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1.  Identification of Immunodominant CD4-Restricted Epitopes Co-Located with Antibody Binding Sites in Individuals Vaccinated with ALVAC-HIV and AIDSVAX B/E 
PLoS ONE  2015;10(2):e0115582.
We performed fine epitope mapping of the CD4+ responses in the ALVAC-HIV-AIDSVAX B/E prime-boost regimen in the Thai Phase III trial (RV144). Non-transformed Env-specific T cell lines established from RV144 vaccinees were used to determine the fine epitope mapping of the V2 and C1 responses and the HLA class II restriction. Data showed that there are two CD4+ epitopes contained within the V2 loop: one encompassing the α4β7 integrin binding site (AA179-181) and the other nested between two previously described genetic sieve signatures (AA169, AA181). There was no correlation between the frequencies of CD4+ fine epitope responses and binding antibody.
PMCID: PMC4321833  PMID: 25665096
2.  HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T-cell epitope 
Journal of hepatology  2013;60(1):10.1016/j.jhep.2013.08.009.
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.
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.
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.
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.
PMCID: PMC3867523  PMID: 23978718
Hepatitis C virus; CD8+ T-cell response; HLA subtypes; HLA-B*27; vaccine design
3.  HLA class I alleles are associated with peptide binding repertoires of different size, affinity and immunogenicity1 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(12):10.4049/jimmunol.1302101.
Prediction of HLA binding affinity is widely utilized to identify candidate T cell epitopes, and an affinity of 500 nM is routinely used as a threshold for peptide selection. However, the fraction (%) of peptides predicted to bind with affinities of 500 nM varies by allele. For example, of a large collection of about 30,000 dengue virus derived peptides only 0.3% were predicted to bind HLA A*0101, while nearly 5% were predicted for A*0201. This striking difference could not be ascribed to variation in accuracy of the algorithms utilized, as predicted values closely correlated with affinity measured in vitro with purified HLA molecules. These data raised the question whether different alleles would also vary in terms of epitope repertoire size, defined as the number of associated epitopes or, alternatively, whether alleles vary drastically in terms of the affinity threshold associated with immunogenicity. To address this issue, strains of HLA transgenic mice with wide (A*0201), intermediate (B*0702) or narrow (A*0101) repertoires were immunized with peptides of varying binding affinity and relative percentile ranking. The results show that absolute binding capacity is a better predictor of immunogenicity, and analysis of epitopes from the Immune Epitope Database (IEDB) revealed that predictive efficacy is increased using allele-specific affinity thresholds. Finally, we investigate the genetic and structural basis of the phenomenon. While no stringent correlate was defined, on average HLA B alleles are associated with significantly narrower repertoires than HLA A alleles.
PMCID: PMC3872965  PMID: 24190657
4.  Conservancy of mAb Epitopes in Ebolavirus Glycoproteins of Previous and 2014 Outbreaks 
PLoS Currents  2014;6:ecurrents.outbreaks.f1a7028a13ce1c5f0bdbb4b0cc0b919b.
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.
PMCID: PMC4234462  PMID: 25642381
disease outbreak; ebola; ebolavirus; EBOV; epitope; mAb
5.  Structure-based design of altered MHC II-restricted peptide ligands with heterogeneous immunogenicity1 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(10):10.4049/jimmunol.1300467.
Insights gained from characterizing MHC-peptide-TCR interactions have held the promise that directed structural modifications can have predictable functional consequences. The ability to manipulate T cell reactivity synthetically or through genetic engineering might thus be translated into new therapies for common diseases such as cancer and autoimmune disorders. In the current study, we determined the crystal structure of HLA-DR4 in complex with the non-mutated dominant gp100 epitope, gp10044–59, associated with many melanomas. Altered peptide ligands (APLs) were designed to enhance MHC binding and hence T cell recognition of gp100 in HLA-DR4+ melanoma patients. Increased MHC binding of several APLs was observed, validating this approach biochemically. Nevertheless, heterogeneous preferences of CD4+ T cells from several HLA-DR4+ melanoma patients for different gp100 APLs suggested highly variable TCR usage, even among six patients who had been vaccinated against the wild type gp100 peptide. This heterogeneity prevented the selection of an APL candidate for developing an improved generic gp100 vaccine in melanoma. Our results are consistent with the idea that even conservative changes in MHC anchor residues may result in subtle, yet crucial, effects on peptide contacts with the TCR or on peptide dynamics, such that alterations intended to enhance immunogenicity may be unpredictable or counterproductive. They also underscore a critical knowledge gap that needs to be filled, before structural and in vitro observations can be used reliably to devise new immunotherapies for cancer and other disorders.
