acute HCV infection. Development of mutations within the epitope coincided with loss of strong ex vivo tetramer and interferon gamma enzyme-linked immunospot responses, and endogenous expression of variant NS3 sequences suggested that the selected mutations altered processing and presentation of the variant epitope. Analysis of NS3 sequences from 30 additional chronic HCV-infected subjects revealed a strong association between sequence variation within this region and expression of HLA-B8, supporting reproducible allele-specific selection pressures at the population level. Interestingly, transmission of an HLA-B8-associated escape mutation to an HLA-B8 negative subject resulted in rapid reversion of the mutation. Together, these data indicate that viral escape from CD8+ T cell responses occurs during human HCV infection and that acute immune selection pressure is of sufficient magnitude to influence HCV evolution.

  19. Positive Selection in CD8+ T-Cell Epitopes of Influenza Virus Nucleoprotein Revealed by a Comparative Analysis of Human and Swine Viral Lineages

    PubMed Central

    Machkovech, Heather M.; Bedford, Trevor; Suchard, Marc A.

    2015-01-01

    ABSTRACT Numerous experimental studies have demonstrated that CD8+ T cells contribute to immunity against influenza by limiting viral replication. It is therefore surprising that rigorous statistical tests have failed to find evidence of positive selection in the epitopes targeted by CD8+ T cells. Here we use a novel computational approach to test for selection in CD8+ T-cell epitopes. We define all epitopes in the nucleoprotein (NP) and matrix protein (M1) with experimentally identified human CD8+ T-cell responses and then compare the evolution of these epitopes in parallel lineages of human and swine influenza viruses that have been diverging since roughly 1918. We find a significant enrichment of substitutions that alter human CD8+ T-cell epitopes in NP of human versus swine influenza virus, consistent with the idea that these epitopes are under positive selection. Furthermore, we show that epitope-altering substitutions in human influenza virus NP are enriched on the trunk versus the branches of the phylogenetic tree, indicating that viruses that acquire these mutations have a selective advantage. However, even in human influenza virus NP, sites in T-cell epitopes evolve more slowly than do nonepitope sites, presumably because these epitopes are under stronger inherent functional constraint. Overall, our work demonstrates that there is clear selection from CD8+ T cells in human influenza virus NP and illustrates how comparative analyses of viral lineages from different hosts can identify positive selection that is otherwise obscured by strong functional constraint. IMPORTANCE There is a strong interest in correlates of anti-influenza immunity that are protective against diverse virus strains. CD8+ T cells provide such broad immunity, since they target conserved viral proteins. An important question is whether T-cell immunity is sufficiently strong to drive influenza virus evolution. Although many studies have shown that T cells limit viral replication in animal

  20. Suppression of Immunodominant Antitumor and Antiviral CD8+ T Cell Responses by Indoleamine 2,3-Dioxygenase

    PubMed Central

    Atef Yekta, Maryam; Szabo, Peter A.; Garg, Nitan; Schell, Todd D.; Jevnikar, Anthony M.; Sharif, Shayan; Singh, Bhagirath; Haeryfar, S. M. Mansour

    2014-01-01

    Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme known to suppress antitumor CD8+ T cells (TCD8). The role of IDO in regulation of antiviral TCD8 responses is far less clear. In addition, whether IDO controls both immunodominant and subdominant TCD8 is not fully understood. This is an important question because the dominance status of tumor- and virus-specific TCD8 may determine their significance in protective immunity and in vaccine design. We evaluated the magnitude and breadth of cross-primed TCD8 responses to simian virus 40 (SV40) large T antigen as well as primary and recall TCD8 responses to influenza A virus (IAV) in the absence or presence of IDO. IDO−/− mice and wild-type mice treated with 1-methyl-D-tryptophan, a pharmacological inhibitor of IDO, exhibited augmented responses to immunodominant epitopes encoded by T antigen and IAV. IDO-mediated suppression of these responses was independent of CD4+CD25+FoxP3+ regulatory T cells, which remained numerically and functionally intact in IDO−/− mice. Treatment with L-kynurenine failed to inhibit TCD8 responses, indicating that tryptophan metabolites are not responsible for the suppressive effect of IDO in our models. Immunodominant T antigen-specific TCD8 from IDO−/− mice showed increased Ki-67 expression, suggesting that they may have acquired a more vigorous proliferative capacity in vivo. In conclusion, IDO suppresses immunodominant TCD8 responses to tumor and viral antigens. Our work also demonstrates that systemic primary and recall TCD8 responses to IAV are controlled by IDO. Inhibition of IDO thus represents an attractive adjuvant strategy in boosting anticancer and antiviral TCD8 targeting highly immunogenic antigens. PMID:24587363

  1. T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen

    PubMed Central

    1995-01-01

    Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self- reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  4. Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

    PubMed Central

    Vasconcelos, Jose R.; Motz, R. Geoffrey; Sullivan, Nicole L.; Gohara, David W.; Blase, Jennifer R.; Di Cera, Enrico; Hoft, Daniel F.

    2016-01-01

    Trypanosoma cruzi infection is controlled but not eliminated by host immunity. The T. cruzi trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4+ and CD8+ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against T. cruzi challenge. We generated a new TS vaccine in which the immunodominant TS CD8+ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8+ T cell epitope enhance subdominant pathogen-specific CD8+ T cell responses. More specifically, CD8+ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8+ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for T. cruzi vaccine development. Of broader significance, we demonstrate that increasing breadth of T

  5. Systematic identification of immunodominant CD8+ T-cell responses to influenza A virus in HLA-A2 individuals

    PubMed Central

    Wu, Chao; Zanker, Damien; Valkenburg, Sophie; Kedzierska, Katherine; Zou, Quan Ming; Doherty, Peter C.; Chen, Weisan

    2011-01-01

    Immunodominant T-cell responses are important for virus clearance. However, the identification of immunodominant T-cell peptide + HLA glycoprotein epitopes has been hindered by the extent of HLA polymorphism and the limitations of predictive algorithms. A simple, systematic approach has been used here to screen for immunodominant CD8+ T-cell specificities. The analysis targeted healthy HLA-A2+ donors to allow comparison with responses to the well-studied influenza matrix protein 1 epitope. Although influenza matrix protein 1 was consistently detected in all individual samples in our study, the response to this epitope was only immunodominant in three of eight, whereas for the other five, prominent CD8+ T-cell responses tended to focus on various peptides from the influenza nucleoprotein that were not presented by HLA-A2. Importantly, with the four immunodominant T-cell epitopes identified here, only one would have been detected by the current prediction programs. The other three peptides would have been either considered too long or classified as not containing typical HLA binding motifs. Our data stress the importance of systematic analysis for discovering HLA-dependent, immunodominant CD8+ T-cell epitopes derived from viruses and tumors. Focusing on HLA-A2 and predictive algorithms may be too limiting as we seek to develop targeted immunotherapy and vaccine strategies that depend on T cell-mediated immunity. PMID:21562214

  6. Conservancy of mAb Epitopes in Ebolavirus Glycoproteins of Previous and 2014 Outbreaks

    PubMed Central

    Ponomarenko, Julia; Vaughan, Kerrie; Sette, Alessandro; Maurer-Stroh, Sebastian

    2014-01-01

    Background: Several monoclonal antibodies (mAb) are being evaluated as treatment options for the current 2014 Ebola outbreak. But they were derived from and tested for protection against the older 1976 Mayinga or 1995 Kikwit Zaire Ebolaviruses (EBOV). The EBOV sequences reported for the current outbreak contain several mutations whose significance remained to be established. Methods: We analyzed sequence and structural conservation of the Ebolavirus glycoprotein (GP) epitopes for all experimentally identified protective mAbs published to date. Results: The conservancy analysis of protective mAb epitopes in the Ebolavirus glycoprotein sequences spanning all Ebola virus lineages since 1976 showed that conservancy within the Zaire EBOV lineage was high, with only one immunodominant epitope of mAb 13F6-1-2 acquiring two novel mutations in the 2014 outbreak that might potentially change the antibody specificity and neutralization activity. However, the conservation to other Ebola viruses was unexpectedly low. Conclusion: Low conservancy of Zaire EBOV mAb epitopes to other EBOV lineages suggests that these epitopes are not indispensable for viral fitness, and that alternative mAbs could be developed to broadly target all EBOV. However, average percent sequence identity of the epitopes for mAbs used in current cocktails to the Zaire EBOV is high with only one epitope differing in the 2014 outbreak. These data bode well for general usefulness of these antibodies in the context of the current outbreak. PMID:25642381

  7. Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.

    PubMed

    Bashyam, Hema S; Green, Sharone; Rothman, Alan L

    2006-03-01

    Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.

  8. A Novel CD8-Independent High-Avidity Cytotoxic T-Lymphocyte Response Directed against an Epitope in the Phosphoprotein of the Paramyxovirus Simian Virus 5

    PubMed Central

    Gray, Peter M.; Parks, Griffith D.; Alexander-Miller, Martha A.

    2001-01-01

    Adoptive transfer studies have shown that cytotoxic T lymphocytes (CTL) of high avidity, capable of recognizing low levels of peptide-MHC I molecules, are more efficient at reducing viral titers than are low-avidity CTL, thus establishing CTL avidity as a critical parameter for the ability of a CTL to clear virus in vivo. It has been well documented that CTL of high avidity are relatively CD8 independent, whereas low-avidity CTL require CD8 engagement in order to become activated. In this study we have analyzed the antiviral CTL response elicited following infection with the paramyxovirus simian virus 5 (SV5). We have identified the immunodominant and subdominant CTL responses and subsequently assessed the avidity of these responses by their CD8 dependence. This is the first study in which the relationship between immunodominance and CTL avidity has been investigated. The immunodominant response was directed against an epitope present in the viral M protein, and subdominant responses were directed against epitopes present in the P, F, and HN proteins. Similarly to other CTL responses we have analyzed, the immunodominant response and the subdominant F and HN responses were comprised of both high- and low-avidity CTL. However, the subdominant response directed against the epitope present in the P protein is novel, as it is exclusively high avidity. This high-avidity response is independent of both the route of infection and expression by recombinant SV5. A further understanding of the inherent properties of P that elicit only high-avidity CTL may allow for the design of more efficacious vaccine vectors that preferentially elicit high-avidity CTL in vivo. PMID:11581375

  9. Variable processing and cross-presentation of HIV by dendritic cells and macrophages shapes CTL immunodominance and immune escape.

    PubMed

    Dinter, Jens; Duong, Ellen; Lai, Nicole Y; Berberich, Matthew J; Kourjian, Georgio; Bracho-Sanchez, Edith; Chu, Duong; Su, Hang; Zhang, Shao Chong; Le Gall, Sylvie

    2015-03-01

    Dendritic cells (DCs) and macrophages (Møs) internalize and process exogenous HIV-derived antigens for cross-presentation by MHC-I to cytotoxic CD8⁺ T cells (CTL). However, how degradation patterns of HIV antigens in the cross-presentation pathways affect immunodominance and immune escape is poorly defined. Here, we studied the processing and cross-presentation of dominant and subdominant HIV-1 Gag-derived epitopes and HLA-restricted mutants by monocyte-derived DCs and Møs. The cross-presentation of HIV proteins by both DCs and Møs led to higher CTL responses specific for immunodominant epitopes. The low CTL responses to subdominant epitopes were increased by pretreatment of target cells with peptidase inhibitors, suggestive of higher intracellular degradation of the corresponding peptides. Using DC and Mø cell extracts as a source of cytosolic, endosomal or lysosomal proteases to degrade long HIV peptides, we identified by mass spectrometry cell-specific and compartment-specific degradation patterns, which favored the production of peptides containing immunodominant epitopes in all compartments. The intracellular stability of optimal HIV-1 epitopes prior to loading onto MHC was highly variable and sequence-dependent in all compartments, and followed CTL hierarchy with immunodominant epitopes presenting higher stability rates. Common HLA-associated mutations in a dominant epitope appearing during acute HIV infection modified the degradation patterns of long HIV peptides, reduced intracellular stability and epitope production in cross-presentation-competent cell compartments, showing that impaired epitope production in the cross-presentation pathway contributes to immune escape. These findings highlight the contribution of degradation patterns in the cross-presentation pathway to HIV immunodominance and provide the first demonstration of immune escape affecting epitope cross-presentation.

  10. Peptide-pulsed dendritic cells induce the hepatitis C viral epitope-specific responses of naïve human T cells.

    PubMed

    Mishra, Sasmita; Losikoff, Phyllis T; Self, Alyssa A; Terry, Frances; Ardito, Matthew T; Tassone, Ryan; Martin, William D; De Groot, Anne S; Gregory, Stephen H

    2014-05-30

    Hepatitis C virus (HCV) is a major cause of liver disease. Spontaneous resolution of infection is associated with broad, MHC class I- (CD8(+)) and class II-restricted (CD4(+)) T cell responses to multiple viral epitopes. Only 20% of patients clear infection spontaneously, however, most develop chronic disease. The response to chemotherapy varies; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success in clinical trials. Vector-mediated vaccination with multi-epitope-expressing DNA constructs provides an improved approach. Highly-conserved, HLA-A2-restricted HCV epitopes and HLA-DRB1-restricted immunogenic consensus sequences (ICS, each composed of multiple overlapping and highly conserved epitopes) were predicted using bioinformatics tools and synthesized as peptides. HLA binding activity was determined in competitive binding assays. Immunogenicity and the ability of each peptide to stimulate naïve human T cell recognition and IFN-γ production were assessed in cultures of total PBMCs and in co-cultures composed of peptide-pulsed dendritic cells (DCs) and purified T lymphocytes, cell populations derived from normal blood donors. Essentially all predicted HLA-A2-restricted epitopes and HLA-DRB1-restricted ICS exhibited HLA binding activity and the ability to elicit immune recognition and IFN-γ production by naïve human T cells. The ability of DCs pulsed with these highly-conserved HLA-A2- and -DRB1-restricted peptides to induce naïve human T cell reactivity and IFN-γ production ex vivo demonstrates the potential efficacy of a multi-epitope-based HCV vaccine targeted to dendritic cells.

  11. Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles.

    PubMed

    Richert, Ludovic; Humbert, Nicolas; Larquet, Eric; Girerd-Chambaz, Yves; Manin, Catherine; Ronzon, Frédéric; Mély, Yves

    2016-10-01

    Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.

  12. Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

    PubMed Central

    Guo, Li; Wu, Chao; Gonzalez, Richard; Paranhos-Baccalà, Gláucia; Vernet, Guy; Wang, Jianwei; Hung, Tao

    2011-01-01

    Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ2 = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs. PMID:21886787

  13. The immunodominant proteins of reticuloendotheliosis virus.

    PubMed

    Davidson, I; Yang, H; Witter, R L; Malkinson, M

    1996-04-01

    The antigenic profiles of three REV prototype strains, CSV, SNV and REV-T and eight Israeli isolates were analysed by SDS-PAGE and immunoblotting with convalescent chicken serum, three mAbs, 11A25, 11C237 and 11C100, a rabbit antiserum to REV-T whole virus (Cui et al., 1986) and a rabbit antiserum to REV-A p30 gag protein (Tsai et al., 1985). Under both reducing (+DTT) and non-reducing conditions of SDS-PAGE, a major immunodominant 75-100 kDa band was shared by all strains examined. In contrast to the chicken serum that recognized both continuous and discontinuous epitopes on the 75-100 kDa band of all the isolates, the mAbs and the two rabbit sera behaved otherwise. Only the DTT-resistant epitopes on the 75-100 kDa band of REV-T were recognized by the rabbit antisera and the mAb 11C237, and only the DTT-labile epitopes of REV-T 75-100 kDa antigen were detected by mAb 11C100. The two mAbs 11A25 and 11C237 detected discontinuous epitopes of all the strains except SNV, while the rabbit antisera recognized the discontinuous epitopes on the 75-100 kDa band of all the 11 strains. The rabbit antisera and mAb 11C237 detected additional lower molecular weight proteins and the mAb 11C237 also detected three proteins of high molecular weight under non-reducing conditions only. The p30 antiserum detected the low molecular weight proteins demonstrating their gag gene-encoded identity. From these results we conclude that the major immunogen of REV is the 75-100 kDa protein that contains both continuous and discontinuous epitopes. With this panel of antibodies the eight new isolates appeared to belong antigenically to REV subtype 3 (Chen et al., 1987).

  14. Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site

    PubMed Central

    Smith, Scott A.; Nivarthi, Usha K.; de Alwis, Ruklanthi; Kose, Nurgun; Sapparapu, Gopal; Bombardi, Robin; Kahle, Kristen M.; Pfaff, Jennifer M.; Lieberman, Sherri; Doranz, Benjamin J.

    2015-01-01

    ABSTRACT The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells. IMPORTANCE Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue pr

  15. A vaccine-elicited, single viral epitope-specific cytotoxic T lymphocyte response does not protect against intravenous, cell-free simian immunodeficiency virus challenge.

    PubMed Central

    Yasutomi, Y; Koenig, S; Woods, R M; Madsen, J; Wassef, N M; Alving, C R; Klein, H J; Nolan, T E; Boots, L J; Kessler, J A

    1995-01-01

    Protection against simian immunodeficiency virus (SIV) challenge was assessed in rhesus monkeys with a vaccine-elicited, single SIV epitope-specific cytotoxic T-lymphocyte (CTL) response in the absence of SIV-specific antibody. Strategies were first explored for eliciting an optimal SIV Gag epitope-specific CTL response. These studies were performed in rhesus monkeys expressing the major histocompatibility complex (MHC) class I gene Mamu-A*01, a haplotype associated with a predominant SIV CTL epitope mapped to residues 182 to 190 of the Gag protein (p11C). We demonstrated that a combined modality immunization strategy using a recombinant Mycobacterium bovis BCG-SIV Gag construct for priming, and peptide formulated in liposome for boosting, elicited a greater p11C-specific CTL response than did a single immunization with peptide-liposome alone. Vaccinated and control monkeys were then challenged with cell-free SIVmne by an intravenous route of inoculation. Despite a vigorous p11C-specific CTL response at the time of virus inoculation, all monkeys became infected with SIV. gag gene sequencing of the virus isolated from these monkeys demonstrated that the established viruses had no mutations in the p11C-coding region. Thus, the preexisting CTL response did not select for a viral variant that might escape T-cell immune recognition. These studies demonstrate that a potent SIV-specific CTL response can be elicited by combining live vector and peptide vaccine modalities. However, a single SIV Gag epitope-specific CTL response in the absence of SIV-specific antibody did not provide protection against a cell-free, intravenous SIV challenge. PMID:7884874

  16. A DNA vaccine encoding foot-and-mouth disease virus B and T-cell epitopes targeted to class II swine leukocyte antigens protects pigs against viral challenge.

    PubMed

    Borrego, Belén; Argilaguet, Jordi M; Pérez-Martín, Eva; Dominguez, Javier; Pérez-Filgueira, Mariano; Escribano, José M; Sobrino, Francisco; Rodriguez, Fernando

    2011-11-01

    Development of efficient and safer vaccines against foot-and-mouth disease virus (FMDV) is a must. Previous results obtained in our laboratory have demonstrated that DNA vaccines encoding B and T cell epitopes from type C FMDV, efficiently controlled virus replication in mice, while they did not protect against FMDV challenge in pigs, one of the FMDV natural hosts. The main finding of this work is the ability to improve the protection afforded in swine using a new DNA-vaccine prototype (pCMV-APCH1BTT), encoding FMDV B and T-cell epitopes fused to the single-chain variable fragment of the 1F12 mouse monoclonal antibody that recognizes Class-II Swine Leukocyte antigens. Half of the DNA-immunized pigs were fully protected upon viral challenge, while the remaining animals were partially protected, showing a delayed, shorter and milder disease than control pigs. Full protection in a given vaccinated-pig correlated with the induction of specific IFNγ-secreting T-cells, detectable prior to FMDV-challenge, together with a rapid development of neutralizing antibodies after viral challenge, pointing towards the relevance that both arms of the immune response can play in protection. Our results open new avenues for developing future FMDV subunit vaccines.

  17. Infection with Trypanosoma cruzi restricts the repertoire of parasite-specific CD8+ T cells leading to immunodominance.

    PubMed

    Tzelepis, Fanny; de Alencar, Bruna C G; Penido, Marcus L O; Claser, Carla; Machado, Alexandre V; Bruna-Romero, Oscar; Gazzinelli, Ricardo T; Rodrigues, Mauricio M

    2008-02-01

    Interference or competition between CD8(+) T cells restricted by distinct MHC-I molecules can be a powerful means to establish an immunodominant response. However, its importance during infections is still questionable. In this study, we describe that following infection of mice with the human pathogen Trypanosoma cruzi, an immunodominant CD8(+) T cell immune response is developed directed to an H-2K(b)-restricted epitope expressed by members of the trans-sialidase family of surface proteins. To determine whether this immunodominance was exerted over other non-H-2K(b)-restricted epitopes, we measured during infection of heterozygote mice, immune responses to three distinct epitopes, all expressed by members of the trans-sialidase family, recognized by H-2K(b)-, H-2K(k)-, or H-2K(d)-restricted CD8(+) T cells. Infected heterozygote or homozygote mice displayed comparably strong immune responses to the H-2K(b)-restricted immunodominant epitope. In contrast, H-2K(k)- or H-2K(d)-restricted immune responses were significantly impaired in heterozygote infected mice when compared with homozygote ones. This interference was not dependent on the dose of parasite or the timing of infection. Also, it was not seen in heterozygote mice immunized with recombinant adenoviruses expressing T. cruzi Ags. Finally, we observed that the immunodominance was circumvented by concomitant infection with two T. cruzi strains containing distinct immunodominant epitopes, suggesting that the operating mechanism most likely involves competition of T cells for limiting APCs. This type of interference never described during infection with a human parasite may represent a sophisticated strategy to restrict priming of CD8(+) T cells of distinct specificities, avoiding complete pathogen elimination by host effector cells, and thus favoring host parasitism.

  18. Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine epidemic diarrhea virus (PEDV) is a pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in newborn piglets. Phage display is a technique with wide application, in particular, the identification of key antigen epitopes for the develop...

  19. Epitope peptides and immunotherapy.

    PubMed

    Tanabe, Soichi

    2007-02-01

    Allergic diseases affect atopic individuals, who synthesize specific Immunoglobulins E (IgE) to environmental allergens, usually proteins or glycoproteins. These allergens include grass and tree pollens, indoor allergens such as house dust mites and animal dander, and various foods. Because allergen-specific IgE antibodies are the main effector molecules in the immune response to allergens, many studies have focused on the identification of IgE-binding epitopes (called B cell epitopes), specific and minimum regions of allergen molecules that binds to IgE. Our initial studies have provided evidence that only four to five amino acid residues are enough to comprise an epitope, since pentapeptide QQQPP in wheat glutenin is minimally required for IgE binding. Afterwards, various kinds of B cell epitope structures have been clarified. Such information contributes greatly not only to the elucidation of the etiology of allergy, but also to the development of strategies for the treatment and prevention of allergy. Allergen-specific T cells also play an important role in allergy and are obvious targets for intervention in the disease. Currently, the principle approach is to modify B cell epitopes to prevent IgE binding while preserving T cell epitopes to retain the capacity for immunotherapy. There is mounting evidence that the administration of peptide(s) containing immunodominant T cell epitopes from an allergen can induce T cell nonresponsiveness (immunotherapy). There have been clinical studies of peptide immunotherapy performed, the most promising being for bee venom sensitivity. Clinical trials of immunotherapy for cat allergen peptide have also received attention. An alternative strategy for the generation of an effective but hypoallergenic preparation for immunotherapy is to modify T cell epitope peptides by, for example, single amino acid substitution. In this article, I will present an overview of epitopes related to allergic disease, particularly stress on

  20. Long-Term Immunity to Trypanosoma cruzi in the Absence of Immunodominant trans-Sialidase-Specific CD8+ T Cells

    PubMed Central

    Rosenberg, Charles S.; Zhang, Weibo; Bustamante, Juan M.

    2016-01-01

    Trypanosoma cruzi infection drives the expansion of remarkably focused CD8+ T cell responses targeting epitopes encoded by variant trans-sialidase (TS) genes. Infection of C57BL/6 mice with T. cruzi results in up to 40% of all CD8+ T cells committed to recognition of the dominant TSKB20 and subdominant TSKB18 TS epitopes. However, despite this enormous response, these mice fail to clear T. cruzi infection and subsequently develop chronic disease. One possible reason for the failure to cure T. cruzi infection is that immunodomination by these TS-specific T cells may interfere with alternative CD8+ T cell responses more capable of complete parasite elimination. To address this possibility, we created transgenic mice that are centrally tolerant to these immunodominant epitopes. Mice expressing TSKB20, TSKB18, or both epitopes controlled T. cruzi infection and developed effector CD8+ T cells that maintained an activated phenotype. Memory CD8+ T cells from drug-cured TSKB-transgenic mice rapidly responded to secondary T. cruzi infection. In the absence of the response to TSKB20 and TSKB18, immunodominance did not shift to other known subdominant epitopes despite the capacity of these mice to expand epitope-specific T cells specific for the model antigen ovalbumin expressed by engineered parasites. Thus, CD8+ T cell responses tightly and robustly focused on a few epitopes within variant TS antigens appear to neither contribute to, nor detract from, the ability to control T. cruzi infection. These data also indicate that the relative position of an epitope within a CD8+ immunodominance hierarchy does not predict its importance in pathogen control. PMID:27354447

  1. Extent of systemic spread determines CD8+ T cell immunodominance for laboratory strains, smallpox vaccines and zoonotic isolates of vaccinia virus1

    PubMed Central

    Flesch, Inge E.A.; Hollett, Natasha A.; Wong, Yik Chun; Quinan, Bárbara Resende; Howard, Debbie; da Fonseca, Flávio G.; Tscharke, David C.

    2015-01-01

    CD8+ T cells that recognize virus-derived peptides presented on MHC class I (pMHC) are vital anti-viral effectors. The pMHC presented by any given virus vary greatly in immunogenicity allowing them to be ranked in an immunodominance hierarchy. However, the full range of parameters that determine immunodominance and the underlying mechanisms remain unknown. Here we show across a range of vaccinia virus (VACV) strains, including the current clonal smallpox vaccine, that the ability of a strain to spread systemically correlated with reduced immunodominance. Reduction in immunodominance was observed both in the lymphoid system and at the primary site of infection. Mechanistically, reduced immunodominance was associated with more robust priming and especially priming in the spleen. Finally, we show this is not just a property of vaccine and laboratory strains of virus, because an association between virulence and immunodominance was also observed in isolates from an outbreak of zoonotic VACV that occurred in Brazil. PMID:26195812

  2. Characterization of the Viral O-Glycopeptidome: a Novel Tool of Relevance for Vaccine Design and Serodiagnosis

    PubMed Central

    Cló, Emiliano; Kračun, Stjepan K.; Nudelman, Aaron S.; Jensen, Knud J.; Liljeqvist, Jan-Åke; Olofsson, Sigvard; Bergström, Tomas

    2012-01-01

    Viral envelope proteins mediate interactions with host cells, leading to internalization and intracellular propagation. Envelope proteins are glycosylated and are known to serve important functions in masking host immunity to viral glycoproteins. However, the viral infectious cycle in cells may also lead to aberrant glycosylation that may elicit immunity. Our knowledge of immunity to aberrant viral glycans and glycoproteins is limited, potentially due to technical limitations in identifying immunogenic glycans and glycopeptide epitopes. This work describes three different complementary methods for high-throughput screening and identification of potential immunodominant O-glycopeptide epitopes on viral envelope glycoproteins: (i) on-chip enzymatic glycosylation of scan peptides, (ii) chemical glycopeptide microarray synthesis, and (iii) a one-bead-one-compound random glycopeptide library. We used herpes simplex virus type 2 (HSV-2) as a model system and identified a simple O-glycopeptide pan-epitope, 501PPA(GalNAc)TAPG507, on the mature gG-2 glycoprotein that was broadly recognized by IgG antibodies in HSV-2-infected individuals but not in HSV-1-infected or noninfected individuals. Serum reactivity to the extended sialyl-T glycoform was tolerated, suggesting that self glycans can participate in immune responses. The methods presented provide new insight into viral immunity and new targets for immunodiagnostic and therapeutic measures. PMID:22491453

  3. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

    PubMed Central

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  4. Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27–restricted human immune response

    PubMed Central

    Dazert, Eva; Neumann-Haefelin, Christoph; Bressanelli, Stéphane; Fitzmaurice, Karen; Kort, Julia; Timm, Jörg; McKiernan, Susan; Kelleher, Dermot; Gruener, Norbert; Tavis, John E.; Rosen, Hugo R.; Shaw, Jaqueline; Bowness, Paul; Blum, Hubert E.; Klenerman, Paul; Bartenschlager, Ralf; Thimme, Robert

    2009-01-01

    There is an association between expression of the MHC class I molecule HLA-B27 and protection following human infection with either HIV or HCV. In both cases, protection has been linked to HLA-B27 presentation of a single immunodominant viral peptide epitope to CD8+ T cells. If HIV mutates the HLA-B27–binding anchor of this epitope to escape the protective immune response, the result is a less-fit virus that requires additional compensatory clustered mutations. Here, we sought to determine whether the immunodominant HLA-B27–restricted HCV epitope was similarly constrained by analyzing the replication competence and immunogenicity of different escape mutants. Interestingly, in most HLA-B27–positive patients chronically infected with HCV, the escape mutations spared the HLA-B27–binding anchor. Instead, the escape mutations were clustered at other sites within the epitope and had only a modest impact on replication competence. Further analysis revealed that the cluster of mutations is required for efficient escape because a combination of mutations is needed to impair T cell recognition of the epitope. Artificially introduced mutations at the HLA-B27–binding anchors were found to be either completely cross-reactive or to lead to substantial loss of fitness. These results suggest that protection by HLA-B27 in HCV infection can be explained by the requirement to accumulate a cluster of mutations within the immunodominant epitope to escape T cell recognition. PMID:19139562

  5. A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B

    PubMed Central

    Becerra-Artiles, Aniuska; Dominguez-Amorocho, Omar; Stern, Lawrence J.; Calvo-Calle, J. Mauricio

    2015-01-01

    Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6

  6. DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV

    PubMed Central

    Hu, Xintao; Valentin, Antonio; Dayton, Frances; Kulkarni, Viraj; Alicea, Candido; Rosati, Margherita; Chowdhury, Bhabadeb; Gautam, Rajeev; Broderick, Kate E.; Sardesai, Niranjan Y.; Martin, Malcolm A.; Mullins, James I.

    2016-01-01

    HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag. Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth. PMID:27733554

  7. Mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and 7b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats.

    PubMed

    Takano, Tomomi; Tomiyama, Yoshika; Katoh, Yasuichiroh; Nakamura, Michiyo; Satoh, Ryoichi; Hohdatsu, Tsutomu

    2011-03-01

    We previously prepared neutralizing monoclonal antibody (MAb)-resistant (mar) mutant viruses using a laboratory strain feline infectious peritonitis virus (FIPV) 79-1146 (Kida et al., 1999). Mar mutant viruses are mutated several amino acids of the neutralizing epitope of Spike protein, compared with the parent strain, FIPV 79-1146. We clarified that MAb used to prepare mar mutant viruses also lost its activity to enhance homologous mar mutant viruses, strongly suggesting that neutralizing and antibody-dependent enhancing epitopes are present in the same region in the strain FIPV 79-1146. We also discovered that amino acid mutation in the neutralizing epitope reduced viral replication in monocytes/macrophages. We also demonstrated that the mutation or deletion of two nucleotides in 7b gene abrogate the virulence of strain FIPV 79-1146.

  8. Insertion of exogenous epitopes in the E3-19K of oncolytic adenoviruses to enhance TAP-independent presentation and immunogenicity.

    PubMed

    Rodríguez-García, A; Svensson, E; Gil-Hoyos, R; Fajardo, C A; Rojas, L A; Arias-Badia, M; Loskog, A S I; Alemany, R

    2015-07-01

    Oncolytic adenoviruses can promote immune responses against tumors by expressing and/or displaying tumor-associated antigens. However, the strong immunodominance of viral antigens mask responses against tumor epitopes. In addition, defects in major histocompatibility complex class I antigen presentation pathway such as the downregulation of the transporter-associated with antigen processing (TAP) are frequently associated with immune evasion of tumor cells. To promote the immunogenicity of exogenous epitopes in the context of an oncolytic adenovirus, we have taken advantage of the ER localization of the viral protein E3-19K. We have inserted tumor-associated epitopes after the N-terminal signal sequence for membrane insertion of this protein and flanked them with linkers cleavable by the protease furin to facilitate their TAP-independent presentation. This strategy allowed an enhanced presentation of the exogenous epitopes in TAP-deficient tumor cells in vitro and the generation of higher specific immune responses in vivo that were able to significantly control tumor growth.

  9. B-cell epitopes of canine parvovirus: distribution on the primary structure and exposure on the viral surface.

    PubMed Central

    Langeveld, J P; Casal, J I; Vela, C; Dalsgaard, K; Smale, S H; Puijk, W C; Meloen, R H

    1993-01-01

    Ten antigenic sites on canine parvovirus (CPV) were mapped with a complete set of overlapping nonapeptides of the capsid proteins VP1 and VP2: five of these sites were recognized by sera from CPV-infected dogs, three were recognized by a rabbit anti-CPV antiserum, and two were recognized by murine monoclonal anti-CPV antibodies. A region covering the first 21 amino-terminal amino acid residues of VP2 was recognized by three sera from infected dogs, one neutralizing rabbit antiserum, and one neutralizing murine monoclonal antibody. Immunoabsorption experiments with full virions indicated that at least 6 of the 10 antigenic sites are located on the surface. Of these six, three sites occur in the amino terminus of VP2. When superimposed on the three-dimensional structure of canine parvovirus (J. Tsao, M. S. Chapman, M. Agbandje, W. Keller, K. Smith, H. Wu, M. Luo, T. J. Smith, M. G. Rossmann, R. W. Compans, and C. R. Parrish, Science 251:1456-1464, 1991), the other three epitopes are located on two loops of VP2 which form the highly exposed "spike" around the threefold-symmetry axis of the virus. Thus, these regions (amino terminus and loops 1 and 3) are of interest as major target sites for induction of neutralizing antibodies. Images PMID:7678305

  10. Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome

    PubMed Central

    Van Dorst, Bieke; Stuyver, Lieven J.

    2017-01-01

    Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed. PMID:28125577

  11. Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome.

    PubMed

    Lagatie, Ole; Van Dorst, Bieke; Stuyver, Lieven J

    2017-01-01

    Understanding the immune response upon infection with the filarial nematode Onchocerca volvulus and the mechanisms that evolved in this parasite to evade immune mediated elimination is essential to expand the toolbox available for diagnostics, therapeutics and vaccines development. Using high-density peptide microarrays we scanned the proteome-wide linear epitope repertoire in Cameroonian onchocerciasis patients and healthy controls from Southern Africa which led to the identification of 249 immunodominant antigenic peptides. Motif analysis learned that 3 immunodominant motifs, encompassing 3 linear epitopes, are present in 70, 43, and 31 of these peptides, respectively and appear to be scattered over the entire proteome in seemingly non-related proteins. These linear epitopes are shown to have an atypical isotype profile dominated by IgG1, IgG3, IgE and IgM, in contrast to the commonly observed IgG4 response in chronic active helminth infections. The identification of these linear epitope motifs may lead to novel diagnostic development but further evaluation of cross-reactivity against common co-infecting human nematode infections will be needed.

  12. The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

    PubMed

    Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

    2015-12-01

    Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased.

  13. Unique epitopes recognized by monoclonal antibodies against HP-PRRSV: deep understanding of antigenic structure and virus-antibody interaction.

    PubMed

    Wang, Qian; Peng, Jinmei; Sun, Yan; Chen, Jiazeng; An, Tongqing; Leng, Chaoliang; Li, Lin; Zhao, Hongyuan; Guo, Xin; Ge, Xinna; Yang, Hanchun; Tian, Zhijun

    2014-01-01

    Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is a member of the genus Arterivirus within the family Arteriviridae. N and GP3 proteins are the immunodominance regions of the PRRSV viral proteins. To identify the B-cell linear antigenic epitopes within HP-PRRSV N and GP3 proteins, two monoclonal antibodies (mAbs) against N and GP3 proteins were generated and characterized, designated as 3D7 and 1F10 respectively. The mAb 3D7 recognized only HuN4-F112 not the corresponding virulent strain (HuN4-F5). It also recognized two other commercial vaccines (JXA1-R and TJM-F92), but not two other HP-PRRSV strains (HNZJJ-F1 and HLJMZ-F2). The B-cell epitope recognized by the mAb 3D7 was localized to N protein amino acids 7-33. Western blot showed that the only difference amino acid between HuN4-F112-N and HuN4-F5-N did not change the mAb 3D7 recognization to N protein. The epitope targeted by the mAb 1F10 was mapped by truncated proteins. We found a new epitope (68-76aa) can be recognized by the mAb. However, the epitope could not be recognized by the positive sera, suggesting the epitope could not induce antibody in pigs. These results should extend our understanding of the antigenic structure of the N protein and antigen-antibody reactions of the GP3 protein in different species.

  14. Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

    PubMed

    Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M

    2014-01-01

    Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

  15. Identification of Continuous Human B-Cell Epitopes in the VP35, VP40, Nucleoprotein and Glycoprotein of Ebola Virus

    PubMed Central

    Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M.

    2014-01-01

    Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7–12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods. PMID:24914933

  16. Identification of Dominant Antibody-Dependent Cell-Mediated Cytotoxicity Epitopes on the Hemagglutinin Antigen of Pandemic H1N1 Influenza Virus

    PubMed Central

    Srivastava, Vikram; Yang, Zheng; Hung, Ivan Fan Ngai; Xu, Jianqing; Zheng, Bojian

    2013-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections in vivo. Hemagglutinin (HA) of influenza virus is highly immunogenic and considered the most important target for immune protection. Several potent cross-reactive HA-specific neutralizing monoclonal antibodies (MAbs) have been reported, and their conserved neutralizing epitopes have been revealed, but there has been no report so far about ADCC epitopes on HA. Here we identified two dominant ADCC epitopes, designated E1 (amino acids [aa] 92 to 117) and E2 (aa 124 to 159), on HA of pandemic H1N1 influenza virus by epitope mapping of convalescent-phase plasma IgG antibodies from six H1N1-infected human subjects in China that exhibited different levels of ADCC activity. The E1 and E2 ADCC epitopes overlapped with immunodominant epitopes of HA. Depletion of purified patient plasma IgG antibodies with EBY100 yeast cells expressing E1 or E2 decreased the ADCC activity of the IgG antibodies. E1 and E2 sequences were found to be highly conserved in H1N1 strains but less so in other subtypes of influenza A viruses. Our study may aid in designing immunogens that can elicit antibodies with high ADCC activity. Vaccine immunogens designed to include the structural determinants of potent broadly neutralizing antibodies and ADCC epitopes may confer comprehensive immune protection against influenza virus infection. PMID:23487456

  17. Identification and characterization of a cross-neutralization epitope of Enterovirus 71.

    PubMed

    Liu, Chia-Chyi; Chou, Ai-Hsiang; Lien, Shu-Pei; Lin, Hsiao-Yu; Liu, Shih-Jen; Chang, Jui-Yuan; Guo, Meng-Shin; Chow, Yen-Hung; Yang, Wun-Syue; Chang, Kate Hsuen-Wen; Sia, Charles; Chong, Pele

    2011-06-10

    Enterovirus 71 (EV71) infections in children manifest as exanthema and are most commonly known as hand-foot-and-mouth disease (HFMD). Because it can cause severe neurological complications like poliomyelitis, EV71 has now emerged as an important neurotropic virus in Asia. EV71 virus has been shown to consist of 3 (A, B and C) genotypes and many subgenotypes. Although EV71 vaccine development has recently yielded promising preclinical results, yet the correlation between the content of antigen(s) in vaccine candidates and the level of protective antibody responses is not established. The neutralization epitope(s) of EV71 antigens could be used as the surrogate biomarker of vaccine potency. Using peptide ELISA, antisera generated from animals immunized with formalin-inactivated EV71 virion vaccine formulated in alum, EV71-specific neutralizing monoclonal antibody (nMAb) and a panel of 153 overlapping synthetic peptides covering the entire sequences of VP1, VP2 and VP3 of EV71, we screened for immunodominant linear neutralization epitope(s). Synthetic peptide VP2-28, corresponding to residues 136-150 of VP2, was found to bind to and inhibit the binding to EV71 of nMAb MAB979 that was found to have cross-neutralizing activity against different genotypes of EV71 virus. In addition, VP2-28 was found to be recognized only by neutralizing antisera generated from rabbits immunized with the formalin-inactivated whole EV71 virion vaccine but not by antisera from immunized mice and rats. During the epitope mapping, a murine EV71 genotype- and strain-specific linear neutralization epitope VP1-43 was identified within residues 211-220 of VP1. Furthermore, based on sequence alignment and structure prediction analysis using poliovirus as the template for molecular modeling, the VP1-43 and VP2-28 epitopes were shown to run in parallel within 0.1 nm and form a rim of the canyon at the junction site of VP1 and VP2 in the viral capsid. In mouse, rat and rabbit immunogenicity studies

  18. Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load.

    PubMed

    Addo, M M; Yu, X G; Rathod, A; Cohen, D; Eldridge, R L; Strick, D; Johnston, M N; Corcoran, C; Wurcel, A G; Fitzpatrick, C A; Feeney, M E; Rodriguez, W R; Basgoz, N; Draenert, R; Stone, David R; Brander, C; Goulder, P J R; Rosenberg, E S; Altfeld, M; Walker, B D

    2003-02-01

    Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

  19. Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis.

    PubMed Central

    Protti, M P; Manfredi, A A; Straub, C; Howard, J F; Conti-Tronconi, B M

    1990-01-01

    In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation. Images PMID:2145582

  20. Synthetic Long Peptide Influenza Vaccine Containing Conserved T and B Cell Epitopes Reduces Viral Load in Lungs of Mice and Ferrets

    PubMed Central

    Rosendahl Huber, S. K.; Camps, M. G. M.; Jacobi, R. H. J.; Mouthaan, J.; van Dijken, H.; van Beek, J.; Ossendorp, F.; de Jonge, J.

    2015-01-01

    Currently licensed influenza vaccines mainly induce antibodies against highly variable epitopes. Due to antigenic drift, protection is subtype or strain-specific and regular vaccine updates are required. In case of antigenic shifts, which have caused several pandemics in the past, completely new vaccines need to be developed. We set out to develop a vaccine that provides protection against a broad range of influenza viruses. Therefore, highly conserved parts of the influenza A virus (IAV) were selected of which we constructed antibody and T cell inducing peptide-based vaccines. The B epitope vaccine consists of the highly conserved HA2 fusion peptide and M2e peptide coupled to a CD4 helper epitope. The T epitope vaccine comprises 25 overlapping synthetic long peptides of 26-34 amino acids, thereby avoiding restriction for a certain MHC haplotype. These peptides are derived from nucleoprotein (NP), polymerase basic protein 1 (PB1) and matrix protein 1 (M1). C57BL/6 mice, BALB/c mice, and ferrets were vaccinated with the B epitopes, 25 SLP or a combination of both. Vaccine-specific antibodies were detected in sera of mice and ferrets and vaccine-specific cellular responses were measured in mice. Following challenge, both mice and ferrets showed a reduction of virus titers in the lungs in response to vaccination. Summarizing, a peptide-based vaccine directed against conserved parts of influenza virus containing B and T cell epitopes shows promising results for further development. Such a vaccine may reduce disease burden and virus transmission during pandemic outbreaks. PMID:26046664

  1. Antigenicity and predefined specificities of the multi-epitope vaccine in candidate consisting of neutralizing epitope and mutated epitopes suggested a new way against HIV-1 mutation.

