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1.  Quantification of HTLV-1 Clonality and TCR Diversity 
PLoS Computational Biology  2014;10(6):e1003646.
Estimation of immunological and microbiological diversity is vital to our understanding of infection and the immune response. For instance, what is the diversity of the T cell repertoire? These questions are partially addressed by high-throughput sequencing techniques that enable identification of immunological and microbiological “species” in a sample. Estimators of the number of unseen species are needed to estimate population diversity from sample diversity. Here we test five widely used non-parametric estimators, and develop and validate a novel method, DivE, to estimate species richness and distribution. We used three independent datasets: (i) viral populations from subjects infected with human T-lymphotropic virus type 1; (ii) T cell antigen receptor clonotype repertoires; and (iii) microbial data from infant faecal samples. When applied to datasets with rarefaction curves that did not plateau, existing estimators systematically increased with sample size. In contrast, DivE consistently and accurately estimated diversity for all datasets. We identify conditions that limit the application of DivE. We also show that DivE can be used to accurately estimate the underlying population frequency distribution. We have developed a novel method that is significantly more accurate than commonly used biodiversity estimators in microbiological and immunological populations.
Author Summary
The “unseen species problem” is ubiquitous in biology and is frequently encountered outside its original setting in population ecology. For example, the human retrovirus HTLV-1 persists within hosts in multiple, genetically identical clones of infected cells. However, the number of clones in one host is unknown; this knowledge is required for an understanding of how the virus survives despite a strong host immune response. The problem arises again in estimating the diversity of the T-cell repertoire, which influences adaptive immunity. For example, the T-cell diversity may influence the outcome of viral challenge. While there have been numerous attempts to address the unseen species problem, there is currently no consensus on how to do so in immunology and microbiology. The aim of this study was to identify a suitable method to estimate the number of species in immunological and microbiological populations. We found that five existing estimators we tested performed poorly across three data sources (HTLV-1 clonality, T cell receptor, and microbial data). We therefore developed a new estimator, DivE, which significantly outperformed the other estimators. Accurate diversity quantification allows better evaluation of the impact on immunity from factors such as ageing and infection.
PMCID: PMC4063693  PMID: 24945836
2.  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
3.  Bcl6 expressing follicular helper CD4 T cells are fate committed early and have the capacity to form memory 
Follicular helper CD4 T cells (Tfh) are a distinct type of differentiated CD4 T cells uniquely specialized for B cell help. Here we examined Tfh cell fate commitment, including distinguishing features of Tfh versus Th1 proliferation and survival. Using cell transfer approaches at early time points after an acute viral infection, we demonstrate that early Tfh cells and Th1 cells are already strongly cell fate committed by day three. Nevertheless, Tfh cell proliferation was tightly regulated in a TCR-dependent manner. The Tfh cells still depend on extrinsic cell fate cues from B cells in their physiological in vivo environment. Unexpectedly, we found that Tfh cells share a number of phenotypic parallels with memory precursor CD8 T cells, including selective upregulation of IL7Rα and a collection of co-regulated genes. As a consequence, the early Tfh cells can progress to robustly form memory cells. These data support the hypothesis that CD4 and CD8 T cells share core aspects of a memory cell precursor gene expression program involving Bcl6, and a strong relationship exists between Tfh cells and memory CD4 T cell development.
PMCID: PMC3626566  PMID: 23487426
Tfh cells; germinal center; LCMV infection; proliferation capacity; IL-7R; vaccine
4.  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
6.  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
7.  Temporal Intra-Individual Variation of Immunological Biomarkers in Type 1 Diabetes Patients: Implications for Future Use in Cross-Sectional Assessment 
PLoS ONE  2013;8(11):e79383.
Multiple immune parameters such as frequencies of autoreactive CD4+, CD8+ T-cells and CD4+CD25+Foxp3+ T-cells have been explored as biomarkers in human T1D. However, intra-individual temporal variation of these parameters has not been assessed systematically over time. We determined the variation in each of these parameters in a cohort of T1D and healthy donors (HDs), at monthly intervals for one year. Despite low intra- and inter-assay co-efficient of variation (CV), mean CVs for each of the immune parameters were 119.1% for CD4+ T-cell-derived IFN-γ, 50.44% for autoreactive CD8+ T-cells, and 31.24% for CD4+CD25+Foxp3+ T-cells. Further, both HDs and T1D donors had similar CVs. The variation neither correlated with BMI, age, disease duration or insulin usage, nor were there detectable cyclical patterns of variation. However, averaging results from multiple visits for an individual provided a better estimate of the CV between visits. Based on our data we predict that by averaging values from three visits a treatment effect on these parameters with a 50% effect size could be detected with the same power using 1.8–4-fold fewer patients within a trial compared to using values from a single visit. Thus, our present data contribute to a more robust, accurate endpoint design for future clinical trials in T1D and aid in the identification of truly efficacious therapies.