PMCID: PMC3888030  PMID: 24108701
6.  Allergy-associated T cell epitope repertoires are surprisingly diverse and include non-IgE reactive antigens 
We recently identified T cell epitopes associated with human allergic responses. In a majority of cases, responses focused on a few immunodominant epitopes which can be predicted on the basis of MHC binding characteristics. Several observations from our studies challenged the assumption that T cell epitopes are derived from the same allergen proteins that bind IgE. Transcriptomic and proteomics analysis identified pollen proteins, not bound by IgE. These novel Timothy Grass proteins elicited vigorous Th2 responses, suggesting that unlinked T cell help is operational in pollen-specific responses. Thus, the repertoire of antigens recognized by T cells is much broader than IgE-binding allergens. Additionally, we evaluated the use of epitopes from these novel antigens to assess immunological changes associated with Specific Immunotherapy (SIT). We found that a marked decrease in IL5 production is associated with clinically efficacious SIT, suggesting that these novel antigens are potential immunomarkers for SIT efficacy.
PMCID: PMC4210551  PMID: 25352946
T cells; Specific immunotherapy; Timothy grass; Cytokine; Epitopes
7.  Human Circulating PD-1+CXCR3−CXCR5+ Memory Tfh Cells Are Highly Functional and Correlate with Broadly Neutralizing HIV Antibody Responses 
Immunity  2013;39(4):758-769.
The vast majority of currently licensed human vaccines work on the basis of long-term protective antibody responses. It is now conceivable that an antibody-dependent HIV vaccine might be possible, given the discovery of HIV broadly neutralizing antibodies (bnAbs) in some HIV-infected individuals. However, these antibodies are difficult to develop and have characteristics indicative of a high degree of affinity maturation in germinal centers (GCs). CD4+ T follicular helper (Tfh) cells are specialized for B cell help and necessary for GCs. Therefore, the development of HIV bnAbs might depend on Tfh cells. Here, we identified in normal individuals a subpopulation of circulating memory PD-1+CXCR5+ CD4+ T cells that are resting memory cells most related to bona fide GC Tfh cells by gene expression profile, cytokine profile, and functional properties. Importantly, the frequency of these cells correlated with the development of bnAbs against HIV in a large cohort of HIV+ individuals.
PMCID: PMC3996844  PMID: 24035365
8.  Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes 
PLoS ONE  2014;9(9):e106241.
Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.
Methodology/Principal Findings
We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.
We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.
PMCID: PMC4161338  PMID: 25211344
9.  Navigating diabetes-related immune epitope data: resources and tools provided by the Immune Epitope Database (IEDB) 
Immunome research  2013;9(1):10.4172/1745-7580.1000063.
The Immune Epitope Database (IEDB), originally focused on infectious diseases, was recently expanded to allergy, transplantation and autoimmunity diseases. Here we focus on diabetes, chosen as a prototype autoimmune disease. We utilize a combined tutorial and meta-analysis format, which demonstrates how common questions, related to diabetes epitopes can be answered.
A total of 409 references are captured in the IEDB describing >2,500 epitopes from diabetes associated antigens. The vast majority of data were derived from GAD, insulin, IA-2/PTPRN, IGRP, ZnT8, HSP, and ICA-1, and the experiments related to T cell epitopes and MHC binding far outnumbers B cell assays. We illustrate how to search by specific antigens, epitopes or host. Other examples include searching for tetramers or epitopes restricted by specific alleles or assays of interest, or searching based on the clinical status of the host.
The inventory of all published diabetes epitope data facilitates its access for the scientific community. While the global collection of primary data from the literature reflects potential investigational biases present in the literature, the flexible search approach allows users to perform queries tailored to their preferences, including or excluding data as appropriate. Moreover, the analysis highlights knowledge gaps and identifies areas for future investigation.
PMCID: PMC4134942  PMID: 25140192
10.  Analysis of HLA A*02 Association with Vaccine Efficacy in the RV144 HIV-1 Vaccine Trial 
Journal of Virology  2014;88(15):8242-8255.
The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02+) participants than in A*02− participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02+ participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02+ participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials.
IMPORTANCE The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at under registration no. NCT00223080.)
PMCID: PMC4135964  PMID: 24829343
11.  Dataset size and composition impact the reliability of performance benchmarks for peptide-MHC binding predictions 
BMC Bioinformatics  2014;15(1):241.