    PubMed

    Tian, H; Xiao, Y; Qin, L; Chen, Y H

    2001-12-01

    A seven-amino acid epitope GPGRAFY located inside the V3 loop on envelope protein gp120 of HIV-1 is the principal neutralizing epitope (PNE), and a subset of anti-V3 antibodies specific for this epitope show a broad range of neutralizing activity. But this epitope undergoes restricted mutation. In this study, three epitope peptides [C-(GPGRAFY)2, C-(GPGQTFY)2 and C-(GPGQAWY)2] that contain neutralizing epitope GPGRAFY and its two mutated epitope GPGQTFY and GPGQAWY, were synthesized and then conjugated to carrier protein KLH (keyhole limpet hemocyanin). the epitope-vaccines C-(GPGRAFY)2-KLH, C-(GPGQTFY)2-KLH and C-(GPGQAWY)2-KLH induced high levels of antibodies to three V3 loop peptides that contain these epitopes respectively, and the antibody response induced by each epitope-vaccine showed predefined epitope-specific. When these three epitope-peptides mixed together and conjugated to carrier protein, or conjugated to carrier protein separately and then mixed together, high levels of epitope-specific antibodies which respectively recognized these epitopes on V3 loop peptide and both mutated peptides all can be induced by both of them. In blotting assay, these epitope-specific antibodies all recognized the neutralizing epitope and mutated epitopes on peptides respectively. In addition, the reactivity of the antibodies with whole gp120 molecule which contained the epitope GPGRAFY was tested. Only the GPGRAFY-epitope-specific antibodies but not the other antibodies recognized the gp120 molecule. These results provide experimental evidence that the candidate multi-epitope-vaccine containing neutralizing epitope and mutated epitopes may bring new hope against viral mutation resulting in HIV-1 immune evasion and may be developed as an effective vaccine with a broad neutralizing activity against HIV-1 infection.

  2. Systematic identification of immunodominant CD4+ T cell responses to HpaA in Helicobacter pylori infected individuals

    PubMed Central

    Yang, Wuchen; Li, Bin; Sun, Heqiang; Wei, Shanshan; He, Yafei; Zhao, Zhuo; Yang, Shiming; Zou, Quanming; Chen, Weisan; Guo, Hong; Wu, Chao

    2016-01-01

    In mice, antigen-specific CD4+ T cell response is indispensible for the protective immunity against Helicobacter pylori (H. pylori). It has been demonstrated that neuraminyllactose-binding hemagglutinin (HpaA) immunization protected mice from H. pylori infection in a CD4+ T cell dependent manner. However, much remains unclear concerning the human CD4+ T cell responses to HpaA. We conducted a systematic study here to explore the immunodominant, HpaA-specific CD4+ T cell responses in H. pylori infected individuals. We found that HpaA-specific CD4+ T cell responses varied remarkably in their magnitude and had broad epitope-specificity. Importantly, the main responses focused on two regions: HpaA76-105 and HpaA130-159. The HLA-DRB1*0901 restricted HpaA142-159 specific CD4+ T cell response was the most immunodominant response at a population level. The immunodominant epitope HpaA142-159 was naturally presented and highly conserved. We also demonstrated that it was not the broad peptide specificity, but the strength of HpaA specific CD4+ T cell responses associated with gastric diseases potentially caused by H. pylori infection. Such investigation will aid development of novel vaccines against H. pylori infection. PMID:27509059

  3. Differential targeting of viral components by CD4+ versus CD8+ T lymphocytes in dengue virus infection.

    PubMed

    Rivino, Laura; Kumaran, Emmanuelle A P; Jovanovic, Vojislav; Nadua, Karen; Teo, En Wei; Pang, Shyue Wei; Teo, Guo Hui; Gan, Victor Chih Hao; Lye, David C; Leo, Yee Sin; Hanson, Brendon J; Smith, Kenneth G C; Bertoletti, Antonio; Kemeny, David M; MacAry, Paul A

    2013-03-01

    Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4(+) and CD8(+) T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4(+) and 21 are CD8(+) T cell epitopes. We observe that whereas CD8(+) T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4(+) epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4(+) T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4(+) and CD8(+) T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.

  4. Protection against Lethal Enterovirus 71 Challenge in Mice by a Recombinant Vaccine Candidate Containing a Broadly Cross-Neutralizing Epitope within the VP2 EF Loop

    PubMed Central

    Xu, Longfa; He, Delei; Li, Zhiqun; Zheng, Jun; Yang, Lisheng; Yu, Miao; Yu, Hai; Chen, Yixin; Que, Yuqiong; Shih, James Wai Kuo; Liu, Gang; Zhang, Jun; Zhao, Qinjian; Cheng, Tong; Xia, Ningshao

    2014-01-01

    Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the “puff” region that forms the “southern rim” of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection. PMID:24669278

  5. Protection against lethal enterovirus 71 challenge in mice by a recombinant vaccine candidate containing a broadly cross-neutralizing epitope within the VP2 EF loop.

    PubMed

    Xu, Longfa; He, Delei; Li, Zhiqun; Zheng, Jun; Yang, Lisheng; Yu, Miao; Yu, Hai; Chen, Yixin; Que, Yuqiong; Shih, James Wai Kuo; Liu, Gang; Zhang, Jun; Zhao, Qinjian; Cheng, Tong; Xia, Ningshao

    2014-01-01

    Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the "puff" region that forms the "southern rim" of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection.

  6. In vivo immunogenicity of Tax(11-19) epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine.

    PubMed

    Sagar, Divya; Masih, Shet; Schell, Todd; Jacobson, Steven; Comber, Joseph D; Philip, Ramila; Wigdahl, Brian; Jain, Pooja; Khan, Zafar K

    2014-05-30

    Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund's adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-β was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses.

  7. In vivo immunogenicity of Tax 11-19 epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine

    PubMed Central

    Sagar, Divya; Masih, Shet; Schell, Todd; Jacobson, Steven; Comber, Joseph D.; Philip, Ramila; Wigdahl, Brian; Jain, Pooja; Khan, Zafar K.

    2014-01-01

    Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund’s adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8 T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-β was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses. PMID:24739247

  8. Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

    PubMed Central

    Dominguez, Mariana R.; Silveira, Eduardo L. V.; de Vasconcelos, José Ronnie C.; de Alencar, Bruna C. G.; Machado, Alexandre V.; Bruna-Romero, Oscar; Gazzinelli, Ricardo T.; Rodrigues, Mauricio M.

    2011-01-01

    During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development. PMID:21779365

  9. Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.

    PubMed

    Loughney, John W; Rustandi, Richard R; Wang, Dai; Troutman, Matthew C; Dick, Lawrence W; Li, Guanghua; Liu, Zhong; Li, Fengsheng; Freed, Daniel C; Price, Colleen E; Hoang, Van M; Culp, Timothy D; DePhillips, Pete A; Fu, Tong-Ming; Ha, Sha

    2015-06-26

    Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.

  10. Identifying Novel B Cell Epitopes within Toxoplasma gondii GRA6

    PubMed Central

    Wang, Yanhua; Wang, Guangxiang; Cai, Jian Ping

    2016-01-01

    The study of antigenic epitopes from Toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens, the reactions between antigens and antibodies, and many other aspects of immunology, but it also plays a significant role in the development of new diagnostic reagents and vaccines. In the present study, T. gondii GRA6 epitopes were identified using bioinformatics tools and a synthetic peptide technique. The potential B cell epitopes of GRA6 predicted by bioinformatics tools concentrated upon 3 regions of GRA6, 1-20 aa, 44-103 aa, and 172-221 aa. Ten shorter peptides from the 3 regions were synthesized and assessed by ELISA using pig sera from different time points after infection. Three of the 10 peptides (amino acids 44-63, 172-191, and 192-211) tested were recognized by all sera and determined to be immunodominant B-cell epitopes of GRA6. The results indicated that we precisely and accurately located the T. gondii GRA6 epitopes using pig sera collected at different time points after infection. The identified epitopes may be very useful for further studies of epitope-based vaccines and diagnostic reagents. PMID:27658594

  11. Identification of linear B-cell epitopes within Tarp of Chlamydia trachomatis.

    PubMed

    Zhu, Shanli; Feng, Yan; Chen, Jun; Lin, Xiaoyun; Xue, Xiangyang; Chen, Shao; Zhong, Xiaozhi; Li, WenShu; Zhang, Lifang

    2014-12-01

    Chlamydia trachomatis is one of the most prevalent sexually transmitted pathogens. There is currently no commercially available vaccine against C. trachomatis. Chlamydial translocated actin-recruiting phosphoprotein (Tarp) can induce cellular and humoral immune responses in murine models and has been regarded as a potential vaccine candidate. In this report, the amino acid sequence of Tarp was analyzed using computer-assisted techniques to scan B-cell epitopes, and six possible linear B-cell epitopes peptides (aa80-95, aa107-123, aa152-170, aa171-186, aa239-253 and aa497-513) with high predicted antigenicity and high conservation were investigated. Sera from mice immunized with these potential immunodominant peptides was analyzed by ELISA, which showed that epitope 152-170 elicited serum immunoglobulin G (IgG) response and epitope 171-186 elicited both serum IgG and mucosal secretory immunoglobulin A response. The response of immune sera of epitope 171-186 to endogenous Tarp antigen obtained from the Hela229 cells infected with C. trachomatis was confirmed by Western blot and indirect fluorescence assay. In addition, binding of the antibodies against epitope 171-186 to endogenous Tarp was further confirmed by competitive ELISA. Our results demonstrated that the putative epitope (aa171-186) was an immunodominant B-cell epitope of Tarp. If proven protective and safe, this epitope, in combination with other well-documented epitopes, might be included into a candidate epitope-based vaccine against C. trachomatis.

  12. A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71.

    PubMed

    Xu, Longfa; He, Delei; Yang, Lisheng; Li, Zhiqun; Ye, Xiangzhong; Yu, Hai; zhao, Huan; Li, Shuxuan; Yuan, Lunzhi; Qian, Hongliu; Que, Yuqiong; Shih, James Wai Kuo; Zhu, Hua; Li, Yimin; Cheng, Tong; Xia, Ningshao

    2015-08-05

    Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops.

  13. A Broadly Cross-protective Vaccine Presenting the Neighboring Epitopes within the VP1 GH Loop and VP2 EF Loop of Enterovirus 71

    PubMed Central

    Xu, Longfa; He, Delei; Yang, Lisheng; Li, Zhiqun; Ye, Xiangzhong; Yu, Hai; zhao, Huan; Li, Shuxuan; Yuan, Lunzhi; Qian, Hongliu; Que, Yuqiong; Kuo Shih, James Wai; Zhu, Hua; Li, Yimin; Cheng, Tong; Xia, Ningshao

    2015-01-01

    Human enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the major etiological agents of hand, foot and mouth disease (HFMD) and are often associated with neurological complications. Currently, several vaccine types are being developed for EV71 and CA16. In this study, we constructed a bivalent chimeric virus-like particle (VLP) presenting the VP1 (aa208-222) and VP2 (aa141-155) epitopes of EV71 using hepatitis B virus core protein (HBc) as a carrier, designated HBc-E1/2. Immunization with the chimeric VLPs HBc-E1/2 induced higher IgG titers and neutralization titers against EV71 and CA16 in vitro than immunization with only one epitope incorporated into HBc. Importantly, passive immunization with the recombinant HBc-E2 particles protected neonatal mice against lethal EV71 and CA16 infections. We demonstrate that anti-VP2 (aa141-155) sera bound authentic CA16 viral particles, whereas anti-VP1 (aa208-222) sera could not. Moreover, the anti-VP2 (aa141-155) antibodies inhibited the binding of human serum to virions, which demonstrated that the VP2 epitope is immunodominant between EV71 and CA16. These results illustrated that the chimeric VLP HBc-E1/2 is a promising candidate for a broad-spectrum HFMD vaccine, and also reveals mechanisms of protection by the neighboring linear epitopes of the VP1 GH and VP2 EF loops. PMID:26243660

  14. Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

    PubMed Central

    Chong, Pele; Guo, Meng-Shin; Lin, Fion Hsiao-Yu; Hsiao, Kuang-Nan; Weng, Shu-Yang; Chou, Ai-Hsiang; Wang, Jen-Ren; Hsieh, Shih-Yang; Su, Ih-Jen; Liu, Chia-Chyi

    2012-01-01

    Background Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available. Principal Finding In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >106 the tissue culture's infectious dose (TCID50) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10−5 was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190. Conclusion These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary. PMID:23226233

  15. HIV-1 CD4-induced (CD4i) gp120 epitope vaccines promote B and T-cell responses that contribute to reduced viral loads in rhesus macaques.

    PubMed

    Thomas, Michael A; Tuero, Iskra; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Musich, Thomas; Xiao, Peng; Venzon, David; LaBranche, Celia; Montefiori, David C; DiPasquale, Janet; Reed, Steven G; DeVico, Anthony; Fouts, Timothy; Lewis, George K; Gallo, Robert C; Robert-Guroff, Marjorie

    2014-12-01

    To target the HIV CD4i envelope epitope, we primed rhesus macaques with replicating Ad-rhFLSC (HIV-1BaLgp120 linked to macaque CD4 D1 and D2), with or without Ad-SIVgag and Ad-SIVnef. Macaques were boosted with rhFLSC protein. Memory T-cells in PBMC, bronchoalveolar lavage and rectal tissue, antibodies with neutralizing and ADCC activity, and Env-specific secretory IgA in rectal secretions were elicited. Although protective neutralizing antibody levels were induced, SHIVSF162P4 acquisition following rectal challenge was not prevented. Rapid declines in serum ADCC activity, Env-specific memory B cells in PBMC and bone marrow, and systemic and mucosal memory T cells were observed immediately post-challenge together with delayed anamnestic responses. Innate immune signaling resulting from persisting Ad replication and the TLR-4 booster adjuvant may have been in conflict and reoriented adaptive immunity. A different adjuvant paired with replicating Ad, or a longer post-prime interval allowing vector clearance before boosting might foster persistent T- and B-cell memory.

  16. Immunodominance: a new hypothesis to explain parasite escape and host/parasite equilibrium leading to the chronic phase of Chagas' disease?

    PubMed

    Rodrigues, M M; Alencar, B C G de; Claser, C; Tzelepis, F

    2009-03-01

    Intense immune responses are observed during human or experimental infection with the digenetic protozoan parasite Trypanosoma cruzi. The reasons why such immune responses are unable to completely eliminate the parasites are unknown. The survival of the parasite leads to a parasite-host equilibrium found during the chronic phase of chagasic infection in most individuals. Parasite persistence is recognized as the most likely cause of the chagasic chronic pathologies. Therefore, a key question in Chagas' disease is to understand how this equilibrium is established and maintained for a long period. Understanding the basis for this equilibrium may lead to new approaches to interventions that could help millions of individuals at risk for infection or who are already infected with T. cruzi. Here, we propose that the phenomenon of immunodominance may be significant in terms of regulating the host-parasite equilibrium observed in Chagas' disease. T. cruzi infection restricts the repertoire of specific T cells generating, in some cases, an intense immunodominant phenotype and in others causing a dramatic interference in the response to distinct epitopes. This immune response is sufficiently strong to maintain the host alive during the acute phase carrying them to the chronic phase where transmission usually occurs. At the same time, immunodominance interferes with the development of a higher and broader immune response that could be able to completely eliminate the parasite. Based on this, we discuss how we can interfere with or take advantage of immunodominance in order to provide an immunotherapeutic alternative for chagasic individuals.

  17. Stochastic humoral expression of human growth hormone epitopes.

    PubMed Central

    Etcheverrigaray, M; Paladini, A C; Retegui, L A

    1988-01-01

    Competition experiments between insolubilized monoclonal antibodies (mAb) and polyclonal antisera has led to the description of the humoral expression of human growth hormone (hGH) epitopes. This study was carried out with sera from mice and hamsters submitted to different immunization schedules: chronic administration of the antigen, secondary response and conventional hyperimmunization. The results indicated the absence of a unique immunodominant epitope in hGH; a significant individual variation of antibody (Ab) population titres with time; changes with time in the relative proportion of one Ab population with respect to the others; and the occurrence of Ab enhancing the 125I-hGH binding to five mAb depending upon the individuals and the time of immunization. Heterocliticity towards non-human GH was also detected. Although most of the animals showed cross-reacting Ab, two out of 12 mice, chronically injected, developed heteroclitic Ab. The data suggest that the humoral response to different epitopes of a protein antigen during the maturation of the immune response is a stochastic process leading to transient humoral immunodominance, enhancing Ab populations and heterocliticity, depending upon individual characteristics, either in outbred or inbred populations. PMID:2452789

  18. Myelin basic protein-specific T lymphocyte repertoire in multiple sclerosis. Complexity of the response and dominance of nested epitopes due to recruitment of multiple T cell clones.

    PubMed Central

    Meinl, E; Weber, F; Drexler, K; Morelle, C; Ott, M; Saruhan-Direskeneli, G; Goebels, N; Ertl, B; Jechart, G; Giegerich, G

    1993-01-01

    The human T cell response to the myelin basic protein (MBP) has been studied with respect to T cell receptor (TCR) usage, HLA class II restriction elements, and epitope specificity using a total of 215 long-term MBP-specific T cell lines (TCL) isolated from the peripheral blood of 13 patients with multiple sclerosis (MS) and 10 healthy donors. In most donors, the anti-MBP response was exceedingly heterogeneous. Using a panel of overlapping synthetic peptides spanning the entire length of human MBP, at least 26 epitopes recognized by human TCL could be distinguished. The MBP domain most commonly recognized was sequence 80-105 (31% of MS TCL, and 24% of control TCL). Sequence 29-48 was recognized more frequently by control-derived TCL (24%) than by TCL from MS patients (5%). The MBP epitopes were recognized in the context of DRB1 *0101, DRB5*0101, DRB1*1501, DRB1*0301, DRB1*0401, DRB1*1402, and DRB3*0102, as demonstrated using a panel of DR gene-transfected L cells. The TCR gene usage was also heterogeneous. V beta 5.2, a peptide of which is currently being used in a clinical trial for treatment of MS patients, was expressed by only one of our TCL. However, within this complex pattern of MBP-specific T cell responses, a minority of MS patients were found to exhibit a more restricted response with respect to their TCL epitope specificity. In these patients 75-87% of the TCL responded to a single, patient-specific cluster of immunodominant T cell epitopes located within a small (20-amino acid) domain of MBP. These nested clusters of immunodominant epitopes were noted within the amino acids 80-105, 108-131, and 131-153. The T cell response to the immunodominant epitopes was not monoclonal, but heterogeneous, with respect to fine specificity, TCR usage, and even HLA restriction. In one patient (H.K.), this restricted epitope profile remained stable for > 2 yr. The TCR beta chain sequences of TCL specific for the immunodominant region of HK are consistent with an

  19. Linear Epitopes in Vaccinia Virus A27 Are Targets of Protective Antibodies Induced by Vaccination against Smallpox

    PubMed Central

    Kaever, Thomas; Matho, Michael H.; Meng, Xiangzhi; Crickard, Lindsay; Schlossman, Andrew; Xiang, Yan; Crotty, Shane; Peters, Bjoern

    2016-01-01

    ABSTRACT Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their protective capacity and their interaction with A27, we generated and subsequently characterized 7 murine monoclonal antibodies (MAbs), which fell into 4 distinct epitope groups (groups I to IV). The MAbs in three groups (groups I, III, and IV) bound to linear peptides, while the MAbs in group II bound only to VACV lysate and recombinant A27, suggesting that they recognized a conformational and discontinuous epitope. Only group I antibodies neutralized the mature virion in a complement-dependent manner and protected against VACV challenge, while a group II MAb partially protected against VACV challenge but did not neutralize the mature virion. The epitope for group I MAbs was mapped to a region adjacent to the GAG binding site, a finding which suggests that group I MAbs could potentially interfere with the cellular adhesion of A27. We further determined the crystal structure of the neutralizing group I MAb 1G6, as well as the nonneutralizing group IV MAb 8E3, bound to the corresponding linear epitope-containing peptides. Both the light and the heavy chains of the antibodies are important in binding to their antigens. For both antibodies, the L1 loop seems to dominate the overall polar interactions with the antigen, while for MAb 8E3, the light chain generally appears to make more contacts with the antigen. IMPORTANCE Vaccinia virus is a powerful model to study antibody responses upon vaccination, since its use as the smallpox vaccine led to the eradication of one of the world's greatest killers. The immunodominant antigens that elicit the protective antibodies are known, yet for many of these antigens, little information about their precise interaction with antibodies is available. In an

  20. Identification of an immunodominant peptide from citrullinated tenascin-C as a major target for autoantibodies in rheumatoid arthritis

    PubMed Central

    Schwenzer, Anja; Jiang, Xia; Mikuls, Ted R; Payne, Jeffrey B; Sayles, Harlan R; Quirke, Anne-Marie; Kessler, Benedikt M; Fischer, Roman; Venables, Patrick J; Lundberg, Karin; Midwood, Kim S

    2016-01-01

    Objectives We investigated whether citrullinated tenascin-C (cTNC), an extracellular matrix protein expressed at high levels in the joints of patients with rheumatoid arthritis (RA), is a target for the autoantibodies in RA. Methods Citrullinated sites were mapped by mass spectrometry in the fibrinogen-like globe (FBG) domain of tenascin-C treated with peptidylarginine deiminases (PAD) 2 and 4. Antibodies to cyclic peptides containing citrullinated sites were screened in sera from patients with RA by ELISA. Potential cross-reactivity with well-established anticitrullinated protein antibody (ACPA) epitopes was tested by inhibition assays. The autoantibody response to one immunodominant cTNC peptide was then analysed in 101 pre-RA sera (median 7 years before onset) and two large independent RA cohorts. Results Nine arginine residues within FBG were citrullinated by PAD2 and PAD4. Two immunodominant peptides cTNC1 (VFLRRKNG-cit-ENFYQNW) and cTNC5 (EHSIQFAEMKL-cit-PSNF-cit-NLEG-cit-cit-KR) were identified. Antibodies to both showed limited cross-reactivity with ACPA epitopes from α-enolase, vimentin and fibrinogen, and no reactivity with citrullinated fibrinogen peptides sharing sequence homology with FBG. cTNC5 antibodies were detected in 18% of pre-RA sera, and in 47% of 1985 Swedish patients with RA and 51% of 287 North American patients with RA. The specificity was 98% compared with 160 healthy controls and 330 patients with osteoarthritis. Conclusions There are multiple citrullination sites in the FBG domain of tenascin-C. Among these, one epitope is recognised by autoantibodies that are detected years before disease onset, and which may serve as a useful biomarker to identify ACPA-positive patients with high sensitivity and specificity in established disease. PMID:26659718

  1. Induction of memory cytotoxic T cells to influenza A virus and subsequent viral clearance is not modulated by PB1-F2-dependent inflammasome activation

    PubMed Central

    Lee, Patricia (Hoi Yee); Bird, Nicola; MacKenzie-Kludas, Charley; Mansell, Ashley; Kedzierska, Katherine; Brown, Lorena; McAuley, Julie

    2016-01-01

    Expression of the viral virulence protein PB1-F2 during infection has been linked to NLRP3 inflammasome complex activation in macrophages and induction of early inflammatory events enhancing immunopathology during influenza disease. We sought to determine whether PB1-F2-specific NLRP3 inflammasome activation influenced the magnitude and/or robustness of the CD8+ T-cell responses specific for conserved viral antigens and subsequent virus elimination. Using murine heterosubtypic viral infection models, we showed that mice infected with virus unable to produce PB1-F2 protein showed no deficit in the overall magnitude and functional memory responses of CD8+ T cells established during the effector phase compared with those infected with wild-type PB1-F2-expressing virus and were equally capable of mounting robust recall responses. These data indicate that while expression of PB1-F2 protein can induce inflammatory events, the capacity to generate memory CD8+ T cells specific for immunodominant viral epitopes remains uncompromised. PMID:26667784

  2. A novel monoclonal antibody to a defined peptide epitope in MUC16.

    PubMed

    Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa; Blixt, Ola; Arigi, Emma; Pereira, Daniela; Høgdall, Estrid; Mandel, Ulla; Bennett, Eric P; Vakhrushev, Sergey Y; David, Leonor; Clausen, Henrik

    2015-11-01

    The MUC16 mucin is overexpressed and aberrantly glycosylated in ovarian carcinomas. Immunodetection of circulating MUC16 is one of the most used cancer biomarker assays, but existing antibodies to MUC16 fail to distinguish normal and aberrant cancer glycoforms. Although all antibodies react with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent MUC16 immunity. Here, we developed a MUC16 vaccine based on a 1.7TR (264 aa) expressed in Escherichia coli and in vitro enzymatically glycosylated to generate the aberrant cancer-associated glycoform Tn. This vaccine elicited a potent serum IgG response in mice and we identified two major immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry of ovarian benign and cancer lesions, 5E11 showed similar reactivity as traditional MUC16 antibodies, suggesting that the epitope is not efficiently glycosylated. The study provides a vaccine design and immunodominant MUC16 TR epitopes.

  3. Bcipep: A database of B-cell epitopes

    PubMed Central

    Saha, Sudipto; Bhasin, Manoj; Raghava, Gajendra PS

    2005-01-01

    Background Bcipep is a database of experimentally determined linear B-cell epitopes of varying immunogenicity collected from literature and other publicly available databases. Results The current version of Bcipep database contains 3031 entries that include 763 immunodominant, 1797 immunogenic and 471 null-immunogenic epitopes. It covers a wide range of pathogenic organisms like viruses, bacteria, protozoa, and fungi. The database provides a set of tools for the analysis and extraction of data that includes keyword search, peptide mapping and BLAST search. It also provides hyperlinks to various databases such as GenBank, PDB, SWISS-PROT and MHCBN. Conclusion A comprehensive database of B-cell epitopes called Bcipep has been developed that covers information on epitopes from a wide range of pathogens. The Bcipep will be source of information for investigators involved in peptide-based vaccine design, disease diagnosis and research in allergy. It should also be a promising data source for the development and evaluation of methods for prediction of B-cell epitopes. The database is available at . PMID:15921533

  4. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses

    PubMed Central

    da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M.; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S.

    2017-01-01

    Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells. PMID:28081174

  5. Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

    PubMed Central

    Liu, Chia-Chyi; Guo, Meng-Shin; Lin, Fion Hsiao-Yu; Hsiao, Kuang-Nan; Chang, Kate Hsuen-Wen; Chou, Ai-Hsiang; Wang, Yu-Chao; Chen, Yu-Ching; Yang, Chung-Shi; Chong, Pele Choi-Sing

    2011-01-01

    Background Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification. PMID:21603631

  6. Proof of principle for epitope-focused vaccine design

    NASA Astrophysics Data System (ADS)

    Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Chris; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

    2014-03-01

    Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

  7. Proof of principle for epitope-focused vaccine design

    PubMed Central

    Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Christopher; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

    2014-01-01

    Summary Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Multiple major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus (RSV), that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for research and development of a human RSV vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets including antigenically highly variable pathogens such as HIV and influenza. PMID:24499818

  8. Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever.

    PubMed

    Huang, J H; Wey, J J; Sun, Y C; Chin, C; Chien, L J; Wu, Y C

    1999-01-01

    Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B-cell epitopes of nonstructural 1 (NS1) glycoprotein and anti-NS1 antibody response in DEN infection, a series of 15-mer synthetic peptides from the predicted B-cell linear epitopes of DEN-2 NS1 protein were prepared. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN-2 NS1, D2 NS1-P1 (amino acids 1-15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1-P1-specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1-P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti-D2 NS1-P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti-DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection.

  9. Mucosal Tolerance Induced by an Immunodominant Peptide from Rat α3(IV)NC1 in Established Experimental Autoimmune Glomerulonephritis

    PubMed Central

    Reynolds, John; Abbott, Danielle S.; Karegli, Julieta; Evans, David J.; Pusey, Charles D.

    2009-01-01

    Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture’s disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the noncollagenous domain of the α 3 chain of type IV collagen, α3(IV)NC1. Recent studies have identified an immunodominant peptide, pCol (24-38), from the N-terminus of rat α3(IV)NC1; this peptide contains the major B- and T-cell epitopes in EAG and can induce crescentic nephritis. In this study, we investigated the mechanisms of mucosal tolerance in EAG by examining the effects of the nasal administration of this peptide after the onset of disease. A dose-dependent effect was observed: a dose of 300 μg had no effect, a dose of 1000 μg resulted in a moderate reduction in EAG severity, and a dose of 3000 μg produced a marked reduction in EAG severity accompanied by diminished antigen-specific, T-cell proliferative responses. These results demonstrate that mucosal tolerance in EAG can be induced by nasal administration of an immunodominant peptide from the N-terminus of α3(IV)NC1 and should be of value in designing new therapeutic strategies for patients with Goodpasture’s disease and other autoimmune disorders. PMID:19406992

  10. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

    PubMed Central

    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  11. Identification of an immunodominant antigenic site involving the capsid protein VP3 of hepatitis A virus.

    PubMed Central

    Ping, L H; Jansen, R W; Stapleton, J T; Cohen, J I; Lemon, S M

    1988-01-01

    Hepatitis A virus, an hepatotropic picornavirus, is a common cause of acute hepatitis in man for which there is no available vaccine. Competitive binding studies carried out in solid phase suggest that neutralizing monoclonal antibodies to hepatitis A virus recognize a limited number of epitopes on the capsid surface, although the polypeptide locations of these epitopes are not well defined. Neutralization-escape mutants, selected for resistance to monoclonal antibodies, demonstrate broad cross-resistance to other monoclonal antibodies. Sequencing of virion RNA from several of these mutants demonstrated that replacement of aspartic acid residue 70 of capsid protein VP3 (residue 3070) with histidine or alanine confers resistance to neutralization by monoclonal antibody K2-4F2 and prevents binding of this antibody and other antibodies with similar solid-phase competition profiles. These results indicate that residue 3070 contributes to an immunodominant antigenic site. Mutation at residue 102 of VP1 (residue 1102) confers partial resistance against antibody B5-B3 and several other antibodies but does not prevent antibody attachment. Both VP3 and VP1 sites align closely in the linear peptide sequences with sites of neutralization-escape mutations in poliovirus and human rhinovirus, suggesting conservation of structure among these diverse picornaviruses. However, because partial neutralization resistance to several monoclonal antibodies (2D2, 3E1, and B5-B3) was associated with mutation at either residue 3070 or residue 1102, these sites appear more closely related functionally in hepatitis A virus than in these other picornaviruses. PMID:2460866

  12. Epitope spreading of the anti-CYP2D6 antibody response in patients with autoimmune hepatitis and in the CYP2D6 mouse model.

    PubMed

    Hintermann, Edith; Holdener, Martin; Bayer, Monika; Loges, Stephanie; Pfeilschifter, Josef M; Granier, Claude; Manns, Michael P; Christen, Urs

    2011-11-01

    Autoimmune hepatitis (AIH) is a serious chronic inflammatory disease of the liver with yet unknown etiology and largely uncertain immunopathology. The hallmark of type 2 AIH is the generation of liver kidney microsomal-1 (LKM-1) autoantibodies, which predominantly react to cytochrome P450 2D6 (CYP2D6). The identification of disease initiating factors has been hampered in the past, since antibody epitope mapping was mostly performed using serum samples collected late during disease resulting in the identification of immunodominant epitopes not necessarily representing those involved in disease initiation. In order to identify possible environmental triggers for AIH, we analyzed for the first time the spreading of the anti-CYP2D6 antibody response over a prolonged period of time in AIH patients and in the CYP2D6 mouse model, in which mice infected with Adenovirus-human CYP2D6 (Ad-h2D6) develop antibodies with a similar specificity than AIH patients. Epitope spreading was analyzed in six AIH-2-patients and in the CYP2D6 mouse model using SPOTs membranes containing peptides covering the entire CYP2D6 protein. Despite of a considerable variation, both mice and AIH patients largely focus their humoral immune response on an immunodominant epitope early after infection (mice) or diagnosis (patients). The CYP2D6 mouse model revealed that epitope spreading is initiated at the immunodominant epitope and later expands to neighboring and remote regions. Sequence homologies to human pathogens have been detected for all identified epitopes. Our study demonstrates that epitope spreading does indeed occur during the pathogenesis of AIH and supports the concept of molecular mimicry as a possible initiating mechanism for AIH.

  13. TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes

    PubMed Central

    Chowell, Diego; Krishna, Sri; Cocita, Clément; Shu, Jack; Tan, Xuefang; Greenberg, Philip D.; Klavinskis, Linda S.; Blattman, Joseph N.; Anderson, Karen S.

    2015-01-01

    Despite the availability of major histocompatibility complex (MHC)-binding peptide prediction algorithms, the development of T-cell vaccines against pathogen and tumor antigens remains challenged by inefficient identification of immunogenic epitopes. CD8+ T cells must distinguish immunogenic epitopes from nonimmunogenic self peptides to respond effectively against an antigen without endangering the viability of the host. Because this discrimination is fundamental to our understanding of immune recognition and critical for rational vaccine design, we interrogated the biochemical properties of 9,888 MHC class I peptides. We identified a strong bias toward hydrophobic amino acids at T-cell receptor contact residues within immunogenic epitopes of MHC allomorphs, which permitted us to develop and train a hydrophobicity-based artificial neural network (ANN-Hydro) to predict immunogenic epitopes. The immunogenicity model was validated in a blinded in vivo overlapping epitope discovery study of 364 peptides from three HIV-1 Gag protein variants. Applying the ANN-Hydro model on existing peptide-MHC algorithms consistently reduced the number of candidate peptides across multiple antigens and may provide a correlate with immunodominance. Hydrophobicity of TCR contact residues is a hallmark of immunogenic epitopes and marks a step toward eliminating the need for empirical epitope testing for vaccine development. PMID:25831525

  14. An immunodominant class I-restricted cytotoxic T lymphocyte determinant of human immunodeficiency virus type 1 induces CD4 class II-restricted help for itself

    PubMed Central

    1990-01-01

    We have observed that a peptide corresponding to an immunodominant epitope of the HIV-1 envelope protein recognized by class I MHC- restricted CD8+ CTL can also induce T cell help for itself. The help is necessary for restimulation of CTL precursors in vitro with peptide alone in the absence of exogenous lymphokines, can be removed by depletion of CD4+ T cells, and can be replaced by exogenous IL-2. Whereas the CTL in BALB/c or B10. D2 mice are restricted by the class I molecule Dd, the Th cells are restricted by the class II molecule Ad, and the help can be blocked by anti-Ad mAb. To examine the genetic regulation of the induction of help, we studied B10.A mice that share the class I Dd molecule, but have different class II molecules, Ak and Ek. Spleen cells of immune B10.A mice behave like CD4-depleted BALB/c spleen cells in that they cannot be restimulated in vitro by the peptide alone, but can with peptide plus IL-2. Therefore, in the absence of exogenous lymphokines, peptide-specific help is necessary for restimulation with this immunodominant CTL epitope peptide, and in H-2d mice, this peptide stimulates help for itself as well as CTL. We speculate on the implications of these findings for the immunodominance of this peptide in H-2d mice, and for the selective advantage of pairing certain class I and class II molecules in an MHC haplotype. PMID:1689366

  15. Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library.

    PubMed

    Wang, Hongjun; Gao, Yaping; Gong, Yumei; Chen, Xiaoling; Liu, Chuan; Zhou, Xuemei; Blackall, P J; Zhang, Peijun; Yang, Hanchun

    2007-01-31

    Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

  16. Conformational studies of immunodominant myelin basic protein 1-11 analogues using NMR and molecular modeling

    NASA Astrophysics Data System (ADS)

    Laimou, Despina; Lazoura, Eliada; Troganis, Anastassios N.; Matsoukas, Minos-Timotheos; Deraos, Spyros N.; Katsara, Maria; Matsoukas, John; Apostolopoulos, Vasso; Tselios, Theodore V.

    2011-11-01

    Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP1-11) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP1-11[4A]) or a tyrosine residue (Ac-MBP1-11[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-Au was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln3 residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-Au complex, has a different orientation in the mutated analogues especially in the Ac-MBP1-11[4A] peptide. In particular the side chain of Gln3 is not solvent exposed as for the native Ac-MBP1-11 and it is not available for interaction with the TCR.

  17. Conformational studies of immunodominant myelin basic protein 1-11 analogues using NMR and molecular modeling.

    PubMed

    Laimou, Despina; Lazoura, Eliada; Troganis, Anastassios N; Matsoukas, Minos-Timotheos; Deraos, Spyros N; Katsara, Maria; Matsoukas, John; Apostolopoulos, Vasso; Tselios, Theodore V

    2011-11-01

    Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP(1-11)) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP(1-11)[4A]) or a tyrosine residue (Ac-MBP(1-11)[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-A(u) was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln(3) residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-A(u) complex, has a different orientation in the mutated analogues especially in the Ac-MBP(1-11)[4A] peptide. In particular the side chain of Gln(3) is not solvent exposed as for the native Ac-MBP(1-11) and it is not available for interaction with the TCR.

  18. Epitope-vaccine strategy against HIV-1: today and tomorrow.

    PubMed

    Liu, Zuqiang; Xiao, Yi; Chen, Ying-Hua

    2003-01-01

    Vaccines play important roles in preventing infectious diseases caused by different pathogens. However, some pathogens such as HIV-1 challenge current vaccine strategy. Poor immunogenicity and the high mutation rate of HIV-1 make great difficulties in inducing potent immune responses strong enough to prevent infection via vaccination. Epitope-vaccine, which could intensively enhance predefined epitope-specific immune responses, was suggested as a new strategy against HIV-1 and HIV-1 mutation. Epitope-vaccines afford powerful approaches to elicit potent, broad and complete immune protection against not only primary homologous viral isolates but also heterologous viral mutants. Although most studies are still preliminary now, epitope-vaccine as a novel strategy against the AIDS epidemic has great developmental potential. To trigger T-cell-dependent IgG antibody responses and improve affinities of the epitope-specific antibodies, approaches such as recombinant multi-epitope-vaccination and prime-boosting vaccination were suggested. Cellular immune responses, especially CTL responses, could also be elicited and enhanced in addition to humoral immune responses. Developed epitope-vaccines activating both arms of the immune system would benefit prevention and immunotherapy not only against HIV but also other chronic infections.

  19. Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

    PubMed Central

    da Silva, Andréa N. M. Rangel; Nascimento, Eduardo J. M.; Cordeiro, Marli Tenório; Gil, Laura H. V. G.; Montenegro, Silvia M. L.; Marques, Ernesto T. A.

    2009-01-01

    Background Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes. Methodology/Principal Findings Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51–65, 71–90, 131–170, 196–210 and 246–260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51–65), 27 and 28 (aa131–150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51–65, 131–170, 196–210 and 246–260 elicited the highest antibody response and epitopes131–170, 196–210 and 246–260, elicited IFN-γ production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively. Conclusions/Significance Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine. PMID:19826631

  20. Identification and characterization of nematode specific protective epitopes of Brugia malayi TRX towards development of synthetic vaccine construct for lymphatic filariasis.

    PubMed

    Madhumathi, Jayaprakasam; Prince, Prabhu Rajaiah; Anugraha, Gandhirajan; Kiran, Pote; Rao, Donthamsetty Nageswara; Reddy, Maryada Venkata Rami; Kaliraj, Perumal

    2010-07-12

    Although multi-epitope vaccines have been evaluated for various diseases, they have not yet been investigated for lymphatic filariasis. Here, we report for the first time identification of two immunodominant B epitopes (TRXP1 and TRXP2) from the antioxidant Brugia malayi thioredoxin by studying their immune responses in mice model and human subjects. TRXP1 was also found to harbor a T epitope recognized by human PBMCs and mice splenocytes. Further, the epitopic peptides were synthesized as a single peptide conjugate (PC1) and their prophylactic efficacy was tested in a murine model of filariasis with L3 larvae. PC1 conferred a significantly high protection (75.14%) (P < 0.0001) compared to control (3.7%) and recombinant TRX (63.03%) (P < 0.018) in experimental filariasis. Our results suggest that multi-epitope vaccines could be a promising strategy in the control of lymphatic filariasis.

  1. Immunodominant fragments of myelin basic protein initiate T cell-dependent pain

    PubMed Central

    2012-01-01

    to the generation of tactile allodynia, neuroinflammation, and the immunodominant MBP digest peptides in nerve. These MBP peptides initiate mechanical allodynia in both a T cell-dependent and -independent manner. In the course of Wallerian degeneration, the repeated exposure of the cryptic MBP epitopes, which are normally sheltered from immunosurveillance, may induce the MBP-specific T cell clones and a self-sustaining immune reaction, which may together contribute to the transition of acute pain into a chronic neuropathic pain state. PMID:22676642

  2. Identification of cytotoxic T lymphocyte epitopes on swine viruses: multi-epitope design for universal T cell vaccine.

    PubMed

    Liao, Yu-Chieh; Lin, Hsin-Hung; Lin, Chieh-Hua; Chung, Wen-Bin

    2013-01-01

    Classical swine fever (CSF), foot-and-mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are the primary diseases affecting the pig industry globally. Vaccine induced CD8(+) T cell-mediated immune response might be long-lived and cross-serotype and thus deserve further attention. Although large panels of synthetic overlapping peptides spanning the entire length of the polyproteins of a virus facilitate the detection of cytotoxic T lymphocyte (CTL) epitopes, it is an exceedingly costly and cumbersome approach. Alternatively, computational predictions have been proven to be of satisfactory accuracy and are easily performed. Such a method enables the systematic identification of genome-wide CTL epitopes by incorporating epitope prediction tools in analyzing large numbers of viral sequences. In this study, we have implemented an integrated bioinformatics pipeline for the identification of CTL epitopes of swine viruses including the CSF virus (CSFV), FMD virus (FMDV) and PRRS virus (PRRSV) and assembled these epitopes on a web resource to facilitate vaccine design. Identification of epitopes for cross protections to different subtypes of virus are also reported in this study and may be useful for the development of a universal vaccine against such viral infections among the swine population. The CTL epitopes identified in this study have been evaluated in silico and possibly provide more and wider protection in compared to traditional single-reference vaccine design. The web resource is free and open to all users through http://sb.nhri.org.tw/ICES.

  3. Analyses of p53 antibodies in sera of patients with lung carcinoma define immunodominant regions in the p53 protein.

    PubMed Central

    Schlichtholz, B.; Trédaniel, J.; Lubin, R.; Zalcman, G.; Hirsch, A.; Soussi, T.

    1994-01-01

    Antibodies specific for human p53 were analysed in sera of lung cancer patients. We detected p53 antibodies in the sera of 24% (10/42) of patients with lung carcinoma. The distribution was as follows: 4/9 small-cell lung carcinomas (SCLCs), 2/18 squamous cell lung carcinomas (SCCs), 2/10 adenocarcinomas (ADCs) and 2/5 large-cell lung carcinomas (LCCs). p53 antibodies were always present at the time of diagnosis and did not appear during progression of the disease. Using an original peptide-mapping procedure, we precisely localised the p53 epitopes recognised by p53 antibodies. Immunodominant epitopes reacting with antibodies were localised in the amino and carboxy termini of the protein, similar to those found in breast carcinoma patients or in animals immunised with p53. In light of these data, we suggest that p53 antibodies occur via a self-immunisation process that is the consequence of p53 accumulation in tumour cells. p53 antibodies were also detected in two patients without detected malignant disease. One of these patients died 6 months later of lung carcinoma, suggesting that p53 antibodies may be a precocious marker of p53 alteration. Images Figure 2 PMID:7514026

  4. Design of immunogenic and effective multi-epitope DNA vaccines for melanoma.

    PubMed

    Cho, Hyun-Il; Celis, Esteban

    2012-03-01

    Plasmid DNA vaccination is an attractive way to elicit T cell responses against infectious agents and tumor cells. DNA constructs can be designed to contain multiple T cell epitopes to generate a diverse immune response to incorporate numerous antigens and to reduce limitations due to MHC restriction into a single entity. We have prepared cDNA plasmid constructs containing several mouse T cell epitopes connected by either furin-sensitive or furin-resistant linkers and studied the effects of a cationic cell-penetrating sequence from HIV-tat. Significant CD8 T cell responses were obtained with multi-epitope DNA vaccines followed by in vivo electroporation regardless of the type of linker used and whether the construct had the HIV-tat sequence. The magnitude of immune responses was very similar to all CD8 T cell epitopes contained within each vaccine construct, indicating the absence of immunodominance. Incorporating a T helper epitope into the constructs increased the T cell responses. Prophylactic and therapeutic antitumor responses against B16 melanoma were obtained using a construct containing epitopes from melanosomal proteins, indicating that this vaccination was successful in generating responses to self-antigens that potentially may be subjected to immune tolerance. These findings are useful for designing DNA vaccines for a multitude of diseases where T lymphocytes play a protective or therapeutic role.