PMCID: PMC3817042  PMID: 24223938
8.  Ex vivo tetramer staining and cell surface phenotyping for early activation markers CD38 and HLA-DR to enumerate and characterize malaria antigen-specific CD8+ T-cells induced in human volunteers immunized with a Plasmodium falciparum adenovirus-vectored malaria vaccine expressing AMA1 
Malaria Journal  2013;12:376.
Malaria is responsible for up to a 600,000 deaths per year; conveying an urgent need for the development of a malaria vaccine. Studies with whole sporozoite vaccines in mice and non-human primates have shown that sporozoite-induced CD8+ T cells targeting liver stage antigens can mediate sterile protection. There is a need for a direct method to identify and phenotype malaria vaccine-induced CD8+ T cells in humans.
Fluorochrome-labelled tetramers consisting of appropriate MHC class I molecules in complex with predicted binding peptides derived from Plasmodium falciparum AMA-1 were used to label ex vivo AMA-1 epitope specific CD8+ T cells from research subjects responding strongly to immunization with the NMRC-M3V-Ad-PfCA (adenovirus-vectored) malaria vaccine. The identification of these CD8+ T cells on the basis of their expression of early activation markers was also investigated.
Analyses by flow cytometry demonstrated that two of the six tetramers tested: TLDEMRHFY: HLA-A*01:01 and NEVVVKEEY: HLA-B*18:01, labelled tetramer-specific CD8+ T cells from two HLA-A*01:01 volunteers and one HLA-B*18:01 volunteer, respectively. By contrast, post-immune CD8+ T cells from all six of the immunized volunteers exhibited enhanced expression of the CD38 and HLA-DRhi early activation markers. For the three volunteers with positive tetramer staining, the early activation phenotype positive cells included essentially all of the tetramer positive, malaria epitope- specific CD8+ T cells suggesting that the early activation phenotype could identify all malaria vaccine-induced CD8+ T cells without prior knowledge of their exact epitope specificity.
The results demonstrated that class I tetramers can identify ex vivo malaria vaccine antigen-specific CD8+ T cells and could therefore be used to determine their frequency, cell surface phenotype and transcription factor usage. The results also demonstrated that vaccine antigen-specific CD8+ T cells could be identified by activation markers without prior knowledge of their antigen-specificity, using a subunit vaccine for proof-of-concept. Whether, whole parasite or adjuvanted protein vaccines will also induce {CD38 and HLA-DRhi}+ CD8+ T cell populations reflective of the antigen-specific response will the subject of future investigations.
PMCID: PMC3819688  PMID: 24168370
Malaria; CD8+ T cells; MHC-I tetramers; Activation markers
9.  Properties of MHC Class I Presented Peptides That Enhance Immunogenicity 
PLoS Computational Biology  2013;9(10):e1003266.
T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.
Author Summary
T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Some peptide-MHC-I complexes (pMHCs) are better recognized by T-cells; we call such pMHCs more immunogenic. For other pMHCs, no recognizing T-cells seem to exist; we call such pMHCs non-immunogenic. We set out to determine which properties of pMHCs cause such differences in immunogenicity, by carefully collecting a large set of immunogenic and non-immunogenic pMHCs, and analysing the difference between these sets. Two important observations were made: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, seem to be better recognized by T-cells as they associate with immunogenicity. Next, this information was combined into a simple model to predict the immunogenicity of new pMHCs (this model is made available at Interestingly, with this model we could show that T-cells are equipped to strongly recognize viral peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.