It is important to accurately determine the performance of peptide:MHC binding predictions, as this enables users to compare and choose between different prediction methods and provides estimates of the expected error rate. Two common approaches to determine prediction performance are cross-validation, in which all available data are iteratively split into training and testing data, and the use of blind sets generated separately from the data used to construct the predictive method. In the present study, we have compared cross-validated prediction performances generated on our last benchmark dataset from 2009 with prediction performances generated on data subsequently added to the Immune Epitope Database (IEDB) which served as a blind set.
We found that cross-validated performances systematically overestimated performance on the blind set. This was found not to be due to the presence of similar peptides in the cross-validation dataset. Rather, we found that small size and low sequence/affinity diversity of either training or blind datasets were associated with large differences in cross-validated vs. blind prediction performances. We use these findings to derive quantitative rules of how large and diverse datasets need to be to provide generalizable performance estimates.
It has long been known that cross-validated prediction performance estimates often overestimate performance on independently generated blind set data. We here identify and quantify the specific factors contributing to this effect for MHC-I binding predictions. An increasing number of peptides for which MHC binding affinities are measured experimentally have been selected based on binding predictions and thus are less diverse than historic datasets sampling the entire sequence and affinity space, making them more difficult benchmark data sets. This has to be taken into account when comparing performance metrics between different benchmarks, and when deriving error estimates for predictions based on benchmark performance.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2105-15-241) contains supplementary material, which is available to authorized users.
PMCID: PMC4111843  PMID: 25017736
Benchmarking of MHC class I predictors; Epitope prediction; Sequence similarity; Cross-validation
12.  Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru 
Infection and Immunity  2014;82(1):124-131.
Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+ T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4+ T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen.
PMCID: PMC3911835  PMID: 24126518
13.  Asymptomatic HLA-A*02:01–Restricted Epitopes from Herpes Simplex Virus Glycoprotein B Preferentially Recall Polyfunctional CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect HLA Transgenic Mice against Ocular Herpes 
Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine.
PMCID: PMC4057607  PMID: 24101547
14.  Sculpting MHC class II-restricted self and non-self peptidome by the class I antigen-processing machinery and its impact on CD4+ TCR repertoire and Th cell responses 
European journal of immunology  2013;43(5):1162-1172.
It is generally assumed that the MHC class I antigen (Ag)-processing (CAP) machinery —which supplies peptides for presentation by class I molecules— plays no role in class II-restricted presentation of cytoplasmic Ags. In striking contrast to this assumption, we previously reported that proteasome inhibition or TAP- or ERAAP-deficiency led to dramatically altered T helper (Th) cell responses to allograft (HY) and microbial (Listeria monocytogenes) Ags. Herein, we tested whether altered Ag processing and presentation, altered CD4+ T cell repertoire, or both underlay the above finding. We found that TAP- and ERAAP-deficiency dramatically altered the quality of class II-associated self peptides suggesting that the CAP machinery impacts class II-restricted Ag processing and presentation. Consistent with altered self peptidomes, the CD4+ T cell receptor repertoire of mice deficient in the CAP machinery substantially differed from that of wildtype animals resulting in altered CD4+ T cell Ag recognition patterns. These data suggest that TAP and ERAAP sculpt the class II-restricted peptidome, impacting the CD4+ T cell repertoire, and ultimately altering Th cell responses. Together with our previous findings, these data suggest multiple CAP machinery components sequester or degrade MHC class II-restricted epitopes that would otherwise be capable of eliciting functional Th cell responses.
PMCID: PMC3798073  PMID: 23386199
antigen presentation; mass spectrometry; T helper cells; self peptidome; MHC
15.  A strategy to determine HLA class II restriction broadly covering the DR, DP and DQ allelic variants most commonly expressed in the general population 
Immunogenetics  2013;65(5):357-370.
Classic ways to determine MHC restriction involve inhibition with locus specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T-cell epitope degeneracy and promiscuity. We describe selection of 46 HLA DR, DQ and DP specificities that provide worldwide population (phenotypic) coverage of almost 90% at each locus, and account for over 66% of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95% of the individuals in four study populations of diverse ethnicity from the US and South Africa. Next, a panel of single HLA class II transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T-cell lines of known restriction. We also show that these HLA class II transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T-cell responses and facilitating the development of HLA tetrameric staining reagents.
PMCID: PMC3633633  PMID: 23392739
HLA Class II; restriction; transfectants; epitopes; population coverage; polymorphism
16.  Peptide binding motifs associated with MHC molecules common in Chinese rhesus macaques are analogous to those of human HLA supertypes, and include HLA-B27-like alleles 
Immunogenetics  2013;65(5):371-386.