  5. B epitope multiplicity and B/T epitope orientation influence immunogenicity of foot-and-mouth disease peptide vaccines.

    PubMed

    Blanco, Esther; Cubillos, Carolina; Moreno, Noelia; Bárcena, Juan; de la Torre, Beatriz G; Andreu, David; Sobrino, Francisco

    2013-01-01

    Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD) vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154) linked to a T-cell epitope (3A 21 to 35) of FMD virus (FMDV) elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T) or four (B4T) copies of the B-cell epitope from type O FMDV (a widespread circulating serotype) were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFN γ . Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

  6. Immunogenicity and specificity of the candidate multi-epitope-vaccines against HIV-1.

    PubMed

    Lu, Y; Ding, J; Chen, Y H

    2001-11-01

    The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)2-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.

  7. Comprehensive, quantitative mapping of T cell epitopes in gluten in celiac disease.

    PubMed

    Tye-Din, Jason A; Stewart, Jessica A; Dromey, James A; Beissbarth, Tim; van Heel, David A; Tatham, Arthur; Henderson, Kate; Mannering, Stuart I; Gianfrani, Carmen; Jewell, Derek P; Hill, Adrian V S; McCluskey, James; Rossjohn, Jamie; Anderson, Robert P

    2010-07-21

    Celiac disease is a genetic condition that results in a debilitating immune reaction in the gut to antigens in grain. The antigenic peptides recognized by the T cells that cause this disease are incompletely defined. Our understanding of the epitopes of pathogenic CD4(+ )T cells is based primarily on responses shown by intestinal T-cells in vitro to hydrolysates or polypeptides of gluten, the causative antigen. A protease-resistant 33-amino acid peptide from wheat alpha-gliadin is the immunodominant antigen, but little is known about the spectrum of T cell epitopes in rye and barley or the hierarchy of immunodominance and consistency of recognition of T-cell epitopes in vivo. We induced polyclonal gluten-specific T cells in the peripheral blood of celiac patients by feeding them cereal and performed a comprehensive, unbiased analysis of responses to all celiac toxic prolamins, a class of plant storage protein. The peptides that stimulated T cells were the same among patients who ate the same cereal, but were different after wheat, barley and rye ingestion. Unexpectedly, a sequence from omega-gliadin (wheat) and C-hordein (barley) but not alpha-gliadin was immunodominant regardless of the grain consumed. Furthermore, T cells specific for just three peptides accounted for the majority of gluten-specific T cells, and their recognition of gluten peptides was highly redundant. Our findings show that pathogenic T cells in celiac disease show limited diversity, and therefore suggest that peptide-based therapeutics for this disease and potentially other strongly HLA-restricted immune diseases should be possible.

  8. Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis.

    PubMed Central

    Salinas-Carmona, M C; Castro-Corona, M A; Sepúlveda-Saavedra, J; Perez, L I

    1997-01-01

    We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained. PMID:9067645

  9. Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis.

    PubMed

    Salinas-Carmona, M C; Castro-Corona, M A; Sepúlveda-Saavedra, J; Perez, L I

    1997-03-01

    We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.

  10. Epitope discovery and their use in peptide based vaccines.

    PubMed

    Dudek, Nadine L; Perlmutter, Patrick; Aguilar, Marie-Isabel; Croft, Nathan P; Purcell, Anthony W

    2010-01-01

    With recent advances in the design and delivery of peptide-based therapeutics there has been a growing interest on the use of peptides in vaccine design. Moreover, functional dissection and proteomic analysis of the immunogenic epitopes of proteins from pathogenic micro-organisms, cancers and self-tissues targeted by autoimmune responses, have broadened the range of target epitopes and given clues to enhancing peptide immunogenicity. Consistent with these observations; peptides can be synthesised with defined chemical modifications to mimic natural epitopes and/or deliberately introduce protease resistant peptide bonds to regulate their processing independent of tissue specific proteolysis and to stabilize these compounds in vivo. We discuss the potential of peptide-based vaccines for the treatment of chronic viral diseases and cancer and review recent developments in the field of epitope discovery and peptide-based vaccines.

  11. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif.

    PubMed

    Molinos-Albert, Luis M; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A; Contreras, F-Xabier; Floor, Martin; Cardona, Pere J; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-13

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants.

  12. Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

    PubMed Central

    Molinos-Albert, Luis M.; Bilbao, Eneritz; Agulló, Luis; Marfil, Silvia; García, Elisabet; Concepción, Maria Luisa Rodríguez de la; Izquierdo-Useros, Nuria; Vilaplana, Cristina; Nieto-Garai, Jon A.; Contreras, F.-Xabier; Floor, Martin; Cardona, Pere J.; Martinez-Picado, Javier; Clotet, Bonaventura; Villà-Freixa, Jordi; Lorizate, Maier; Carrillo, Jorge; Blanco, Julià

    2017-01-01

    The HIV-1 gp41 Membrane Proximal External Region (MPER) is recognized by broadly neutralizing antibodies and represents a promising vaccine target. However, MPER immunogenicity and antibody activity are influenced by membrane lipids. To evaluate lipid modulation of MPER immunogenicity, we generated a 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC)-based proteoliposome collection containing combinations of phosphatidylserine (PS), GM3 ganglioside, cholesterol (CHOL), sphingomyelin (SM) and the TLR4 agonist monophosphoryl lipid A (MPLA). A recombinant gp41-derived miniprotein (gp41-MinTT) exposing the MPER and a tetanus toxoid (TT) peptide that favors MHC-II presentation, was successfully incorporated into lipid mixtures (>85%). Immunization of mice with soluble gp41-MinTT exclusively induced responses against the TT peptide, while POPC proteoliposomes generated potent anti-gp41 IgG responses using lower protein doses. The combined addition of PS and GM3 or CHOL/SM to POPC liposomes greatly increased gp41 immunogenicity, which was further enhanced by the addition of MPLA. Responses generated by all proteoliposomes targeted the N-terminal moiety of MPER overlapping the 2F5 neutralizing epitope. Our data show that lipids impact both, the epitope targeted and the magnitude of the response to membrane-dependent antigens, helping to improve MPER-based lipid carriers. Moreover, the identification of immunodominant epitopes allows for the redesign of immunogens targeting MPER neutralizing determinants. PMID:28084464

  13. Epitope mapping of Campylobacter jejuni flagellar capping protein (FliD) by chicken (Gallus gallus domesticus) sera.

    PubMed

    Yeh, Hung-Yueh; Telli, Arife Ezgi; Jagne, Jarra F; Benson, Christopher L; Hiett, Kelli L; Line, John E

    2016-12-01

    Campylobacter jejuni, a Gram-negative rod, is a zoonotic pathogen associated with human acute bacterial gastroenteritis worldwide. The flagellum, composed of more than 35 proteins, is responsible for colonization of C. jejuni in the host gastrointestinal tract as well as inducing protective antibodies against the homologous serotype. In our previous study, we demonstrated that the flagellar capping protein (FliD) is an immunodominant protein that reacted strongly to sera from field chickens. In this communication, we mapped linear immunoreactive epitopes on FliD using a set of 158 synthetic peptides of 15-mer overlapping with 11 amino acid residues on peptide microarrays with sera from field chickens. The results from peptide microarrays showed (1) no cross-reactivity of the immobilized peptides with the secondary anti-chicken antibody in the control incubation, and (2) heterogeneous patterns of sera reacting to the immobilized peptides. The peptides that reacted to more than three chicken sera and had higher averages of fluorescence units were selected for further validation by the peptide ELISA. The results showed peptides 24, 91 and 92 had relatively high reactivity and less variation among 64 individual serum samples, indicating these peptides represented the shared immunodominant epitopes on the C. jejuni FliD protein. These peptides were also recognized by sera from chickens immunized with the purified recombinant FliD protein. The findings of the specific shared linear immunodominant epitopes on FliD in this study provide a rationale for further evaluation to determine their utility as epitope vaccines covering multiple serotypes for chicken immunization, and subsequently, for providing safer poultry products for human consumption.

  14. Quantifying and imaging NY-ESO-1/LAGE-1-derived epitopes on tumor cells using high affinity T cell receptors.

    PubMed

    Purbhoo, Marco A; Sutton, Deborah H; Brewer, Joanna E; Mullings, Rebecca E; Hill, Maxine E; Mahon, Tara M; Karbach, Julia; Jäger, Elke; Cameron, Brian J; Lissin, Nikolai; Vyas, Paresh; Chen, Ji-Li; Cerundolo, Vincenzo; Jakobsen, Bent K

    2006-06-15

    Presentation of intracellular tumor-associated Ags (TAAs) in the context of HLA class I molecules offers unique cancer-specific cell surface markers for the identification and targeting of tumor cells. For most peptide Ags, the levels of and variations in cell surface presentation remain unknown, yet these parameters are of crucial importance when considering specific TAAs as targets for anticancer therapy. Here we use a soluble TCR with picomolar affinity for the HLA-A2-restricted 157-165 epitope of the NY-ESO-1 and LAGE-1 TAAs to investigate presentation of this immunodominant epitope on the surface of a variety of cancer cells. By single molecule fluorescence microscopy, we directly visualize HLA-peptide presentation for the first time, demonstrating that NY-ESO-1/LAGE-1-positive tumor cells present 10-50 NY-ESO-1/LAGE-1(157-165) epitopes per cell.

  15. Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response.

    PubMed Central

    McLachlan, S M; Rapoport, B

    1995-01-01

    TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with VH gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production. PMID:7544244

  16. Antibody biomarker discovery through in vitro directed evolution of consensus recognition epitopes.

    PubMed

    Ballew, John T; Murray, Joseph A; Collin, Pekka; Mäki, Markku; Kagnoff, Martin F; Kaukinen, Katri; Daugherty, Patrick S

    2013-11-26

    To enable discovery of serum antibodies indicative of disease and simultaneously develop reagents suitable for diagnosis, in vitro directed evolution was applied to identify consensus peptides recognized by patients' serum antibodies. Bacterial cell-displayed peptide libraries were quantitatively screened for binders to serum antibodies from patients with celiac disease (CD), using cell-sorting instrumentation to identify two distinct consensus epitope families specific to CD patients (PEQ and (E)/DxFV(Y)/FQ). Evolution of the (E)/DxFV(Y)/FQ consensus epitope identified a celiac-specific epitope, distinct from the two CD hallmark antigens tissue transglutaminase-2 and deamidated gliadin, exhibiting 71% sensitivity and 99% specificity (n = 231). Expansion of the first-generation PEQ consensus epitope via in vitro evolution yielded octapeptides QPEQAFPE and PFPEQxFP that identified ω- and γ-gliadins, and their deamidated forms, as immunodominant B-cell epitopes in wheat and related cereal proteins. The evolved octapeptides, but not first-generation peptides, discriminated one-way blinded CD and non-CD sera (n = 78) with exceptional accuracy, yielding 100% sensitivity and 98% specificity. Because this method, termed antibody diagnostics via evolution of peptides, does not require prior knowledge of pathobiology, it may be broadly useful for de novo discovery of antibody biomarkers and reagents for their detection.

  17. Induction of protective anti-CTL epitope responses against HER-2-positive breast cancer based on multivalent T7 phage nanoparticles.

    PubMed

    Pouyanfard, Somayeh; Bamdad, Taravat; Hashemi, Hamidreza; Bandehpour, Mojgan; Kazemi, Bahram

    2012-01-01

    We report here the development of multivalent T7 bacteriophage nanoparticles displaying an immunodominant H-2k(d)-restricted CTL epitope derived from the rat HER2/neu oncoprotein. The immunotherapeutic potential of the chimeric T7 nanoparticles as anti-cancer vaccine was investigated in BALB/c mice in an implantable breast tumor model. The results showed that T7 phage nanoparticles confer a high immunogenicity to the HER-2-derived minimal CTL epitope, as shown by inducing robust CTL responses. Furthermore, the chimeric nanoparticles protected mice against HER-2-positive tumor challenge in both prophylactic and therapeutic setting. In conclusion, these results suggest that CTL epitope-carrying T7 phage nanoparticles might be a promising approach for development of T cell epitope-based cancer vaccines.

  18. Common antiviral cytotoxic t-lymphocyte epitope for diverse arenaviruses.

    PubMed

    Oldstone, M B; Lewicki, H; Homann, D; Nguyen, C; Julien, S; Gairin, J E

    2001-07-01

    Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

  19. Expanding specificity of class 1 restricted CD8+ T cells for viral epitopes following multiple inoculations of swine with a human adenivorus vectored foot-and-mouth disease virus (FMDV) vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The immune response to the highly acute foot-and-mouth disease virus (FMDV) is routinely reported as a measure of serum antibody. However, a critical effector function of immune responses combating viral infection of mammals is the cytotoxic T lymphocyte (CTL) response, mediated by virus specific ...

  20. Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

    SciTech Connect

    McLellan, Jason S.; Correia, Bruno E.; Chen, Man; Yang, Yongping; Graham, Barney S.; Schief, William R.; Kwong, Peter D.

    2012-06-28

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.

  1. A new EV71 VP3 epitope in norovirus P particle vector displays neutralizing activity and protection in vivo in mice.

    PubMed

    Jiang, Liping; Fan, Rongjun; Sun, Shiyang; Fan, Peihu; Su, Weiheng; Zhou, Yan; Gao, Feng; Xu, Fei; Kong, Wei; Jiang, Chunlai

    2015-11-27

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16), as the main agents causing hand, foot and mouth disease (HFMD), have become a serious public health concern in the Asia-Pacific region. Recently, various neutralizing B cell epitopes of EV71 were identified as targets for promising vaccine candidates. Structural studies of Picornaviridae indicated that potent immunodominant epitopes typically lie in the hypervariable loop of capsid surfaces. However, cross-neutralizing antibodies and cross-protection between EV71 and CVA16 have not been observed. Therefore, we speculated that divergent sequences of the two viruses are key epitopes for inducing protective neutralizing responses. In this study, we selected 10 divergent epitope candidates based on alignment of the EV71 and CVA16 P1 amino acid sequences using the Multalin interface page, and these epitopes are conserved among all subgenotypes of EV71. Simultaneously, by utilizing the norovirus P particle as a novel vaccine delivery carrier, we identified the 71-6 epitope (amino acid 176-190 of VP3) as a conformational neutralizing epitope against EV71 in an in vitro micro-neutralization assay as well as an in vivo protection assay in mice. Altogether, these results indicated that the incorporation of the 71-6 epitope into the norovirus P domain can provide a promising candidate for an effective synthetic peptide-based vaccine against EV71.

  2. Vaccines targeting the cancer-testis antigen SSX-2 elicit HLA-A2 epitope-specific cytolytic T cells.

    PubMed

    Smith, Heath A; McNeel, Douglas G

    2011-10-01

    The cancer-testis antigen synovial sarcoma X breakpoint-2 (SSX-2) is a potentially attractive target for tumor immunotherapy based upon its tissue-restricted expression to germline cells and its frequent expression in malignancies. The goal of this study was to evaluate genetic vaccine encoding SSX-2 to prioritize human leukocyte antigen (HLA)-A2-specific epitopes and determine if a DNA vaccine can elicit SSX-2-specific cytotoxic T lymphocytes (CTLs) capable of lysing prostate cancer cells. HLA-A2-restricted epitopes were identified based on their in vitro binding affinity for HLA-A2 and by the ability of a genetic vaccine to elicit peptide-specific CTL in A2/DR1 (HLA-A2.1+/HLA-DR1+/H-2 class I-/class II-knockout) transgenic mice. We found that SSX-2 peptides p41-49 (KASEKIFYV) and p103-111 (RLQGISPKI) had high affinity for HLA-A2 and were immunogenic in vivo; however, peptide p103-111 was immunodominant with robust peptide-specific immune responses elicited in mice vaccinated with a plasmid DNA vaccine encoding SSX-2. Furthermore, p103-111-specific CTLs were able to lyse an HLA-A2+ prostate cancer cell line. The immunodominance of this epitope was found not to be due to a putative HLA-DR1 epitope (p98-112) flanking p103-111. Finally, we demonstrated that SSX-2 epitope-specific CTLs could be detected and cultured from the peripheral blood of HLA-A2+ prostate cancer patients, notably patients with advanced prostate cancer. Overall, we conclude that SSX-2 peptide p103-111 is an immunodominant HLA-A2-restricted epitope, and epitope-specific CD8 T cells can be detected in patients with prostate cancer, suggesting that tolerance to SSX-2 can be circumvented in vivo. Together, these findings suggest that SSX-2 may be a relevant target antigen for prostate cancer vaccine approaches.

  3. Beyond Viral Neutralization.

    PubMed

    Lewis, George K; Pazgier, Marzena; Evans, David; Ferrari, Guido; Bournazos, Stylianos; Parsons, Matthew S; Bernard, Nicole F; Finzi, Andrés

    2017-01-13

    It has been known for more than 30 years that Human Immunodeficiency Virus 1 (HIV-1) infection drives a very potent B cell response resulting in the production of anti-HIV-1 antibodies targeting several viral proteins, particularly its envelope glycoproteins (Env). Env epitopes are exposed on the surfaces of viral particles and infected cells where they are targets of potentially protective antibodies. These antibodies can interdict infection by neutralization and there is strong evidence suggesting that Fc-mediated effector function can also contribute to protection. Current evidence suggests that Fc-mediated effector function plays a role in protection against infection by broadly neutralizing antibodies (bnAbs) and it might be important for protection by non-neutralizing antibodies. Fc-mediated effector function includes diverse mechanisms that include antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated complement activation (ADC), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cell-mediated virus inhibition (ADCVI), antibody-mediated trancytosis inhibition, and antibody-mediated virus opsonization. All these functions could be beneficial in fighting viral infections including HIV-1. In this perspective, we discuss the latest developments for ADCC responses discussed at the HIVR4P satellite session on non-neutralizing antibodies, with emphasis on the mechanisms of ADCC resistance employed by HIV-1, the structural basis of epitopes recognized by antibodies that mediate ADCC, NK-cell education and ADCC, and murine models to study ADCC against HIV-1.

  4. Identification of Mycobacterium tuberculosis PPE68-specific HLA-A*0201-restricted epitopes for tuberculosis diagnosis.

    PubMed

    Duan, Zhi-Liang; Li, Qiang; Wang, Sina; Chen, Xin-Yu; Liu, Hui-Fang; Chen, Bo-Kun; Li, De-Zhou; Huang, Xi; Wen, Jin-Sheng

    2015-06-01

    PPE68 is a Mycobacterium tuberculosis-specific protein which is absent from the vaccine strains of BCG. A panel of 14 PPE68-derived peptides predicted to bind to HLA-A*0201 was synthesized. The HLA-A*0201 restriction of these peptides was determined in T2 cell line and HLA-A*0201 transgenic mice. The specificity of peptides was assessed in pulmonary tuberculosis (TB) patients using IFN-γ enzyme-linked immunospot (ELISPOT) assay, and immunodominant peptides were further used to evaluate their diagnostic potential in HLA-A*0201-positive pulmonary TB patients. 13 out of 14 peptides were identified as high-affinity binders. Of these peptides, 12 peptides induced significant IFN-γ-secreting T cell response in transgenic mice and 9 peptides were efficiently recognized by peripheral blood mononuclear cells of 10 HLA-A*0201-positive TB patients. Four immunodominant HLA-A*0201-restricted epitopes (PPE68126-134, PPE68133-141, PPE68140-148, and PPE68148-156) were recognized by the most of 80 HLA-A*0201-positive TB patients (81, 86, 74, and 84 %, respectively). These epitopes may be used for a potential diagnosis of M. tuberculosis infection.

  5. Viral Hepatitis

    MedlinePlus

    ... Public Home » For Veterans and the Public Viral Hepatitis Menu Menu Viral Hepatitis Viral Hepatitis Home For ... the Public Veterans and Public Home How is Hepatitis C Treated? Find the facts about the newest ...

  6. Evaluation of a multi-epitope subunit vaccine against avian leukosis virus subgroup J in chickens.

    PubMed

    Xu, Qingqing; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Zhao, Xiaomin

    2015-12-02

    The intricate sequence and antigenic variability of avian leukosis virus subgroup J (ALV-J) have led to unprecedented difficulties in the development of vaccines. Much experimental evidence demonstrates that ALV-J mutants have caused immune evasion and pose a challenge for traditional efforts to develop effective vaccines. To investigate the potential of a multi-epitope vaccination strategy to prevent chickens against ALV-J infections, a recombinant chimeric multi-epitope protein X (rCMEPX) containing both immunodominant B and T epitope concentrated domains selected from the major structural protein of ALV-J using bioinformatics approach was expressed in Escherichia coli Rosetta (DE3). Its immunogenicity and protective efficacy was studied in chickens. The results showed that rCMEPX could elicit neutralizing antibodies and cellular responses, and antibodies induced by rCMEPX could specifically recognize host cell naturally expressed ALV-J proteins, which indicated that the rCMEPX is a good immunogen. Challenge experiments showed 80% chickens that received rCMEPX were well protected against ALV-J challenge. This is the first report of a chimeric multi-epitope protein as a potential immunogen against ALV-J.

  7. Conserved B-Cell Epitopes among Human Bocavirus Species Indicate Potential Diagnostic Targets

    PubMed Central

    Wang, Yaying; Zhou, Hongli; Wu, Chao; Paranhos-Baccalà, Gláucia; Vernet, Guy; Guo, Li; Wang, Jianwei

    2014-01-01

    Background Human bocavirus species 1–4 (HBoV1–4) have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs. Methodology/Principal Findings We generated HBoV1–3 VP2 gene fragment phage display libraries (GFPDLs) and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides–P1 (1MSDTDIQDQQPDTVDAPQNT20), and P2 (162EHAYPNASHPWDEDVMPDL180)–that are conserved among HBoV1–4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1–4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4). Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide. Conclusions/Significance The identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools. PMID:24475201

  8. A Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326

    PubMed Central

    Luthra, Amit; Anand, Arvind; Hawley, Kelly L.; LeDoyt, Morgan; La Vake, Carson J.; Caimano, Melissa J.; Cruz, Adriana R.; Salazar, Juan C.

    2015-01-01

    ABSTRACT We recently demonstrated that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses characteristic BamA bipartite topology. Herein, we used immunofluorescence analysis (IFA) to show that only the β-barrel domain of TP_0326 contains surface-exposed epitopes in intact T. pallidum. Using the solved structure of Neisseria gonorrhoeae BamA, we generated a homology model of full-length TP_0326. Although the model predicts a typical BamA fold, the β-barrel harbors features not described in other BamAs. Structural modeling predicted that a dome comprised of three large extracellular loops, loop 4 (L4), L6, and L7, covers the barrel's extracellular opening. L4, the dome's major surface-accessible loop, contains mainly charged residues, while L7 is largely neutral and contains a polyserine tract in a two-tiered conformation. L6 projects into the β-barrel but lacks the VRGF/Y motif that anchors L6 within other BamAs. IFA and opsonophagocytosis assay revealed that L4 is surface exposed and an opsonic target. Consistent with B cell epitope predictions, immunoblotting and enzyme-linked immunosorbent assay (ELISA) confirmed that L4 is an immunodominant loop in T. pallidum-infected rabbits and humans with secondary syphilis. Antibody capture experiments using Escherichia coli expressing OM-localized TP_0326 as a T. pallidum surrogate further established the surface accessibility of L4. Lastly, we found that a naturally occurring substitution (Leu593 → Gln593) in the L4 sequences of T. pallidum strains affects antibody binding in sera from syphilitic patients. Ours is the first study to employ a “structure-to-pathogenesis” approach to map the surface topology of a T. pallidum OMP within the context of syphilitic infection. IMPORTANCE Previously, we reported that TP_0326 is a bona fide rare outer membrane protein (OMP) in Treponema pallidum and that it possesses the bipartite topology characteristic of a BamA ortholog

  9. Mapping of a Mycoplasma-Neutralizing Epitope on the Mycoplasmal p37 Protein

    PubMed Central

    Kim, Min Kyu; Kim, Won-Tae; Lee, Hyun Min; Choi, Hong Seo; Jo, Yu Ra; Lee, Yangsoon; Jeong, Jaemin; Choi, Dongho; Chang, Hee Jin; Kim, Dae Shick; Jang, Young-Joo; Ryu, Chun Jeih

    2016-01-01

    Many studies have shown that the mycoplasmal membrane protein p37 enhances cancer cell migration, invasion, and metastasis. Previously, we generated 6 monoclonal antibodies (MAbs) against the mycoplasmal protein p37 and showed the presence of mycoplasma-infected circulating tumor cells in the blood of hepatocellular carcinoma patients by using CA27, one of the six MAbs. When mycoplasmas were incubated with cancer cells in the presence of CA27, mycoplasma infection was completely inhibited, suggesting that CA27 is a neutralizing antibody inhibiting mycoplasma infection. To examine the neutralizing epitope of CA27, we generated a series of glutathione S-transferase (GST)-fused p37 deletion mutant proteins in which p37 was partly deleted. To express p37-coding sequences in E.coli, mycoplasmal TGA codons were substituted with TGG in the p37 deletion mutant genes. GST-fused p37 deletion mutant proteins were then screened to identify the epitope targeted by CA27. Western blots showed that CA27 bound to the residues 216–246 on the middle part of the p37 protein while it did not bind to the residues 183–219 and 216–240. Fine mapping showed that CA27 was able to bind to the residues 226–246, but its binding activity was relatively weakened as compared to that to the residues 216–246, suggesting that the residues 226–246 is essential for optimal binding activity of CA27. Interestingly, the treatment of the purified GST-tagged epitopes with urea showed that CA27 binding to the epitope was sodium dodecyl sulfate-resistant but urea-sensitive. The same 226–246 residues were also recognized by two other anti-p37 MAbs, suggesting that the epitope is immunodominant. The identification of the novel neutralizing epitope may provide new insight into the interaction between the p37 protein and host receptors. PMID:28036384

  10. Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein.

    PubMed

    Parra-López, Carlos; Calvo-Calle, J Mauricio; Cameron, Thomas O; Vargas, Luis E; Salazar, Luz Mary; Patarroyo, Manuel E; Nardin, Elizabeth; Stern, Lawrence J

    2006-05-26

    A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.

  11. Evaluation of conformational epitopes on thyroid peroxidase by antipeptide antibody binding and mutagenesis

    PubMed Central

    GORA, M; GARDAS, A; WIKTOROWICZ, W; HOBBY, P; WATSON, P F; WEETMAN, A P; SUTTON, B J; BANGA, J P

    2004-01-01

    Autoantibodies to thyroid peroxidase (TPO) recognize predominantly conformational epitopes, which are restricted to two distinct determinants, termed immunodominant domain region (IDR) A and B. These dominant determinants reside in the region with structural homology to myeloperoxidase (MPO)-like domain and may extend into the adjacent complement control protein (CCP) domain. We have explored the location of these determinants on the MPO-like domain of the structural model of TPO, by identifying exposed hydrophilic loops that are potential candidates for the autoantigenic sites, generating rabbit antipeptide antisera, and competing with well characterized murine monoclonal antibodies (mabs) specific for these two IDRs. We recently defined the location of IDR-B, and here report our findings on the location of IDR-A and its relationship to IDR-B, defined with a new panel of 15 antipeptide antisera. Moreover, in combination with single amino acid replacements by in vitro mutagenesis, we have defined the limits of the IDR-B region on the TPO model. The combination of antisera to peptides P12 (aa 549–563), P14 (aa 599–617) and P18 (aa 210–225) inhibited the binding of the mab specific for IDR-A (mab 2) by 75. The same combination inhibited the binding of autoantibodies to native TPO from 67 to 94% (mean 81·5%) at autoantibody levels of 5 IU. Fabs prepared from the antipeptide IgG and pooled in this combination were also effective in competition assays, thus defining the epitopes more precisely. IDR-A was found to lie immediately adjacent to IDR-B and thus the two immunodominant epitopes form an extended patch on the surface of TPO. Finally, by single amino acid mutagenesis, we show that IDR-B extends to residue N642, thus further localizing the boundary of this autoantigenic region on the structural model. PMID:15030525

  12. Conjugated anionic PEG-citrate G2 dendrimer with multi-epitopic HIV-1 vaccine candidate enhance the cellular immune responses in mice.

    PubMed

    Abdoli, Asghar; Radmehr, Nina; Bolhassani, Azam; Eidi, Akram; Mehrbod, Parvaneh; Motevalli, Fatemeh; Kianmehr, Zahra; Chiani, Mohsen; Mahdavi, Mehdi; Yazdani, Shaghayegh; Ardestani, Mehdi Shafiee; Kandi, Mohammad Reza; Aghasadeghi, Mohammad Reza

    2017-02-20

    Multi-epitope vaccines might cause immunity against multiple antigenic targets. Four immunodominant epitopes of HIV-1 genome were used to construct a polytope vaccine, formulated by dendrimer. Two regimens of polytopes mixture with dendrimer were utilized to immunize BALB/c mice. Adjuvants were also used to boost immune responses. The conjugated polytope could arouse significant cellular immune responses (P < 0.05) and Th1 response showed higher intensity compared to Th2 (P < 0.05). Our study depicted that conjugated dendrimer with multi-epitopic rHIVtop4 would efficiently induce cell-mediated immune responses and might be considered as promising delivery system for vaccines formulation.

  13. Viral induced demyelination.

    PubMed

    Stohlman, S A; Hinton, D R

    2001-01-01

    oligodendroglia or the myelin sheath; or 4) infection initiates activation of an immune response specific for either oligodendroglia or myelin components. Virus-induced inflammation may be associated with the processing of myelin or oligodendroglial components and their presentation to the host's own T cell compartment. Alternatively, antigenic epitopes derived from the viral proteins may exhibit sufficient homology to host components that the immune response to the virus activates autoreactive T cells, i.e. molecular mimicry. Although it is not clear that each of these potential mechanisms participates in the pathogenesis of human demyelinating disease, analysis of the diverse demyelinating viral infections of both humans and rodents provides examples of many of these potential mechanisms.

  14. Self-adjuvanting influenza candidate vaccine presenting epitopes for cell-mediated immunity on a proteinaceous multivalent nanoplatform.

    PubMed

    Szurgot, Inga; Szolajska, Ewa; Laurin, David; Lambrecht, Benedicte; Chaperot, Laurence; Schoehn, Guy; Chroboczek, Jadwiga

    2013-09-13

    We exploit the features of a virus-like particle, adenoviral dodecahedron (Ad Dd), for engineering a multivalent vaccination platform carrying influenza epitopes for cell-mediated immunity. The delivery platform, Ad Dd, is a proteinaceous, polyvalent, and biodegradable nanoparticle endowed with remarkable endocytosis activity that can be engineered to carry 60 copies of a peptide. Influenza M1 is the most abundant influenza internal protein with the conserved primary structure. Two different M1 immunodominant epitopes were separately inserted in Dd external positions without destroying the particles' dodecahedric structure. Both kinds of DdFluM1 obtained through expression in baculovirus system were properly presented by human dendritic cells triggering efficient activation of antigen-specific T cells responses. Importantly, the candidate vaccine was able to induce cellular immunity in vivo in chickens. These results warrant further investigation of Dd as a platform for candidate vaccine, able to stimulate cellular immune responses.

  15. Significance of the immune response to a major, conformational B-cell epitope on the hepatitis C virus NS3 region defined by a human monoclonal antibody.

    PubMed Central

    Mondelli, M U; Cerino, A; Boender, P; Oudshoorn, P; Middeldorp, J; Fipaldini, C; La Monica, N; Habets, W

    1994-01-01

    The nonstructural protein NS3 of hepatitis C virus (HCV) possesses two enzymatic domains which are thought to be essential for the virus life cycle: an N-terminal serine-type proteinase, responsible for the processing of nonstructural polypeptides, and a C-terminal nucleoside triphosphatase/helicase, presumably involved in the unwinding of the viral genome. The human antibody response to NS3 usually appears early in the course of HCV infection and is predominantly directed against the carboxyl-terminal portion; however, its fine specificity and clinical significance are largely unknown. We have generated a human monoclonal antibody (hMAb), designated CM3.B6, from a cloned B-cell line obtained from the peripheral blood of a patient with chronic HCV infection, which selectively recognized the purified NS3 protein expressed in bacteria or in eukaryotic cells transfected with full-length or NS3 cDNA. Fine-specificity studies revealed that CM3.B6 recognized a 92-amino-acid sequence (clone 8, amino acids 1363 to 1454) selected from an NS3 DNase fragment library but failed to bind to 12-mer peptides synthesized from the same region, suggesting recognition of a conformational B-cell epitope. Experiments using deletion mutants of clone 8 and competitive inhibition studies using a panel of NS3 peptide-specific murine MAbs indicated that limited N-terminal and C-terminal deletions resulted in a significant reduction of hMAb binding to clone 8, thus identifying a minimal antibody binding domain within clone 8. Competition experiments showed that binding of CM3.B6 to the NS3 protein was efficiently inhibited by 39 of 44 (89%) sera from HCV-infected patients, suggesting that the hMAb recognized an immunodominant epitope within the NS3 region. More importantly, recognition of the sequence defined by CM3.B6 appeared to accurately discriminate between viremic and nonviremic anti-HCV positive sera, suggesting potentially relevant clinical applications in the diagnosis and treatment

  16. Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding

    NASA Astrophysics Data System (ADS)

    He, Linling; Cheng, Yushao; Kong, Leopold; Azadnia, Parisa; Giang, Erick; Kim, Justin; Wood, Malcolm R.; Wilson, Ian A.; Law, Mansun; Zhu, Jiang

    2015-08-01

    Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314-324) and E2 (residues 412-423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

  17. Epitope structure of the Bordetella pertussis protein P.69 pertactin, a major vaccine component and protective antigen.

    PubMed

    Hijnen, Marcel; Mooi, Frits R; van Gageldonk, Pieter G M; Hoogerhout, Peter; King, Audrey J; Berbers, Guy A M

    2004-07-01

    Bordetella pertussis is reemerging in several countries with a traditionally high vaccine uptake. An analysis of clinical isolates revealed antigenic divergence between vaccine strains and circulating strains with respect to P.69 pertactin. Polymorphisms in P.69 pertactin are mainly limited to regions comprised of amino acid repeats, designated region 1 and region 2. Region 1 flanks the RGD motif, which is involved in adherence. Although antibodies against P.69 pertactin are implicated in protective immunity, little is known about the structure and location of its epitopes. Here we describe the identification by pepscan analysis of the locations of mainly linear epitopes recognized by human sera and mouse monoclonal antibodies (MAbs). A total of 24 epitopes were identified, and of these only 2 were recognized by both MAbs and human antibodies in serum. A number of immunodominant epitopes were identified which were recognized by 78 to 93% of the human sera tested. Blocking experiments indicated the presence of high-avidity human antibodies against conformational epitopes. Human antibodies against linear epitopes had much lower avidities, as they were unable to block MAbs. Pepscan analyses revealed several MAbs which bound to both region 1 and region 2. The two regions are separated by 289 amino acids in the primary structure, and we discuss the possibility that they form a single conformational epitope. Thus, both repeat regions may serve to deflect the immune response targeted to the functional domain of P.69 pertactin. This may explain why the variation in P.69 pertactin is so effective, despite the fact that it is limited to only two small segments of the molecule.

  18. Synthesis and comparison of antibody recognition of conjugates containing herpes simplex virus type 1 glycoprotein D epitope VII.

    PubMed

    Mezö, Gábor; de Oliveira, Eliandre; Krikorian, Dimitrios; Feijlbrief, Matty; Jakab, Annamária; Tsikaris, Vassilios; Sakarellos, Constantinos; Welling-Wester, Sytske; Andreu, David; Hudecz, Ferenc

    2003-01-01

    Synthetic oligopeptides comprising linear or continuous topographic B-cell epitope sequences of proteins might be considered as specific and small size antigens. It has been demonstrated that the strength and specificity of antibody binding could be altered by conjugation to macromolecules or by modification in the flanking regions. However, no systematic studies have been reported to describe the effect of different carrier macromolecules in epitope conjugates. To this end, the influence of carrier structure and topology on antibody recognition of attached epitope has been studied by comparing the antibody binding properties of a new set of conjugates with tetratuftsin analogue (H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2), T20) sequential oligopeptide carrier (SOC(n)), branched chain polypeptide, poly[Lys(Ser(i)-DL-Ala(m))] (SAK), multiple antigenic peptide (MAP), and keyhole limpet hemocyanine (KLH). In these novel constructs, peptide (9)LKNleADPNRFRGKDL(22) ([Nle(11)]-9-22) representing an immunodominant B cell epitope of herpes simplex virus type 1 glycoprotein D (HSV-1 gD) was conjugated to polypeptides through a thioether or amide bond. Here we report on the preparation of sequential and polymeric polypeptides possessing chloroacetyl groups in multiple copies at the alpha- and/or epsilon-amino group of the polypeptides and its use for the conjugation of epitope peptides possessing Cys at C-terminal position. We have performed binding studies (direct and competitive ELISA) with monoclonal antibody (Mab) A16, recognizing the HSV gD-related epitope, [Nle(11)]-9-22, and conjugates containing identical and uniformly oriented epitope peptide in multiple copies attached to five different macromolecules as carrier. Data suggest that the chemical nature of the carrier and the degree of substitution have marked influence on the strength of antibody binding.

  19. Viral Meningitis

    MedlinePlus

    ... have severe illness from viral meningitis. Causes Non-polio enteroviruses are the most common cause of viral ... following viruses spread by visiting CDC’s websites: Non-polio enteroviruses Mumps virus Herpesviruses, including Epstein-Barr virus , ...

  20. Viral Infections

    MedlinePlus

    ... from medicines, which usually move through your bloodstream. Antibiotics do not work for viral infections. There are a few antiviral medicines available. Vaccines can help prevent you from getting many viral diseases. NIH: National Institute of Allergy and Infectious Diseases

  1. Chaperonin GroEL a Brucella immunodominant antigen identified using Nanobody and MALDI-TOF-MS technologies.

    PubMed

    Abbady, A Q; Al-Daoude, A; Al-Mariri, A; Zarkawi, M; Muyldermans, S

    2012-05-15

    The deployment of today's antibodies that are able to distinguish Brucella from the closely similar pathogens, such as Yersinia, is still considered a great challenge since both pathogens share identical LPS (lipopolysaccharide) O-ring epitopes. In addition, because of the great impact of Brucella on health and economy in many countries including Syria, much effort is going to the development of next generation vaccines, mainly on the identification of new immunogenic proteins of this pathogen. In this context, Brucella-specific nanobodies (Nbs), camel genetic engineered heavy-chain antibody fragments, could be of great value. Previously, a large Nb library was constructed from a camel immunized with heat-killed Brucella. Phage display panning of this 'immune' library with Brucella total lysate resulted in a remarkable fast enrichment for a Nb referred to as NbBruc02. In the present work, we investigated the main characteristics of this Nb that can efficiently distinguish under well-defined conditions the Brucella from other bacteria including Yersinia. NbBruc02 showed a strong and specific interaction with its antigen within the crude lysate as tested by a surface plasmon resonance (SPR) biosensor and it was also able to pull down its cognate antigen from such lysate by immuno-capturing. Using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), NbBruc02 specific antigen was identified as chaperonin GroEL, also known as heat shock protein of 60 kDa (HSP-60), which represents a Brucella immunodominant antigen responsible of maintaining proteins folding during stress conditions. Interestingly, the antigen recognition by NbBruc02 was found to be affected by the state of GroEL folding. Thus, the Nb technology applied in the field of infectious diseases, e.g. brucellosis, yields two outcomes: (1) it generates specific binders that can be used for diagnosis, and perhaps treatment, and (2) it identifies the immunogenic candidate

  2. Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution

    PubMed Central

    Haddon, David James; Jarrell, Justin Ansel; Diep, Vivian K.; Wand, Hannah E.; Price, Jordan V.; Tangsombatvisit, Stephanie; Credo, Grace M.; Mackey, Sally; Dekker, Cornelia L.; Baechler, Emily C.; Liu, Chih Long; Varma, Madoo; Utz, Paul J.

    2016-01-01

    The mechanisms underlying development of ribonucleoprotein (RNP) autoantibodies are unclear. The U1-70K protein is the predominant target of RNP autoantibodies, and the RNA binding domain has been shown to be the immunodominant autoantigenic region of U1-70K, although the specific epitopes are not known. To precisely map U1-70K epitopes, we developed silicon-based peptide microarrays with >5700 features, corresponding to 843 unique peptides derived from the U1-70K protein. The microarrays feature overlapping peptides, with single-amino acid resolution in length and location, spanning amino acids 110–170 within the U1-70K RNA binding domain. We evaluated the serum IgG of a cohort of patients with systemic lupus erythematosus (SLE; n = 26) using the microarrays, and identified multiple reactive epitopes, including peptides 116–121 and 143–148. Indirect peptide ELISA analysis of the sera of patients with SLE (n = 88) revealed that ~14% of patients had serum IgG reactivity to 116–121, while reactivity to 143–148 appeared to be limited to a single patient. SLE patients with serum reactivity to 116–121 had significantly lower SLE Disease Activity Index (SLEDAI) scores at the time of sampling, compared to non-reactive patients. Minimal reactivity to the peptides was observed in the sera of healthy controls (n = 92). Competitive ELISA showed antibodies to 116–121 bind a common epitope in U1-70K (68–72) and the matrix protein M1 of human influenza B viruses. Institutional Review Boards approved this study. Knowledge of the precise epitopes of U1-70K autoantibodies may provide insight into the mechanisms of development of anti-RNP, identify potential clinical biomarkers and inform ongoing clinical trails of peptide-based therapeutics. PMID:26333287

  3. Viral pneumonia

    MedlinePlus

    ... Names Pneumonia - viral; Walking pneumonia - viral Images Lungs Respiratory system References Lee FE, Treanor JJ. Viral infections. In: Broaddus VC, Mason RJ, Ernst JD, et al, eds. Murray and Nadel's Textbook of Respiratory Medicine . 6th ed. Philadelphia, PA: Elsevier Saunders; 2016: ...

  4. Diversity of PR3-ANCA epitope specificity in Wegener's granulomatosis. Analysis using the biosensor technology.

    PubMed

    Rarok, Agnieszka A; van der Geld, Ymke M; Stegeman, Coen A; Limburg, Pieter C; Kallenberg, Cees G M

    2003-11-01

    Wegener's granulomatosis is a systemic disease characterized by the presence of antineutrophil cytoplasm autoantibodies specific for proteinase 3 (PR3-ANCA). The functional characteristics of PR3-ANCA differ between quiescent and active disease, suggesting changes in the properties of the autoantibodies in time. Using biosensor technology, we found that PR3-ANCA of different patients (n = 8) recognize a limited number of overlapping regions on PR3 at the time of diagnosis of Wegener's granulomatosis. This area might cover an immunodominant epitope, common for PR3-ANCA from all patients, irrespective of the size of the total area recognized by an individual autoantibody. Experiments with sera (n = 4) collected at the moment of diagnosis and at the time of relapse showed that the individual epitope specificities of PR3-ANCA change during the course of the disease. These changes in epitope specificity of PR3-ANCA may be responsible for the differences in functional properties of these autoantibodies between various stages of the disease.