PMCID: PMC3808449  PMID: 24204222
10.  Evaluating the Immunogenicity of Protein Drugs by Applying In Vitro MHC Binding Data and the Immune Epitope Database and Analysis Resource 
The immune system has evolved to become highly specialized in recognizing and responding to pathogens and foreign molecules. Specifically, the function of HLA class II is to ensure that a sufficient sample of peptides derived from foreign molecules is presented to T cells. This leads to an important concern in human drug development as the possible immunogenicity of biopharmaceuticals, especially those intended for chronic administration, can lead to reduced efficacy and an undesired safety profile for biological therapeutics. As part of this review, we will highlight the molecular basis of antigen presentation as a key step in the induction of T cell responses, emphasizing the events associated with peptide binding to polymorphic and polygenic HLA class II molecules. We will further review methodologies that predict HLA class II binding peptides and candidate epitopes. We will focus on tools provided by the Immune Epitope Database and Analysis Resource, discussing the basic features of different prediction methods, the objective evaluation of prediction quality, and general guidelines for practical use of these tools. Finally the use, advantages, and limitations of the methodology will be demonstrated in a review of two previous studies investigating the immunogenicity of erythropoietin and timothy grass pollen.
PMCID: PMC3816028  PMID: 24222776
11.  T cell responses to known allergen proteins are differently polarized and account for a variable fraction of total response to allergen extracts1 
A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds and indoor allergens, was surveyed utilizing prediction of HLA class II binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens where the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for “low epitope coverage” allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γresponses were measured as prototype Th2 and Th1 responses, respectively. While in some cases (e.g., Orchard Grass, Alternaria, Cypress, and Russian Thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and Alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of SIT treatment or varying disease severity (asthma or rhinitis).
PMCID: PMC3411923  PMID: 22786768
12.  Unusual Features of Vaccinia Virus Extracellular Virion Form Neutralization Resistance Revealed in Human Antibody Responses to the Smallpox Vaccine 
Journal of Virology  2013;87(3):1569-1585.
The extracellular virion form (EV) of vaccinia virus (VACV) is essential for viral pathogenesis and is difficult to neutralize with antibodies. Why this is the case and how the smallpox vaccine overcomes this challenge remain incompletely understood. We previously showed that high concentrations of anti-B5 antibodies are insufficient to directly neutralize EV (M. R. Benhnia, et al., J. Virol. 83:1201–1215, 2009). This allowed for at least two possible interpretations: covering the EV surface is insufficient for neutralization, or there are insufficient copies of B5 to allow anti-B5 IgG to cover the whole surface of EV and another viral receptor protein remains active. We endeavored to test these possibilities, focusing on the antibody responses elicited by immunization against smallpox. We tested whether human monoclonal antibodies (MAbs) against the three major EV antigens, B5, A33, and A56, could individually or together neutralize EV. While anti-B5 or anti-A33 (but not anti-A56) MAbs of appropriate isotypes were capable of neutralizing EV in the presence of complement, a mixture of anti-B5, anti-A33, and anti-A56 MAbs was incapable of directly neutralizing EV, even at high concentrations. This remained true when neutralizing the IHD-J strain, which lacks a functional version of the fourth and final known EV surface protein, A34. These immunological data are consistent with the possibility that viral proteins may not be the active component of the EV surface for target cell binding and infectivity. We conclude that the protection afforded by the smallpox vaccine anti-EV response is predominantly mediated not by direct neutralization but by isotype-dependent effector functions, such as complement recruitment for antibodies targeting B5 and A33.
PMCID: PMC3554146  PMID: 23152530
13.  Analysis of T cell responses to the major allergens from German cockroach: epitope specificity and relationship to IgE production1 
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 (SIT). 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 over 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, while 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 co-presented immunomodulators. In comparing antibody 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-B help might support development of IgE responses. Finally, SIT 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.
PMCID: PMC3392449  PMID: 22706084
14.  Identification of minimal human MHC-restricted CD8+ T-cell epitopes within the Plasmodium falciparum circumsporozoite protein (CSP) 
Malaria Journal  2013;12:185.
Plasmodium falciparum circumsporozoite protein (CSP) is a leading malaria vaccine candidate antigen, known to elicit protective antibody responses in humans (RTS,S vaccine). Recently, a DNA prime / adenovirus (Ad) vector boost vaccine encoding CSP and a second P. falciparum antigen, apical membrane antigen-1, also elicited sterile protection, but in this case associated with interferon gamma ELISpot and CD8+ T cell but not antibody responses. The finding that CSP delivered by an appropriate vaccine platform likely elicits protective cell-mediated immunity provided a rationale for identifying class I-restricted epitopes within this leading vaccine candidate antigen.