Chinese rhesus macaques are of particular interest in SIV/HIV research as these animals have prolonged kinetics of disease progression to AIDS, compared to their Indian counterparts, suggesting that they may be a better model for HIV. Nevertheless, the specific mechanism(s) accounting for these kinetics remains unclear. The study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide binding motifs, provides valuable information for measuring cellular immune responses and deciphering outcomes of infection and vaccine efficacy. In this study, we have provided detailed characterization of six prevalent Chinese rhesus macaque MHC class I alleles, yielding a combined phenotypic frequency of 29%. The peptide binding specificity of two of these alleles, Mamu-A2*01:02 and -B*010:01, as well as the previously characterized allele Mamu-B*003:01 (and Indian rhesus Mamu-B*003:01), was found to be analogous to that of alleles in the HLA-B27 supertype family. Specific alleles in the HLA-B27 supertype family, including HLA-B*27:05, have been associated with long-term non-progression to AIDS in humans. All six alleles characterized in the present study were found to have specificities analogous to HLA-supertype alleles. These data contribute to the concept that Chinese rhesus macaque MHC immunogenetics is more similar to HLA than their Indian rhesus macaque counterparts, and thereby warrant further studies to decipher the role of these alleles in the context of SIV infection.
PMCID: PMC3633659  PMID: 23417323
MHC; non-human primate; Chinese rhesus macaques; MHC:peptide binding motif
17.  Relapse or eradication of cancer is predicted by peptide-MHC affinity 
Cancer cell  2013;23(4):516-526.
Cancers often relapse after adoptive therapy, even though specific T cells kill cells from the same cancer efficiently in vitro. We found that tumor eradication by T cells required high affinities of the targeted peptides for MHC class I. Affinities of at least 10 nM were required for relapse-free regression. Only high-affinity peptide-MHC interactions led to efficient cross-presentation of antigen, thereby stimulating cognate T cells to secrete cytokines. These findings highlight the importance of targeting peptides with high affinity for MHC class I when designing T cell-based immunotherapy.
PMCID: PMC3658176  PMID: 23597565
18.  Association of HLA-DRB1-restricted CD4+ T cell responses with HIV immune control 
Nature medicine  2013;19(7):930-933.
The contribution of HLA class II-restricted CD4+ T cell responses to HIV immune control is poorly defined. Here, we delineated novel peptide-DRB1 restrictions in functional assays and analyzed the host genetic effects of HLA-DRB1 alleles on HIV viremia in a large cohort of HIV controllers and progressors (n=1085). We found distinct stratifications in the effect of HLA-DRB1 alleles on HIV viremia, with DRB1*15:02 significantly associated with low viremia (P=0.003, q=0.04) and DRB1*03:01 significantly associated with high viremia (P=0.004, q=0.04). Interestingly, a sub-group of HLA-DRB1 alleles linked with low viremia showed the ability to promiscuously present a larger breadth of peptides with lower functional avidity when compared to HLA-DRB1 alleles linked with high viremia (p=0.018). Our data provide systematic evidence that HLA-DRB1 allele expression significantly impacts the durable control of HIV replication, an effect that appears to be mediated primarily by the protein-specificity of HIV-specific CD4+ T cell responses to Gag and Nef.
PMCID: PMC3974408  PMID: 23793098
19.  Immunization with Apical Membrane Antigen 1 Confers Sterile Infection-Blocking Immunity against Plasmodium Sporozoite Challenge in a Rodent Model 
Infection and Immunity  2013;81(10):3586-3599.
Apical membrane antigen 1 (AMA-1) is a leading blood-stage malaria vaccine candidate. Consistent with a key role in erythrocytic invasion, AMA-1-specific antibodies have been implicated in AMA-1-induced protective immunity. AMA-1 is also expressed in sporozoites and in mature liver schizonts where it may be a target of protective cell-mediated immunity. Here, we demonstrate for the first time that immunization with AMA-1 can induce sterile infection-blocking immunity against Plasmodium sporozoite challenge in 80% of immunized mice. Significantly higher levels of gamma interferon (IFN-γ)/interleukin-2 (IL-2)/tumor necrosis factor (TNF) multifunctional T cells were noted in immunized mice than in control mice. We also report the first identification of minimal CD8+ and CD4+ T cell epitopes on Plasmodium yoelii AMA-1. These data establish AMA-1 as a target of both preerythrocytic- and erythrocytic-stage protective immune responses and validate vaccine approaches designed to induce both cellular and humoral immunity.