  5. T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.

    PubMed

    Widhe, M; Ekerfelt, C; Jarefors, S; Skogman, B H; Peterson, E M; Bergström, S; Forsberg, P; Ernerudh, J

    2009-08-01

    We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-gamma secreting cells were detected by ELISPOT. The peptides OspA(17-36), OspA(49-68), OspA(105-124), OspA(137-156), OspA(193-212) and OspA(233-252) showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls (P = 0.004). CSF cells more often showed positive responses to these peptides than blood cells (P = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.

  6. Characterization of Monoclonal Antibodies to Terminal and Internal O-Antigen Epitopes of Francisella tularensis Lipopolysaccharide

    PubMed Central

    Roche, Marly I.; Lu, Zhaohua; Hui, Julia H.

    2011-01-01

    The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia. PMID:21466282

  7. Analysis of amino acid sequence variations and immunoglobulin E-binding epitopes of German cockroach tropomyosin.

    PubMed

    Jeong, Kyoung Yong; Lee, Jongweon; Lee, In-Yong; Ree, Han-Il; Hong, Chein-Soo; Yong, Tai-Soon

    2004-09-01

    The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.

  8. Kinetics of HIV-1 CTL epitopes recognized by HLA I alleles in HIV-infected individuals at times near primary infection: the Provir/Latitude45 study.

    PubMed

    Papuchon, Jennifer; Pinson, Patricia; Guidicelli, Gwenda-Line; Bellecave, Pantxika; Thomas, Réjean; LeBlanc, Roger; Reigadas, Sandrine; Taupin, Jean-Luc; Baril, Jean Guy; Routy, Jean Pierre; Wainberg, Mark; Fleury, Hervé

    2014-01-01

    In patients responding successfully to ART, the next therapeutic step is viral cure. An interesting strategy is antiviral vaccination, particularly involving CD8 T cell epitopes. However, attempts at vaccination are dependent on the immunogenetic background of individuals. The Provir/Latitude 45 project aims to investigate which CTL epitopes in proviral HIV-1 will be recognized by the immune system when HLA alleles are taken into consideration. A prior study (Papuchon et al, PLoS ONE 2013) showed that chronically-infected patients under successful ART exhibited variations of proviral CTL epitopes compared to a reference viral strain (HXB2) and that a generic vaccine may not be efficient. Here, we investigated viral and/or proviral CTL epitopes at different time points in recently infected individuals of the Canadian primary HIV infection cohort and assessed the affinity of these epitopes for HLA alleles during the study period. An analysis of the results confirms that it is not possible to fully predict which epitopes will be recognized by the HLA alleles of the patients if the reference sequences and epitopes are taken as the basis of simulation. Epitopes may be seen to vary in circulating RNA and proviral DNA. Despite this confirmation, the overall variability of the epitopes was low in these patients who are temporally close to primary infection.

  9. Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

    PubMed

    Kalaiselvan, Sagadevan; Sankar, Sathish; Ramamurthy, Mageshbabu; Ghosh, Asit Ranjan; Nandagopal, Balaji; Sridharan, Gopalan

    2017-01-20

    Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 9999: 1-5, 2017. © 2017 Wiley Periodicals, Inc.

  10. Epitope mapping of B-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidum (Tpp15) with synthetic peptides.

    PubMed Central

    Baughn, R E; Demecs, M; Taber, L H; Musher, D M

    1996-01-01

    The antigenicity of the 15-kDa lipoprotein of Treponema pallidum (Tpp15 or TpN15) was comprehensively evaluated in epitope-scanning studies with overlapping deca- and octapeptides and polygonal rabbit and human infant immunoglobulins (Igs) and antisera. This approach enabled us to identify potentially important regions and to determine the optimal dilutions of Igs or antisera for use in further studies. IgM and IgG from both species were capable of recognizing multiple, continuous epitopes. A total of 13 peptides, principally clustered in the central regions of the protein, were recognized by all syphilitic sera and Ig fractions. On the basis of window analyses, frequency profiles, and alanine substitution studies, five heptapeptides were selected for mimetic studies. Two of these five immunodominant, continuous epitopes initially appeared to be species specific; however, antisera elicited against mimetics of all five epitopes were polyspecific, recognizing similar motifs on several other treponemal proteins, including those of avirulent organisms. The only mimetic which yielded positive reactions with infant IgM and syphilitic sera in the absence of cross-reactions with rabbit antisera to avirulent treponemes was the variant of the VMYASSG motif. These findings are relevant to the development of simple, inexpensive assays for the serodiagnosis of active syphilis. PMID:8698467

  11. Vaccines 85: Molecular and chemical basis of resistance to parasitic, bacterial, and viral diseases

    SciTech Connect

    Lerner, R.A.; Chanock, R.M.; Brown, F.

    1985-01-01

    This book contains 70 selections. Some of the selection titles are: Structure of the Gene Encoding of Immunodominant Surface Antigen on the Sprozoite of the Human Malaria Parasite Plasmodium falciparum; Cloning and Expression in Bacteria of the Genes for Merozite-specific Antigens from the Malaria Parasite Plasmodium falciparum; A Major Surface Antigen of Plasmodium falciparum in Merozoites: Studies on the Protein and its Gene; Genetic Construction of Cholera Vaccine Prototypes; and Viral Genes, Cytotoxic T Lymphocytes and Immunity.

  12. Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches.

    PubMed

    Nezafat, Navid; Karimi, Zeinab; Eslami, Mahboobeh; Mohkam, Milad; Zandian, Sanam; Ghasemi, Younes

    2016-06-01

    Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics

  13. Computational Prediction of Broadly Neutralizing HIV-1 Antibody Epitopes from Neutralization Activity Data

    PubMed Central

    Ferguson, Andrew L.; Falkowska, Emilia; Walker, Laura M.; Seaman, Michael S.; Burton, Dennis R.; Chakraborty, Arup K.

    2013-01-01

    Broadly neutralizing monoclonal antibodies effective against the majority of circulating isolates of HIV-1 have been isolated from a small number of infected individuals. Definition of the conformational epitopes on the HIV spike to which these antibodies bind is of great value in defining targets for vaccine and drug design. Drawing on techniques from compressed sensing and information theory, we developed a computational methodology to predict key residues constituting the conformational epitopes on the viral spike from cross-clade neutralization activity data. Our approach does not require the availability of structural information for either the antibody or antigen. Predictions of the conformational epitopes of ten broadly neutralizing HIV-1 antibodies are shown to be in good agreement with new and existing experimental data. Our findings suggest that our approach offers a means to accelerate epitope identification for diverse pathogenic antigens. PMID:24312481

  14. Viral arthritides.

    PubMed

    Outhred, Alexander C; Kok, Jen; Dwyer, Dominic E

    2011-05-01

    Viral infections may manifest as acute or chronic arthritis. Joint involvement arises from either direct infection of the joint, through an immunological response directed towards the virus or autoimmunity. Epidemiological clues to the diagnosis include geographic location and exposure to vector-borne, blood-borne or sexually transmitted viruses. Although not always possible, it is important to diagnose the pathogenic virus, usually by serology, nucleic acid tests or rarely, viral culture. In general, viral arthritides are self-limiting and treatment is targeted at symptomatic relief. This article focuses on the causes, clinical features, diagnosis and treatment of viral arthritides.

  15. Immunodominant regions of a Chlamydia trachomatis type III secretion effector protein, Tarp.

    PubMed

    Wang, Jie; Zhang, Yingqian; Yu, Ping; Zhong, Guangming

    2010-09-01

    We have previously shown that individuals infected with Chlamydia trachomatis can develop a robust antibody response to a Chlamydia type III secretion effector protein called Tarp and that immunization with Tarp induces protection against challenge infection in mice. The current study aimed to map the immunodominant regions of the Tarp protein by expressing 11 fragments of Tarp as glutathione S-transferase (GST) fusion proteins and detecting the reactivity of these fusion proteins with antisera from patients infected with C. trachomatis in the urogenital tract or in the ocular tissue and from rabbits immunized with C. trachomatis organisms. A major immunodominant region was strongly recognized by all antibodies. This region covers amino acids 152 to 302, consisting of three repeats (amino acids 152 to 201, 202 to 251, and 252 to 302). Each of the repeats contains multiple tyrosine residues that are phosphorylated by host cell kinases when Tarp is injected into host cells. Several other minor immunodominant regions were also identified, including those comprising amino acids 1 to 156, 310 to 431, and 582 to 682 (recognized by antisera from both humans and rabbits), that comprising amino acids 425 to 581 (recognized only by human antisera), and that comprising amino acids 683 to 847 (preferentially recognized by rabbit antisera). This immunodominance was also confirmed by the observations that six out of the nine monoclonal antibodies (MAbs) bound to the major immunodominant region and that the other three each bound to one of the minor fragments, comprising amino acids 1 to 119, 120 to 151, and 310 to 431. The antigenicity analyses have provided important information for further understanding the structure and function of Tarp.

  16. Epitope specific T-cell responses against influenza A in a healthy population.

    PubMed

    Savic, Miloje; Dembinski, Jennifer L; Kim, Yohan; Tunheim, Gro; Cox, Rebecca J; Oftung, Fredrik; Peters, Bjoern; Mjaaland, Siri

    2016-02-01

    Pre-existing human CD4(+) and CD8(+) T-cell-mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross-reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database (www.iedb.org), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super-type coverage. Optimized libraries of CD4(+) and CD8(+) T-cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T-cell responses in healthy donors using interferon-γ ELISPOT assays. Upon stimulation, significant CD4(+) and CD8(+) T-cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4(+) and CD8(+) T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T-cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.

  17. Conservation of HIV-1 T cell epitopes across time and clades: validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine.

    PubMed

    Levitz, Lauren; Koita, Ousmane A; Sangare, Kotou; Ardito, Matthew T; Boyle, Christine M; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D; De Groot, Anne S

    2012-12-14

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the "Achilles' heel" of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine.

  18. Conservation of HIV-1 T cell epitopes across time and clades: Validation of immunogenic HLA-A2 epitopes selected for the GAIA HIV vaccine

    PubMed Central

    Levitz, Lauren; Koita, Ousmane A.; Sangare, Kotou; Ardito, Matthew T.; Boyle, Christine M.; Rozehnal, John; Tounkara, Karamoko; Dao, Sounkalo M.; Koné, Youssouf; Koty, Zoumana; Buus, Soren; Moise, Leonard; Martin, William D.; De Groot, Anne S.

    2012-01-01

    HIV genomic sequence variability has complicated efforts to generate an effective globally relevant vaccine. Regions of the viral genome conserved in sequence and across time may represent the “Achilles’ heel” of HIV. In this study, highly conserved T-cell epitopes were selected using immunoinformatics tools combining HLA-A2 supertype binding predictions with relative global conservation. Analysis performed in 2002 on 10,803 HIV-1 sequences, and again in 2009, on 43,822 sequences, yielded 38 HLA-A2 epitopes. These epitopes were experimentally validated for HLA binding and immunogenicity with PBMCs from HIV-infected patients in Providence, Rhode Island, and/or Bamako, Mali. Thirty-five (92%) stimulated an IFNγ response in PBMCs from at least one subject. Eleven of fourteen peptides (79%) were confirmed as HLA-A2 epitopes in both locations. Validation of these HLA-A2 epitopes conserved across time, clades, and geography supports the hypothesis that such epitopes could provide effective coverage of virus diversity and would be appropriate for inclusion in a globally relevant HIV vaccine. PMID:23102976

  19. Identification of Two New HLA-A*1101-Restricted Tax Epitopes Recognized by Cytotoxic T Lymphocytes in an Adult T-Cell Leukemia Patient after Hematopoietic Stem Cell Transplantation

    PubMed Central

    Harashima, Nanae; Tanosaki, Ryuji; Shimizu, Yukiko; Kurihara, Kiyoshi; Masuda, Takao; Okamura, Jun; Kannagi, Mari

    2005-01-01

    We previously reported that Tax-specific CD8+ cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo. PMID:16014972

  20. Identification of two new HLA-A*1101-restricted tax epitopes recognized by cytotoxic T lymphocytes in an adult T-cell leukemia patient after hematopoietic stem cell transplantation.

    PubMed

    Harashima, Nanae; Tanosaki, Ryuji; Shimizu, Yukiko; Kurihara, Kiyoshi; Masuda, Takao; Okamura, Jun; Kannagi, Mari

    2005-08-01

    We previously reported that Tax-specific CD8(+) cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.

  1. Antibody responses to the immunodominant Cryptosporidium gp15 antigen and gp15 polymorphisms in a case-control study of cryptosporidiosis in children in Bangladesh.

    PubMed

    Allison, Genève M; Rogers, Kathleen A; Borad, Anoli; Ahmed, Sabeena; Karim, Mohammad Mahbubul; Kane, Anne V; Hibberd, Patricia L; Naumova, Elena N; Calderwood, Stephen B; Ryan, Edward T; Khan, Wasif A; Ward, Honorine D

    2011-07-01

    Although Cryptospridium hominis is the dominant Cryptosporidium species infecting humans, immune responses to cognate antigens in C. hominis-infected persons have not been reported. We investigated antibody responses to the immunodominant gp15 antigen from C. hominis and C. parvum, in C. hominis-infected Bangladeshi children less than five years of age with diarrhea (cases) and uninfected children with diarrhea (controls). We also investigated polymorphisms in the C. hominis gp15 sequence from cases. Serum IgG responses to gp15 from both species were significantly greater in cases than controls. In spite of polymorphisms in the gp15 sequence, there was a significant correlation between antibody levels to gp15 from both species, indicating cross-reactivity to conserved epitopes. Cases with acute diarrhea had a significantly greater serum IgA response to gp15 compared with those with persistent diarrhea, suggesting that this response may be associated with protection from prolonged disease. These findings support further investigation of gp15 as a vaccine candidate.

  2. Construction and immunological evaluation of truncated hepatitis B core particles carrying HBsAg amino acids 119-152 in the major immunodominant region (MIR).

    PubMed

    Su, Qiudong; Yi, Yao; Guo, Minzhuo; Qiu, Feng; Jia, Zhiyuan; Lu, Xuexin; Meng, Qingling; Bi, Shengli

    2013-09-13

    Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire 'α' antigenic determinant amino acids (aa) 119-152 of HBsAg into the truncated HBc (aa 1-144), between Asp(78) and Pro(79). Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH4)2SO4 precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire 'α' antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.

  3. Mapping of T cell epitopes of the 30-kDa {alpha} antigen of Mycobacterium bovis strain bacillus Calmette-Guerin in Purified Protein Derivative (PPD)-positive individuals

    SciTech Connect

    Silver, R.F.; Wallis, R.S.; Ellner, J.J.

    1995-05-01

    The fibronectin-binding 30-kDa {alpha} Ag is a major secretory protein of growing mycobacteria that stimulates in vitro lymphocyte blastogenesis in most healthy purified protein derivative-positive individuals, but only a minority of patients with active tuberculosis. T cell epitopes of the {alpha} Ag were assessed using blastogenic responses of PBMC from 12 healthy purified protein derivative-positive subjects to a set of synthetic peptides based on the 325-amino acid sequence of the {alpha} Ag of Mycobacterium bovis BCG. Because epitope-specific precursor cells are infrequent and randomly distributed, we used Poisson analysis to determine positive responses to 10 {mu}g/ml of each peptide in 12 replicate culture wells. Seven immunodominant regions of the {alpha} Ag were identified. Each subject responded to at least one of the two most dominant epitopes, which correspond to amino acids 131-155 and 233-257 (from N terminus). Peptides of these two epitopes induced production of IFN-{gamma} by sorted CD4{sup +} T cells. The immuno-dominant peptides may have use as components of a vaccine and as tools to study the evolution of the immune response to M. tuberculosis. The two most dominant epitopes both occur in regions of the {alpha} Ag that differ from those of the atypical pathogens M. avium and M. kansasii. In addition, the M. bovis epitope of amino acids 133-155 differs from that of M. tuberculosis by a single amino acid. It may be possible to exploit the sequence differences for development of diagnostic tests with increased specificity. 39 refs., 4 figs., 1 tab.

  4. In Vivo Validation of Predicted and Conserved T Cell Epitopes in a Swine Influenza Model

    PubMed Central

    Gutiérrez, Andres H.; Loving, Crystal; Moise, Leonard; Terry, Frances E.; Brockmeier, Susan L.; Hughes, Holly R.; Martin, William D.; De Groot, Anne S.

    2016-01-01

    Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs. PMID:27411061

  5. Molecular construction of HIV-gp120 discontinuous epitope mimics by assembly of cyclic peptides on an orthogonal alkyne functionalized TAC-scaffold.

    PubMed

    Werkhoven, P R; Elwakiel, M; Meuleman, T J; Quarles van Ufford, H C; Kruijtzer, J A W; Liskamp, R M J

    2016-01-14

    Mimics of discontinuous epitopes of for example bacterial or viral proteins may have considerable potential for the development of synthetic vaccines, especially if conserved epitopes can be mimicked. However, due to the structural complexity and size of discontinuous epitopes molecular construction of these mimics remains challeging. We present here a convergent route for the assembly of discontinuous epitope mimics by successive azide alkyne cycloaddition on an orthogonal alkyne functionalized scaffold. Here the synthesis of mimics of the HIV gp120 discontinuous epitope that interacts with the CD4 receptor is described. The resulting protein mimics are capable of inhibition of the gp120-CD4 interaction. The route is convergent, robust and should be applicable to other discontinuous epitopes.

  6. Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

    PubMed Central

    Keck, Zhen-Yong; Enterlein, Sven G.; Howell, Katie A.; Vu, Hong; Shulenin, Sergey; Warfield, Kelly L.; Froude, Jeffrey W.; Araghi, Nazli; Douglas, Robin; Biggins, Julia; Lear-Rooney, Calli M.; Wirchnianski, Ariel S.; Lau, Patrick; Wang, Yong; Herbert, Andrew S.; Dye, John M.; Glass, Pamela J.; Holtsberg, Frederick W.; Foung, Steven K. H.

    2015-01-01

    ABSTRACT Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus

  7. Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein

    PubMed Central

    Jagessar, S. Anwar; Holtman, Inge R.; Hofman, Sam; Morandi, Elena; Heijmans, Nicole; Laman, Jon D.; Gran, Bruno; Faber, Bart W.; van Kasteren, Sander I.; Eggen, Bart J. L.

    2016-01-01

    EBV is the major infectious environmental risk factor for multiple sclerosis (MS), but the underlying mechanisms remain obscure. Patient studies do not allow manipulation in vivo. We used the experimental autoimmune encephalomyelitis (EAE) models in the common marmoset and rhesus monkey to model the association of EBV and MS. We report that B cells infected with EBV-related lymphocryptovirus (LCV) are requisite APCs for MHC-E–restricted autoaggressive effector memory CTLs specific for the immunodominant epitope 40-48 of myelin oligodendrocyte glycoprotein (MOG). These T cells drive the EAE pathogenesis to irreversible neurologic deficit. The aim of this study was to determine why LCV infection is important for this pathogenic role of B cells. Transcriptome comparison of LCV-infected B cells and CD20+ spleen cells from rhesus monkeys shows increased expression of genes encoding elements of the Ag cross-presentation machinery (i.e., of proteasome maturation protein and immunoproteasome subunits) and enhanced expression of MHC-E and of costimulatory molecules (CD70 and CD80, but not CD86). It was also shown that altered expression of endolysosomal proteases (cathepsins) mitigates the fast endolysosomal degradation of the MOG40–48 core epitope. Finally, LCV infection also induced expression of LC3-II+ cytosolic structures resembling autophagosomes, which seem to form an intracellular compartment where the MOG40–48 epitope is protected against proteolytic degradation by the endolysosomal serine protease cathepsin G. In conclusion, LCV infection induces a variety of changes in B cells that underlies the conversion of destructive processing of the immunodominant MOG40–48 epitope into productive processing and cross-presentation to strongly autoaggressive CTLs. PMID:27412414

  8. Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

    PubMed

    Lottersberger, J; Salvetti, J L; Beltramini, L M; Tonarelli, G

    2004-01-01

    The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

  9. Localization of key amino acid residues in the dominant conformational epitopes on thyroid peroxidase recognized by mouse monoclonal antibodies.

    PubMed

    Godlewska, Marlena; Czarnocka, Barbara; Gora, Monika

    2012-09-01

    Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders.

  10. Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

    PubMed

    Yin, Ying; Zhang, Sheng; Cai, Chenguang; Zhang, Jun; Dong, Dayong; Guo, Qiang; Fu, Ling; Xu, Junjie; Chen, Wei

    2014-02-01

    Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2β2-2β3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2β2-2β3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine.

  11. Identification of the Neutralizing Epitopes of Merkel Cell Polyomavirus Major Capsid Protein within the BC and EF Surface Loops

    PubMed Central

    Fleury, Maxime J. J.; Nicol, Jérôme T. J.; Samimi, Mahtab; Arnold, Françoise; Cazal, Raphael; Ballaire, Raphaelle; Mercey, Olivier; Gonneville, Hélène; Combelas, Nicolas; Vautherot, Jean-Francois; Moreau, Thierry; Lorette, Gérard; Coursaget, Pierre; Touzé, Antoine

    2015-01-01

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops. PMID:25812141

  12. Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

    PubMed

    Blaher, B; Suphioglu, C; Knox, R B; Singh, M B; McCluskey, J; Rolland, J M

    1996-07-01

    T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

  13. Evolution of viral life-cycle in response to cytotoxic T lymphocyte-mediated immunity.

    PubMed

    Louzoun, Yoram; Ganusov, Vitaly V

    2012-10-07

    Viruses in mammals are constantly faced with the problem of elimination by the host immunity. Cytotoxic T lymphocyte (CTL) responses are thought to play a major role in the control and clearance of several viral infections in mice and humans. It is therefore expected that over evolutionary time, viruses would be forced to evolve to avoid recognition by CTLs. Indeed, a number of studies have documented the accumulation of viral variants with escape mutations. These mutations allow viruses to hide from CTL responses common in the host population. CTLs recognize viruses by short protein sequences, named epitopes, derived from viral proteins. The efficiency of viral recognition by epitope-specific CTL responses depends on the expression pattern of the proteins carrying these epitopes, and the total amount of that protein (and thus epitopes) in the cell. When a virus replicates in a cell, some viral genes are expressed early in the life cycle of the virus, while other proteins are expressed late. For example, HIV infected cells first express Rev and Tat proteins, and the Gag proteins are expressed late. Here we propose a dynamical model of the viral life cycle to study how expression level of early vs. late genes may affect viral dynamics within the host and virus transmission over the course of infection. We find that for acute and chronic viral infections lower expression of early genes than that of the late genes is expected to give selective advantage and higher transmission to viruses.

  14. Viral arthritis

    PubMed Central

    Marks, Michael; Marks, Jonathan L

    2016-01-01

    Acute-onset arthritis is a common clinical problem facing both the general clinician and the rheumatologist. A viral aetiology is though to be responsible for approximately 1% of all cases of acute arthritis with a wide range of causal agents recognised. The epidemiology of acute viral arthritis continues to evolve, with some aetiologies, such as rubella, becoming less common due to vaccination, while some vector-borne viruses have become more widespread. A travel history therefore forms an important part of the assessment of patients presenting with an acute arthritis. Worldwide, parvovirus B19, hepatitis B and C, HIV and the alphaviruses are among the most important causes of virally mediated arthritis. Targeted serological testing may be of value in establishing a diagnosis, and clinicians must also be aware that low-titre autoantibodies, such as rheumatoid factor and antinuclear antibody, can occur in the context of acute viral arthritis. A careful consideration of epidemiological, clinical and serological features is therefore required to guide clinicians in making diagnostic and treatment decisions. While most virally mediated arthritides are self-limiting some warrant the initiation of specific antiviral therapy. PMID:27037381

  15. Viral quasispecies.

    PubMed

    Andino, Raul; Domingo, Esteban

    2015-05-01

    New generation sequencing is greatly expanding the capacity to examine the composition of mutant spectra of viral quasispecies in infected cells and host organisms. Here we review recent progress in the understanding of quasispecies dynamics, notably the occurrence of intra-mutant spectrum interactions, and implications of fitness landscapes for virus adaptation and de-adaptation. Complementation or interference can be established among components of the same mutant spectrum, dependent on the mutational status of the ensemble. Replicative fitness relates to an optimal mutant spectrum that provides the molecular basis for phenotypic flexibility, with implications for antiviral therapy. The biological impact of viral fitness renders particularly relevant the capacity of new generation sequencing to establish viral fitness landscapes. Progress with experimental model systems is becoming an important asset to understand virus behavior in the more complex environments faced during natural infections.

  16. An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction

    PubMed Central

    Potocnakova, Lenka

    2016-01-01

    Identification of B-cell epitopes is a fundamental step for development of epitope-based vaccines, therapeutic antibodies, and diagnostic tools. Epitope-based antibodies are currently the most promising class of biopharmaceuticals. In the last decade, in-depth in silico analysis and categorization of the experimentally identified epitopes stimulated development of algorithms for epitope prediction. Recently, various in silico tools are employed in attempts to predict B-cell epitopes based on sequence and/or structural data. The main objective of epitope identification is to replace an antigen in the immunization, antibody production, and serodiagnosis. The accurate identification of B-cell epitopes still presents major challenges for immunologists. Advances in B-cell epitope mapping and computational prediction have yielded molecular insights into the process of biorecognition and formation of antigen-antibody complex, which may help to localize B-cell epitopes more precisely. In this paper, we have comprehensively reviewed state-of-the-art experimental methods for B-cell epitope identification, existing databases for epitopes, and novel in silico resources and prediction tools available online. We have also elaborated new trends in the antibody-based epitope prediction. The aim of this review is to assist researchers in identification of B-cell epitopes. PMID:28127568

  17. An Introduction to B-Cell Epitope Mapping and In Silico Epitope Prediction.

    PubMed

    Potocnakova, Lenka; Bhide, Mangesh; Pulzova, Lucia Borszekova

    2016-01-01

    Identification of B-cell epitopes is a fundamental step for development of epitope-based vaccines, therapeutic antibodies, and diagnostic tools. Epitope-based antibodies are currently the most promising class of biopharmaceuticals. In the last decade, in-depth in silico analysis and categorization of the experimentally identified epitopes stimulated development of algorithms for epitope prediction. Recently, various in silico tools are employed in attempts to predict B-cell epitopes based on sequence and/or structural data. The main objective of epitope identification is to replace an antigen in the immunization, antibody production, and serodiagnosis. The accurate identification of B-cell epitopes still presents major challenges for immunologists. Advances in B-cell epitope mapping and computational prediction have yielded molecular insights into the process of biorecognition and formation of antigen-antibody complex, which may help to localize B-cell epitopes more precisely. In this paper, we have comprehensively reviewed state-of-the-art experimental methods for B-cell epitope identification, existing databases for epitopes, and novel in silico resources and prediction tools available online. We have also elaborated new trends in the antibody-based epitope prediction. The aim of this review is to assist researchers in identification of B-cell epitopes.

  18. Epitope-Specific Evolution of Human B Cell Responses to Borrelia burgdorferi VlsE Protein from Early to Late Stages of Lyme Disease.

    PubMed

    Jacek, Elzbieta; Tang, Kevin S; Komorowski, Lars; Ajamian, Mary; Probst, Christian; Stevenson, Brian; Wormser, Gary P; Marques, Adriana R; Alaedini, Armin

    2016-02-01

    Most immunogenic proteins of Borrelia burgdorferi, the causative agent of Lyme disease, are known or expected to contain multiple B cell epitopes. However, the kinetics of the development of human B cell responses toward the various epitopes of individual proteins during the course of Lyme disease has not been examined. Using the highly immunogenic VlsE as a model Ag, we investigated the evolution of humoral immune responses toward its immunodominant sequences in 90 patients with a range of early to late manifestations of Lyme disease. The results demonstrate the existence of asynchronous, independently developing, Ab responses against the two major immunogenic regions of the VlsE molecule in the human host. Despite their strong immunogenicity, the target epitopes were inaccessible to Abs on intact spirochetes, suggesting a lack of direct immunoprotective effect. These observations document the association of immune reactivity toward specific VlsE sequences with different phases of Lyme disease, demonstrating the potential use of detailed epitope mapping of Ags for staging of the infection, and offer insights regarding the pathogen's possible immune evasion mechanisms.

  19. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope

    PubMed Central

    1994-01-01

    Mutations that abrogate recognition of a viral epitope by class I- restricted cytotoxic T lymphocyte (CTL) can lead to viral escape if the CTL response against that epitope is crucial for viral clearance. The likelihood of this type of event is low when the CTL response is simultaneously directed against multiple viral epitopes, as has been recently reported for patients with acute self-limited hepatitis B virus (HBV) infection. The CTL response to HBV is usually quite weak, however, during chronic HBV infection, and it is generally acknowledged that this is a major determinant of viral persistence in this disease. If such individuals were to produce a mono- or oligospecific CTL response, however, negative selection of the corresponding mutant viruses might occur. We have recently studied two HLA-A2-positive patients with chronic hepatitis B who, atypically, developed a strong HLA-A2-restricted CTL response against an epitope (FLPSDFFPSV) that contains an HLA-A2-binding motif located between residues 18-27 of the viral nucleocapsid protein, hepatitis B core antigen (HBcAg). These patients failed, however, to respond to any of other HLA-A2-restricted HBV-derived peptides that are generally immunogenic in acutely infected patients who successfully clear the virus. Interestingly, DNA sequence analysis of HBV isolates from these two patients demonstrated alternative residues at position 27 (V --> A and V --> I) and position 21 (S --> N, S --> A, and S --> V) that reduced the HLA and T cell receptor-binding capacities of the variant sequences, respectively. Synthetic peptides containing these alternative sequences were poorly immunogenic compared to the prototype HBc18-27 sequence, and they could not be recognized by CTL clones specific for the prototype peptide. While we do not know if the two patients were originally infected by these variant viruses or if the variants emerged subsequent to infection because of immune selection, the results are most consistent with

  20. Characterization and Epitope Mapping of the Polyclonal Antibody Repertoire Elicited by Ricin Holotoxin-Based Vaccination

    PubMed Central

    Cohen, Ofer; Mechaly, Adva; Sabo, Tamar; Alcalay, Ron; Aloni-Grinstein, Ronit; Seliger, Nehama; Kronman, Chanoch

    2014-01-01

    Ricin, one of the most potent and lethal toxins known, is classified by the Centers for Disease Control and Prevention (CDC) as a select agent. Currently, there is no available antidote against ricin exposure, and the most promising therapy is based on neutralizing antibodies elicited by active vaccination or that are given passively. The aim of this study was to characterize the repertoire of anti-ricin antibodies generated in rabbits immunized with ricin toxoid. These anti-ricin antibodies exhibit an exceptionally high avidity (thiocyanate-based avidity index, 9 M) toward ricin and an apparent affinity of 1 nM. Utilizing a novel tissue culture-based assay that enables the determination of ricin activity within a short time period, we found that the anti-ricin antibodies also possess a very high neutralizing titer. In line with these findings, these antibodies conferred mice with full protection against pulmonary ricinosis when administered as a passive vaccination. Epitope mapping analysis using phage display random peptide libraries revealed that the polyclonal serum contains four immunodominant epitopes, three of which are located on the A subunit and one on the B subunit of ricin. Only two of the four epitopes were found to have a significant role in ricin neutralization. To the best of our knowledge, this is the first work that characterizes these immunological aspects of the polyclonal response to ricin holotoxin-based vaccination. These findings provide useful information and a possible strategy for the development and design of an improved ricin holotoxin-based vaccine. PMID:25209559

  1. Identification of Two Major Conformational Aquaporin-4 Epitopes for Neuromyelitis Optica Autoantibody Binding*

    PubMed Central

    Pisani, Francesco; Mastrototaro, Mauro; Rossi, Andrea; Nicchia, Grazia Paola; Tortorella, Carla; Ruggieri, Maddalena; Trojano, Maria; Frigeri, Antonio; Svelto, Maria

    2011-01-01

    Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. We recently reported that these autoantibodies are able to bind AQP4 when organized in the supramolecular structure called the orthogonal array of particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multiple alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C, and E), and the binding capacity of NMO sera was tested on AQP4 mutants. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence 146GVT(T/M)V150. A second group of sera was characterized by a predominant role of loop A. Deletion of four AQP4-specific amino acids (61G(S/T)E(N/K)64) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E. Finally, a series of AQP0 mutants were produced in which the extracellular loops were progressively changed to make them identical to AQP4. Results showed that none of the mutants was able to reproduce in AQP0 the NMO-IgG epitopes, indicating that the extracellular loop sequence by itself was not sufficient to determine the rearrangement required to create the epitopes. Although our data highlight the complexity of the disease, this study identifies key immunodominant epitopes and provides direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops. PMID:21212277

  2. Identification of two major conformational aquaporin-4 epitopes for neuromyelitis optica autoantibody binding.

    PubMed

    Pisani, Francesco; Mastrototaro, Mauro; Rossi, Andrea; Nicchia, Grazia Paola; Tortorella, Carla; Ruggieri, Maddalena; Trojano, Maria; Frigeri, Antonio; Svelto, Maria

    2011-03-18

    Neuromyelitis optica (NMO) is an autoimmune demyelinating disease characterized by the presence of anti-aquaporin-4 (AQP4) antibodies in the patient sera. We recently reported that these autoantibodies are able to bind AQP4 when organized in the supramolecular structure called the orthogonal array of particles (OAP). To map the antigenic determinants, we produced a series of AQP4 mutants based on multiple alignment sequence analysis between AQP4 and other OAP-forming AQPs. Mutations were introduced in the three extracellular loops (A, C, and E), and the binding capacity of NMO sera was tested on AQP4 mutants. Results indicate that one group of sera was able to recognize a limited portion of loop C containing the amino acid sequence (146)GVT(T/M)V(150). A second group of sera was characterized by a predominant role of loop A. Deletion of four AQP4-specific amino acids ((61)G(S/T)E(N/K)(64)) in loop A substantially affected the binding of this group of sera. However, the binding capacity was further reduced when amino acids in loop A were mutated together with those in loop E or when those in loop C were mutated in combination with loop E. Finally, a series of AQP0 mutants were produced in which the extracellular loops were progressively changed to make them identical to AQP4. Results showed that none of the mutants was able to reproduce in AQP0 the NMO-IgG epitopes, indicating that the extracellular loop sequence by itself was not sufficient to determine the rearrangement required to create the epitopes. Although our data highlight the complexity of the disease, this study identifies key immunodominant epitopes and provides direct evidence that the transition from AQP4 tetramers to AQP4-OAPs involves conformational changes of the extracellular loops.

  3. Mutant MHC class II epitopes drive therapeutic immune responses to cancer

    PubMed Central

    Kreiter, Sebastian; Vormehr, Mathias; van de Roemer, Niels; Diken, Mustafa; Löwer, Martin; Diekmann, Jan; Boegel, Sebastian; Schrörs, Barbara; Vascotto, Fulvia; Castle, John C.; Tadmor, Arbel D.; Schoenberger, Stephen P.; Huber, Christoph; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope

  4. Characterization and epitope mapping of the polyclonal antibody repertoire elicited by ricin holotoxin-based vaccination.

    PubMed

    Cohen, Ofer; Mechaly, Adva; Sabo, Tamar; Alcalay, Ron; Aloni-Grinstein, Ronit; Seliger, Nehama; Kronman, Chanoch; Mazor, Ohad

    2014-11-01

    Ricin, one of the most potent and lethal toxins known, is classified by the Centers for Disease Control and Prevention (CDC) as a select agent. Currently, there is no available antidote against ricin exposure, and the most promising therapy is based on neutralizing antibodies elicited by active vaccination or that are given passively. The aim of this study was to characterize the repertoire of anti-ricin antibodies generated in rabbits immunized with ricin toxoid. These anti-ricin antibodies exhibit an exceptionally high avidity (thiocyanate-based avidity index, 9 M) toward ricin and an apparent affinity of 1 nM. Utilizing a novel tissue culture-based assay that enables the determination of ricin activity within a short time period, we found that the anti-ricin antibodies also possess a very high neutralizing titer. In line with these findings, these antibodies conferred mice with full protection against pulmonary ricinosis when administered as a passive vaccination. Epitope mapping analysis using phage display random peptide libraries revealed that the polyclonal serum contains four immunodominant epitopes, three of which are located on the A subunit and one on the B subunit of ricin. Only two of the four epitopes were found to have a significant role in ricin neutralization. To the best of our knowledge, this is the first work that characterizes these immunological aspects of the polyclonal response to ricin holotoxin-based vaccination. These findings provide useful information and a possible strategy for the development and design of an improved ricin holotoxin-based vaccine.

  5. Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

    PubMed Central

    Bian, Yixin; Zhao, Shuoxian; Zhu, Shaomei; Zeng, Jinfeng; Li, Tingting; Fu, Yongshui; Wang, Yuanzhan; Zheng, Xin; Zhang, Ling; Wang, Wenjing; Yang, Baocheng; Zhou, Yuanping; Allain, Jean-Pierre; Li, Chengyao

    2013-01-01

    Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope 1231PTGSGKSTK1239 (EP05) or core motif 1373IPFYGKAI1380 (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection. PMID:23894620

  6. Viral Hepatitis

    MedlinePlus

    ... with hepatitis? How does a pregnant woman pass hepatitis B virus to her baby? If I have hepatitis B, what does my baby need so that she ... Can I breastfeed my baby if I have hepatitis B? More information on viral hepatitis What is hepatitis? ...

  7. A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response.

    PubMed Central

    Yoshimoto, K; Rosenfeld, S; Frickhofen, N; Kennedy, D; Hills, R; Kajigaya, S; Young, N S

    1991-01-01

    We used 18 monoclonal antibodies against B19 parvovirus to identify neutralizing epitopes on the viral capsid. Of the 18 antibodies, 9 had in vitro neutralizing activity in a bone marrow colony culture assay. The overlapping polypeptide fragments spanning the B19 structural proteins were produced in a pMAL-c Escherichia coli expression system and used to investigate the binding sites of the neutralizing antibodies. One of the nine neutralizing antibodies reacted with both VP1 and VP2 capsid proteins and a single polypeptide fragment on an immunoblot, identifying a linear neutralizing epitope between amino acids 57 and 77 of the VP2 capsid protein. Eight of nine neutralizing antibodies failed to react with either of the capsid proteins or any polypeptide fragments, despite reactivities with intact virions in a radioimmunoassay, suggesting that additional conformationally dependent neutralizing epitopes exist. Images PMID:1719240

  8. Acyclovir Therapy Reduces the CD4+ T Cell Response against the Immunodominant pp65 Protein from Cytomegalovirus in Immune Competent Individuals.

    PubMed

    Pachnio, Annette; Begum, Jusnara; Fox, Ashini; Moss, Paul

    2015-01-01

    Cytomegalovirus (CMV) infects the majority of the global population and leads to the development of a strong virus-specific immune response. The CMV-specific CD4+ and CD8+ T cell immune response can comprise between 10 and 50% of the T cell pool within peripheral blood and there is concern that this may impair immunity to other pathogens. Elderly individuals with the highest magnitude of CMV-specific immune response have been demonstrated to be at increased risk of mortality and there is increasing interest in interventions that may serve to moderate this. Acyclovir is an anti-viral drug with activity against a range of herpes viruses and is used as long term treatment to suppress reactivation of herpes simplex virus. We studied the immune response to CMV in patients who were taking acyclovir to assess if therapy could be used to suppress the CMV-specific immune response. The T cell reactivity against the immunodominant late viral protein pp65 was reduced by 53% in people who were taking acyclovir. This effect was seen within one year of therapy and was observed primarily within the CD4+ response. Acyclovir treatment only modestly influenced the immune response to the IE-1 target protein. These data show that low dose acyclovir treatment has the potential to modulate components of the T cell response to CMV antigen proteins and indicate that anti-viral drugs should be further investigated as a means to reduce the magnitude of CMV-specific immune response and potentially improve overall immune function.

  9. Identification of a linear B-cell epitope on the avian leukosis virus P27 protein using monoclonal antibodies.

    PubMed

    Li, Xiaofei; Qin, Liting; Zhu, Haibo; Sun, Yingjun; Cui, Xuezhi; Gao, Yadong; Qi, Xiaole; Wang, Yongqiang; Gao, Honglei; Gao, Yulong; Wang, Xiaomei

    2016-10-01

    Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce various clinical tumors. The capsid protein P27 is the group-specific antigen of ALV and has many viral antigen sites that are easy to detect. In this study, we produced a monoclonal antibody (mAb), 3A9, that is specific for the P27 protein. A series of partially overlapping peptides were screened to define (181)PPSAR(185) as the minimal linear epitope recognized by mAb 3A9. The identified epitope could be recognized by chicken anti-ALV and mouse anti-ALV P27 sera. The epitope was highly conserved among a number of ALV-A, ALV-B and ALV-J strains. MAb 3A9 might be a valuable tool for the development of new immunodiagnostic approaches for ALV, and the defined linear epitope might help further our understanding of the antigenic structure of the P27 protein.

  10. Identification of class I HLA T cell control epitopes for West Nile virus.

    PubMed

    Kaabinejadian, Saghar; Piazza, Paolo A; McMurtrey, Curtis P; Vernon, Stephen R; Cate, Steven J; Bardet, Wilfried; Schafer, Fredda B; Jackson, Kenneth W; Campbell, Diana M; Buchli, Rico; Rinaldo, Charles R; Hildebrand, William H

    2013-01-01

    The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.

  11. Presence of celiac disease epitopes in modern and old hexaploid wheat varieties: wheat breeding may have contributed to increased prevalence of celiac disease

    PubMed Central

    de Jong, Hein C.; Salentijn, Elma M. J.; Dekking, Liesbeth; Bosch, Dirk; Hamer, Rob J.; Gilissen, Ludovicus J. W. J.; van der Meer, Ingrid M.; Smulders, Marinus J. M.

    2010-01-01

    Gluten proteins from wheat can induce celiac disease (CD) in genetically susceptible individuals. Specific gluten peptides can be presented by antigen presenting cells to gluten-sensitive T-cell lymphocytes leading to CD. During the last decades, a significant increase has been observed in the prevalence of CD. This may partly be attributed to an increase in awareness and to improved diagnostic techniques, but increased wheat and gluten consumption is also considered a major cause. To analyze whether wheat breeding contributed to the increase of the prevalence of CD, we have compared the genetic diversity of gluten proteins for the presence of two CD epitopes (Glia-α9 and Glia-α20) in 36 modern European wheat varieties and in 50 landraces representing the wheat varieties grown up to around a century ago. Glia-α9 is a major (immunodominant) epitope that is recognized by the majority of CD patients. The minor Glia-α20 was included as a technical reference. Overall, the presence of the Glia-α9 epitope was higher in the modern varieties, whereas the presence of the Glia-α20 epitope was lower, as compared to the landraces. This suggests that modern wheat breeding practices may have led to an increased exposure to CD epitopes. On the other hand, some modern varieties and landraces have been identified that have relatively low contents of both epitopes. Such selected lines may serve as a start to breed wheat for the introduction of ‘low CD toxic’ as a new breeding trait. Large-scale culture and consumption of such varieties would considerably aid in decreasing the prevalence of CD. PMID:20664999

  12. Automatic Generation of Validated Specific Epitope Sets.

    PubMed

    Carrasco Pro, Sebastian; Sidney, John; Paul, Sinu; Lindestam Arlehamn, Cecilia; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro

    2015-01-01

    Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue) were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.

  13. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    DOE PAGES

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; ...

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

  14. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    SciTech Connect

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; Kamin-Lewis, Roberta; Al-Darmarki, Salma; Flinko, Robin; Lovo, Elena; Wu, Xueji; Robinson, James E.; Seaman, Michael S.; Fouts, Timothy R.; Gallo, Robert C.; DeVico, Anthony L.; Lewis, George K.