Limited samples of peripheral blood mononuclear cells from clinical trials of the Ad vaccine were used to identify CD8+ T cell epitopes within pools of overlapping 15mer peptides spanning portions of CSP that stimulated recall responses. Computerized algorithms (NetMHC) predicted 17 minimal class I-restricted 9-10mer epitopes within fifteen 15mers positive in ELISpot assay using PBMC from 10 HLA-matched study subjects. Four additional epitopes were subsequently predicted using NetMHC, matched to other study subjects without initial 15mer ELISpot screening. Nine of the putative epitopes were synthesized and tested by ELISpot assay, and six of these nine were further tested for CD8+ T cell responses by ELISpot CD4+ and CD8+ T cell-depletion and flow cytometry assays for evidence of CD8+ T cell dependence.
Each of the nine putative epitopes, all sequence-conserved, recalled responses from HLA-matched CSP-immunized research subjects. Four shorter sequences contained within these sequences were identified using NetMHC predictions and may have contributed to recall responses. Five (9-10mer) epitopes were confirmed to be targets of CD8+ T cell responses using ELISpot depletion and ICS assays. Two 9mers among these nine epitopes were each restricted by two HLA supertypes (A01/B07; A01A24/A24) and one 9mer was restricted by three HLA supertypes (A01A24/A24/B27) indicating that some CSP class I-restricted epitopes, like DR epitopes, may be HLA-promiscuous.
This study identified nine and confirmed five novel class I epitopes restricted by six HLA supertypes, suggesting that an adenovirus-vectored CSP vaccine would be immunogenic and potentially protective in genetically diverse populations.
PMCID: PMC3683343  PMID: 23738590
Malaria; Vaccine; Circumsporozoite protein; ELISpot; Flow cytometry; NetMHC; Epitope mapping; Class I restriction; Localization
15.  Dissecting Mechanisms of Immunodominance to the Common TB Antigens ESAT-6, CFP10, Rv2031c (hspX), Rv2654c (TB7.7) and Rv1038c (EsxJ)1 
Diagnosis of tuberculosis often relies on the ex vivo interferon gamma release assays QuantiFERON-TB Gold In-Tube and T-SPOT.TB. However, understanding of the immunological mechanisms underlying their diagnostic utility is still incomplete. Accordingly, we investigated T cell responses for the TB antigens included in the these assays and other commonly studied antigens; ESAT-6, CFP10, Rv2031c, Rv2654c, and Rv1038c. PBMC from latently infected individuals were tested in ex vivo ELISPOT assays with overlapping peptides spanning the entirety of these antigens. We found striking variations in prevalence and magnitude of ex vivo reactivity, with CFP10 being most dominant, followed by ESAT-6 and Rv2654c being virtually inactive. Rv2031c and Rv1038c were associated with intermediate patterns of reactivity. Further studies showed that low reactivity was not due to lack of HLA binding peptides, and high reactivity was associated with recognition of a few discrete dominant antigenic regions. Different donors recognized the same core sequence in a given epitope. In some cases the identified epitopes were restricted by a single specific common HLA molecule (selective restriction), while in other cases promiscuous restriction of the same epitope by multiple HLA molecules was apparent. Definition of the specific restricting HLA allowed to produce tetrameric reagents and show that epitope-specific T cells recognizing either selectively or promiscuously restricted epitopes were predominantly T effector memory (TEM). In conclusion, these results highlight the feasibility of more clearly defined TB diagnostic reagent.
PMCID: PMC3345088  PMID: 22504645
16.  Human Germline Antibody Gene Segments Encode Polyspecific Antibodies 
PLoS Computational Biology  2013;9(4):e1003045.
Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three VH gene segments. Panels of somatically mutated antibodies encoded by a common VH gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the VH gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.
Author Summary
Human antibodies are critical for eradication of viral and bacterial infections, while providing the basis for immunological memory. Antibody protein molecules are encoded by several recombined germline gene segments prior to antigen exposure. The initial set of antibodies that are generated by recombination in the bone marrow is the antigen-naïve antibody repertoire. It is of great interest to know how a finite set of such germline gene-encoded antibodies can recognize the large number of possible foreign antigens. A current hypothesis in the field suggests that antibodies encoded by germline gene segments are structurally flexible and able to accommodate binding to many antigens, much like one glove fitting the shape of many hands. The phenomenon of one structure binding to many targets is known as polyspecificity. Here we further support this hypothesis by showing that entire antibody protein variable regions encoded by germline gene segments are close to ideal for polyspecificity. We used computational design algorithms to explore antibody sequence space rapidly and predict optimal sequences to achieve polyspecificity. The resulting designed sequences recapitulated the germline gene segment sequences and highlighted residues critical for achieving polyspecificity. These results suggest how a finite set of antibody germline gene segments can encode antibodies that can engage a large number of antigens.