PMCID: PMC3811765  PMID: 23836827
20.  Broadly Reactive Human CD8 T Cells that Recognize an Epitope Conserved between VZV, HSV and EBV 
PLoS Pathogens  2014;10(3):e1004008.
Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV), we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1) along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a “pan-herpesvirus” vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.
Author Summary
Human herpesviruses can cause a wide range of serious infections. They are extremely common and individuals remain latently infected lifelong, with reactivations often causing recurrent or severe disease. T-cells are important in controlling herpesvirus infections and preventing their reactivation, so vaccines that induce T-cells are likely to improve control. Here, we examined human T-cells against VZV that might allow focused vaccine development. We identified a dominant target against which the majority of subjects had mounted a CD8 T-cell response. We found that very similar targets also exist in three other important herpesviruses, HSV-1, HSV-2 and EBV. We showed that CD8 T-cells recognizing the VZV target could also recognize the others and we hypothesized that recurrent encounter with these viruses could boost this common response. In some individuals, immunization with a VZV vaccine did cause activation of these cells, but in most it did not. This reflects the variable efficacy of the currently available VZV vaccine. Our findings suggest that T-cell targets may be shared between herpesvirus species and may therefore contribute to a novel “pan-herpesvirus” vaccine. However, current VZV vaccines cannot reliably stimulate these T-cells and new strategies will be necessary to achieve this goal.
PMCID: PMC3968128  PMID: 24675761
21.  CD8+ T-Cell Epitope Mapping for Pneumonia Virus of Mice in H-2b Mice 
Journal of Virology  2013;87(17):9949-9952.
The paramyxovirus pneumonia virus of mice (PVM) is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8+ T-cell repertoire in susceptible C57BL/6 mice. In total, 15 PVM-specific CD8+ T-cell epitopes restricted by H-2Db and/or H-2Kb were identified. These data open the door for using widely profiled, genetically manipulated C57BL/6 mice to study the contribution of epitope-specific CD8+ T cells to PVM pathogenesis.
PMCID: PMC3754129  PMID: 23824814
22.  Measurement of MHC/Peptide Interactions by Gel Filtration or Monoclonal Antibody Capture 
This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.
PMCID: PMC3626435  PMID: 23392640
MHC class I; MHC class II; T cell epitope; peptide ligand; binding affinity; CTL; epitope recognition
23.  Prolonged Exposure to HIV Reinforces a Poised Epigenetic Program for PD-1 Expression in Virus-specific CD8 T Cells 
Antigen-specific CD8 T cells play a critical role in controlling HIV infection but eventually lose antiviral functions in part because of expression and signaling through the inhibitory PD-1 receptor. To better understand the impact of prolonged TCR ligation on regulation of PD-1 expression in HIV-specific CD8 T cells we investigated the capacity of virus-specific CD8 T cells to modify the PD-1 epigenetic program following reduction in viral load. We observed that the transcriptional regulatory region was unmethylated in the PD-1hi HIV-specific CD8 T cells while it remained methylated in donor matched naïve cells at acute and chronic stages of infection. Surprisingly, the PD-1 promoter remained unmethylated in HIV-specific CD8 T cells from subjects with a viral load controlled by antiviral therapy for greater than 2 years or from elite controllers. Together these data demonstrate that the epigenetic program at the PD-1 locus becomes fixed following prolonged exposure to HIV virus.
PMCID: PMC3702641  PMID: 23772031
25.  Strategies to Query and Display Allergy-Derived Epitope Data from the Immune Epitope Database 
The recognition of specific epitopes on allergens by antibodies and T cells is a key element in allergic processes. Analysis of epitope data may be of interest for basic immunopathology or for potential application in diagnostics or immunotherapy. The Immune Epitope Database (IEDB) is a freely available repository of epitope data from infectious disease agents, as well as epitopes defined for allergy, autoimmunity, and transplantation. The IEDB curates the experiments associated with each epitope and thus provides a variety of different ways to search the data. This review aims to demonstrate the utility of the IEDB and its query strategies, including searching by epitope structure (peptidic/nonpeptidic), by assay methodology, by host, by the allergen itself, or by the organism from which the allergen was derived. Links to tools for visualization of 3-D structures, epitope prediction, and analyses of B and T cell reactivity by host response frequency score are also highlighted.
PMCID: PMC3629817  PMID: 23172234
Allergy; Allergen; Epitope; Database

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