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

  15. Ligand-induced Epitope Masking

    PubMed Central

    Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

    2016-01-01

    We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

  16. Location and dynamics of the immunodominant CD8 T cell response to SIVΔnef immunization and SIVmac251 vaginal challenge.

    PubMed

    Sasikala-Appukuttan, Arun K; Kim, Hyeon O; Kinzel, Nikilyn J; Hong, Jung Joo; Smith, Anthony J; Wagstaff, Reece; Reilly, Cavan; Piatak, Michael; Lifson, Jeffrey D; Reeves, R Keith; Johnson, R Paul; Haase, Ashley T; Skinner, Pamela J

    2013-01-01

    Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission.

  17. Construction and immunological evaluation of truncated hepatitis B core particles carrying HBsAg amino acids 119–152 in the major immunodominant region (MIR)

    SciTech Connect

    Su, Qiudong; Yi, Yao; Guo, Minzhuo; Qiu, Feng; Jia, Zhiyuan; Lu, Xuexin; Meng, Qingling; Bi, Shengli

    2013-09-13

    Highlights: •The conformational HBV neutralization antigen domain was successfully displayed on the surface of truncated HBc particles. •Appropriate dialysis procedures to support the renaturing environment for the protein refolding. •Efficient purification procedures to obtain high purity and icosahedral particles of mosaic HBV antigen. •Strong immune responses not only including neutralization antibody response but also Th1 cell response were induced in mice. -- Abstract: Hepatitis B capsid protein expressed in Escherichia coli can reassemble into icosahedral particles, which could strongly enhance the immunogenicity of foreign epitopes, especially those inserted into its major immunodominant region. Herein, we inserted the entire ‘α’ antigenic determinant amino acids (aa) 119–152 of HBsAg into the truncated HBc (aa 1–144), between Asp{sup 78} and Pro{sup 79}. Prokaryotic expression showed that the mosaic HBc was mainly in the form of inclusion bodies. After denaturation with urea, it was dialyzed progressively for protein renaturation. We observed that before and after renaturation, mosaic HBc was antigenic as determined by HBsAg ELISA and a lot of viruslike particles were observed after renaturation. Thus, we further purified the mosaic viruslike particles by (NH{sub 4}){sub 2}SO{sub 4} precipitation, DEAE chromatography, and Sepharose 4FF chromatography. Negative staining electron microscopy demonstrated the morphology of the viruslike particles. Immunization of Balb/c mice with mosaic particles induced the production of anti-HBs antibody and Th1 cell immune response supported by ELISPOT and CD4/CD8 proportions assay. In conclusion, we constructed mosaic hepatitis core particles displaying the entire ‘α’ antigenic determinant on the surface and laid a foundation for researching therapeutic hepatits B vaccines.

  18. Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides.

    PubMed

    Hardy, Melinda Y; Tye-Din, Jason A; Stewart, Jessica A; Schmitz, Frederike; Dudek, Nadine L; Hanchapola, Iresha; Purcell, Anthony W; Anderson, Robert P

    2015-01-01

    Celiac disease (CD) is a common CD4(+) T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin-specific T cell response in vivo. We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLA-DQ2.5(+) CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye. Avenin-specific responses were observed in 6/73 HLA-DQ2.5(+) CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability. Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.

  19. Towards the knowledge-based design of universal influenza epitope ensemble vaccines

    PubMed Central

    Sheikh, Qamar M.; Gatherer, Derek; Reche, Pedro A; Flower, Darren R.

    2016-01-01

    Motivation: Influenza A viral heterogeneity remains a significant threat due to unpredictable antigenic drift in seasonal influenza and antigenic shifts caused by the emergence of novel subtypes. Annual review of multivalent influenza vaccines targets strains of influenza A and B likely to be predominant in future influenza seasons. This does not induce broad, cross protective immunity against emergent subtypes. Better strategies are needed to prevent future pandemics. Cross-protection can be achieved by activating CD8+ and CD4+ T cells against highly conserved regions of the influenza genome. We combine available experimental data with informatics-based immunological predictions to help design vaccines potentially able to induce cross-protective T-cells against multiple influenza subtypes. Results: To exemplify our approach we designed two epitope ensemble vaccines comprising highly conserved and experimentally verified immunogenic influenza A epitopes as putative non-seasonal influenza vaccines; one specifically targets the US population and the other is a universal vaccine. The USA-specific vaccine comprised 6 CD8+ T cell epitopes (GILGFVFTL, FMYSDFHFI, GMDPRMCSL, SVKEKDMTK, FYIQMCTEL, DTVNRTHQY) and 3 CD4+ epitopes (KGILGFVFTLTVPSE, EYIMKGVYINTALLN, ILGFVFTLTVPSERG). The universal vaccine comprised 8 CD8+ epitopes: (FMYSDFHFI, GILGFVFTL, ILRGSVAHK, FYIQMCTEL, ILKGKFQTA, YYLEKANKI, VSDGGPNLY, YSHGTGTGY) and the same 3 CD4+ epitopes. Our USA-specific vaccine has a population protection coverage (portion of the population potentially responsive to one or more component epitopes of the vaccine, PPC) of over 96 and 95% coverage of observed influenza subtypes. The universal vaccine has a PPC value of over 97 and 88% coverage of observed subtypes. Availability and Implementation: http://imed.med.ucm.es/Tools/episopt.html. Contact: d.r.flower@aston.ac.uk PMID:27402904

  20. Characterization of the neutralizing epitopes of VP7 of the Gottfried strain of porcine rotavirus.

    PubMed

    Kang, S Y; Benfield, D A; Gorziglia, M; Saif, L J

    1993-09-01

    The neutralization epitopes of the outer capsid protein VP7 of a porcine group A rotavirus were studied by using neutralizing monoclonal antibodies (N-MAbs). Six N-MAbs which were specific for the VP7 protein of the Gottfried strain of porcine rotavirus (serotype G4) were used for analyzing the antigenic sites of VP7. Three different approaches were used for this analysis: testing the serological reactivity of each N-MAb against different G serotypes of human and animal rotaviruses, analyzing N-MAb-resistant viral antigenic variants, and performing a nucleotide sequence analysis of the VP7 gene of each of the viral antigenic variants generated. From the serological analyses, three different reactivity patterns were recognized by plaque reduction virus neutralization and cell culture immunofluorescence tests. A single MAb (RG36H9) reacted with animal rotavirus serotypes G3 and G4 but not with human serotypes G3 and G4. The MAb 57/8 (D. A. Benfield, E. A. Nelson, and Y. Hoshino, p. 111, in Abstr. VIIth Internat. Congr. Virol., 1987, and E. R. Mackow, R. D. Shaw, S. M. Matsui, P. T. Vo, D. A. Benfield, and H. B. Greenberg, Virology 165:511-517, 1988) reacted with animal and human rotavirus serotypes G3 and G4 and also with human serotype G9 and bovine serotype G6. The other four MAbs reacted only with the porcine rotavirus serotype G4. The epitope defined by MAb 57/8 and the epitope defined by the other five MAbs appeared to be partially overlapping or close to each other, as identified by viral antigenic variant analysis. However, data from nucleotide and deduced amino acid sequence analyses of the VP7 of each of the viral antigenic variants showed that these two epitopes constituted a large, single neutralization domain.

  1. Viral epigenetics.

    PubMed

    Milavetz, Barry I; Balakrishnan, Lata

    2015-01-01

    DNA tumor viruses including members of the polyomavirus, adenovirus, papillomavirus, and herpes virus families are presently the subject of intense interest with respect to the role that epigenetics plays in control of the virus life cycle and the transformation of a normal cell to a cancer cell. To date, these studies have primarily focused on the role of histone modification, nucleosome location, and DNA methylation in regulating the biological consequences of infection. Using a wide variety of strategies and techniques ranging from simple ChIP to ChIP-chip and ChIP-seq to identify histone modifications, nuclease digestion to genome wide next generation sequencing to identify nucleosome location, and bisulfite treatment to MeDIP to identify DNA methylation sites, the epigenetic regulation of these viruses is slowly becoming better understood. While the viruses may differ in significant ways from each other and cellular chromatin, the role of epigenetics appears to be relatively similar. Within the viral genome nucleosomes are organized for the expression of appropriate genes with relevant histone modifications particularly histone acetylation. DNA methylation occurs as part of the typical gene silencing during latent infection by herpesviruses. In the simple tumor viruses like the polyomaviruses, adenoviruses, and papillomaviruses, transformation of the cell occurs via integration of the virus genome such that the virus's normal regulation is disrupted. This results in the unregulated expression of critical viral genes capable of redirecting cellular gene expression. The redirected cellular expression is a consequence of either indirect epigenetic regulation where cellular signaling or transcriptional dysregulation occurs or direct epigenetic regulation where epigenetic cofactors such as histone deacetylases are targeted. In the more complex herpersviruses transformation is a consequence of the expression of the viral latency proteins and RNAs which again can

  2. Variable Constraints on the Principal Immunodominant Domain of the Transmembrane Glycoprotein of Human Immunodeficiency Virus Type 1

    PubMed Central

    Merat, Rastine; Raoul, Herve; Leste-Lasserre, Thierry; Sonigo, Pierre; Pancino, Gianfranco

    1999-01-01

    Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits

  3. First report on the antibody verification of MICA epitopes recorded in the HLA epitope registry.

    PubMed

    Duquesnoy, R J; Mostecki, J; Marrari, M; da Silva, A S; da Mata Sousa, L C D; do Monte, S J H

    2014-10-01

    The International Registry of HLA Epitopes (http://epregistry.com.br) has been recently established as a tool to understand antibody responses to HLA mismatches. These epitopes are defined structurally by three-dimensional molecular modelling and amino acid sequence differences between HLA antigens. A major goal was to identify HLA epitopes that have been verified experimentally with informative antibodies. This report addresses the identification of MICA epitopes. Our analysis included published information about MICA antibody reactivity in sera from sensitized patients as well as data from our own laboratories. This report describes twenty-one MICA epitopes verified with antibodies which have primarily been tested in Luminex assays with single alleles. The epitopes correspond to distinct eplets that are often defined by single residues. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

  4. Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope.

    PubMed

    Gao, Huiguang; Fu, Weiling; Li, Rongfen; Chen, Linfeng; Ji, Qing; Zhang, Li; Huang, Gang; He, Fengtian

    2006-10-01

    The DNA encoding soluble B lymphocyte stimulator (134-285 amino acids, sBLyS) mutant with residues 217-224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5'-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5alpha after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.

  5. Ethylenecarbodiimide-Treated Splenocytes Carrying Male CD4 Epitopes Confer Hya Transplant Protection by inhibiting CD154 upregulation1

    PubMed Central

    Martin, Aaron J.; McCarthy, Derrick; Waltenbaugh, Carl; Luo, Xunrong; Goings, Gwen; Miller, Stephen D.

    2010-01-01

    In humans and certain strains of laboratory mice, male tissue is recognized as non-self and destroyed by the female immune system via recognition of histocompatibility-Y chromosome-encoded antigens (Hya). Male tissue destruction is thought to be accomplished by cytotoxic T lymphocytes in a helper-dependent manner. We show that graft protection induced with the immunodominant Hya-encoded CD4 epitope (Dby) attached to female splenic leukocytes (Dby-SP) with the chemical cross-linker ethylene-carbodiimide (ECDI) significantly, and often indefinitely, prolongs the survival of male skin graft transplants in an antigen-specific manner. In contrast, treatments with the Hya CD8 epitopes (Uty- and Smcy-SP) failed to prolong graft survival. Dby-SP-tolerized CD4+ T cells fail to proliferate, secrete IFN-γ, or effectively prime a CD8 response in recipients of male grafts. Ag-SP treatment is associated with defective CD40-CD40L interactions as evidenced by the observation that CD4 cells from treated animals exhibit a defect in CD40L upregulation following in vitro antigen challenge. Furthermore, treatment with an agonistic anti-CD40 antibody at the time of transplantation abrogates protection from graft rejection. Interestingly, anti-CD40 treatment completely restores the function of Dby-specific CD4 cells, but not Uty- or Smcy-specific CD8 cells. PMID:20713889

  6. Dominant recognition of a cross-reactive B-cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy

    PubMed Central

    Hussain, R; Dockrell, H M; Chiang, T J

    1999-01-01

    Mycobacterium leprae-specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (ρ=0·748; P < 0002) suggesting that IgG1 B-cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat-shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n = 26) or tuberculoid form (BT/TT, n = 39) of the disease as well as in healthy households (HC, n = 14) and endemic controls (EC = 19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross-reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15-mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41–55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded. PMID:10233750

  7. Identification and characterization of CD4⁺ T-cell epitopes on GapC protein of Streptococcus dysgalactiae.

    PubMed

    Yao, Di; Zhang, Hua; Wang, Xintong; Yu, Simiao; Wei, Yuhua; Liu, Wei; Wang, Jiannan; Chen, Xiaoting; Zhang, Zhenghai; Sun, Hunan; Yu, Liquan; Ma, Jinzhu; Tong, Chunyu; Song, Baifen; Cui, Yudong

    2016-02-01

    The GapC protein is highly conserved surface dehydrogenase among Streptococcus dysgalactiae (S. dysgalactiae) and is shown to be involved in bacterial virulence. Immunization of GapC protein can induce specific CD4(+) T-cell immune responses and protect against S. dysgalactiae infection. However, there are no studies to identify immunodominant CD4(+) T-cell epitopes on GapC protein. In this study, in silico MHC affinity measurement method was firstly used to predict potential CD4(+) T-cell epitopes on GapC protein. Six predictive 15-mer peptides were synthesized and two novel GapC CD4(+) T-cell epitopes, GapC63-77 and GapC96-110, were for the first time identified using CD4(+) T-cells obtained from GapC-immunized BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice spleen based on cell proliferation and cytokines response. The results showed that peptides containing 63-77 and 96-110 induced significant antigen-specific CD4(+) T-cells proliferation response in vivo. At the same time, high levels of IFN-γ and IL-17A, as well as moderate levels of IL-10 and IL-4 were detected in CD4(+) T-cells isolated from both GapC and peptide-immunized mice in vivo, suggesting that GapC63-77 and GapC96-110 preferentially elicited polarized Th1/Th17-type responses. The characterization of GapC CD4(+) T-cell epitopes not only helps us understand its protective immunity, but also contributes to design effective T-cell epitope-based vaccine against S. dysgalactiae infection.

  8. Viral Miniproteins

    PubMed Central

    DiMaio, Daniel

    2015-01-01

    Many viruses encode short transmembrane proteins that play vital roles in virus replication or virulence. Because these proteins are often less than 50 amino acids long and not homologous to cellular proteins, their open reading frames were often overlooked during the initial annotation of viral genomes. Some of these proteins oligomerize in membranes and form ion channels. Other miniproteins bind to cellular transmembrane proteins and modulate their activity, whereas still others have an unknown mechanism of action. Based on the underlying principles of transmembrane miniprotein structure, it is possible to build artificial small transmembrane proteins that modulate a variety of biological processes. These findings suggest that short transmembrane proteins provide a versatile mechanism to regulate a wide range of cellular activities, and we speculate that cells also express many similar proteins that have not yet been discovered. PMID:24742054

  9. Viral Carcinogenesis.

    PubMed

    Smith, A J; Smith, L A

    2016-01-01

    Cancer has been recognized for thousands of years. Egyptians believed that cancer occurred at the will of the gods. Hippocrates believed human disease resulted from an imbalance of the four humors: blood, phlegm, yellow bile, and black bile with cancer being caused by excess black bile. The lymph theory of cancer replaced the humoral theory and the blastema theory replaced the lymph theory. Rudolph Virchow was the first to recognize that cancer cells like all cells came from other cells and believed chronic irritation caused cancer. At the same time there was a belief that trauma caused cancer, though it never evolved after many experiments inducing trauma. The birth of virology occurred in 1892 when Dimitri Ivanofsky demonstrated that diseased tobacco plants remained infective after filtering their sap through a filter that trapped bacteria. Martinus Beijerinck would call the tiny infective agent a virus and both Dimitri Ivanofsky and Marinus Beijerinck would become the fathers of virology. Not to long thereafter, Payton Rous founded the field of tumor virology in 1911 with his discovery of a transmittable sarcoma of chickens by what would come to be called Rous sarcoma virus or RSV for short. The first identified human tumor virus was the Epstein-Barr virus (EBV), named after Tony Epstein and Yvonne Barr who visualized the virus particles in Burkitt's lymphoma cells by electron microscopy in 1965. Since that time, many viruses have been associated with carcinogenesis including the most studied, human papilloma virus associated with cervical carcinoma, many other anogenital carcinomas, and oropharyngeal carcinoma. The World Health Organization currently estimates that approximately 22% of worldwide cancers are attributable to infectious etiologies, of which viral etiologies is estimated at 15-20%. The field of tumor virology/viral carcinogenesis has not only identified viruses as etiologic agents of human cancers, but has also given molecular insights to all human

  10. Immunogenicity of recombinant proteins consisting of Plasmodium vivax circumsporozoite protein allelic variant-derived epitopes fused with Salmonella enterica Serovar Typhimurium flagellin.

    PubMed

    Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida; Rodrigues, Mauricio M

    2013-09-01

    A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.

  11. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    SciTech Connect

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  12. Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

    PubMed Central

    Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

    2016-01-01

    ABSTRACT The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit

  13. Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1.

    PubMed

    Li, Hua; Liu, Zu-Qiang; Ding, Jian; Chen, Ying-Hua

    2002-11-01

    Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low. Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli. After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice. These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not. Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine. These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.

  14. Immunoproteasome LMP2 60HH Variant Alters MBP Epitope Generation and Reduces the Risk to Develop Multiple Sclerosis in Italian Female Population

    PubMed Central

    Mishto, Michele; Bellavista, Elena; Ligorio, Claudia; Textoris-Taube, Kathrin; Santoro, Aurelia; Giordano, Mara; D'Alfonso, Sandra; Listì, Florinda; Nacmias, Benedetta; Cellini, Elena; Leone, Maurizio; Grimaldi, Luigi M.E.; Fenoglio, Chiara; Esposito, Federica; Martinelli-Boneschi, Filippo; Galimberti, Daniela; Scarpini, Elio; Seifert, Ulrike; Amato, Maria Pia; Caruso, Calogero; Foschini, Maria P.; Kloetzel, Peter M.; Franceschi, Claudio

    2010-01-01

    Background Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. Methodology/Principal Findings Immunoproteasomes and PA28-αβ regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP111–119. Conclusion/Significance The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis. PMID:20174631

  15. 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.

  16. Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

    PubMed

    Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

    2014-11-04

    Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

  17. Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes

    PubMed Central

    Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi

    2015-01-01

    ABSTRACT Identification and characterization of CD8+ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8+ T cells have been only partially identified. In this study, we sought to identify CD8+ T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8+ T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10−11) and positively associated with CD4 count (P = 1.2 × 10−11), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8+ T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. IMPORTANCE HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52

  18. Malondialdehyde epitopes as mediators of sterile inflammation.

    PubMed

    Busch, Clara J; Binder, Christoph J

    2017-04-01

    Enhanced lipid peroxidation occurs during oxidative stress and results in the generation of lipid peroxidation end products such as malondialdehyde (MDA), which can attach to autologous biomolecules, thereby generating neo-self epitopes capable of inducing potentially undesired biological responses. Therefore, the immune system has developed mechanisms to protect from MDA epitopes by binding and neutralizing them through both cellular and soluble effectors. Here, we briefly discuss innate immune responses targeting MDA epitopes and their pro-inflammatory properties, followed by a review of physiological carriers of MDA epitopes that are relevant in homeostasis and disease. Then we discuss in detail the evidence for cellular responses towards MDA epitopes mainly in lung, liver and the circulation as well as signal transduction mechanisms and receptors implicated in the response to MDA epitopes. Last, we hypothesize on the role of MDA epitopes as mediators of inflammation in diseases and speculate on their contribution to disease pathogenesis. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.

  19. The Immune Epitope Database 2.0

    PubMed Central

    Vita, Randi; Zarebski, Laura; Greenbaum, Jason A.; Emami, Hussein; Hoof, Ilka; Salimi, Nima; Damle, Rohini; Sette, Alessandro; Peters, Bjoern

    2010-01-01

    The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course of 4 years, the data from 180 978 experiments were curated manually from the literature, which covers ∼99% of all publicly available information on peptide epitopes mapped in infectious agents (excluding HIV) and 93% of those mapped in allergens. In addition, data that would otherwise be unavailable to the public from 129 186 experiments were submitted directly by investigators. The curation of epitopes related to autoimmunity is expected to be completed by the end of 2010. The database can be queried by epitope structure, source organism, MHC restriction, assay type or host organism, among other criteria. The database structure, as well as its querying, browsing and reporting interfaces, was completely redesigned for the IEDB 2.0 release, which became publicly available in early 2009. PMID:19906713

  20. Variable Lymphocyte Receptor Recognition of the Immunodominant Glycoprotein of Bacillus anthracis Spores

    SciTech Connect

    Kirchdoerfer, Robert N.; Herrin, Brantley R.; Han, Byung Woo; Turnbough, Jr., Charles L.; Cooper, Max D.; Wilson, Ian A.

    2012-07-25

    Variable lymphocyte receptors (VLRs) are the adaptive immune receptors of jawless fish, which evolved adaptive immunity independent of other vertebrates. In lieu of the immunoglobulin fold-based T and B cell receptors, lymphocyte-like cells of jawless fish express VLRs (VLRA, VLRB, or VLRC) composed of leucine-rich repeats and are similar to toll-like receptors (TLRs) in structure, but antibodies (VLRB) and T cell receptors (VLRA and VLRC) in function. Here, we present the structural and biochemical characterization of VLR4, a VLRB, in complex with BclA, the immunodominant glycoprotein of Bacillus anthracis spores. Using a combination of crystallography, mutagenesis, and binding studies, we delineate the mode of antigen recognition and binding between VLR4 and BclA, examine commonalities in VLRB recognition of antigens, and demonstrate the potential of VLR4 as a diagnostic tool for the identification of B. anthracis spores.

  1. Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240.

    PubMed

    Gohain, Neelakshi; Tolbert, William D; Orlandi, Chiara; Richard, Jonathan; Ding, Shilei; Chen, Xishan; Bonsor, Daniel A; Sundberg, Eric J; Lu, Wuyuan; Ray, Krishanu; Finzi, Andrés; Lewis, George K; Pazgier, Marzena

    2016-11-09

    Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement.

  2. Molecular basis for epitope recognition by non-neutralizing anti-gp41 antibody F240

    PubMed Central

    Gohain, Neelakshi; Tolbert, William D.; Orlandi, Chiara; Richard, Jonathan; Ding, Shilei; Chen, Xishan; Bonsor, Daniel A.; Sundberg, Eric J.; Lu, Wuyuan; Ray, Krishanu; Finzi, Andrés; Lewis, George K.; Pazgier, Marzena

    2016-01-01

    Antibody-dependent cell-mediated cytotoxicity (ADCC) by non-neutralizing antibodies (nnAbs) specific to the HIV envelope (Env) glycoproteins present at the surface of virus sensitized or infected cells plays a role in the effective adaptive immune response to HIV. Here, we explore the molecular basis for the epitope at the disulfide loop region (DLR) of the principal immunodominant domain of gp41, recognized by the well-known nnAb F240. Our structural studies reveal details of the F240-gp41 interface and describe a structure of DLR that is distinct from known conformations of this region studied in the context of either CD4-unliganded Env trimer or the gp41 peptide in the unbound state. These data coupled with binding and functional analyses indicate that F240 recognizes non-trimeric Env forms which are significantly overexpressed on intact virions but poorly represented at surfaces of cells infected with infectious molecular clones and endogenously-infected CD4 T cells from HIV-1-infected individuals. Furthermore, although we detect ADCC activities of F240 against cells spinoculated with intact virions, our data suggest that these activities result from F240 recognition of gp41 stumps or misfolded Env variants present on virions rather than its ability to recognize functional gp41 transition structures emerging on trimeric Env post CD4 receptor engagement. PMID:27827447

  3. Evaluation of immunodominant proteins of Mycobacterium avium paratuberculosis cell wall by Western blot analysis.

    PubMed

    Hashemi, Maryam; Madani, Rasool; Razmi, Nematollah

    2014-04-01

    Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a slow growing mycobactin, whose dependence on mycobacterial species is known to be the causative agent of Johne's disease (paratuberculosis) in all species of domestic ruminants worldwide. The organism is transmitted via close contact, ingestion, or transplacentally from mother to fetus and occurs commonly in grazing domestic animals. Johne's disease (JD) is characterized by gradual weight loss, decreased milk production, and diarrhea due to the chronic, progressive, granulomatous enteritis and lymphadenitis. The disease can cause serious economic damage to the dairy industry due to the loss of milk production and early culling of infected animals. In recent years, researchers have focused on the identification of a specific antigen of M. paratuberculosis to use in diagnosis test and preparation of effective vaccine. The goal of this study is evaluation of the immunodominant proteins of M. paratuberculosis cell wall. The amount of protein was determined with a Lowry assay (22.68 μg/100 μL). For production of polyclonal antibody against proteins of M. paratuberculosis cell wall, a New Zealand white rabbit was immunized with antigen and Freund's adjuvant. After immunization, the rabbit was bled to produce enriched serum. Antibodies were purified from serum with ion exchange chromatography. In the Ouchterlony test, the reactions between antigen and antibodies were seen in dilutions of one quarter for serum, one quarter for Ig, and one half for IgG by clear precipitation lines due to the well immunization of the rabbit. Electrophoresis and Western blot analysis were used and subsequently a sharp band appeared in nitrocellulose paper; these bands were about 25, 37, 50, 75, and 150 kDa molecular weight, which indicated immunodominant proteins.

  4. Sculpting the Immunological Response against Viral Disease: Statistical Mechanics and Network Theory

    NASA Astrophysics Data System (ADS)

    Zhou, Hao; Deem, Michael

    2007-03-01

    The twin challenges of immunodominance and heterologous immunity have hampered discovery of an effective vaccine against all four dengue viruses. Here we develop a generalized NK, or spin glass, theory of T cell original antigenic sin and immunodominance. The theory we develop predicts dengue vaccine clinical trial data well. From the insights that we gain by this theory, we propose two new ideas for design of epitope-based T cell vaccines against dengue. The H5N1 strain of avian influenza first appeared in Hong Kong in 1997. Since then, it has spread to at least eight other Asian countries, Romania, and Russia, and it is widely expected to enter the rest of Europe through migratory birds. Various countries around the world have started to create stockpiles of avian influenza vaccines. However, since the avian influenza is mutating, how many and which strains should be stockpiled? Here we use a combination of statistical physics and network theory to simulate the bird flu transmission and evolution. From the insights that we gain by the theory, we propose new strategies to improve the vaccine efficacy.

  5. Characterization of a Novel Conformational GII.4 Norovirus Epitope: Implications for Norovirus-Host Interactions

    PubMed Central

    Carmona-Vicente, Noelia; Vila-Vicent, Susana; Allen, David; Gozalbo-Rovira, Roberto; Iturriza-Gómara, Miren

    2016-01-01

    ABSTRACT Human noroviruses (NoVs) are the main etiological agents of acute gastroenteritis worldwide. While NoVs are highly diverse (more than 30 genotypes have been detected in humans), during the last 40 years most outbreaks and epidemics have been caused by GII.4 genotype strains, raising questions about their persistence in the population. Among other potential explanations, immune evasion is considered to be a main driver of their success. In order to study antibody recognition and evasion in detail, we analyzed a conformational epitope recognized by a monoclonal antibody (3C3G3) by phage display, site-directed mutagenesis, and surface plasmon resonance. Our results show that the predicted epitope is composed of 11 amino acids within the P domain: P245, E247, I389, Q390, R397, R435, G443, Y444, P445, N446, and D448. Only two of them, R397 and D448, differ from the homologous variant (GII.4 Den-Haag_2006b) and from a previous variant (GII.4 VA387_1996) that is not recognized by the antibody. A double mutant derived from the VA387_1996 variant containing both changes, Q396R and N447D, is recognized by the 3C3G3 monoclonal antibody, confirming the participation of the two sites in the epitope recognized by the antibody. Furthermore, a single change, Q396R, is able to modify the histo-blood group antigen (HBGA) recognition pattern. These results provide evidence that the epitope recognized by the 3C3G3 antibody is involved in the virus-host interactions, both at the immunological and at the receptor levels. IMPORTANCE Human noroviruses are the main cause of viral diarrhea worldwide in people of all ages. Noroviruses can infect individuals who had been previously exposed to the same or different norovirus genotypes. Norovirus genotype GII.4 has been reported to be most prevalent during the last 40 years. In the present study, we describe a novel viral epitope identified by a monoclonal antibody and located within the highly diverse P domain of the capsid protein

  6. Viral Gastroenteritis (Stomach Flu)

    MedlinePlus

    Diseases and Conditions Viral gastroenteritis (stomach flu) By Mayo Clinic Staff Viral gastroenteritis is an intestinal infection marked by watery diarrhea, abdominal cramps, nausea or vomiting, and ...

  7. Identification of protective linear B-cell epitopes on the subolesin/akirin orthologues of Ornithodoros spp. soft ticks.

    PubMed

    Manzano-Román, Raúl; Díaz-Martín, Verónica; Oleaga, Ana; Pérez-Sánchez, Ricardo

    2015-02-18

    Subolesin/akirin is a protective antigen that is highly conserved across hematophagous vector species and is therefore potentially useful for the development of a universal vaccine for vector control, including soft ticks. Recent results have shown that in Ornithodoros erraticus and O. moubata soft ticks, RNAi-mediated subolesin gene knockdown inhibits tick oviposition and fertility by more than 90%; however, vaccination with recombinant subolesins resulted in remarkably low protective efficacies (5-24.5% reduction in oviposition). Here we report that vaccination with subolesin recombinants induces non-protective antibodies mainly directed against immunodominant linear B-cell epitopes located on highly structured regions of the subolesin protein, probably unrelated to its biological activity, while leaving the unstructured/disordered regions unrecognized. Accordingly, for a new vaccine trial we designed four synthetic peptides (OE1, OE2, OM1 and OM2) from the unrecognized/disordered regions of the Ornithodoros subolesin sequences and coupled them to keyhole limpet haemocyanin (KLH). These KLH-peptide conjugates induced the synthesis of antibodies that recognized linear B-cell epitopes located on the unstructured loops of the subolesin protein and provided up to 70.1% and 83.1% vaccine efficacies in O. erraticus and O. moubata, respectively. These results show that the protective effect of subolesin-based vaccines is highly dependent on the particular epitope recognized by antibodies on the subolesin sequence and strongly suggest that the biological activity of subolesin is exerted through its unstructured regions. The results reported here contribute to our understanding of the mechanism of protection of subolesin-based vaccines and reveal novel protective peptides that could be included among the array of candidate antigens useful for developing anti-vector vaccines based on subolesin/akirin.

  8. Analysis of TCR antagonism and molecular mimicry of an HLA-A0201-restricted CTL epitope in primary biliary cirrhosis.

    PubMed

    Kita, Hiroto; Matsumura, Shuji; He, Xiao-Song; Ansari, Aftab A; Lian, Zhe-Xiong; Van de Water, Judy; Coppel, Ross L; Kaplan, Marshall M; Gershwin, M Eric

    2002-10-01

    Although the etiology and mechanism of primary biliary cirrhosis (PBC) is unknown, growing evidence suggests a major role for T cells. We have recently identified the first CD8 T-cell epitope, amino acid 159-167 of the E2 component of pyruvate dehydrogenase complexes (PDC-E2). To seek for analogue peptide-antagonizing effector function of CTLs specific for this autoantigen, we examined the effector functions of the PDC-E2-specific CTLs against alanine substituted peptides. Furthermore, because molecular mimicry has been postulated as a possible cause of initiating PBC, we carried out studies aimed at identifying naturally occurring peptides for the 159-167 peptide of PDC-E2 that may serve as agonists. An alanine substitution at position 5 of this epitope significantly reduced peptide-specific effector functions of CTLs. Moreover, this analogue peptide inhibited effector functions of the CTLs to the prototype peptide, including cytotoxicity and IFN-gamma production. We also identified a peptide derived from Pseudomonas aeruginosa, which showed a higher binding affinity to the HLA-A*0201 than the prototype peptide. This homologous peptide was recognized by CTLs specific for the prototype epitope on PDC-E2. In conclusion, a modification of the immunodominant autoepitope can be utilized to manipulate the CD8 T-cell responses against the autoantigen PDC-E2. Our finding also supports the thesis that molecular mimicry may be implicated in the initiation of the autoreactive CD8 T-cell responses and has implications for the use of such peptides for immunotherapy.

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  10. Influence of High Hydrostatic Pressure on Epitope Mapping of Tobacco Mosaic Virus Coat Protein

    PubMed Central

    Bonafe, Carlos Francisco Sampaio; Arns, Clarice Weis

    2014-01-01

    Abstract In this study, we investigated the effect of high hydrostatic pressure (HHP) on tobacco mosaic virus (TMV), a model virus in immunology and one of the most studied viruses to date. Exposure to HHP significantly altered the recognition epitopes when compared to sera from mice immunized with native virus. These alterations were studied further by combining HHP with urea or low temperature and then inoculating the altered virions into Balb-C mice. The antibody titers and cross-reactivity of the resulting sera were determined by ELISA. The antigenicity of the viral particles was maintained, as assessed by using polyclonal antibodies against native virus. The antigenicity of canonical epitopes was maintained, although binding intensities varied among the treatments. The patterns of recognition determined by epitope mapping were cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations had occurred. These findings suggest that different cleavage sites were exposed after the treatments and this was confirmed by epitope mapping using sera from mice immunized with virus previously exposed to HHP. PMID:24605789

  11. Analysis of neutralizing epitopes on foot-and-mouth disease virus.

    PubMed Central

    Pfaff, E; Thiel, H J; Beck, E; Strohmaier, K; Schaller, H

    1988-01-01

    For the investigation of the antigenic determinant structure of foot-and-mouth disease virus (FMDV), neutralizing monoclonal antibodies (MAbs) against complete virus were characterized by Western blot (immunoblot), enzyme immunoassay, and competition experiments with a synthetic peptide, isolated coat protein VP1, and viral particles as antigens. Two of the four MAbs reacted with each of these antigens, while the other two MAbs recognized only complete viral particles and reacted only very poorly with the peptide. The four MAbs showed different neutralization patterns with a panel of 11 different FMDV strains. cDNA-derived VP1 protein sequences of the different strains were compared to find correlations between the primary structure of the protein and the ability of virus to be neutralized. Based on this analysis, it appears that the first two MAbs recognized overlapping sequential epitopes in the known antigenic site represented by the peptide, whereas the two other MAbs recognized conformational epitopes. These conclusions were supported and extended by structural analyses of FMDV mutants resistant to neutralization by an MAb specific for a conformational epitope. These results demonstrate that no amino acid exchanges had occurred in the primary antigenic site of VP1 but instead in the other coat proteins VP2 and VP3, which by themselves do not induce neutralizing antibodies. Images PMID:2835507

  12. Molecular mimicry between the immunodominant ribosomal protein P0 of Trypanosoma cruzi and a functional epitope on the human beta 1- adrenergic receptor

    PubMed Central

    1995-01-01

    Sera from chagasic patients possess antibodies recognizing the carboxy- terminal part of the ribosomal P0 protein of Trypanosoma cruzi and the second extracellular loop of the human beta 1-adrenergic receptor. Comparison of both peptides showed that they contain a pentapeptide with very high homology (AESEE in P0 and AESDE in the human beta 1- adrenergic receptor). Using a competitive immunoenzyme assay, recognition of the peptide corresponding to the second extracellular loop (H26R) was inhibited by both P0-14i (AAAESEEEDDDDDF) and P0-beta (AESEE). Concomitantly, recognition of P0-beta was inhibited with the H26R peptide. Recognition of P0 in Western blots was inhibited by P0- 14i, P0-beta, and H26R, but not by a peptide corresponding to the second extracellular loop of the human beta 2-adrenergic receptor or by an unrelated peptide. Autoantibodies affinity purified with the immobilized H26R peptide were shown to exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats. This effect was blocked by both the specific beta 1 blocker bisoprolol and the peptide P0-beta. These results unambiguously prove that T. cruzi is able to induce a functional autoimmune response against the cardiovascular human beta 1-adrenergic receptor through a molecular mimicry mechanism. PMID:7790824

  13. Molecular mimicry between the immunodominant ribosomal protein P0 of Trypanosoma cruzi and a functional epitope on the human beta 1-adrenergic receptor.

    PubMed

    Ferrari, I; Levin, M J; Wallukat, G; Elies, R; Lebesgue, D; Chiale, P; Elizari, M; Rosenbaum, M; Hoebeke, J

    1995-07-01

    Sera from chagasic patients possess antibodies recognizing the carboxy-terminal part of the ribosomal P0 protein of Trypanosoma cruzi and the second extracellular loop of the human beta 1-adrenergic receptor. Comparison of both peptides showed that they contain a pentapeptide with very high homology (AESEE in P0 and AESDE in the human beta 1-adrenergic receptor). Using a competitive immunoenzyme assay, recognition of the peptide corresponding to the second extracellular loop (H26R) was inhibited by both P0-14i (AAAESEEEDDDDDF) and P0-beta (AESEE). Concomitantly, recognition of P0-beta was inhibited with the H26R peptide. Recognition of P0 in Western blots was inhibited by P0-14i, P0-beta, and H26R, but not by a peptide corresponding to the second extracellular loop of the human beta 2-adrenergic receptor or by an unrelated peptide. Autoantibodies affinity purified with the immobilized H26R peptide were shown to exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats. This effect was blocked by both the specific beta 1 blocker bisoprolol and the peptide P0-beta. These results unambiguously prove that T. cruzi is able to induce a functional autoimmune response against the cardiovascular human beta 1-adrenergic receptor through a molecular mimicry mechanism.

  14. Epitope mapping: the first step in developing epitope-based vaccines.

    PubMed

    Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael

    2007-01-01

    Antibodies are an effective line of defense in preventing infectious diseases. Highly potent neutralizing antibodies can intercept a virus before it attaches to its target cell and, thus, inactivate it. This ability is based on the antibodies' specific recognition of epitopes, the sites of the antigen to which antibodies bind. Thus, understanding the antibody/epitope interaction provides a basis for the rational design of preventive vaccines. It is assumed that immunization with the precise epitope, corresponding to an effective neutralizing antibody, would elicit the generation of similarly potent antibodies in the vaccinee. Such a vaccine would be a 'B-cell epitope-based vaccine', the implementation of which requires the ability to backtrack from a desired antibody to its corresponding epitope. In this article we discuss a range of methods that enable epitope discovery based on a specific antibody. Such a reversed immunological approach is the first step in the rational design of an epitope-based vaccine. Undoubtedly, the gold standard for epitope definition is x-ray analyses of crystals of antigen:antibody complexes. This method provides atomic resolution of the epitope; however, it is not readily applicable to many antigens and antibodies, and requires a very high degree of sophistication and expertise. Most other methods rely on the ability to monitor the binding of the antibody to antigen fragments or mutated variations. In mutagenesis of the antigen, loss of binding due to point modification of an amino acid residue is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping are also useful. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For

  15. Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis

    PubMed Central

    Bastos, Luciana M.; Macêdo, Arlindo G.; Silva, Murilo V.; Santiago, Fernanda M.; Ramos, Eliezer L. P.; Santos, Fabiana A. A.; Pirovani, Carlos P.; Goulart, Luiz R.; Mineo, Tiago W. P.; Mineo, José R.

    2016-01-01

    Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-α and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii. PMID:27313992

  16. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1

    PubMed Central

    El-Diwany, Ramy; Mankowski, Madeleine C.; Wasilewski, Lisa N.; Brady, Jillian K.; Snider, Anna E.; Osburn, William O.; Murrell, Ben; Ray, Stuart C.

    2017-01-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. PMID:28235087

  17. Human viral cardiomyopathy.

    PubMed

    Maisch, Bernhard; Ristic, Arsen D; Portig, Irene; Pankuweit, Sabine

    2003-01-01

    Viral infection of the heart is relatively common, usually asymptomatic and has a spontaneous and complete resolution. It can, however, in rare cases, lead to substantial cardiac damage, development of viral cardiomyopathy and congestive heart failure. Viral cardiomyopathy is defined as viral persistence in a dilated heart. It may be accompanied by myocardial inflammation and then termed inflammatory viral cardiomyopathy (or viral myocarditis with cardiomegaly). If no inflammation is observed in the biopsy of a dilated heart (<14 lymphocytes and macrophages/mm ) the term viral cardiomyopathy or viral persistence in dilated cardiomyopathy should be applied. The diagnosis of myocarditis and viral cardiomyopathy can be made only by endomyocardial biopsy, implementing the WHO/WHF criteria, and PCR techniques for identification of viral genome. The most frequent cardiotropic viruses detected by endomyocardial biopsy are Parvo B19, enteroviruses, adenoviruses, cytomegalovirus, and less frequently Epstein-Barr virus, and influenza virus.

  18. Diversification of the celiac disease α-gliadin complex in wheat: a 33-mer peptide with six overlapping epitopes, evolved following polyploidization.

    PubMed

    Ozuna, Carmen V; Iehisa, Julio C M; Giménez, María J; Alvarez, Juan B; Sousa, Carolina; Barro, Francisco

    2015-06-01

    The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6-8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31-43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1-6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31-43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides.

  19. Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice.

    PubMed

    Santos, Paula S; Sena, Angela A S; Nascimento, Rafael; Araújo, Thaise G; Mendes, Mirian M; Martins, João R S; Mineo, Tiago W P; Mineo, José R; Goulart, Luiz R

    2013-01-01

    Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

  20. Immunoglobulin M Capture Assay for Serologic Confirmation of Early Lyme Disease: Analysis of Immune Complexes with Biotinylated Borrelia burgdorferi Sonicate Enhanced with Flagellin Peptide Epitope

    PubMed Central

    Brunner, Michael; Stein, Stanley; Mitchell, Paul D.; Sigal, Leonard H.

    1998-01-01

    We previously reported on the efficacy of the enzyme-linked immunoglobulin M capture immune complex (IC) biotinylated antigen assay (EMIBA) for the seroconfirmation of early Lyme disease and active infection with Borrelia burgdorferi. In earlier work we identified non-cross-reacting epitopes of a number of B. burgdorferi proteins, including flagellin. We now report on an improvement in the performance of EMIBA with the addition of a biotinylated form of a synthetic non-cross-reacting immunodominant flagellin peptide to the biotinylated B. burgdorferi B31 sonicate antigen source with the avidin-biotinylated peroxidase complex detection system used in our recently developed indirect IgM-capture immune complex-based assay (EMIBA). As in our previous studies, the enzyme-linked immunosorbent assay (ELISA) reactivities of antibodies liberated from circulating ICs (by EMIBA) were compared with those of antibodies in unprocessed serum (antibodies found free in the serum, thus as an IgM-capture ELISA, but not EMIBA, because the antibodies were not liberated from ICs), the sample usually used in standard ELISAs and Western blot assays. The addition of the flagellin epitope enhanced the ELISA signal obtained with untreated sera from many Lyme disease patients but not from healthy controls. In tests with both free antibodies and ICs, with or without the addition of the flagellin epitope to the sonicate, we found the most advantageous combination was IC as the source of antibodies and sonicate plus the flagellin epitope as the antigen. In a blinded study of sera obtained from patients with early and later-phase Lyme disease, EMIBA with the enhanced antigenic preparation compared favorably with other serologic assays, especially for the confirmation of early disease. PMID:9542940

  1. Systematic Bioinformatic Approach for Prediction of Linear B-Cell Epitopes on Dengue E and prM Protein.

    PubMed

    Nadugala, Mahesha N; Premaratne, Prasad H; Goonasekara, Charitha L

    2016-01-01

    B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48-55), epitope number 4 of E protein at amino acids 48-55, EP9/E (165-182), EP11/E (218-233), EP20/E (322-349), EP21/E (326-353), EP23/E (356-365), and EP25/E (380-386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30-41) located in domain-I and EP26/E (393-409), EP27/E (416-435), EP28/E (417-430) located in the stem region of E protein, and EP8/prM (93-112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16-21), EP5/E (62-123), EP6/E (63-89), EP19/E (310-329), and EP24/E (371-402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes.

  2. Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

    PubMed Central

    1996-01-01

    An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

  3. Systematic Bioinformatic Approach for Prediction of Linear B-Cell Epitopes on Dengue E and prM Protein

    PubMed Central

    Nadugala, Mahesha N.

    2016-01-01

    B-cell epitopes on the envelope (E) and premembrane (prM) proteins of dengue virus (DENV) were predicted using bioinformatics tools, BepiPred, Ellipro, and SVMTriP. Predicted epitopes, 32 and 17 for E and prM proteins, respectively, were then characterized for their level of conservations. The epitopes, EP4/E (48–55), epitope number 4 of E protein at amino acids 48–55, EP9/E (165–182), EP11/E (218–233), EP20/E (322–349), EP21/E (326–353), EP23/E (356–365), and EP25/E (380–386), showed a high intraserotype conservancy with very low pan-serotype conservancy, demonstrating a potential target as serotype specific diagnostic markers. EP3 (30–41) located in domain-I and EP26/E (393–409), EP27/E (416–435), EP28/E (417–430) located in the stem region of E protein, and EP8/prM (93–112) from the prM protein have a pan-serotype conservancy higher than 70%. These epitopes indicate a potential use as universal vaccine candidates, subjected to verification of their potential in viral neutralization. EP2/E (16–21), EP5/E (62–123), EP6/E (63–89), EP19/E (310–329), and EP24/E (371–402), which have more than 50% pan-serotype conservancies, were found on E protein regions that are important in host cell attachment. Previous studies further show evidence for some of these epitopes to generate cross-reactive neutralizing antibodies, indicating their importance in antiviral strategies for DENV. This study suggests that bioinformatic approaches are attractive first line of screening for identification of linear B-cell epitopes. PMID:27688753

  4. Identification of a novel canine distemper virus B-cell epitope using a monoclonal antibody against nucleocapsid protein.

    PubMed

    Yi, Li; Cheng, Yuening; Zhang, Miao; Cao, Zhigang; Tong, Mingwei; Wang, Jianke; Zhao, Hang; Lin, Peng; Cheng, Shipeng

    2016-02-02

    Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses.

  5. Rabbit hemorrhagic disease virus capsid, a versatile platform for foreign B-cell epitope display inducing protective humoral immune responses

    PubMed Central

    Moreno, Noelia; Mena, Ignacio; Angulo, Iván; Gómez, Yolanda; Crisci, Elisa; Montoya, María; Castón, José R.; Blanco, Esther; Bárcena, Juan

    2016-01-01

    Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge. PMID:27549017

  6. Intramolecular epitope spreading in Heymann nephritis.

    PubMed

    Shah, Pallavi; Tramontano, Alfonso; Makker, Sudesh P

    2007-12-01

    Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.

  7. Expression of bovine viral diarrhea virus glycoprotein E2 as a soluble secreted form in a Mammalian cell line.

    PubMed

    Donofrio, Gaetano; Bottarelli, Ezio; Sandro, Cavirani; Flammini, Cesidio Filippo

    2006-06-01

    Bovine viral diarrhea virus (BVDV) membrane-anchored type I glycoprotein E2 is an approximately 53-kDa immunodominant glycoprotein inducing the production of neutralizing antibodies in the animal host after natural infection or following immunization with live or killed vaccines. The E2 coding region lacking the transmembrane domain was constructed in a soluble secreted form (secE2) and expressed in the medium of a transiently transfected human cell line. The crude conditioned medium containing secE2 can be potentially employed to develop an enzyme-linked immunosorbent assay antigen for the diagnosis of BVDV infection or for vaccine purposes.

  8. Immunodominant antigens in Naegleria fowleri excretory--secretory proteins were potential pathogenic factors.

    PubMed

    Kim, Jong-Hyun; Yang, Ae-Hee; Sohn, Hae-Jin; Kim, Daesik; Song, Kyoung-Ju; Shin, Ho-Joon

    2009-11-01

    Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

  9. Age-specific seroprevalence to an immunodominant Cryptosporidium sporozoite antigen in a Brazilian population.

    PubMed Central

    Cox, M. J.; Elwin, K.; Massad, E.; Azevedo, R. S.

    2005-01-01

    The seroepidemiology of Cryptosporidium infection was investigated in a representative sample of a normal population in the State of Sao Paulo, Brazil using a recombinant form of the immunodominant 27-kDa sporozoite antigen. IgG seropositivity was low in infants following loss of maternal antibody but quickly increased to approximately 60% by 5 years, then 80% by the age of 10 years, after which prevalence remained constant. The broad range of antibody concentrations is consistent with previous reports that the IgG response to C. parvum is short-lived. There is also evidence that average antibody concentrations increase with age. Results suggest that the recombinant antigen may be a more sensitive method of measuring seroprevalence than the native antigen in Western blot. Although cross-sectional studies can provide an insight into the epidemiology of C. parvum in normal populations, further studies investigating the dynamics of the humoral immune responses to Cryptosporidium and the use of serology in epidemiological studies are required. PMID:16181518

  10. The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus.

    PubMed

    Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A; David, Chella S; Admon, Arie; López, Daniel

    2015-04-01

    The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.

  11. The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

    PubMed Central

    Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A.; David, Chella S.; Admon, Arie; López, Daniel

    2015-01-01

    The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. PMID:25635267

  12. The Immune Epitope Database and Analysis Resource in Epitope Discovery and Synthetic Vaccine Design

    PubMed Central

    Fleri, Ward; Paul, Sinu; Dhanda, Sandeep Kumar; Mahajan, Swapnil; Xu, Xiaojun; Peters, Bjoern; Sette, Alessandro

    2017-01-01

    The task of epitope discovery and vaccine design is increasingly reliant on bioinformatics analytic tools and access to depositories of curated data relevant to immune reactions and specific pathogens. The Immune Epitope Database and Analysis Resource (IEDB) was indeed created to assist biomedical researchers in the development of new vaccines, diagnostics, and therapeutics. The Analysis Resource is freely available to all researchers and provides access to a variety of epitope analysis and prediction tools. The tools include validated and benchmarked methods to predict MHC class I and class II binding. The predictions from these tools can be combined with tools predicting antigen processing, TCR recognition, and B cell epitope prediction. In addition, the resource contains a variety of secondary analysis tools that allow the researcher to calculate epitope conservation, population coverage, and other relevant analytic variables. The researcher involved in vaccine design and epitope discovery will also be interested in accessing experimental published data, relevant to the specific indication of interest. The database component of the IEDB contains a vast amount of experimentally derived epitope data that can be queried through a flexible user interface. The IEDB is linked to other pathogen-specific and immunological database resources. PMID:28352270

  13. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  14. The Immune Epitope Database and Analysis Resource in Epitope Discovery and Synthetic Vaccine Design.

    PubMed

    Fleri, Ward; Paul, Sinu; Dhanda, Sandeep Kumar; Mahajan, Swapnil; Xu, Xiaojun; Peters, Bjoern; Sette, Alessandro

    2017-01-01

    The task of epitope discovery and vaccine design is increasingly reliant on bioinformatics analytic tools and access to depositories of curated data relevant to immune reactions and specific pathogens. The Immune Epitope Database and Analysis Resource (IEDB) was indeed created to assist biomedical researchers in the development of new vaccines, diagnostics, and therapeutics. The Analysis Resource is freely available to all researchers and provides access to a variety of epitope analysis and prediction tools. The tools include validated and benchmarked methods to predict MHC class I and class II binding. The predictions from these tools can be combined with tools predicting antigen processing, TCR recognition, and B cell epitope prediction. In addition, the resource contains a variety of secondary analysis tools that allow the researcher to calculate epitope conservation, population coverage, and other relevant analytic variables. The researcher involved in vaccine design and epitope discovery will also be interested in accessing experimental published data, relevant to the specific indication of interest. The database component of the IEDB contains a vast amount of experimentally derived epitope data that can be queried through a flexible user interface. The IEDB is linked to other pathogen-specific and immunological database resources.

  15. Conservation and Diversity of Influenza A H1N1 HLA-Restricted T Cell Epitope Candidates for Epitope-Based Vaccines

    PubMed Central

    Tan, Paul ThiamJoo; Heiny, A. T.; Miotto, Olivo; Salmon, Jerome; Marques, Ernesto T. A.; Lemonnier, Francois; August, J. Thomas

    2010-01-01

    Background The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. Methodology/Principal Findings HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54) peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-γ ELISpot assay. The 54 peptides were compared to the 2007–2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. Conclusions/Significance Seventeen (17) T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus. PMID:20090904

  16. Mapping of T cell epitopes of the major fraction of rye grass using peripheral blood mononuclear cells from atopics and non-atopics. II. Isoallergen clone 5A of Lolium perenne group I (Lol p I).

    PubMed

    Bungy, G A; Rodda, S; Roitt, I; Brostoff, J

    1994-09-01

    Rye grass is the major cause of hay fever which currently affects 20% of the population. Lolium perenne group I (Lol p I) is a glycoprotein of 240 amino acid residues, representing the main allergen of rye grass. We have used peripheral blood mononuclear cells (PBMC) from controls and subjects allergic to rye grass and cultured them with L. perenne extract (LPE) and Lol p I and measured lymphocyte activation using thymidine incorporation. Patients were further studied against the 115 overlapping peptides of the iso-allergen clone 5A of Lol p I to see whether the 4 amino acid residue differences between clone 1A and clone 5A affect the T cell epitope and thus, lymphocyte activation. There are 24 peptide differences between isoallergen clone 1A and clone 5A occurring in pools 4, 13, 16 and 19 each one of which could be an immunodominant epitope. The PBMC from all allergic patients studied showed a strong proliferative response to LPE and Lol p I. Five immunogenic peptide pools, pool 6, 15, 16, 17 and 19 of the isoallergen clone 5A were also identified. Most of these pools are in the C-terminal region of Lol p I. Out of 20 pools tested in vitro 1 pool (pool-17) induced PBMC proliferation in five out of six patients who were not restricted to an HLA class II DR gene product. However, three out of the six subjects responded to various other peptide pools in addition to the immunodominant pool. In spite of the amino acid differences between the two clones, pool 17 still remains the immunodominant T cell epitope. Control subjects showed only weak responses to LPE and no detectable response to either Lol p I or peptide pools. From within the most active pool we have defined two peptides of the isoallergen clone 5A (identical in sequence with clone 1A) which stimulate lymphocytes from rye grass-sensitive patients in vitro. Previous studies with the two continuous sequences (193WGAVWRIDTPDK204 and 195AVWRIDTPDKLT206) tested in vivo by intradermal skin testing have shown

  17. The Relationship between B-cell Epitope and Mimotope Sequences.

    PubMed

    Zhang, Chunhua; Li, Yunyun; Tang, Weina; Zhou, Zhiguo; Sun, Pingping; Ma, Zhiqiang

    2016-01-01

    B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint.

  18. Modules for C-terminal epitope tagging of Tetrahymena genes

    PubMed Central

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  19. Viral Hemorrhagic Fevers

    MedlinePlus

    ... The CDC Cancel Submit Search The CDC Viral Hemorrhagic Fevers (VHFs) Note: Javascript is disabled or is not ... please visit this page: About CDC.gov . Viral Hemorrhagic Fevers (VHFs) Virus Families Arenaviruses Old World/New World ...

  20. Alternative Recognition of the Conserved Stem Epitope in Influenza A Virus Hemagglutinin by a VH3-30-Encoded Heterosubtypic Antibody

    PubMed Central

    Wyrzucki, Arkadiusz; Dreyfus, Cyrille; Kohler, Ines; Steck, Marco

    2014-01-01

    ABSTRACT A human monoclonal heterosubtypic antibody, MAb 3.1, with its heavy chain encoded by VH3-30, was isolated using phage display with immobilized hemagglutinin (HA) from influenza virus A/Japan/305/1957(H2N2) as the target. Antibody 3.1 potently neutralizes influenza viruses from the H1a clade (i.e., H1, H2, H5, H6) but has little neutralizing activity against the H1b clade. Its crystal structure in complex with HA from a pandemic H1N1 influenza virus, A/South Carolina/1/1918(H1N1), revealed that like other heterosubtypic anti-influenza virus antibodies, MAb 3.1 contacts a hydrophobic groove in the HA stem, primarily using its heavy chain. However, in contrast to the closely related monoclonal antibody (Mab) FI6 that relies heavily on HCDR3 for binding, MAb 3.1 utilizes residues from HCDR1, HCDR3, and framework region 3 (FR3). Interestingly, HCDR1 of MAb 3.1 adopts an α-helical conformation and engages in hydrophobic interactions with the HA very similar to those of the de novo in silico-designed and affinity-matured synthetic protein HB36.3. These findings improve our understanding of the molecular requirements for binding to the conserved epitope in the stem of the HA protein and, therefore, aid the development of more universal influenza vaccines targeting these epitopes. IMPORTANCE Influenza viruses rapidly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by acquiring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by immunization or infection protect only against the immunizing or closely related strains. Here, we describe a novel monoclonal antibody that recognizes the conserved heterosubtypic epitope in the stem of influenza A virus hemagglutinin. This antibody, referred to as MAb 3.1, recognizes its epitope in a manner that resembles recognition of a similar epitope by the de novo in silico-designed and affinity-matured synthetic

  1. Viral Evolution and Cytotoxic T Cell Restricted Selection in Acute Infant HIV-1 Infection

    PubMed Central

    Garcia-Knight, Miguel A.; Slyker, Jennifer; Payne, Barbara Lohman; Pond, Sergei L. Kosakovsky; de Silva, Thushan I.; Chohan, Bhavna; Khasimwa, Brian; Mbori-Ngacha, Dorothy; John-Stewart, Grace; Rowland-Jones, Sarah L.; Esbjörnsson, Joakim

    2016-01-01

    Antiretroviral therapy-naive HIV-1 infected infants experience poor viral containment and rapid disease progression compared to adults. Viral factors (e.g. transmitted cytotoxic T- lymphocyte (CTL) escape mutations) or infant factors (e.g. reduced CTL functional capacity) may explain this observation. We assessed CTL functionality by analysing selection in CTL-targeted HIV-1 epitopes following perinatal infection. HIV-1 gag, pol and nef sequences were generated from a historical repository of longitudinal specimens from 19 vertically infected infants. Evolutionary rate and selection were estimated for each gene and in CTL-restricted and non-restricted epitopes. Evolutionary rate was higher in nef and gag vs. pol, and lower in infants with non-severe immunosuppression vs. severe immunosuppression across gag and nef. Selection pressure was stronger in infants with non-severe immunosuppression vs. severe immunosuppression across gag. The analysis also showed that infants with non-severe immunosuppression had stronger selection in CTL-restricted vs. non-restricted epitopes in gag and nef. Evidence of stronger CTL selection was absent in infants with severe immunosuppression. These data indicate that infant CTLs can exert selection pressure on gag and nef epitopes in early infection and that stronger selection across CTL epitopes is associated with favourable clinical outcomes. These results have implications for the development of paediatric HIV-1 vaccines. PMID:27403940

  2. Viral immunology. Comprehensive serological profiling of human populations using a synthetic human virome.

    PubMed

    Xu, George J; Kula, Tomasz; Xu, Qikai; Li, Mamie Z; Vernon, Suzanne D; Ndung'u, Thumbi; Ruxrungtham, Kiat; Sanchez, Jorge; Brander, Christian; Chung, Raymond T; O'Connor, Kevin C; Walker, Bruce; Larman, H Benjamin; Elledge, Stephen J

    2015-06-05

    The human virome plays important roles in health and immunity. However, current methods for detecting viral infections and antiviral responses have limited throughput and coverage. Here, we present VirScan, a high-throughput method to comprehensively analyze antiviral antibodies using immunoprecipitation and massively parallel DNA sequencing of a bacteriophage library displaying proteome-wide peptides from all human viruses. We assayed over 10(8) antibody-peptide interactions in 569 humans across four continents, nearly doubling the number of previously established viral epitopes. We detected antibodies to an average of 10 viral species per person and 84 species in at least two individuals. Although rates of specific virus exposure were heterogeneous across populations, antibody responses targeted strongly conserved "public epitopes" for each virus, suggesting that they may elicit highly similar antibodies. VirScan is a powerful approach for studying interactions between the virome and the immune system.

  3. Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice.

    PubMed

    Mishra, Sasmita; Lavelle, Bianca J; Desrosiers, Joe; Ardito, Matt T; Terry, Frances; Martin, William D; De Groot, Anne S; Gregory, Stephen H

    2014-01-01

    Hepatitis C virus (HCV) is the etiologic agent of chronic liver disease, hepatitis C. Spontaneous resolution of viral infection is associated with vigorous HLA class I- and class II-restricted T cell responses to multiple viral epitopes. Unfortunately, only 20% of patients clear infection spontaneously, most develop chronic disease and require therapy. The response to chemotherapy varies, however; therapeutic vaccination offers an additional treatment strategy. To date, therapeutic vaccines have demonstrated only limited success. Vector-mediated vaccination with multi-epitope-expressing DNA constructs alone or in combination with chemotherapy offers an additional treatment approach. Gene sequences encoding validated HLA-A2- and HLA-DRB1-restricted epitopes were synthesized and cloned into an expression vector. Dendritic cells (DCs) derived from humanized, HLA-A2/DRB1 transgenic (donor) mice were transfected with these multi-epitope-expressing DNA constructs. Recipient HLA-A2/DRB1 mice were vaccinated s.c. with transfected DCs; control mice received non-transfected DCs. Peptide-specific IFN-γ production by splenic T cells obtained at 5 weeks post-immunization was quantified by ELISpot assay; additionally, the production of IL-4, IL-10 and TNF-α were quantified by cytokine bead array. Splenocytes derived from vaccinated HLA-A2/DRB1 transgenic mice exhibited peptide-specific cytokine production to the vast majority of the vaccine-encoded HLA class I- and class II-restricted T cell epitopes. A multi-epitope-based HCV vaccine that targets DCs offers an effective approach to inducing a broad immune response and viral clearance in chronic, HCV-infected patients.

  4. Epitope specificity of rabbit immunoglobulin G (IgG) elicited by pneumococcal type 23F synthetic oligosaccharide- and native polysaccharide-protein conjugate vaccines: comparison with human anti-polysaccharide 23F IgG.

    PubMed Central

    Alonso de Velasco, E; Verheul, A F; van Steijn, A M; Dekker, H A; Feldman, R G; Fernández, I M; Kamerling, J P; Vliegenthart, J F; Verhoef, J; Snippe, H

    1994-01-01

    Streptococcus pneumoniae type 23F capsular polysaccharide (PS23F) consitss of a repeating glycerol-phosphorylated branched tetrasaccharide. The immunogenicities of the following related antigens were investigated: (i) a synthetic trisaccharide comprising the backbone of one repeating unit, (ii) a synthetic tetrasaccharide comprising the complete repeating unit, and (iii) native PS23F (all three conjugated to keyhole limpet hemocyanin [KLH]) and (iv) formalin-killed S. pneumoniae 23F. All antigens except the trisaccharide-KLH conjugate induced relatively high anti-PS23F antibody levels in rabbits. The epitope specificity of such antibodies was then studied by means of an inhibition immunoassay. The alpha(1-->2)-linked L-rhamnose branch was shown to be immunodominant for immunoglobulin G (IgG) induced by tetrasaccharide-KLH, PS23F-KLH, and killed S. pneumoniae 23F: in most sera L-rhamnose totally inhibited the binding of IgG to PS23F. Thus, there appears to be no major difference in epitope specificity between IgG induced by tetrasaccharide-KLH and that induced by antigens containing the polymeric form of PS23F. Human anti-PS23F IgG (either vaccine induced or naturally acquired) had a different epitope specificity: none of the inhibitors used, including L-rhamnose and tetrasaccharide-KLH, exhibited substantial inhibition. These observations suggest that the epitope recognized by human IgG on PS23F is larger than the epitope recognized by rabbit IgG. Both human and rabbit antisera efficiently opsonized type 23F pneumococci, as measured in a phagocytosis assay using human polymorphonuclear leukocytes. PMID:7509318

  5. Japanese encephalitis protein vaccine candidates expressing neutralizing epitope and M.T hsp70 induce virus-specific memory B cells and long-lasting antibodies in swine.

    PubMed

    Fei-fei, Ge; Jian, Wang; Feng, Xu; Li-ping, Sheng; Quan-yun, Sun; Jin-ping, Zhou; Pu-yan, Chen; Pei-hong, Liu

    2008-10-16

    Swine are an important amplifier of Japanese encephalitis (JE) virus in the paradomestic environment. In this study, two JE protein vaccine candidates were evaluated for immunogenicity in swine. Both vaccine plasmids are based on a prokaryotic vector pET-32a(+). One plasmid, designated pET-32a(+)-epitope, encode a cassette consisting of a neutralizing epitope on envelope (E) protein of JE virus, whereas the other plasmid, designated pET-32a(+)-epitope-hsp70, express the fusion protein of the epitope and M.T hsp70. Some differences were detected in the immunogenicity of these two proteins in swine. Swine immunized twice with 2000pmol of the neutralizing epitope or the fusion protein developed neutralizing antibody titers of respectively, 154 and 300, and anti-neutralizing epitope antibody titers of 10(4.25) and 10(6.0) by 3 weeks after the second immunization. In addition, swine immunized with the neutralizing epitope emulsified with adjuvant S206 or with imported mineral oil and Tween-80 induced neutralizing antibody titers of 196 and 244, and anti-neutralizing epitope antibody titers of 10(5.25) or 10(5.6) at the same time point. However, swine administered two doses of a commercial JE vaccine (attenuated virus preparation; JEV SA14-14-2 strain) developed less favorable antibody responses with neutralizing antibody titer 40 and anti-neutralizing epitope antibody titers 10(3.7). The anamnestic response was followed by monitoring titers 1 week after boosting with a viral antigen; swine immunized twice with the fusion protein showed a 177-fold increase in anti-neutralizing epitope titer, indicating a strong recall of the antibody response. The animals maintained detectable levels of anti-neutralizing epitope antibody for at least 105 days after two immunizations, indicating that these four protein antigens are able to stimulate virus-specific memory B cells and long-lasting antibodies at higher levels than is achieved using a current commercial attenuated JEV vaccine

  6. Epitope Mapping with Random Phage Display Library

    PubMed Central

    Midoro-Horiuti, Terumi; Goldblum, Randall M.

    2017-01-01

    Random phage display library is used to map conformational as well as linear epitopes. These libraries are available in varying lengths and with circularization. We provide here a protocol conveying our experience using a commercially available peptide phage display library, which in our hands provides good results. PMID:24515483

  7. Relevant B Cell Epitopes in Allergic Disease

    PubMed Central

    Pomés, Anna

    2010-01-01

    The 3-dimensional structure of an allergen defines the accessible parts on the surface of the molecule or epitopes that interact with antibodies. Mapping the antigenic determinants for IgE antibody binding has been pursued through strategies based on the use of overlapping synthetic peptides, recombinant allergenic fragments or unfolded allergens. These approaches led to the identification of mostly linear epitopes and are useful for food allergens that undergo digestion or food processing. For inhaled allergens, conformational epitopes appear to be the primary targets of IgE responses. Knowledge of the molecular structure of allergens alone and in complex with antibodies that interfere with IgE antibody binding is important to understand the immune recognition of B cell-antigenic determinants on allergens and the design of recombinant allergens for immunotherapy. Starting with the molecular cloning and expression of allergens, and with the advent of X-ray crystallography and nuclear magnetic resonance techniques, we have been able to visualize conformational epitopes on allergens. PMID:19940500

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

    SciTech Connect

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

    1995-08-01

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

  9. Whole genome protein microarrays for serum profiling of immunodominant antigens of Bacillus anthracis

    PubMed Central

    Kempsell, Karen E.; Kidd, Stephen P.; Lewandowski, Kuiama; Elmore, Michael J.; Charlton, Sue; Yeates, Annemarie; Cuthbertson, Hannah; Hallis, Bassam; Altmann, Daniel M.; Rogers, Mitch; Wattiau, Pierre; Ingram, Rebecca J.; Brooks, Tim; Vipond, Richard

    2015-01-01

    A commercial Bacillus anthracis (Anthrax) whole genome protein microarray has been used to identify immunogenic Anthrax proteins (IAP) using sera from groups of donors with (a) confirmed B. anthracis naturally acquired cutaneous infection, (b) confirmed B. anthracis intravenous drug use-acquired infection, (c) occupational exposure in a wool-sorters factory, (d) humans and rabbits vaccinated with the UK Anthrax protein vaccine and compared to naïve unexposed controls. Anti-IAP responses were observed for both IgG and IgA in the challenged groups; however the anti-IAP IgG response was more evident in the vaccinated group and the anti-IAP IgA response more evident in the B. anthracis-infected groups. Infected individuals appeared somewhat suppressed for their general IgG response, compared with other challenged groups. Immunogenic protein antigens were identified in all groups, some of which were shared between groups whilst others were specific for individual groups. The toxin proteins were immunodominant in all vaccinated, infected or other challenged groups. However, a number of other chromosomally-located and plasmid encoded open reading frame proteins were also recognized by infected or exposed groups in comparison to controls. Some of these antigens e.g., BA4182 are not recognized by vaccinated individuals, suggesting that there are proteins more specifically expressed by live Anthrax spores in vivo that are not currently found in the UK licensed Anthrax Vaccine (AVP). These may perhaps be preferentially expressed during infection and represent expression of alternative pathways in the B. anthracis “infectome.” These may make highly attractive candidates for diagnostic and vaccine biomarker development as they may be more specifically associated with the infectious phase of the pathogen. A number of B. anthracis small hypothetical protein targets have been synthesized, tested in mouse immunogenicity studies and validated in parallel using human sera from

  10. Identification of protective Lassa virus epitopes that are restricted by HLA-A2.

    PubMed

    Botten, Jason; Alexander, Jeff; Pasquetto, Valerie; Sidney, John; Barrowman, Polly; Ting, Joey; Peters, Bjoern; Southwood, Scott; Stewart, Barbara; Rodriguez-Carreno, Maria P; Mothe, Bianca; Whitton, J Lindsay; Sette, Alessandro; Buchmeier, Michael J

    2006-09-01

    Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8(+) T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC(42-50), GLVGLVTFL; GPC(60-68), SLYKGVYEL; and GPC(441-449), YLISIFLHL) that displayed high-affinity binding (< or =98 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC(42-50) or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.

  11. Delivery of a foreign epitope by sharing amino acid residues with the carrier matrix.

    PubMed

    Cheong, Wan-Shoo; Drummer, Heidi Edelgard; Netter, Hans-Jürgen

    2009-06-01

    A broad range of structural viral proteins has the ability to assemble into virus-like particles (VLPs). Under the condition that modified subunits are still competent to assemble into VLPs, they are epitope delivery platforms suitable for vaccination purposes. The insertion of foreign sequences can be detrimental for the formation of chimeric VLPs as a result of misfolded subunit proteins. Hence, a strategy was adopted to screen for locations allowing the use of shared residues between the wildtype subunit sequence and the foreign insert. The insertion of a cysteine-containing sequence of hepatitis C virus (HCV) envelope protein 2 (E2) without adding an additional cysteine residue retained the ability of recombinant small hepatitis B surface antigen (HBsAg-S) to form secretion competent VLPs. A cysteine residue shared by the insert and the template protein avoided the formation of non-native disulfide bonds, and allowed the formation of VLPs. The chimeric HBsAg-S VLPs were similar to wildtype VLPs in density exposing the inserted foreign epitope and being immunogenic. Overall, the use of shared sequences between the insert and the subunit will facilitate the design of chimeric VLPs carrying multiple epitopes.

  12. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals

    PubMed Central

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2015-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines. PMID:25668665

  13. Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

    PubMed

    Comber, Joseph D; Karabudak, Aykan; Huang, Xiaofang; Piazza, Paolo A; Marques, Ernesto T A; Philip, Ramila

    2014-01-01

    Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines.

  14. Genetic mapping of a highly variable norovirus GII.4 blockade epitope: potential role in escape from human herd immunity.

    PubMed

    Debbink, Kari; Donaldson, Eric F; Lindesmith, Lisa C; Baric, Ralph S

    2012-01-01

    Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.

  15. B cell epitope spreading: mechanisms and contribution to autoimmune diseases.

    PubMed

    Cornaby, Caleb; Gibbons, Lauren; Mayhew, Vera; Sloan, Chad S; Welling, Andrew; Poole, Brian D

    2015-01-01

    While a variety of factors act to trigger or initiate autoimmune diseases, the process of epitope spreading is an important contributor in their development. Epitope spreading is a diversification of the epitopes recognized by the immune system. This process happens to both T and B cells, with this review focusing on B cells. Such spreading can progress among multiple epitopes on a single antigen, or from one antigenic molecule to another. Systemic lupus erythematosus, multiple sclerosis, pemphigus, bullous pemphigoid and other autoimmune diseases, are all influenced by intermolecular and intramolecular B cell epitope spreading. Endocytic processing, antigen presentation, and somatic hypermutation act as molecular mechanisms that assist in driving epitope spreading and broadening the immune response in autoimmune diseases. The purpose of this review is to summarize our current understanding of B cell epitope spreading with regard to autoimmunity, how it contributes during the progression of various autoimmune diseases, and treatment options available.

  16. Efficient epitope mapping by bacteriophage {lambda} surface display

    SciTech Connect

    Kuwabara, I.; Maruyama, H.; Zuberi, R.I.

    1997-01-01

    A bacteriophage {lambda} surface expression system, {lambda}foo, was used for epitope mapping of human galectin-3. We constructed random epitope and peptide libraries and compared their efficiencies in the mapping. The galectin-3 cDNA was randomly digested by DNase I to make random epitope libraries. The libraries were screened by affinity selection using a microtiter plate coated with monoclonal antibodies. Direct DNA sequencing of the selected clones defined two distinct epitope sites consisting of nine and 11 amino-acid residues. Affinity selection of random peptide libraries recovered a number of sequences that were similar to each other but distinct from the galectin-3 sequence. These results demonstrate that a single affinity selection of epitope libraries with antibodies is able to define an epitope determinant as small as nine residues long and is more efficient in epitope mapping than random peptide libraries. 25 refs., 4 figs., 1 tab.

  17. A novel approach to the identification of T-cell epitopes in Mycobacterium tuberculosis using human T-lymphocyte clones.

    PubMed Central

    Lamb, J R; Young, D B

    1987-01-01

    Current approaches to the analysis of antigens involved in the cellular immune response to mycobacterial infection rely on the initial identification and isolation of molecular components using monoclonal antibodies. In order to overcome the constraints of this approach, we have utilized a procedure involving T-cell recognition of antigens fractionated by polyacrylamide gel electrophoresis (SDS-PAGE) and added to proliferation assays after blotting onto nitrocellulose membranes. Analysis of human T-cell responses to Mycobacterium tuberculosis and Mycobacterium bovis BCG by this procedure revealed distinctive patterns of reactivity to different molecular weight components indicative of the selective recognition of immunodominant and species-specific determinants. Human T-cell clones were subsequently derived, and SDS-PAGE immunoblotting was used to identify the antigen recognized by each clone. Three epitopes defined by individual T-cell clones were identified on separate polypeptides with molecular weights 16,000-18,000 (clone P53), 18,000-20,000 (clone P57) and 52,000-55,000 (clone P35). This study demonstrates the potential application of T-cell cloning in conjunction with SDS-PAGE immunoblotting for the dissection and analysis of the cellular immune response to pathogenic agents during human infection. Images Figure 1 PMID:2434413

  18. Epitope-specificities of HLA antibodies: the effect of epitope structure on Luminex technique-dependent antibody reactivity.

    PubMed

    Resse, Marianna; Paolillo, Rossella; Minucci, Biagio Pellegrino; Cavalca, Francesco; Casamassimi, Amelia; Napoli, Claudio

    2015-04-01

    The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity.

  19. Epitope mapping of Canine distemper virus phosphoprotein by monoclonal antibodies.

    PubMed

    Sugai, Akihiro; Kooriyama, Takanori; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

    2009-12-01

    The gene for phosphoprotein (P) of CDV encodes three different proteins, P, V, and C. The P protein is involved in viral gene transcription and replication. In the present study, we produced MAbs against a unique domain of the CDV-P protein, from aa 232 to 507, and determined their antigenic sites. By immunizing BALB/c mice with the recombinant P protein-specific fragment, we obtained six MAbs. Competitive binding inhibition assays revealed that they recognized two distinct regions of the P protein. Western blot analysis and immunofluorescence assays using deletion mutants of the unique C-terminus of the CDV-P protein revealed that all MAbs recognized a central short region (aa 233-303) of the CDV-P protein. In addition, linear and conformational epitopes have been determined, and at least four antigenic sites exist in the P protein central region. Furthermore, four of the MAbs were found to react with the P protein of recent Japanese field isolates but not with that of the older CDV strains, including a vaccine strain. Thus, these MAbs could be clinically useful for quick diagnosis during the CDV outbreaks.

  20. Computational design of high-affinity epitope scaffolds by backbone grafting of a linear epitope.

    PubMed

    Azoitei, Mihai L; Ban, Yih-En Andrew; Julien, Jean-Philippe; Bryson, Steve; Schroeter, Alexandria; Kalyuzhniy, Oleksandr; Porter, Justin R; Adachi, Yumiko; Baker, David; Pai, Emil F; Schief, William R

    2012-01-06

    Computational grafting of functional motifs onto scaffold proteins is a promising way to engineer novel proteins with pre-specified functionalities. Typically, protein grafting involves the transplantation of protein side chains from a functional motif onto structurally homologous regions of scaffold proteins. Using this approach, we previously transplanted the human immunodeficiency virus 2F5 and 4E10 epitopes onto heterologous proteins to design novel "epitope-scaffold" antigens. However, side-chain grafting is limited by the availability of scaffolds with compatible backbone for a given epitope structure and offers no route to modify backbone structure to improve mimicry or binding affinity. To address this, we report here a new and more aggressive computational method-backbone grafting of linear motifs-that transplants the backbone and side chains of linear functional motifs onto scaffold proteins. To test this method, we first used side-chain grafting to design new 2F5 epitope scaffolds with improved biophysical characteristics. We then independently transplanted the 2F5 epitope onto three of the same parent scaffolds using the newly developed backbone grafting procedure. Crystal structures of side-chain and backbone grafting designs showed close agreement with both the computational models and the desired epitope structure. In two cases, backbone grafting scaffolds bound antibody 2F5 with 30- and 9-fold higher affinity than corresponding side-chain grafting designs. These results demonstrate that flexible backbone methods for epitope grafting can significantly improve binding affinities over those achieved by fixed backbone methods alone. Backbone grafting of linear motifs is a general method to transplant functional motifs when backbone remodeling of the target scaffold is necessary.

  1. Selection of Peptide Mimics of HIV-1 Epitope Recognized by Neutralizing Antibody VRC01

    PubMed Central

    Chikaev, Anton N.; Bakulina, Anastasiya Yu.; Burdick, Ryan C.; Karpenko, Larisa I.; Pathak, Vinay K.; Ilyichev, Alexander A.

    2015-01-01

    The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies. PMID:25785734

  2. Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus

    PubMed Central

    Klausberger, Miriam; Tscheliessnig, Rupert; Neff, Silke; Nachbagauer, Raffael; Wohlbold, Teddy John; Wilde, Monika; Palmberger, Dieter; Krammer, Florian; Jungbauer, Alois; Grabherr, Reingard

    2016-01-01

    Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection. PMID:27088239

  3. Construction of a bovine enterovirus-based vector expressing a foot-and-mouth disease virus epitope.

    PubMed

    Chu, Jia-Qi; Lee, Yeo-Joo; Park, Jeong-Nam; Kim, Su-Mi; Lee, Kwang-Nyeong; Ko, Young-Joon; Lee, Hyang-Sim; Cho, In-Soo; Kim, Byounghan; Park, Jong-Hyeon

    2013-04-01

    A recombinant infectious bovine enterovirus (BEV) vector was constructed to express a foot-and-mouth disease virus (FMDV) capsid protein (VP1) epitope. Sequences encoding the VP1 epitope (amino acid residues 141-160) of FMDV (vaccine strain O1/Manisa/Turkey/69) were inserted into pBLUBEV at the VP1/2A junction. The growth characteristics of the parental virus and viruses derived from recombinant plasmids (pBLUBEV, pBLUBEV-Manisa-epi) were determined by plaque assay and one-step growth curve analysis. There were no significant differences in the growth kinetics and plaque morphologies between transfectant viruses and their parental virus. The expressed VP1 epitope was detected successfully by using indirect immunofluorescence assay with a polyclonal antibody against the FMDV VP1 epitope from Madin Darby bovine kidney (MDBK) cells infected with BEV-Manisa-epi transfectant virus. This study demonstrated a novel alternative live viral vector that may be utilized as a candidate vaccine vector for veterinary applications.

  4. Influenza, but not HIV-specific CTL epitopes, elicits delayed-type hypersensitivity (DTH) reactions in HIV-infected patients.

    PubMed

    Ruiz-Riol, Marta; Mothe, Beatriz; Gandhi, Rajesh T; Bhardwaj, Nina; Scadden, David T; Sanchez-Merino, Victor; Brander, Christian

    2013-06-01

    The induction of cytotoxic T lymphocytes (CTLs) is believed to be an important defense mechanism against viral infections. The availability of simple, sensitive, specific and physiologically informative in vivo tests, applicable to humans, would greatly elucidate the nature of protective immune responses and facilitate immune monitoring in large vaccine trials. Here we studied the possibility of using defined HLA-A*02:01-restricted CTL epitopes from influenza matrix protein (GL9, GILGFVFTL) and HIV Gag p17 (SL9, SLYNTVATL) to elicit a cutaneous delayed-type hypersensitivity (DTH) reaction. Our results show that the GL9 but not the SL9 epitope was able to induce a DTH reaction. HIV infection status, HIV RNA level and CD4(+) T-cell counts were not predictive of the extent of DTH reactions. However, a markedly reduced expression of skin homing markers CD103 and cutaneous lymphocyte associated Ag (CLA) on epitope-specific CTL populations was associated with a lack of SL9 DTH reactivity. These data demonstrate that DTH reactions can be elicited by optimally defined CTL epitopes per se and point towards specific homing markers that are required for such reactions. These data may offer new insights into the immune pathogenesis of HIV infection and provide the basis of novel immune monitoring approaches for large-scale HIV vaccine trials.

  5. Broad epitope coverage of a human in vitro antibody library.

    PubMed

    Sivasubramanian, Arvind; Estep, Patricia; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Niles, Rebecca Hurley; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes.

  6. A continuous infusion of a minor histocompatibility antigen-immunodominant peptide induces a delay of male skin graft rejection.

    PubMed

    Sireci, Guido; Barera, Annalisa; Macaluso, Pasquale; Di Sano, Caterina; Bonanno, Cesira T; Pio La Manna, Marco; Di Liberto, Diana; Dieli, Francesco; Salerno, Alfredo

    2009-01-01

    We previously reported that an inhibition of antigen-specific Interferon-gamma release and cytotoxicity occurs after a continuous infusion of an HY immunodominant peptide although this treatment is not able to cause a significant delay of male skin grafts rejection. In vivo administration of high doses of an HY peptide, through mini-osmotic pumps, in naïve female mice was used to study the effects on the male skin grafts rejection. A continuous infusion of 1mg of an HY peptide induces a significant delay of male skin graft rejection. In vitro HY-specific Interferon-gamma release was inhibited adding peptide-specific suppressor cells: the ability to inhibit Interferon-gamma release was evident when two HY peptides were present on the same dendritic cells indicating that the suppressor cells exert "linked-suppression". The phenotype of the suppressor cells is CD8(+)CD28(-) and these cells express more CD62 ligand and FOXP3 than controls. Suppressor cells were able to cause a significant delay of rejection of male skin grafts when injected in naive female mice. The inhibitory effects of these suppressor cells seem to be due to the impairment of antigen presentation; down-regulation of B7 molecules on dendritic cells occurred. Taken all together, our data demonstrate that a continuous infusion of an immunodominant HY peptide induces a T CD8 suppressor subset able to inhibit immune responses to male tissues and cells.

  7. Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes.

    PubMed

    Hui, Daniel J; Basner-Tschakarjan, Etiena; Chen, Yifeng; Davidson, Robert J; Buchlis, George; Yazicioglu, Mustafa; Pien, Gary C; Finn, Jonathan D; Haurigot, Virginia; Tai, Alex; Scott, David W; Cousens, Leslie P; Zhou, Shangzhen; De Groot, Anne S; Mingozzi, Federico

    2013-09-01

    Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.

  8. Proteolytic Cleavage of the Immunodominant Outer Membrane Protein rOmpA in Rickettsia rickettsii.

    PubMed

    Noriea, Nicholas F; Clark, Tina R; Mead, David; Hackstadt, Ted

    2017-03-15

    Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, contains two immunodominant proteins, rOmpA and rOmpB, in the outer membrane. Both rOmpA and rOmpB are conserved throughout spotted fever group rickettsiae as members of a family of autotransporter proteins. Previously, it was demonstrated that rOmpB is proteolytically processed, with the cleavage site residing near the autotransporter domain at the carboxy-terminal end of the protein, cleaving the 168-kDa precursor into apparent 120-kDa and 32-kDa fragments. The 120- and 32-kDa fragments remain noncovalently associated on the surface of the bacterium, with implications that the 32-kDa fragment functions as the membrane anchor domain. Here we present evidence for a similar posttranslational processing of rOmpA. rOmpA is expressed as a predicted 224-kDa precursor yet is observed on SDS-PAGE as a 190-kDa protein. A small rOmpA fragment of ∼32 kDa was discovered during surface proteome analysis and identified as the carboxy-terminal end of the protein. A rabbit polyclonal antibody was generated to the autotransporter region of rOmpA and confirmed a 32-kDa fragment corresponding to the calculated mass of a proteolytically cleaved rOmpA autotransporter region. N-terminal amino acid sequencing revealed a cleavage site on the carboxy-terminal side of Ser-1958 in rOmpA. An avirulent strain of R. rickettsii Iowa deficient in rOmpB processing was also defective in the processing of rOmpA. The similarities of the cleavage sites and the failure of R. rickettsii Iowa to process either rOmpA or rOmpB suggest that a single enzyme may be responsible for both processing events.IMPORTANCE Members of the spotted fever group of rickettsiae, including R. rickettsii, the etiologic agent of Rocky Mountain spotted fever, express at least four autotransporter proteins that are protective antigens or putative virulence determinants. One member of this class of proteins, rOmpB, is proteolytically processed to a

  9. The use of multiple-pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti-(recombinant bovine growth hormone) sera. Comparison with predicted regions of immunogenicity and location within the three-dimensional structure of the molecule.

    PubMed

    Beattie, J; Fawcett, H A; Flint, D J

    1992-11-15

    A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth-hormone (rbGH) molecule recognised by three polyclonal guinea-pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea-pig antisera, 3 or 4, very-well-defined major continuous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea-pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring alpha-helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea-pig antiserum. Comparison of the four guinea-pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson-Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods. In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous

  10. Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV

    PubMed Central

    Luytjes, Willem; Leenhouts, Kees; Rottier, Peter J. M.; van Kuppeveld, Frank J. M.; Haijema, Bert Jan

    2016-01-01

    ABSTRACT Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better

  11. Computational approach for predicting the conserved B-cell epitopes of hemagglutinin H7 subtype influenza virus

    PubMed Central

    Wang, Xiangyu; Sun, Qi; Ye, Zhonghua; Hua, Ying; Shao, Na; Du, Yanli; Zhang, Qiwei; Wan, Chengsong

    2016-01-01

    An avian-origin influenza H7N9 virus epidemic occurred in China in 2013–2014, in which >422 infected people suffered from pneumonia, respiratory distress syndrome and septic shock. H7N9 viruses belong to the H7 subtype of avian-origin influenza viruses (AIV-H7). Hemagglutinin (HA) is a vital membrane protein of AIV that has an important role in host recognition and infection. The epitopes of HA are significant determinants of the regularity of epidemic and viral mutation and recombination mechanisms. The present study aimed to predict the conserved B-cell epitopes of AIV-H7 HA using a bioinformatics approach, including the three most effective epitope prediction softwares available online: Artificial Neural Network based B-cell Epitope Prediction (ABCpred), B-cell Epitope Prediction (BepiPred) and Linear B-cell Epitope Prediction (LBtope). A total of 24 strains of Euro-Asiatic AIV-H7 that had been associated with a serious poultry pandemic or had infected humans in the past 30 years were selected to identify the conserved regions of HA. Sequences were obtained from the National Center for Biotechnology Information and Global Initiative on Sharing Avian Influenza Data databases. Using a combination of software prediction and sequence comparisons, the conserved epitopes of AIV-H7 were predicted and clarified. A total of five conserved epitopes [amino acids (aa) 37–52, 131–142, 215–234, 465–484 and 487–505] with a suitable length, high antigenicity and minimal variation were predicted and confirmed. Each obtained a score of >0.80 in ABCpred, 60% in LBtope and a level of 0.35 in Bepipred. In addition, a representative amino acid change (glutamine235-to-leucine235) in the HA protein of the 2013 AIV-H7N9 was discovered. The strategy adopted in the present study may have profound implications on the rapid diagnosis and control of infectious disease caused by H7N9 viruses, as well as by other virulent viruses, such as the Ebola virus. PMID:27703505

  12. A unique and conserved neutralization epitope in H5N1 influenza viruses identified by an antibody against the A/Goose/Guangdong/1/96 hemagglutinin.

    PubMed

    Zhu, Xueyong; Guo, Yong-Hui; Jiang, Tao; Wang, Ya-Di; Chan, Kwok-Hung; Li, Xiao-Feng; Yu, Wenli; McBride, Ryan; Paulson, James C; Yuen, Kwok-Yung; Qin, Cheng-Feng; Che, Xiao-Yan; Wilson, Ian A

    2013-12-01

    Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab' fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.

  13. Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein

    PubMed Central

    2011-01-01

    Background The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. Results In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. Conclusion The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex. PMID:21375771

  14. Identification of a dominant epitope of glutamic acid decarboxylase (GAD-65) recognized by autoantibodies in stiff-man syndrome

    PubMed Central

    1993-01-01

    Glutamic acid decarboxylase (GAD) is the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA) in neurons and in pancreatic beta cells. It is a major target of autoimmunity in Stiff- Man syndrome (SMS), a rare neurological disease, and in insulin- dependent diabetes mellitus. The two GAD isoforms, GAD-65 and GAD-67, are the products of two different genes. GAD-67 and GAD-65 are very similar to each other in amino acid sequence and differ substantially only at their NH2-terminal region. We have investigated the reactivity of autoantibodies of 30 Stiff-Man syndrome patients to GAD. All patient sera contained antibodies that recognize strongly GAD-65, but also GAD- 67, when tested by immunoprecipitation on brain extracts and by immunoprecipitation or immunocytochemistry on cells transfected with either the GAD-65 or the GAD-67 gene. When tested by Western blotting, all patient sera selectively recognized GAD-65. Western blot analysis of deletion mutants of GAD-65 demonstrated that autoantibodies are directed predominantly against two regions of the GAD-65 molecule. All SMS sera strongly recognized a fragment contained between amino acid 475 and the COOH terminus (amino acid 585). Within this region, amino acids 475-484 and 571-585 were required for reactivity. The requirement of these two discontinuous segments implies that the epitope is influenced by conformation. This reactivity is similar to that displayed by the monoclonal antibody GAD 6, suggesting the presence of a single immunodominant epitope (SMS-E1) in this region of GAD-65. In addition, most SMS sera recognized at least one epitope (SMS-E2) in the NH2-terminal domain of GAD-65 (amino acids 1-95). The demonstration in SMS patients of a strikingly homogeneous humoral autoimmune response against GAD and the identification of dominant autoreactive target regions may help to elucidate the molecular mechanisms of GAD processing and presentation involved in GAD autoimmunity. Moreover, the

  15. Internal epitope tagging informed by relative lack of sequence conservation

    PubMed Central

    Burg, Leonard; Zhang, Karen; Bonawitz, Tristan; Grajevskaja, Viktorija; Bellipanni, Gianfranco; Waring, Richard; Balciunas, Darius

    2016-01-01

    Many experimental techniques rely on specific recognition and stringent binding of proteins by antibodies. This can readily be achieved by introducing an epitope tag. We employed an approach that uses a relative lack of evolutionary conservation to inform epitope tag site selection, followed by integration of the tag-coding sequence into the endogenous locus in zebrafish. We demonstrate that an internal epitope tag is accessible for antibody binding, and that tagged proteins retain wild type function. PMID:27892520

  16. An influenza A virus hemagglutinin (HA) epitope inserted in and expressed from several loci of the infectious bursal disease virus genome induces HA-specific antibodies.

    PubMed

    Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

    2014-08-01

    The N-terminus of infectious bursal disease virus (IBDV) VP5 has been shown to be capable of tolerating the insertion of small epitopes. The objective of the present study was to determine if IBDV genomic sites, including the 5' end of vp5, could carry an influenza A virus hemagglutinin (HA) epitope. HA-expressing IBDVs were generated when the HA epitope was fused to the N-terminus of VP5 (HA5-IBDV) or VP4 (HA4-IBDV) or the C-terminus of VP1 (1HA-IBDV). Viral titers obtained after co-transfection with cDNA from the ha-containing segment and the complementary genomic segment were 1.3 × 10(4), 3.7 × 10(3) and 3.8 × 10(4) pfu/ml for HA5-IBDV, HA4-IBDV and 1HA-IBDV, respectively. The HA tag expression remained stable after 10 passages when the tag gene was inserted into the vp4 and vp1 genes. HA-IBDVs did not cause pathogenicity in specific-pathogen-free (SPF) chickens. However, only HA4-IBDV and 1HA-IBDV induced HA-specific antibodies, which were measured by ELISA with a maximum optical density (OD) value of 0.701 and 0.769, respectively, at 24 days after infection. Thus, IBDV can potentially be employed as a bivalent viral vector when the epitope is fused with VP4 or VP1.

  17. Bats as Viral Reservoirs.

    PubMed

    Hayman, David T S

    2016-09-29

    Bats are hosts of a range of viruses, including ebolaviruses, and many important human viral infections, such as measles and mumps, may have their ancestry traced back to bats. Here, I review viruses of all viral families detected in global bat populations. The viral diversity in bats is substantial, and viruses with all known types of genomic structures and replication strategies have been discovered in bats. However, the discovery of viruses is not geographically even, with some apparently undersampled regions, such as South America. Furthermore, some bat families, including those with global or wide distributions such as Emballonuridae and Miniopteridae, are underrepresented on viral databases. Future studies, including those that address these sampling gaps along with those that develop our understanding of viral-host relationships, are highlighted.

  18. Viral Disease Networks?

    NASA Astrophysics Data System (ADS)

    Gulbahce, Natali; Yan, Han; Vidal, Marc; Barabasi, Albert-Laszlo

    2010-03-01

    Viral infections induce multiple perturbations that spread along the links of the biological networks of the host cells. Understanding the impact of these cascading perturbations requires an exhaustive knowledge of the cellular machinery as well as a systems biology approach that reveals how individual components of the cellular system function together. Here we describe an integrative method that provides a new approach to studying virus-human interactions and its correlations with diseases. Our method involves the combined utilization of protein - protein interactions, protein -- DNA interactions, metabolomics and gene - disease associations to build a ``viraldiseasome''. By solely using high-throughput data, we map well-known viral associated diseases and predict new candidate viral diseases. We use microarray data of virus-infected tissues and patient medical history data to further test the implications of the viral diseasome. We apply this method to Epstein-Barr virus and Human Papillomavirus and shed light into molecular development of viral diseases and disease pathways.

  19. Immunoinformatics prediction of linear epitopes from Taenia solium TSOL18

    PubMed Central

    Zimic, Mirko; Gutiérrez, Andrés Hazaet; Gilman, Robert Hugh; López, César; Quiliano, Miguel; Evangelista, Wilfredo; Gonzales, Armando; García, Héctor Hugo; Sheen, Patricia

    2011-01-01

    Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18. PMID:21738328

  20. Immunoinformatics prediction of linear epitopes from Taenia solium TSOL18.

    PubMed

    Zimic, Mirko; Gutiérrez, Andrés Hazaet; Gilman, Robert Hugh; López, César; Quiliano, Miguel; Evangelista, Wilfredo; Gonzales, Armando; García, Héctor Hugo; Sheen, Patricia

    2011-01-01

    Cysticercosis is a public health problem in several developing countries. The oncosphere protein TSOL18 is the most immunogenic and protective antigen ever reported against porcine cysticercosis, although no specific epitope has been identified to account for these properties. Recent evidence suggests that protection might be associated with conformational epitopes. Linear epitopes from TSOL18 were computationally predicted and evaluated for immunogenicity and protection against porcine cysticercosis. A synthetic peptide was designed based on predicted linear B cell and T cell epitopes that are exposed on the surface of the theoretically modeled structure of TSOL18. Three surface epitopes from TSOL18 were predicted as immunogenic. A peptide comprising a linear arrangement of these epitopes was chemically synthesized. The capacity of the synthetic peptide to protect pigs against an oral challenge with Taenia solium proglottids was tested in a vaccine trial. The synthetic peptide was able to produce IgG antibodies in pigs and was associated to a reduction of the number of cysts, although was not able to provide complete protection, defined as the complete absence of cysts in necropsy. This study demonstrated that B cell and T cell predicted epitopes from TSOL18 were not able to completely protect pigs against an oral challenge with Taenia solium proglottids. Therefore, other linear epitopes or eventually conformational epitopes may be responsible for the protection conferred by TSOL18.

  1. Reflections on HLA Epitope-Based Matching for Transplantation

    PubMed Central

    Duquesnoy, Rene J.

    2016-01-01

    HLA antibodies are primary causes of transplant rejection; they recognize epitopes that can be structurally defined by eplets. There are many reviews about HLA epitope-based matching in transplantation. This article describes some personal reflections about epitopes including a historical perspective of HLA typing at the antigen and allele levels, the repertoires of antibody-verified HLA epitopes, the use of HLAMatchmaker in determining the specificities of antibodies tested in different assays, and, finally, possible strategies to control HLA antibody responses. PMID:27965660

  2. Conservation of Babesia bovis small heat shock protein (Hsp20) among strains and definition of T helper cell epitopes recognized by cattle with diverse major histocompatibility complex class II haplotypes.

    PubMed

    Norimine, Junzo; Mosqueda, Juan; Palmer, Guy H; Lewin, Harris A; Brown, Wendy C

    2004-02-01

    Babesia bovis small heat shock protein (Hsp20) is recognized by CD4+ T lymphocytes from cattle that have recovered from infection and are immune to challenge. This candidate vaccine antigen is related to a protective antigen of Toxoplasma gondii, Hsp30/bag1, and both are members of the alpha-crystallin family of proteins that can serve as molecular chaperones. In the present study, immunofluorescence microscopy determined that Hsp20 is expressed intracellularly in all merozoites. Importantly, Hsp20 is also expressed by tick larval stages, including sporozoites, so that natural tick-transmitted infection could boost a vaccine-induced response. The predicted amino acid sequence of Hsp20 from merozoites is completely conserved among different B. bovis strains. To define the location of CD4+ T-cell epitopes for inclusion in a multiepitope peptide or minigene vaccine construct, truncated recombinant Hsp20 proteins and overlapping peptides were tested for their ability to stimulate T cells from immune cattle. Both amino-terminal (amino acids [aa] 1 to 105) and carboxy-terminal (aa 48 to 177) regions were immunogenic for the majority of cattle in the study, stimulating strong proliferation and IFN-gamma production. T-cell lines from all individuals with distinct DRB3 haplotypes responded to aa 11 to 62 of Hsp20, which contained one or more immunodominant epitopes for each animal. One epitope, DEQTGLPIKS (aa 17 to 26), was identified by T-cell clones. The presence of strain-conserved T helper cell epitopes in aa 11 to 62 of the ubiquitously expressed Hsp20 that are presented by major histocompatibility complex class II molecules represented broadly in the Holstein breed supports the inclusion of this region in vaccine constructs to be tested in cattle.

  3. Plasmid DNA and protein vaccination of mice to the outer surface protein A of Borrelia burgdorferi leads to induction of T helper cells with specificity for a major epitope and augmentation of protective IgG antibodies in vivo.

    PubMed

    Zhong, W; Wiesmüller, K H; Kramer, M D; Wallich, R; Simon, M M

    1996-11-01

    Plasmid DNA-based vaccination is an efficient way to evoke various forms of protective immunity in laboratory animals. Our previous experiments have shown that mice immunized with either plasmid DNA encoding the outer surface lipoprotein A (pOspA) of Borrelia burgdorferi or the respective lipoprotein (Lip-OspA) produce protective antibodies against subsequent challenge with virulent spirochetes. In the present study, we compared the specificity and function of T cells generated in AKR/N mice previously immunized to either pOspA or Lip-OspA. T cell populations derived by either of the two protocols consistently responded by proliferation in vitro to one (residues 186-203; B4) out of a panel of 27 overlapping 20-mer peptides spanning the entire OspA molecule of strain ZS7. B4 was shown to express allele-specific ligand motifs for I-Ek. Most of the other peptides produced variable and much less pronounced or marginal proliferative T cell responses. T cells reactive to B4 as well as to some minor epitopes were CD4+CD8- T cells which produced IFN-gamma but no detectable IL-4 upon antigen stimulation in vitro. Priming of AKR/N mice with B4 but not with inactive peptides of OspA led to an enhanced production of IgG antibodies, mainly of the IgG1 isotype, including those to a prominent protective epitope (LA-2) upon subsequent challenge with Lip-OspA or intact spirochetes. The data demonstrate that both plasmid DNA and protein immunization with OspA results in T cell responses with specificity for a dominant OspA epitope and suggest that priming of mice with immunodominant peptides accelerates the appearance of protective antibodies in vivo. The identification of T helper cell epitopes relevant for the induction of protective antibodies will also facilitate the design of more potent vaccines against Lyme disease.

  4. Quantitation of Anaplasma marginale major surface protein (MSP)1a and MSP2 epitope-specific CD4+ T lymphocytes using bovine DRB3*1101 and DRB3*1201 tetramers.

    PubMed

    Norimine, Junzo; Han, Sushan; Brown, Wendy C

    2006-09-01

    Antigen-specific CD4+ T cells play a critical role in protective immunity to many infectious pathogens. Although the antigen-specific CD4+ T cells can be measured by functional assays such as proliferation or cytokine enzyme-linked immunospot, such assays are limited to a specific function and cannot quantify anergic or suppressed T cells. In contrast, major histocompatiblity complex (MHC) class II tetramers can enumerate epitope-specific CD4+ T cells independent of function. In this paper, we report the construction of bovine leukocyte antigen MHC class II tetramers using a novel mammalian cell system to express soluble class II DRA/DRB3 molecules and defined immunodominant peptide epitopes of Anaplasma marginale major surface proteins (MSPs). Phycoerythrin-labeled tetramers were either loaded with exogenous peptide or constructed with the peptide epitope linked to the N terminus of the DRB3 chain. A DRB3*1101 tetramer loaded with MSP1a peptide F2-5B (ARSVLETLAGHVDALG) and DRB3*1201 tetramers loaded with MSP1a peptide F2-1-1b (GEGYATYLAQAFA) or MSP2 peptide P16-7 (NFAYFGGELGVRFAF) specifically stained antigen-specific CD4+ T cell lines and clones. Tetramers constructed with the T-cell epitope linked to the DRB3 chain were slightly better at labeling CD4+ T cells. In one cell line, the number of tetramer-positive T cells increased to approximately 94% of the CD4+ T cells after culture for 21 weeks with specific antigen. This novel technology should be useful to track the fate of antigen-specific CD4+ T-cell responses in cattle after immunization or infection with persistent pathogens, such as A. marginale, that modulate the host immune response.

  5. Defining the immunogenicity and antigenicity of HLA epitopes is crucial for optimal epitope matching in clinical renal transplantation.

    PubMed

    Kramer, C S M; Roelen, D L; Heidt, S; Claas, F H J

    2017-04-05

    Transplantation of an human leukocyte antigen (HLA) mismatched graft can lead to the development of donor-specific antibodies (DSA), which can result in antibody mediated rejection and graft loss as well as complicate repeat transplantation. These DSA are induced by foreign epitopes present on the mismatched HLA antigens of the donor. However, not all epitopes appear to be equally effective in their ability to induce DSA. Understanding the characteristics of HLA epitopes is crucial for optimal epitope matching in clinical transplantation. In this review, the latest insights on HLA epitopes are described with a special focus on the definition of immunogenicity and antigenicity of HLA epitopes. Furthermore, the use of this knowledge to prevent HLA antibody formation and to select the optimal donor for sensitised transplant candidates will be discussed.

  6. First report on the antibody verification of HLA-ABC epitopes recorded in the website-based HLA Epitope Registry.

    PubMed

    Duquesnoy, R J; Marrari, M; Mulder, A; Sousa, L C D da Mata; da Silva, A S; do Monte, S J H

    2014-06-01

    The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to human leukocyte antigen (HLA) mismatches. These epitopes are defined structurally by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. So-called eplets represent essential components of HLA epitopes and they are defined by polymorphic residues. A major goal is to identify HLA epitopes that have been verified experimentally with informative antibodies. Our analysis has also included data in many publications. As of 1 November 2013, 95 HLA-ABC antibody-verified epitopes have been recorded, 62 correspond to eplets and 33 are defined by eplets paired with other residue configurations. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

  7. Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells

    SciTech Connect

    Kleinow, Tatjana; Tanwir, Fariha; Kocher, Cornelia; Krenz, Bjoern; Wege, Christina; Jeske, Holger

    2009-09-01

    The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

  8. Immunoglobulin class and subclass distribution of antibodies reactive with the immunodominant antigen of Actinobacillus actinomycetemcomitans serotype b.

    PubMed Central

    Lu, H; Califano, J V; Schenkein, H A; Tew, J G

    1993-01-01

    The aims of this study were to determine the immunodominant antigens of Actinobacillus actinomycetemcomitans serotype b (Aab) for the different immunoglobulin (Ig) classes and subclasses and to determine the relative levels of these different Igs in serum. Seropositive early-onset periodontitis patients were sampled, and the Ig classes IgG, IgA, and IgM and subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 were studied. Reactivity with Aab antigens was assessed by using the Western blot (immunoblot) in limiting dilution analysis and radioimmunoassay with sera from 13 early-onset periodontitis subjects. A smeared antigen in the upper portion of the immunoblots, typical of high-molecular-weight LPS, was immunodominant for IgG, IgA, IgM, IgG1, IgG2, IgG3, IgA1, and IgA2. This smeared antigen was present in every patient for all of these Igs at the endpoint. A few additional antigens were also present at the endpoint in some patients, but none were present in more than half of the subjects. The distribution of antibody titers by Ig classes reactive with the Aab immunodominant antigen was IgG > IgA > IgM. The distribution of antibody titers by IgG subclass was IgG2 > IgG1 approximately IgG3. Further quantitation by radioimmunoassay revealed that the mean concentration of IgG2 (65.7 micrograms/ml) was significantly greater than that of IgG1 (8.8 micrograms/ml). The IgA subclass distribution was IgA1 >> IgA2, with IgA1 apparently being second only to IgG2. Therefore, the Aab antigen eliciting the highest antibody level in virtually all Ig classes and subclasses appeared to be lipopolysaccharide, and IgG2 was markedly elevated over all other serum Ig classes or subclasses reactive with Aab. Images PMID:8500879

  9. Salivary Antigen SP32 Is the Immunodominant Target of the Antibody Response to Phlebotomus papatasi Bites in Humans

    PubMed Central

    Marzouki, Soumaya; Abdeladhim, Maha; Abdessalem, Chaouki Ben; Oliveira, Fabiano; Ferjani, Beya; Gilmore, Dana; Louzir, Hechmi; Valenzuela, Jesus G.; Ahmed, Mélika Ben

    2012-01-01

    Background Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is highly prevalent in Tunisia and is transmitted by a hematophagous vector Phlebotomus papatasi (P. papatasi). While probing for a blood meal, the sand fly injects saliva into the host's skin, which contains a variety of compounds that are highly immunogenic. We recently showed that the presence of anti-saliva antibodies was associated with an enhanced risk for leishmaniasis and identified the immunodominant salivary protein of Phlebotomus papatasi as a protein of approximately 30 kDa. Methodology/Principal Findings We cloned and expressed in mammalian cells two salivary proteins PpSP30 and PpSP32 with predicted molecular weights close to 30 kDa from the Tunisian strain of P. papatasi. The two recombinant salivary proteins were purified by two-step HPLC (High-Performance Liquid Chromatography) and tested if these proteins correspond to the immunodominant antigen of 30 kDa previously shown to be recognized by human sera from endemic areas for ZCL and exposed naturally to P. papatasi bites. While recombinant PpSP30 (rPpSP30) was poorly recognized by human sera from endemic areas for ZCL, rPpSP32 was strongly recognized by the tested sera. The binding of human IgG antibodies to native PpSP32 was inhibited by the addition of rPpSP32. Consistently, experiments in mice showed that PpSP32 induced the highest levels of antibodies compared to other P. papatasi salivary molecules while PpSP30 did not induce any detectable levels of antibodies. Conclusions Our findings demonstrate that PpSP32 is the immunodominant target of the antibody response to P. papatasi saliva. They also indicate that the recombinant form of PpSP32 is similar to the native one and represents a good candidate for large scale testing of human exposure to P. papatasi bites and perhaps for assessing the risk of contracting the disease. PMID:23209854

  10. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

    PubMed Central

    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-01-01

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes. PMID:26230706

  11. Epitope Addition and Ablation via Manipulation of a Dengue Virus Serotype 1 Infectious Clone

    PubMed Central

    Gallichotte, Emily N.; Menachery, Vineet D.; Yount, Boyd L.; Widman, Douglas G.; Dinnon, Kenneth H.; Hartman, Steven; de Silva, Aravinda M.

    2017-01-01

    ABSTRACT Despite the clinical relevance, dengue virus (DENV) research has been hampered by the absence of robust reverse genetic systems to manipulate the viral serotypes for propagation and generation of mutant viruses. In this article, we describe application of an infectious clone system for DENV serotype 1 (DENV1). Similar to previous clones in both flaviviruses and coronaviruses, the approach constructs a panel of contiguous cDNAs that span the DENV genome and can be systematically and directionally assembled to produce viable, full-length viruses. Comparison of the virus derived from the infectious clone with the original viral isolate reveals identical sequence, comparable endpoint titers, and similar focus staining. Both focus-forming assays and percent infection by flow cytometry revealed overlapping replication levels in two different cell types. Moreover, serotype-specific monoclonal antibodies (MAbs) bound similarly to infectious clone and the natural isolate. Using the clone, we were able to insert a DENV4 type-specific epitope recognized by primate MAb 5H2 into envelope (E) protein domain I (EDI) of DENV1 and recover a viable chimeric recombinant virus. The recombinant DENV1 virus was recognized and neutralized by the DENV4 type-specific 5H2 MAb. The introduction of the 5H2 epitope ablated two epitopes on DENV1 EDI recognized by human MAbs (1F4 and 14C10) that strongly neutralize DENV1. Together, the work demonstrates the utility of the infectious clone and provides a resource to rapidly manipulate the DENV1 serotype for generation of recombinant and mutant viruses. IMPORTANCE Dengue viruses (DENVs) are significant mosquito-transmitted pathogens that cause widespread infection and can lead to severe infection and complications. Here we further characterize a novel and robust DENV serotype 1 (DENV1) infectious clone system that can be used to support basic and applied research. We demonstrate how the system can be used to probe the antigenic

  12. Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

    PubMed Central

    Ye, Xiaohua; Ku, Zhiqiang; Liu, Qingwei; Wang, Xiaoli; Shi, Jinping; Zhang, Yunfang; Kong, Liangliang; Cong, Yao

    2014-01-01

    Enterovirus 71 (EV71) is a major causative agent of hand, food, and mouth disease, which frequently occurs in young children. Since there are 11 subgenotypes (A, B1 to B5, and C1 to C5) within EV71, an EV71 vaccine capable of protecting against all of these subgenotypes is desirable. We report here the vaccine potential and protective mechanism of two chimeric virus-like particles (VLPs) presenting conserved neutralizing epitopes of EV71. We show that fusions of hepatitis B core antigen (HBc) with the SP55 or SP70 epitope of EV71, designated HBcSP55 and HBcSP70, respectively, can be rapidly generated and self-assembled into VLPs with the epitopes displayed on the surface. Immunization with the chimeric VLPs induced carrier- and epitope-specific antibody responses in mice. Anti-HBcSP55 and anti-HBcSP70 sera, but not anti-HBc sera, were able to neutralize in vitro multiple genotypes and strains of EV71. Importantly, passive immunization with anti-HBcSP55 or anti-HBcSP70 sera protected neonatal mice against lethal EV71 infections. Interestingly, anti-HBcSP70 sera could inhibit EV71 attachment to susceptible cells, whereas anti-HBcSP55 sera could not. However, both antisera were able to neutralize EV71 infection in vitro at the postattachment stage. The divergent mechanism of neutralization and protection conferred by anti-SP70 and anti-SP55 sera is in part attributed to their respective ability to bind authentic viral particles. Collectively, our study not only demonstrates that chimeric VLPs displaying the SP55 and SP70 epitopes are promising candidates for a broad-spectrum EV71 vaccine but also reveals distinct mechanisms of neutralization by the SP55- and SP70-targeted antibodies. PMID:24131712

  13. Screening and Identification of an H-2Kb-Restricted CTL Epitope within the Glycoprotein of Hantaan Virus

    PubMed Central

    Ma, Rui-xue; Cheng, Lin-feng; Ying, Qi-kang; Liu, Rong-rong; Ma, Tie-jun; Zhang, Xiao-xiao; Liu, Zi-yu; Zhang, Liang; Ye, Wei; Zhang, Fang-lin; Xu, Zhi-kai; Wang, Fang; Wu, Xing-an

    2016-01-01

    The cytotoxic T lymphocyte (CTL) response plays a key role in controlling viral infection, but only a few epitopes within the HTNV glycoprotein (GP) that are recognized by CTLs have been reported. In this study, we identified one murine HTNV GP-derived H2-Kb-restricted CTL epitope in C57BL/6 mice, which could be used to design preclinical studies of vaccines for HTNV infection. First, 15 8-mer peptides were selected from the HTNV GP amino acid sequence based on a percentile rank of <=1% by IEDB which is the most comprehensive collection of epitope prediction and analysis tool. A lower percentile rank indicates higher affinity and higher immune response. In the case of the consensus method, we also evaluated the binding score of peptide-binding affinity by the BIMAS software to confirm that all peptides were able to bind H2-Kb. Second, one novel GP-derived CTL epitope, GP6 aa456-aa463 (ITSLFSLL), was identified in the splenocytes of HTNV-infected mice using the IFN-γ ELISPOT assay. Third, a single peptide vaccine was administered to C57BL/6 mice to evaluate the immunogenic potential of the identified peptides. ELISPOT and cell-mediated cytotoxicity assays showed that this peptide vaccine induced a strong IFN-γ response and potent cytotoxicity in immunized mice. Last, we demonstrated that the peptide-vaccinated mice had partial protection from challenge with HTNV. In conclusion, we identified an H2-Kb-restricted CTL epitope with involvement in the host immune response to HTNV infection. PMID:27933274

  14. Altered Peptide Ligands Revisited: Vaccine Design through Chemically Modified HLA-A2–Restricted T Cell Epitopes

    PubMed Central

    Hoppes, Rieuwert; Oostvogels, Rimke; Luimstra, Jolien J.; Wals, Kim; Toebes, Mireille; Bies, Laura; Ekkebus, Reggy; Rijal, Pramila; Celie, Patrick H. N.; Huang, Julie H.; Emmelot, Maarten E.; Spaapen, Robbert M.; Lokhorst, Henk; Schumacher, Ton N. M.; Mutis, Tuna; Ovaa, Huib

    2014-01-01

    Virus or tumor Ag–derived peptides that are displayed by MHC class I molecules are attractive starting points for vaccine development because they induce strong protective and therapeutic cytotoxic T cell responses. In thus study, we show that the MHC binding and consequent T cell reactivity against several HLA-A*02 restricted epitopes can be further improved through the incorporation of nonproteogenic amino acids at primary and secondary anchor positions. We screened more than 90 nonproteogenic, synthetic amino acids through a range of epitopes and tested more than 3000 chemically enhanced altered peptide ligands (CPLs) for binding affinity to HLA-A*0201. With this approach, we designed CPLs of viral epitopes, of melanoma-associated Ags, and of the minor histocompatibility Ag UTA2-1, which is currently being evaluated for its antileukemic activity in clinical dendritic cell vaccination trials. The crystal structure of one of the CPLs in complex with HLA-A*0201 revealed the molecular interactions likely responsible for improved binding. The best CPLs displayed enhanced affinity for MHC, increasing MHC stability and prolonging recognition by Ag-specific T cells and, most importantly, they induced accelerated expansion of antitumor T cell frequencies in vitro and in vivo as compared with the native epitope. Eventually, we were able to construct a toolbox of preferred nonproteogenic residues with which practically any given HLA-A*02 restricted epitope can be readily optimized. These CPLs could improve the therapeutic outcome of vaccination strategies or can be used for ex vivo enrichment and faster expansion of Ag-specific T cells for transfer into patients. PMID:25311806

  15. Resistance-associated epitopes of HIV-1C-highly probable candidates for a multi-epitope vaccine.

    PubMed

    Sundaramurthi, Jagadish Chandrabose; Swaminathan, Soumya; Hanna, Luke Elizabeth

    2012-10-01

    Earlier studies have identified a large number of immunogenic epitopes in HIV-1. Efforts are required to prioritize these epitopes in order to identify the best candidates for formulating an effective multi-epitope vaccine for HIV. We modeled 155 known cytotoxic T lymphocyte epitopes of HIV-1 subtype C on the 3D structure of HLA-A*0201, HLA-B*2705, and HLA-B*5101 using MODPROPEP, as these alleles are known to be associated with resistance to HIV/slow progression to AIDS. Thirty-six epitopes were identified to bind to all the three HLA alleles with better binding affinity than the control peptides complexed with each HLA allele but not to any of the HLA alleles reported to be associated with susceptibility to HIV infection/rapid progression to disease. As increase in stability of the epitope-HLA complex results in increased immunogenicity, the short-listed epitopes could be suitable candidates for vaccine development. Twenty of the 36 epitopes were polyfunctional in nature adding to their immunological relevance for vaccine design. Further, 9 of the 20 polyfunctional epitopes were found to bind to all three resistance-associated HLA alleles using an additional method, adding worth to their potential as candidates for a vaccine formulation for HIV-1C.

  16. Human antibodies reveal a protective epitope that is highly conserved among human and nonhuman influenza A viruses.

    PubMed

    Grandea, Andres G; Olsen, Ole A; Cox, Thomas C; Renshaw, Mark; Hammond, Philip W; Chan-Hui, Po-Ying; Mitcham, Jennifer L; Cieplak, Witold; Stewart, Shaun M; Grantham, Michael L; Pekosz, Andrew; Kiso, Maki; Shinya, Kyoko; Hatta, Masato; Kawaoka, Yoshihiro; Moyle, Matthew

    2010-07-13

    Influenza remains a serious public health threat throughout the world. Vaccines and antivirals are available that can provide protection from infection. However, new viral strains emerge continuously because of the plasticity of the influenza genome, which necessitates annual reformulation of vaccine antigens, and resistance to antivirals can appear rapidly and become entrenched in circulating virus populations. In addition, the spread of new pandemic strains is difficult to contain because of the time required to engineer and manufacture effective vaccines. Monoclonal antibodies that target highly conserved viral epitopes might offer an alternative protection paradigm. Herein we describe the isolation of a panel of monoclonal antibodies derived from the IgG(+) memory B cells of healthy, human subjects that recognize a previously unknown conformational epitope within the ectodomain of the influenza matrix 2 protein, M2e. This antibody binding region is highly conserved in influenza A viruses, being present in nearly all strains detected to date, including highly pathogenic viruses that infect primarily birds and swine, and the current 2009 swine-origin H1N1 pandemic strain (S-OIV). Furthermore, these human anti-M2e monoclonal antibodies protect mice from lethal challenges with either H5N1 or H1N1 influenza viruses. These results suggest that viral M2e can elicit broadly cross-reactive and protective antibodies in humans. Accordingly, recombinant forms of these human antibodies may provide useful therapeutic agents to protect against infection from a broad spectrum of influenza A strains.

  17. Characterization and specificity of the linear epitope of the enterovirus 71 VP2 protein

    PubMed Central

    2012-01-01

    Background Enterovirus 71 (EV71) has emerged as a major causative agent of hand, foot and mouth disease in the Asia-Pacific region over the last decade. Hand, foot and mouth disease can be caused by different etiological agents from the enterovirus family, mainly EV71 and coxsackieviruses, which are genetically closely related. Nevertheless, infection with EV71 may occasionally lead to high fever, neurologic complications and the emergence of a rapidly fatal syndrome of pulmonary edema associated with brainstem encephalitis. The rapid progression and high mortality of severe EV71 infection has highlighted the need for EV71-specific diagnostic and therapeutic tools. Monoclonal antibodies are urgently needed to specifically detect EV71 antigens from patient specimens early in the infection process. Furthermore, the elucidation of viral epitopes will contribute to the development of targeted therapeutics and vaccines. Results We have identified the monoclonal antibody 7C7 from a screen of hybridoma cells derived from mice immunized with the EV71-B5 strain. The linear epitope of 7C7 was mapped to amino acids 142-146 (EDSHP) of the VP2 capsid protein and was characterized in detail. Mutational analysis of the epitope showed that the aspartic acid to asparagine mutation of the EV71 subgenogroup A (BrCr strain) did not interfere with antibody recognition. In contrast, the serine to threonine mutation at position 144 of VP2, present in recently emerged EV71-C4 China strains, abolished antigenicity. Mice injected with this virus strain did not produce any antibodies against the VP2 protein. Immunofluorescence and Western blotting confirmed that 7C7 specifically recognized EV71 subgenogroups and did not cross-react to Coxsackieviruses 4, 6, 10, and 16. 7C7 was successfully used as a detection antibody in an antigen-capture ELISA assay. Conclusions Detailed mapping showed that the VP2 protein of Enterovirus 71 contains a single, linear, non-neutralizing epitope, spanning

  18. Viruses and viral proteins.

    PubMed

    Verdaguer, Nuria; Ferrero, Diego; Murthy, Mathur R N

    2014-11-01

    For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

  19. Viruses and viral proteins

    PubMed Central

    Verdaguer, Nuria; Ferrero, Diego; Murthy, Mathur R. N.

    2014-01-01

    For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes. PMID:25485129

  20. Investigating the immunodominance of carbohydrate antigens in a bivalent unimolecular glycoconjugate vaccine against serogroup A and C meningococcal disease.

    PubMed

    Adamo, Roberto; Nilo, Alberto; Harfouche, Carole; Brogioni, Barbara; Pecetta, Simone; Brogioni, Giulia; Balducci, Evita; Pinto, Vittoria; Filippini, Sara; Mori, Elena; Tontini, Marta; Romano, Maria Rosaria; Costantino, Paolo; Berti, Francesco

    2014-12-01

    Multicomponent constructs, obtained by coupling different glycans to the carrier protein, have been proposed as a way to co-deliver multiple surface carbohydrates targeting different strains of one pathogen and reduce the number of biomolecules in the formulation of multivalent vaccines. To assess the feasibility of this approach for anti-microbial vaccines and investigate the potential immunodominance of one carbohydrate antigen over the others in these constructs, we designed a bivalent unimolecular vaccine against serogroup A (MenA) and C (MenC) meningococci, with the two different oligomers conjugated to same molecule of carrier protein (CRM197). The immune response elicited in mice by the bivalent MenAC construct was compared with the ones induced by the monovalent MenA and MenC vaccines and their combinations. After the second dose, the bivalent construct induced good levels of anti-MenA and anti-MenC antibodies with respect to the controls. However, the murine sera from the MenAC construct exhibited good anti-MenC bactericidal activity, and very low anti-MenA functionality when compared to the monovalent controls. This result was explained with the diverse relative avidities against MenA and MenC polysaccharides, which were measured in the generated sera. The immunodominant effect of the MenC antigen was fully overcome following the third immunization, when sera endowed with higher avidity and excellent bactericidal activity against both MenA and MenC expressing strains were elicited. Construction of multicomponent glycoconjugate vaccines against microbial pathogens is a feasible approach, but particular attention should be devoted to study and overcome possible occurrence of immune interference among the carbohydrates.

  1. Viral hemorrhagic septicemia

    USGS Publications Warehouse

    Batts, William N.; Winton, James R.

    2012-01-01

    Viral hemorrhagic septicemia (VHS) is one of the most important viral diseases of finfish worldwide. In the past, VHS was thought to affect mainly rainbow trout Oncorhynchus mykiss reared at freshwater facilities in Western Europe where it was known by various names including Egtved disease and infectious kidney swelling and liver degeneration (Wolf 1988). Today, VHS is known as an important source of mortality for cultured and wild fish in freshwater and marine environments in several regions of the northern hemisphere (Dixon 1999; Gagné et al. 2007; Kim and Faisal 2011; Lumsden et al. 2007; Marty et al. 1998, 2003; Meyers and Winton 1995; Skall et al. 2005b; Smail 1999; Takano et al. 2001). Viral hemorrhagic septicemia is caused by the fish rhabdovirus, viral hemorrhagic septicemia virus (VHSV), a member of the genus Novirhabdovirus of the family Rhabdoviridae

  2. Viral quasispecies complexity measures.

    PubMed

    Gregori, Josep; Perales, Celia; Rodriguez-Frias, Francisco; Esteban, Juan I; Quer, Josep; Domingo, Esteban

    2016-06-01

    Mutant spectrum dynamics (changes in the related mutants that compose viral populations) has a decisive impact on virus behavior. The several platforms of next generation sequencing (NGS) to study viral quasispecies offer a magnifying glass to study viral quasispecies complexity. Several parameters are available to quantify the complexity of mutant spectra, but they have limitations. Here we critically evaluate the information provided by several population diversity indices, and we propose the introduction of some new ones used in ecology. In particular we make a distinction between incidence, abundance and function measures of viral quasispecies composition. We suggest a multidimensional approach (complementary information contributed by adequately chosen indices), propose some guidelines, and illustrate the use of indices with a simple example. We apply the indices to three clinical samples of hepatitis C virus that display different population heterogeneity. Areas of virus biology in which population complexity plays a role are discussed.

  3. Protective Efficacy of Individual CD8+ T Cell Specificities in Chronic Viral Infection §§

    PubMed Central

    Johnson, Susan; Bergthaler, Andreas; Graw, Frederik; Flatz, Lukas; Bonilla, Weldy V.; Siegrist, Claire-Anne; Lambert, Paul-Henri; Regoes, Roland R.; Pinschewer, Daniel D.

    2014-01-01

    Specific CD8+ T cells (CTLs) play an important role in resolving protracted infection with hepatitis B and C virus in humans and lymphocytic choriomeningitis virus (LCMV) in mice. The contribution of individual CTL specificities to chronic virus control, as well as epitope-specific patterns in timing and persistence of antiviral selection pressure remain, however, incompletely defined. To monitor and characterize the antiviral efficacy of individual CTL specificities throughout the course of chronic infection, we co-inoculated mice with a mixture of wildtype LCMV and genetically engineered CTL epitope-deficient mutant virus. A quantitative longitudinal assessment of viral competition revealed that mice continuously exerted CTL selection pressure on the persisting virus population. The timing of selection pressure characterized individual epitope specificities, and its magnitude varied considerably between individual mice. This longitudinal assessment of “antiviral efficacy” provides a novel parameter to characterize CTL responses in chronic viral infection. It demonstrates remarkable perseverance of all antiviral CTL specificities studied, thus raising hope for therapeutic vaccination in the treatment of persistent viral diseases. PMID:25567678

  4. Immigration and viral hepatitis.

    PubMed

    Sharma, Suraj; Carballo, Manuel; Feld, Jordan J; Janssen, Harry L A

    2015-08-01

    WHO estimates reveal that the global prevalence of viral hepatitis may be as high as 500 million, with an annual mortality rate of up to 1.3 million individuals. The majority of this global burden of disease is borne by nations of the developing world with high rates of vertical and iatrogenic transmission of HBV and HCV, as well as poor access to healthcare. In 2013, 3.2% of the global population (231 million individuals) migrated into a new host nation. Migrants predominantly originate from the developing countries of the south, into the developed economies of North America and Western Europe. This mass migration of individuals from areas of high-prevalence of viral hepatitis poses a unique challenge to the healthcare systems of the host nations. Due to a lack of universal standards for screening, vaccination and treatment of viral hepatitis, the burden of chronic liver disease and hepatocellular carcinoma continues to increase among migrant populations globally. Efforts to increase case identification and treatment among migrants have largely been limited to small outreach programs in urban centers, such that the majority of migrants with viral hepatitis continue to remain unaware of their infection. This review summarizes the data on prevalence of viral hepatitis and burden of chronic liver disease among migrants, current standards for screening and treatment of immigrants and refugees, and efforts to improve the identification and treatment of viral hepatitis among migrants.

  5. NCBI viral genomes resource.

    PubMed

    Brister, J Rodney; Ako-Adjei, Danso; Bao, Yiming; Blinkova, Olga

    2015-01-01

    Recent technological innovations have ignited an explosion in virus genome sequencing that promises to fundamentally alter our understanding of viral biology and profoundly impact public health policy. Yet, any potential benefits from the billowing cloud of next generation sequence data hinge upon well implemented reference resources that facilitate the identification of sequences, aid in the assembly of sequence reads and provide reference annotation sources. The NCBI Viral Genomes Resource is a reference resource designed to bring order to this sequence shockwave and improve usability of viral sequence data. The resource can be accessed at http://www.ncbi.nlm.nih.gov/genome/viruses/ and catalogs all publicly available virus genome sequences and curates reference genome sequences. As the number of genome sequences has grown, so too have the difficulties in annotating and maintaining reference sequences. The rapid expansion of the viral sequence universe has forced a recalibration of the data model to better provide extant sequence representation and enhanced reference sequence products to serve the needs of the various viral communities. This, in turn, has placed increased emphasis on leveraging the knowledge of individual scientific communities to identify important viral sequences and develop well annotated reference virus genome sets.

  6. Autoantibody recognition mechanisms of p53 epitopes

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2016-06-01

    There is an urgent need for economical blood based, noninvasive molecular biomarkers to assist in the detection and diagnosis of cancers in a cost-effective manner at an early stage, when curative interventions are still possible. Serum autoantibodies are attractive biomarkers for early cancer detection, but their development has been hindered by the punctuated genetic nature of the ten million known cancer mutations. A landmark study of 50,000 patients (Pedersen et al., 2013) showed that a few p53 15-mer epitopes are much more sensitive colon cancer biomarkers than p53, which in turn is a more sensitive cancer biomarker than any other protein. The function of p53 as a nearly universal "tumor suppressor" is well established, because of its strong immunogenicity in terms of not only antibody recruitment, but also stimulation of autoantibodies. Here we examine dimensionally compressed bioinformatic fractal scaling analysis for identifying the few sensitive epitopes from the p53 amino acid sequence, and show how it could be used for early cancer detection (ECD). We trim 15-mers to 7-mers, and identify specific 7-mers from other species that could be more sensitive to aggressive human cancers, such as liver cancer. Our results could provide a roadmap for ECD.

  7. Broad epitope coverage of a human in vitro antibody library

    PubMed Central

    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  8. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  9. Antibody responses to Four Corners hantavirus infections in the deer mouse (Peromyscus maniculatus): identification of an immunodominant region of the viral nucleocapsid protein.

    PubMed Central

    Yamada, T; Hjelle, B; Lanzi, R; Morris, C; Anderson, B; Jenison, S

    1995-01-01

    Antibody responses to Four Corners hantavirus (FCV) infections in the deer mouse (Peromyscus maniculatus) were characterized by using FCV nucleocapsid protein (N), glycoprotein 1 (G1), and glycoprotein 2 (G2) recombinant polypeptides in Western immunoblot assays. Strong immunoglobulin G reactivities to FCV N were observed among FCV-infected wild P. maniculatus mice (n = 34) and in laboratory-infected P. maniculatus mice (n = 11). No immunoglobulin G antibody reactivities to FCV G1 or G2 linear determinants were detected. The strongest N responses were mapped to an amino-proximal segment between amino acids 17 and 59 (QLVTARQKLKDAERAVELDPDDVNKSTLQSRRAAVSALETKLG). FCV N antibodies cross-reacted with recombinant N proteins encoded by Puumala, Seoul, and Hantaan viruses. PMID:7853538

  10. Identification of two neutralization epitopes on the capsid protein of avian hepatitis E virus.

    PubMed

    Zhou, E-M; Guo, H; Huang, F F; Sun, Z F; Meng, X J

    2008-02-01

    Avian hepatitis E virus (avian HEV) is genetically and antigenically related to human HEV, the causative agent of hepatitis E. To identify the neutralizing epitopes on the capsid (ORF2) protein of avian HEV, four mAbs (7B2, 1E11, 10A2 and 5G10) against recombinant avian HEV ORF2 protein were generated. mAbs 7B2, 1E11 and 10A2 blocked each other for binding to avian HEV ORF2 protein in a competitive ELISA, whereas 5G10 did not block the other mAbs, suggesting that 7B2, 1E11 and 10A2 recognize the same or overlapping epitopes and 5G10 recognizes a different one. The epitopes recognized by 7B2, 1E11 and 10A2, and by 5G10 were mapped by Western blotting between aa 513 and 570, and between aa 476 and 513, respectively. mAbs 1E11, 10A2 and 5G10 were shown to bind to avian HEV particles in vitro, although only 5G10 reacted to viral antigens in transfected LMH cells. To assess the neutralizing activities of the mAbs, avian HEV was incubated in vitro with each mAb before inoculation into specific-pathogen-free chickens. Both viraemia and faecal virus shedding were delayed in chickens inoculated with the mixtures of avian HEV and 1E11, 10A2 or 5G10, suggesting that these three mAbs partially neutralize avian HEV.

  11. Immune-driven adaptation of hepatitis B virus genotype D involves preferential alteration in B-cell epitopes and replicative attenuation--an insight from human immunodeficiency virus/hepatitis B virus coinfection.

    PubMed

    Mondal, R K; Khatun, M; Ghosh, S; Banerjee, P; Datta, S; Sarkar, S; Saha, B; Santra, A; Banerjee, S; Chowdhury, A; Datta, S

    2015-07-01

    An important driving force behind the sequence diversity of hepatitis B virus (HBV) is viral adaptation to host immune responses. To gain an insight into the impact of host immunity on genetic diversification and properties of HBV, we characterized HBV of genotype D from treatment-naive hepatitis B e antigen-positive (EP) and hepatitis B e antigen-negative (EN) patients with chronic hepatitis B (CHB), where HBV is under stronger immune pressure, with that of HBV derived from human immunodeficiency virus (HIV)/HBV-coinfected individuals, where HIV infection has significantly weakened the immune system. Full-length sequence analysis showed that HBV heterogeneity was most extensive in EN-CHB followed by EP-CHB and HIV/HBV coinfection. The relative magnitude of non-synonymous changes within B-cell epitopes was greater than that in T-cell epitopes of HBV open reading frames (ORFs) in both EP-CHB and EN-CHB. Nine amino acid substitutions were identified in B-cell epitopes and one in a T-cell epitope of HBV in EN-CHB, most of which resulted in altered hydrophobicities, as determined using the Kyte and Doolittle method, relative to wild-type residues found in HBV from the HIV-positive group. Additionally, 19 substitutions occurred at significantly higher frequencies in non-epitope regions of HBV ORF-P in EN-CHB than HIV/HBV-coinfected patients. In vitro replication assay demonstrated that the substitutions, particularly in reverse transcriptase and RNaseH domains of ORF-P, resulted in a decline in replication capacity of HBV. Hence, our results indicate that HBV adapts to increasing immune pressure through preferential mutations in B-cell epitopes and by replicative attenuation. The viral epitopes linked to immune response identified in this study bear important implications for future HBV vaccine studies.

  12. Efficient control of chronic LCMV infection by a CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model.

    PubMed

    He, Ran; Yang, Xinxin; Liu, Cheng; Chen, Xiangyu; Wang, Lin; Xiao, Minglu; Ye, Jianqiang; Wu, Yuzhang; Ye, Lilin

    2017-03-13

    CD4(+) T cells are essential for sustaining CD8(+) T cell responses during a chronic infection. The adoptive transfer of virus-specific CD4(+) T cells has been shown to efficiently rescue exhausted CD8(+) T cells. However, the question of whether endogenous virus-specific CD4(+) T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8(+) T cells remains unexplored. In this study, we developed a CD4(+) T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We primed chronically LCMV-infected mice with a Listeria monocytogenes vector that expressed the LCMV glycoprotein-specific I-A(b)-restricted CD4(+) T cell epitope GP61-80 (LM-GP61) and subsequently boosted the primed mice with an influenza virus A (PR8 strain) vector that expressed the same CD4(+) T cell epitope (IAV-GP61). This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4(+) T cell responses, which further improved both the quantity and quality of the virus-specific CD8(+) T cells and led to better control of the viral loads. The combination of this strategy and the blockade of the programmed cell death-1 (PD-1) inhibitory pathway further enhanced the anti-viral CD8(+) T cell responses and viral clearance. Thus, a heterologous prime-boost immunization that selectively induces virus-specific CD4(+) T cell responses in conjunction with blockade of the inhibitory pathway may represent a promising therapeutic approach to treating patients with chronic viral infections.Cellular & Molecular Immunology advance online publication, 13 March 2017; doi:10.1038/cmi.2017.3.

  13. In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential.

    PubMed

    Muñoz-Medina, José Esteban; Sánchez-Vallejo, Carlos Javier; Méndez-Tenorio, Alfonso; Monroy-Muñoz, Irma Eloísa; Angeles-Martínez, Javier; Santos Coy-Arechavaleta, Andrea; Santacruz-Tinoco, Clara Esperanza; González-Ibarra, Joaquín; Anguiano-Hernández, Yu-Mei; González-Bonilla, César Raúl; Ramón-Gallegos, Eva; Díaz-Quiñonez, José Alberto

    2015-01-01

    The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an in silico analysis of the reported epitopes of the 4 IAV proteins that are antigenically most significant (HA, NA, NP, and M2) in the 3 strains with the greatest world circulation in the last century (H1N1, H2N2, and H3N2) and in one of the main aviary subtypes responsible for zoonosis (H5N1). For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques.

  14. Ex vivo detection of adenovirus specific CD4{sup +} T-cell responses to HLA-DR-epitopes of the Hexon protein show a contracted specificity of T{sub HELPER} cells following stem cell transplantation

    SciTech Connect

    Serangeli, Celine; Bicanic, Oliver; Scheible, Michael H.; Lang, Peter; Handgretinger, Rupert

    2010-02-20

    Human adenovirus (HAdV) is a cause of significant morbidity and mortality in immunocompromised patients, especially after stem cell transplantation (SCT). Viral clearance has been attributed to CD4{sup +} T-cell responses against the Hexon-protein, but the frequency of specific T{sub HELPER} cells is extremely low or not detectable ex vivo and preference for different CD4{sup +} T-cell epitopes is variable among individuals. We therefore analyzed 44 healthy donors and 6 SCT-recipients for Hexon-specific CD4{sup +}-responses ex vivo, to identify epitopes which would be broadly applicable. We selected 19 candidate epitopes with predicted restriction to HLA-DR1/DR3/DR4/DR7; 16 were located within the highly conserved regions, indicating cross-reactivity of T cells among HAdV-subspecies. Ten epitopes induced CD4{sup +}-proliferation in >50% of individuals, confirmed by intracellular IFN-gamma detection. Three SCT recipients who recovered from an infection with HAdV displayed reactivity towards only a single hexon epitope, whereas healthy individuals were responsive to two to eight epitopes (median 3). The ex vivo detection of Hexon-specific CD4{sup +} T-cells, without any long-term culture in vitro, enables the detection and generation of HAdV-specific CD4{sup +} T cells for adoptive T-cell transfer against HAdV-infection post SCT.

  15. In Silico Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential

    PubMed Central

    Muñoz-Medina, José Esteban; Sánchez-Vallejo, Carlos Javier; Méndez-Tenorio, Alfonso; Monroy-Muñoz, Irma Eloísa; Angeles-Martínez, Javier; Santos Coy-Arechavaleta, Andrea; Santacruz-Tinoco, Clara Esperanza; González-Ibarra, Joaquín; Anguiano-Hernández, Yu-Mei; González-Bonilla, César Raúl; Ramón-Gallegos, Eva; Díaz-Quiñonez, José Alberto

    2015-01-01

    The unpredictable, evolutionary nature of the influenza A virus (IAV) is the primary problem when generating a vaccine and when designing diagnostic strategies; thus, it is necessary to determine the constant regions in viral proteins. In this study, we completed an in silico analysis of the reported epitopes of the 4 IAV proteins that are antigenically most significant (HA, NA, NP, and M2) in the 3 strains with the greatest world circulation in the last century (H1N1, H2N2, and H3N2) and in one of the main aviary subtypes responsible for zoonosis (H5N1). For this purpose, the HMMER program was used to align 3,016 epitopes reported in the Immune Epitope Database and Analysis Resource (IEDB) and distributed in 34,294 stored sequences in the Pfam database. Eighteen epitopes were identified: 8 in HA, 5 in NA, 3 in NP, and 2 in M2. These epitopes have remained constant since they were first identified (~91 years) and are present in strains that have circulated on 5 continents. These sites could be targets for vaccination design strategies based on epitopes and/or as markers in the implementation of diagnostic techniques. PMID:26346523

  16. Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection.

    PubMed

    Rodrigo, Chaturaka; Walker, Melanie R; Leung, Preston; Eltahla, Auda A; Grebely, Jason; Dore, Gregory J; Applegate, Tanya; Page, Kimberly; Dwivedi, Sunita; Bruneau, Julie; Morris, Meghan D; Cox, Andrea L; Osburn, William; Kim, Arthur Y; Schinkel, Janke; Shoukry, Naglaa H; Lauer, Georg M; Maher, Lisa; Hellard, Margaret; Prins, Maria; Luciani, Fabio; Lloyd, Andrew R; Bull, Rowena A

    2017-04-01

    Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute (<180days) and chronic (>180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p<0.05), and at position 422 (p<0.001) with a significant difference in variability (entropy). The majority of experimentally-described escape variants do not occur frequently in nature. The observed differences between acute and chronically isolated sequences suggest constraints on residue usage early in infection.

  17. Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection

    PubMed Central

    Chen, Chien-Wen; Wu, Ming-Shan; Huang, Yi-Jen; Cheng, Chao-An; Chang, Chi-Yao

    2015-01-01

    Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development. PMID:25938761

  18. Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

    PubMed Central

    Han, Thomas; Sui, Jianhua; Bennett, Andrew S.; Liddington, Robert C.; Donis, Ruben O.; Zhu, Quan; Marasco, Wayne A.

    2011-01-01

    Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines. PMID:21569761

  19. Concepts in viral pathogenesis II

    SciTech Connect

    Notkins, A.L.; Oldstone, M.B.A.

    1986-01-01

    This paper contains papers divided among 10 sections. The section titles are: Viral Structure and Function; Viral Constructs; Oncogenes, Transfection, and Differentiation; Viral Tropism and Entry into Cells; Immune Recognition of Viruses; Evolving Concepts in Viral Pathogenesis Illustrated by Selected Plant and Animal Models; Evolving Concepts in Viral Pathogenesis Illustrated by Selected Diseases in Humans; New Trends in Diagnosis and Epidemiology; and Vaccines and Antiviral Therapy.

  20. Bypass of carrier-induced epitope-specific suppression using a T-helper epitope.

    PubMed Central

    Sad, S; Rao, K; Arora, R; Talwar, G P; Raghupathy, R

    1992-01-01

    A gonadotropin-releasing hormone (GnRH)-based vaccine is being developed as a method for non-surgical immunotherapy as immunization with this vaccine results in atrophy of the prostate. This vaccine, a conjugate of GnRH and diphtheria toxoid (DT), provides a unique hapten-carrier system for investigating the influence of carrier presensitization on antibody responses to self haptens. In a recent communication we showed that preimmunization with carriers diphtheria toxoid and tetanus toxoid results in a strain-dependent inhibition of anti-GnRH responses in mice and that T cells from carrier-presensitized mice are responsible for anti-haptenic suppression. In the present report we describe a strategy for bypassing DT-induced epitopic suppression using a T-helper epitope from DT. PMID:1383134

  1. Analysis of T cell responses to the major allergens from German cockroach: epitope specificity and relationship to IgE production.

    PubMed

    Oseroff, Carla; Sidney, John; Tripple, Victoria; Grey, Howard; Wood, Robert; Broide, David H; Greenbaum, Jason; Kolla, Ravi; Peters, Bjoern; Pomés, Anna; Sette, Alessandro

    2012-07-15

    Bla g allergens are major targets of IgE responses associated with cockroach allergies. However, little is known about corresponding T cell responses, despite their potential involvement in immunopathology and the clinical efficacy of specific immunotherapy. Bioinformatic predictions of the capacity of Bla g 1, 2, 4, 5, 6, and 7 peptides to bind HLA-DR, -DP, and -DQ molecules, and PBMC responses from 30 allergic donors, identified 25 T cell epitopes. Five immunodominant epitopes accounted for more than half of the response. Bla g 5, the most dominant allergen, accounted for 65% of the response, and Bla g 6 accounted for 20%. Bla g 5 induced both IL-5 and IFN-γ responses, whereas Bla g 6 induced mostly IL-5, and, conversely, Bla g 2 induced only IFN-γ. Thus, responses to allergens within a source are independently regulated, suggesting a critical role for the allergen itself, and not extraneous stimulation from other allergens or copresented immunomodulators. In comparing Ab with T cell responses for several donor/allergen combinations, we detected IgE titers in the absence of detectable T cell responses, suggesting that unlinked T cell-B cell help might support development of IgE responses. Finally, specific immunotherapy resulted in IL-5 down modulation, which was not associated with development of IFN-γ or IL-10 responses to any of the Bla g-derived peptides. In summary, the characteristics of T cell responses to Bla g allergens appear uncorrelated with IgE responses. Monitoring these responses may therefore yield important information relevant to understanding cockroach allergies and their treatment.

  2. Characterizing affinity epitopes between prion protein and β-amyloid using an epitope mapping immunoassay

    PubMed Central

    Kang, Mino; Kim, Su Yeon; An, Seong Soo A; Ju, Young Ran

    2013-01-01

    Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that β-amyloid1–42 oligomer causes neurotoxicity associated with Alzheimer's disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer's disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in β-amyloid. Residues 23–39 and 93–119 in the prion protein were involved in binding to β-amyloid1–40 and 1–42, and monomers of this protein interacted with prion protein residues 93–113 and 123–166. Furthermore, β-amyloid antibodies against the C-terminus detected bound β-amyloid1–42 at residues 23–40, 104–122 and 159–175. β-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to β-amyloid1–40 and 1–42. The 3D structure appears to be necessary for β-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer's disease. PMID:23907583

  3. Building Classifier Ensembles for B-Cell Epitope Prediction

    PubMed Central

    EL-Manzalawy, Yasser; Honavar, Vasant

    2015-01-01

    Identification of B-cell epitopes in target antigens is a critical step in epitope-driven vaccine design, immunodiagnostic tests, and antibody production. B-cell epitopes could be linear, i.e., a contiguous amino acid sequence fragment of an antigen, or conformational, i.e., amino acids that are often not contiguous in the primary sequence but appear in close proximity within the folded 3D antigen structure. Numerous computational methods have been proposed for predicting both types of B-cell epitopes. However, the development of tools for reliably predicting B-cell epitopes remains a major challenge in immunoinformatics. Classifier ensembles a promising approach for combining a set of classifiers such that the overall performance of the resulting ensemble is better than the predictive performance of the best individual classifier. In this chapter, we show how to build a classifier ensemble for improved prediction of linear B-cell epitopes. The method can be easily adapted to build classifier ensembles for predicting conformational epitopes. PMID:25048130

  4. Dissecting Antibodies with Regards to Linear and Conformational Epitopes

    PubMed Central

    Forsström, Björn; Bisławska Axnäs, Barbara; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

    2015-01-01

    An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

  5. The challenges and opportunities for the development of a T-cell epitope-based herpes simplex vaccine.

    PubMed

    Kuo, Tiffany; Wang, Christine; Badakhshan, Tina; Chilukuri, Sravya; BenMohamed, Lbachir

    2014-11-28

    Herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) infections have been prevalent since the ancient Greek times. To this day, they still affect a staggering number of over a billion individuals worldwide. HSV-1 infections are predominant than HSV-2 infections and cause potentially blinding ocular herpes, oro-facial herpes and encephalitis. HSV-2 infections cause painful genital herpes, encephalitis, and death in newborns. While prophylactic and therapeutic HSV vaccines remain urgently needed for centuries, their development has been difficult. During the most recent National Institute of Health (NIH) workshop titled "Next Generation Herpes Simplex Virus Vaccines: The Challenges and Opportunities", basic researchers, funding agencies, and pharmaceutical representatives gathered: (i) to assess the status of herpes vaccine research; and (ii) to identify the gaps and propose alternative approaches in developing a safe and efficient herpes vaccine. One "common denominator" among previously failed clinical herpes vaccine trials is that they either used a whole virus or a whole viral protein, which contain both "pathogenic symptomatic" and "protective asymptomatic" antigens and epitopes. In this report, we continue to advocate developing "asymptomatic" epitope-based sub-unit vaccine strategies that selectively incorporate "protective asymptomatic" epitopes which: (i) are exclusively recognized by effector memory CD4(+) and CD8(+) T cells (TEM cells) from "naturally" protected seropositive asymptomatic individuals; and (ii) protect human leukocyte antigen (HLA) transgenic animal models of ocular and genital herpes. We review the role of animal models in herpes vaccine development and discuss their current status, challenges, and prospects.

  6. Identification of human viral protein-derived ligands recognized by individual MHCI-restricted T-cell receptors

    PubMed Central

    Szomolay, Barbara; Liu, Jie; Brown, Paul E; Miles, John J; Clement, Mathew; Llewellyn-Lacey, Sian; Dolton, Garry; Ekeruche-Makinde, Julia; Lissina, Anya; Schauenburg, Andrea J; Sewell, Andrew K; Burrows, Scott R; Roederer, Mario; Price, David A; Wooldridge, Linda; van den Berg, Hugo A

    2016-01-01

    Evidence indicates that autoimmunity can be triggered by virus-specific CD8+ T cells that crossreact with self-derived peptide epitopes presented on the cell surface by major histocompatibility complex class I (MHCI) molecules. Identification of the associated viral pathogens is challenging because individual T-cell receptors can potentially recognize up to a million different peptides. Here, we generate peptide length-matched combinatorial peptide library (CPL) scan data for a panel of virus-specific CD8+ T-cell clones spanning different restriction elements and a range of epitope lengths. CPL scan data drove a protein database search limited to viruses that infect humans. Peptide sequences were ranked in order of likelihood of recognition. For all anti-viral CD8+ T-cell clones examined in this study, the index peptide was either the top-ranked sequence or ranked as one of the most likely sequences to be recognized. Thus, we demonstrate that anti-viral CD8+ T-cell clones are highly focused on their index peptide sequence and that ‘CPL-driven database searching' can be used to identify the inciting virus-derived epitope for a given CD8+ T-cell clone. Moreover, to augment access to CPL-driven database searching, we have created a publicly accessible webtool. Application of these methodologies in the clinical setting may clarify the role of viral pathogens in the etiology of autoimmune diseases. PMID:26846725

  7. Early Gag Immunodominance of the HIV-Specific T-Cell Response during Acute/Early Infection Is Associated with Higher CD8+ T-Cell Antiviral Activity and Correlates with Preservation of the CD4+ T-Cell Compartment

    PubMed Central

    Ghiglione, Yanina; Falivene, Juliana; Socias, María Eugenia; Laufer, Natalia; Coloccini, Romina Soledad; Rodriguez, Ana María; Ruiz, María Julia; Pando, María Ángeles; Giavedoni, Luis David; Cahn, Pedro; Sued, Omar; Salomon, Horacio; Gherardi, María Magdalena

    2013-01-01

    The important role of the CD8+ T-cell response on HIV control is well established. Moreover, the acute phase of infection represents a proper scenario to delineate the antiviral cellular functions that best correlate with control. Here, multiple functional aspects (specificity, ex vivo viral inhibitory activity [VIA] and polyfunctionality) of the HIV-specific CD8+ T-cell subset arising early after infection, and their association with disease progression markers, were examined. Blood samples from 44 subjects recruited within 6 months from infection (primary HIV infection [PHI] group), 16 chronically infected subjects, 11 elite controllers (EC), and 10 healthy donors were obtained. Results indicated that, although Nef dominated the anti-HIV response during acute/early infection, a higher proportion of early anti-Gag T cells correlated with delayed progression. Polyfunctional HIV-specific CD8+ T cells were detected at early time points but did not associate with virus control. Conversely, higher CD4+ T-cell set points were observed in PHI subjects with higher HIV-specific CD8+ T-cell VIA at baseline. Importantly, VIA levels correlated with the magnitude of the anti-Gag cellular response. The advantage of Gag-specific cells may result from their enhanced ability to mediate lysis of infected cells (evidenced by a higher capacity to degranulate and to mediate VIA) and to simultaneously produce IFN-γ. Finally, Gag immunodominance was associated with elevated plasma levels of interleukin 2 (IL-2) and macrophage inflammatory protein 1β (MIP-1β). All together, this study underscores the importance of CD8+ T-cell specificity in the improved control of disease progression, which was related to the capacity of Gag-specific cells to mediate both lytic and nonlytic antiviral mechanisms at early time points postinfection. PMID:23616666

  8. New salivary anti-haemostatics containing protective epitopes from Ornithodoros moubata ticks: Assessment of their individual and combined vaccine efficacy.

    PubMed

    Díaz-Martín, Verónica; Manzano-Román, Raúl; Oleaga, Ana; Pérez-Sánchez, Ricardo

    2015-09-15

    Ornithodoros moubata is the main vector of the pathogens causing African swine fever and human relapsing fever in Africa. The development of an efficient vaccine against this tick would facilitate its control and the prevention of the diseases it transmits to a considerable extent. Previous efforts to identify vaccine target candidates led us to the discovery of novel salivary proteins that probably act as anti-haemostatics at the host-tick interface, including a secreted phospholipase A2 (PLA2), a 7DB-like protein (7DB-like), a riboprotein 60S L10 (RP-60S), an apyrase (APY), and a new platelet aggregation inhibitor peptide, designated mougrin (MOU). In this work, the corresponding recombinant proteins were expressed in Escherichia coli and their individual vaccine efficacy was tested in rabbit vaccination trials. All of them, except the less immunogenic RP-60S, induced strong humoral responses that reduced tick feeding and survival, providing vaccine efficacies of 44.2%, 43.2% and 27.2%, 19.9% and 17.3% for PLA2, APY, MOU, RP-60S and 7DB-like, respectively. In the case of the more protective recombinant antigens (PLA2, APY and MOU), the immunodominant protective linear B-cell epitopes were identified and their combined vaccine efficacy was tested in a second vaccine trial using different adjuvants. In comparison with the best efficacy of individual antigens, the multicomponent vaccine increased vaccine efficacy by 13.6%, indicating additive protective effects rather than a synergistic effect. Tick saliva inoculated during natural tick-host contacts had a boosting effect on vaccinated animals, increasing specific antibody levels and protection.

  9. Differential presentation of tumor antigen-derived epitopes by MHC-class I and antigen-positive tumor cells.

    PubMed

    Held, Gerhard; Neumann, Frank; Sturm, Christine; Kaestner, Lars; Dauth, Nina; de Bruijn, Diederik R; Renner, Christoph; Lipp, Peter; Pfreundschuh, Michael

    2008-10-15

    SSX2 is a member of the family of cancer/testis antigens. The SSX2 derived peptide SSX2(103-111) has been shown to be presented to cytotoxic T-lymphocytes (CTL) by Major-Histocompatibility (MHC) Class-I complexes after endogenous processing, more precisely by the allele HLA-A*0201. The HLA-A*0201- and SSX2-positive melanoma cell line SK-Mel-37 but not Me275 had been shown to elicit reactivity in SSX2(103-111) specific cytotoxic T-lymphocytes. To analyze the correlation between SSX2(103-111) presentation and T-cell stimulation, we intended to visualize presentation of SSX2(103-111) in these melanoma cell lines. Fab-antibodies were established from a human phage library with specificity for SSX2(103-111)/HLA-A*0201 complexes (but non-reactive with HLA-A*0201 or SSX2(103-111) alone) and used to visualize the presentation of SSX2(103-111) in the context of HLA-A*0201 by fluorescence microscopy. Presentation of SSX2(103-111) the context of HLA-A*0201 was demonstrated for the majority of SK-Mel-37, but for only a small fraction (<1%) of Me275 as indicated by a clear membrane-staining pattern in fluorescence microscopy. The presentation of SSX2(103-111) on SK-Mel37 and Me275, but not the expression of the SSX2 protein correlated with the capability of these cells to stimulate cells of an SSX2(103-111)-specific T-cell clone. MHC-peptide specific antibodies are a valuable tool for the analysis of antigenic peptides in the context of MHC-I molecules and for the structural definition of immunodominant epitopes.

  10. Acute viral myocarditis

    PubMed Central

    Dennert, Robert; Crijns, Harry J.; Heymans, Stephane

    2008-01-01

    Acute myocarditis is one of the most challenging diagnosis in cardiology. At present, no diagnostic gold standard is generally accepted, due to the insensitivity of traditional diagnostic tests. This leads to the need for new diagnostic approaches, which resulted in the emergence of new molecular tests and a more detailed immunohistochemical analysis of endomyocardial biopsies. Recent findings using these new diagnostic tests resulted in increased interest in inflammatory cardiomyopathies and a better understanding of its pathophysiology, the recognition in overlap of virus-mediated damage, inflammation, and autoimmune dysregulation. Novel results also pointed towards a broader spectrum of viral genomes responsible for acute myocarditis, indicating a shift of enterovirus and adenovirus to parvovirus B19 and human herpes virus 6. The present review proposes a general diagnostic approach, focuses on the viral aetiology and associated autoimmune processes, and reviews treatment options for patients with acute viral myocarditis. PMID:18617482

  11. [Vasculitis and viral infection].

    PubMed

    Martínez Aguilar, N E; Guido Bayardo, R; Vargas Camaño, M E; Compañ González, D; Miranda Feria, A J

    1997-01-01

    Viruses have been implicated in vasculitis. To determine activity of viral infection associated with vasculitis. 17 patients with vasculitis had been in immunological and antiviral antibodies evaluation. Twenty five healthy controls sex and age matched with hematic biometry (BH) and AA. All subjects were negative to HIV and HBV. Viral activity was demonstrated in eight patients; vascular purpura (5), Takayasu disease (1), polyarteritis nodosa (1), erythema nodosum (1). None subject of control group had IgM activity. Antibodies response of IgG in patients were of lesser intensity than in control group. 14 abnormalities in BH were found in patients and 4 in control group. Immune response in patients, measured by lymphocyte subpopulations and circulating immune complexes was abnormal. In conclusion 47% showed viral activity, but the dominant feature was abnormal immune response in 82%.

  12. Viral infections and allergies.

    PubMed

    Xepapadaki, Paraskevi; Papadopoulos, Nikolaos G

    2007-01-01

    Respiratory viral infections have been implicated in the origin of, protection from and exacerbation of allergy-related symptoms in a variety of ways. Viral infections are closely linked to infantile wheezing. Severe bronchiolitis in early infancy may predispose to chronic childhood asthma as well as allergic sensitization; alternatively it could represent a marker of susceptible individuals. In contrast, repeated mild infections in early life may have a protective role in the development of asthma or atopy by driving the immune system towards Th1 responses. However, evidence on this hypothesis is not consistent as far as respiratory viruses are concerned. Several factors, including the presence of an atopic environment, timing of exposure and severity of the infection, interactively contribute to the allergy-infection relationship. In the present report, recent data on the role of viral infections in the development and progression of allergy and asthma are reviewed.

  13. Modeling Viral Capsid Assembly

    PubMed Central

    2014-01-01

    I present a review of the theoretical and computational methodologies that have been used to model the assembly of viral capsids. I discuss the capabilities and limitations of approaches ranging from equilibrium continuum theories to molecular dynamics simulations, and I give an overview of some of the important conclusions about virus assembly that have resulted from these modeling efforts. Topics include the assembly of empty viral shells, assembly around single-stranded nucleic acids to form viral particles, and assembly around synthetic polymers or charged nanoparticles for nanotechnology or biomedical applications. I present some examples in which modeling efforts have promoted experimental breakthroughs, as well as directions in which the connection between modeling and experiment can be strengthened. PMID:25663722

  14. Identification of an immunodominant mouse minor histocompatibility antigen (MiHA). T cell response to a single dominant MiHA causes graft-versus-host disease.

    PubMed Central

    Perreault, C; Jutras, J; Roy, D C; Filep, J G; Brochu, S

    1996-01-01

    T cell responses to non-MHC antigens are targeted to a restricted number of immunodominant minor histocompatibility antigens whose identity remains elusive. Here we report isolation and sequencing of a novel immunodominant minor histocompatibility antigen presented by H-2Db on the surface of C57BL/6 mouse cells. This nonapeptide (AAPDNRETF) shows strong biologic activity in cytotoxic T lymphocyte sensitization assays at concentrations as low as 10 pM. C3H.SW mice primed with AAPDNRETF in incomplete Freund's adjuvant generated a potent anti-C57BL/6 T cell-mediated cytotoxic activity, and T lymphocytes from AAPDNRETF-primed mice caused graft-versus-host disease when transplanted in irradiated C57BL/6 recipients. These results (a) provide molecular characterization of a mouse dominant minor histocompatibility antigen, (b) identify this peptide as a potential target of graft-versus-host disease and, (c) more importantly, demonstrate that a single dominant minor antigen can cause graft-versus-host disease. These findings open new avenues for the prevention of graft-versus-host disease and should further our understanding of the mechanisms of immunodominance in T cell responses to minor histocompatibility antigens. PMID:8698852

  15. Viral apoptotic mimicry.

    PubMed

    Amara, Ali; Mercer, Jason

    2015-08-01

    As opportunistic pathogens, viruses have evolved many elegant strategies to manipulate host cells for infectious entry and replication. Viral apoptotic mimicry, defined by the exposure of phosphatidylserine - a marker for apoptosis - on the pathogen surface, is emerging as a common theme used by enveloped viruses to promote infection. Focusing on the four best described examples (vaccinia virus, dengue virus, Ebola virus and pseudotyped lentivirus), we summarize our current understanding of apoptotic mimicry as a mechanism for virus entry, binding and immune evasion. We also describe recent examples of non-enveloped viruses that use this mimicry strategy, and discuss future directions and how viral apoptotic mimicry could be targeted therapeutically.

  16. Advances in viral oncology

    SciTech Connect

    Klein, G.

    1987-01-01

    Volume 6 of Advances in Viral Oncology presents experimental approaches to multifactorial interactions in tumor development. Included are in-depth analyses of malignant phenotypes by oncogene complementation, as well as studies of complementary interactions among DNA viral oncogenes; multiple cell-derived sequences in single retroviral genomes; and sequences that influence the transforming activity and expression of the mos oncogene. The genetic regulation of tumorigenic expression in somatic cell hybrids, the inhibition of oncogenes by cellular genes, and the interaction of genes that favor and genes that suppress tumorigenesis are examined in detail. The book concludes with a study of the relationship of oncogenes to the evolution of the metastatic phenotype.

  17. Measles Virus Epitope Presentation by HLA: Novel Insights into Epitope Selection, Dominance, and Microvariation

    PubMed Central

    Schellens, Ingrid M.; Meiring, Hugo D.; Hoof, Ilka; Spijkers, Sanne N.; Poelen, Martien C. M.; van Gaans-van den Brink, Jacqueline A. M.; Costa, Ana I.; Vennema, Harry; Keşmir, Can; van Baarle, Debbie; van Els, Cécile A. C. M.

    2015-01-01

    Immunity to infections with measles virus (MV) can involve vigorous human leukocyte antigen (HLA) class I-restricted CD8+ cytotoxic T cell (CTL) responses. MV, albeit regarded monotypic, is known to undergo molecular evolution across its RNA genome. To address which regions of the MV proteome are eligible for recognition by CD8+ CTLs and how different HLA class I loci contribute to the epitope display, we interrogated the naturally processed and presented MV peptidome extracted from cell lines expressing in total a broad panel of 16 different common HLA-A, -B, and -C molecules. The repertoire and abundance of MV peptides were bona fide identified by nanoHPLC–MS/MS. ­Eighty-nine MV peptides were discovered and assignment to an HLA-A, -B, or -C allele, based on HLA-peptide affinity prediction, was in most cases successful. Length variation and presentation by multiple HLA class I molecules was common in the MV peptidome. More than twice as many unique MV epitopes were found to be restricted by HLA-B than by HLA-A, while MV peptides with supra-abundant expression rates (>5,000 cc) were rather associated with HLA-A and HLA-C. In total, 59 regions across the whole MV proteome were identified as targeted by HLA class I. Sequence coverage by epitopes was highest for internal proteins transcribed from the MV-P/V/C and -M genes and for hemagglutinin. At the genome level, the majority of the HLA class I-selected MV epitopes represented codons having a higher non-synonymous mutation rate than silent mutation rate, as established by comparison of a set of 58 unique full length MV genomes. Interestingly, more molecular variation was seen for the epitopes expressed at rates ≥1,000 cc. These data for the first time indicate that HLA class I broadly samples the MV proteome and that CTL pressure may contribute to the genomic evolution of MV. PMID:26579122

  18. Immunodominant Dengue Virus-Specific CD8+ T Cell Responses Are Associated with a Memory PD-1+ Phenotype

    PubMed Central

    de Alwis, Ruklanthi; Bangs, Derek J.; Angelo, Michael A.; Cerpas, Cristhiam; Fernando, Anira; Sidney, John; Peters, Bjoern; Gresh, Lionel; Balmaseda, Angel; de Silva, Aruna D.; Harris, Eva; Sette, Alessandro

    2016-01-01

    ABSTRACT Dengue disease is a large public health problem that mainly afflicts tropical and subtropical regions. Understanding of the correlates of protection against dengue virus (DENV) is poor and hinders the development of a successful human vaccine. The present study aims to define DENV-specific CD8+ T cell responses in general and those of HLA alleles associated with dominant responses in particular. In human blood donors in Nicaragua, we observed a striking dominance of HLA B-restricted responses in general and of the allele B*35:01 in particular. Comparing these patterns to those in the general population of Sri Lanka, we found a strong correlation between restriction of the HLA allele and the breadth and magnitude of CD8+ T cell responses, suggesting that HLA genes profoundly influence the nature of responses. The majority of gamma interferon (IFN-γ) responses were associated with effector memory phenotypes, which were also detected in non-B*35:01-expressing T cells. However, only the B*35:01 DENV-specific T cells were associated with marked expression of the programmed death 1 protein (PD-1). These cells did not coexpress other inhibitory receptors and were able to proliferate in response to DENV-specific stimulation. Thus, the expression of particular HLA class I alleles is a defining characteristic influencing the magnitude and breadth of CD8 responses, and a distinct, highly differentiated phenotype is specifically associated with dominant CD8+ T cells. These results are of relevance for both vaccine design and the identification of robust correlates of protection in natural immunity. IMPORTANCE Dengue is an increasingly significant public health problem as its mosquito vectors spread over greater areas; no vaccines against the virus have yet been approved. An important step toward vaccine development is defining protective immune responses; toward that end, we here characterize the phenotype of the immunodominant T cell responses. These DENV-reactive T

  19. BIOMARKERS OF VIRAL EXPOSURE

    EPA Science Inventory

    Viral and protozoan pathogens associated with raw sludge can cause encephalitis, gastroenteritis, hepatitis, myocarditis, and a number of other diseases. Raw sludge that has been treated to reduce these pathogens can be used for land application according to the regulations spec...

  20. Leafhopper viral pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Four newly discovered viral pathogens in leafhopper vectors of Pierce’s disease of grapes, have been shown to replicate in sharpshooter leafhoppers; the glassy-winged sharpshooter, GWSS, Homalodisca vitripennis, and Oncometopia nigricans (Hemiptera: Cicadellidae). The viruses were classified as memb...

  1. Marine Viral Pathogens.

    DTIC Science & Technology

    2007-11-02

    toxin producing microalgae (Raphidophyceae). Although we have not definitively shown that the pathogen is viral, it has many characteristics that...Society America, Miami, FL, June 1994. 40.Hennes, K.P. and C.A. Suttle. 1994. The use of cyanine dyes for quantifying free viruses in natural water

  2. Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

    PubMed Central

    Sarkar, Pampi; Labranche, Celia; Go, Eden P.; Clark, Daniel F.; Sun, Yide; Nandi, Avishek; Hartog, Karin; Desaire, Heather; Montefiori, David; Carfi, Andrea; Srivastava, Indresh K.; Barnett, Susan W.

    2013-01-01

    Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules. PMID:24146829

  3. Structure of allergens and structure based epitope predictions☆

    PubMed Central

    Dall’Antonia, Fabio; Pavkov-Keller, Tea; Zangger, Klaus; Keller, Walter

    2014-01-01

    The structure determination of major allergens is a prerequisite for analyzing surface exposed areas of the allergen and for mapping conformational epitopes. These may be determined by experimental methods including crystallographic and NMR-based approaches or predicted by computational methods. In this review we summarize the existing structural information on allergens and their classification in protein fold families. The currently available allergen-antibody complexes are described and the experimentally obtained epitopes compared. Furthermore we discuss established methods for linear and conformational epitope mapping, putting special emphasis on a recently developed approach, which uses the structural similarity of proteins in combination with the experimental cross-reactivity data for epitope prediction. PMID:23891546

  4. Immunogenicity of a multi-epitope DNA vaccine against hantavirus.

    PubMed

    Zhao, Chen; Sun, Ying; Zhao, Yujie; Wang, Si; Yu, Tongtong; Du, Feng; Yang, X Frank; Luo, Enjie

    2012-02-01

    Hemorrhagic fever with renal syndrome (HFRS) is a severe epidemic disease caused by hantaviruses including Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus. Three of the four HFRS hantaviruses, HTNV, SEOV, and PUUV are found in China. Currently, there is no effective strategy available to reduce infection risk. In this study, we constructed a multi-epitope chimeric DNA vaccine that encodes expressing 25 glycoprotein epitopes from SEOV, HTNV and PUUV (designated as SHP chimeric gene). Vaccination of BALb/c mice with SHP multi-epitope chimeric DNA vaccine led to a dramatic augmentation of humoral and cellular responses. The SHP vaccine DNA was detected in many organs but not for more than 60 d. There was no risk of mutation due to integration. Thus, the SHP multi-epitope chimeric DNA vaccine is a potential effective and safe DNA vaccine against infection by SEOV, HTNV, and PUUV.

  5. Protection of rhesus macaques against disease progression from pathogenic SHIV-89.6PD by vaccination with phage-displayed HIV-1 epitopes.

    PubMed

    Chen, X; Scala, G; Quinto, I; Liu, W; Chun, T W; Justement, J S; Cohen, O J; vanCott, T C; Iwanicki, M; Lewis, M G; Greenhouse, J; Barry, T; Venzon, D; Fauci, A S

    2001-11-01

    The antigenic polymorphism of HIV-1 is a major obstacle in developing an effective vaccine. Accordingly, we screened random peptide libraries (RPLs) displayed on phage with antibodies from HIV-infected individuals and identified an array of HIV-specific epitopes that behave as antigenic mimics of conformational epitopes of gp120 and gp41 proteins. We report that the selected epitopes are shared by a collection of HIV-1 isolates of clades A-F. The phage-borne epitopes are immunogenic in rhesus macaques, where they elicit envelope-specific antibody responses. Upon intravenous challenge with 60 MID50 of pathogenic SHIV-89.6PD, all monkeys became infected; however, in contrast to the naive and mock-immunized monkeys, four of five mimotope-immunized monkeys experienced lower levels of peak viremia, followed by viral set points of undetectable or transient levels of viremia and a mild decline of CD4+ T cells, and were protected from progression to AIDS-like illness. These results provide a new approach to the design of broadly protective HIV-1 vaccines.

  6. Cellular immunogenicity of a multi-epitope peptide vaccine candidate based on hepatitis C virus NS5A, NS4B and core proteins in HHD-2 mice.

    PubMed

    Huang, Xiao-Jun; Lü, Xin; Lei, Ying-Feng; Yang, Jing; Yao, Min; Lan, Hai-Yun; Zhang, Jian-Min; Jia, Zhan-Sheng; Yin, Wen; Xu, Zhi-Kai

    2013-04-01

    To develop a vaccine against hepatitis C virus (HCV), a multi-epitope peptide was synthesized from nonstructural proteins containing HLA-A2 epitopes inducing mainly responses in natural infection. The engineered vaccine candidate, VAL-44, consists of multiple epitopes from the HCV NS5A, NS4B and core proteins. Immunization with the VAL-44 peptide induced higher CTL responses than those by the smaller VL-20 peptide. VAL-44 induced antigen-specific IFN-γ-producing CD4+ T cells and CD8+ T cells. VAL-44 elicited a Th1-biased immune response with secretion of high amounts of IFN-γ and IL-2, compared with VL-20. These results suggest that VAL-44 can elicit strong cellular immune responses. The VAL-44 peptide stimulated IFN-γ production from viral-specific peripheral blood mononuclear cells (PBMCs) of patients infected with HCV. These results suggest that VAL-44 could be developed as a potential HCV multi-epitope peptide vaccine.

  7. Mimotope vaccines: epitope mimics induce anti-cancer antibodies.

    PubMed

    Riemer, Angelika B; Jensen-Jarolim, Erika

    2007-10-31

    Mimotopes are epitope-mimicking structures. When applied for immunizations they induce desired antibody specificities exclusively based on the principle of molecular mimicry. This is important as antibodies directed against tumor-associated antigens may harbor diverse biological effects depending on their epitope specificity. Thus they may inhibit or promote tumor growth. This review gives an update on different vaccination strategies based on the mimotope concept.

  8. Bioinformatic prediction of the epitopes of Echinococcus granulosus antigen 5

    PubMed Central

    Pan, Wei; Chen, De-Sheng; Lu, Yun-Juan; Sun, Fen-Fen; Xu, Hui-Wen; Zhang, Ya-Wen; Yan, Chao; Fu, Lin-Lin; Zheng, Kui-Yang; Tang, Ren-Xian

    2017-01-01

    The aim of the present study was to predict and analyze the secondary structure, and B and T cell epitopes of Echinococcus granulosus antigen 5 (Ag5) using online software in order t