PMCID: PMC3636087  PMID: 23637590
17.  Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes 
Immunogenetics  2011;63(6):325-335.
Previous studies have attempted to define human leukocyte antigen (HLA) class II supertypes, analogous to the case for class I, on the basis of shared peptide-binding motifs or structure. In the present study, we determined the binding capacity of a large panel of non-redundant peptides for a set of 27 common HLA DR, DQ, and DP molecules. The measured binding data were then used to define class II supertypes on the basis of shared binding repertoires. Seven different supertypes (main DR, DR4, DRB3, main DQ, DQ7, main DP, and DP2) were defined. The molecules associated with the respective supertypes fell largely along lines defined by MHC locus and reflect, in broad terms, commonalities in reported peptide-binding motifs. Repertoire overlaps between molecules within the same class II supertype were found to be similar in magnitude to what has been observed for HLA class I supertypes. Surprisingly, however, the degree to which repertoires between molecules in the different class II supertypes also overlapped was found to be five to tenfold higher than repertoire overlaps noted between molecules in different class I supertypes. These results highlight a high degree of repertoire overlap amongst all HLA class II molecules, perhaps reflecting binding in multiple registers, and more pronounced dependence on backbone interactions rather than peptide anchor residues. This fundamental difference between HLA class I and class II would not have been predicted on the basis of analysis of either binding motifs or the sequence/predicted structures of the HLA molecules.
PMCID: PMC3626422  PMID: 21305276
MHC; HLA class I; HLA class II; Peptide binding; T cell epitopes
18.  Query enhancement through the practical application of ontology: the IEDB and OBI 
Journal of Biomedical Semantics  2013;4(Suppl 1):S6.
Ontologies categorize entities, express relationships between them, and provide standardized definitions. Thus, they can be used to present and enforce the specific relationships between database components. The Immune Epitope Database (IEDB, utilizes the Ontology for Biomedical Investigations (OBI) and several additional ontologies to represent immune epitope mapping experiments. Here, we describe our experiences utilizing this representation in order to provide enhanced database search functionality. We applied a simple approach to incorporate the benefits of the information captured in a formal ontology directly into the user web interface, resulting in an improved user experience with minimal changes to the database itself. The integration is easy to maintain, provides standardized terms and definitions, and allows for subsumption queries. In addition to these immediate benefits, our long-term goal is to enable true semantic integration of data and knowledge in the biomedical domain. We describe our progress towards that goal and what we perceive as the main obstacles.
PMCID: PMC3633001  PMID: 23734660
19.  Structural and Biochemical Characterization of the Vaccinia Virus Envelope Protein D8 and Its Recognition by the Antibody LA5 
Journal of Virology  2012;86(15):8050-8058.
Smallpox vaccine is considered a gold standard of vaccines, as it is the only one that has led to the complete eradication of an infectious disease from the human population. B cell responses are critical for the protective immunity induced by the vaccine, yet their targeted epitopes recognized in humans remain poorly described. Here we describe the biochemical and structural characterization of one of the immunodominant vaccinia virus (VACV) antigens, D8, and its binding to the monoclonal antibody LA5, which is capable of neutralizing VACV in the presence of complement. The full-length D8 ectodomain was found to form a tetramer. We determined the crystal structure of the LA5 Fab-monomeric D8 complex at a resolution of 2.1 Å, as well as the unliganded structures of D8 and LA5-Fab at resolutions of 1.42 Å and 1.6 Å, respectively. D8 features a carbonic anhydrase (CAH) fold that has evolved to bind to the glycosaminoglycan (GAG) chondroitin sulfate (CS) on host cells. The central positively charged crevice of D8 was predicted to be the CS binding site by automated docking experiments. Furthermore, sequence alignment of various poxvirus D8 orthologs revealed that this crevice is structurally conserved. The D8 epitope is formed by 23 discontinuous residues that are spread across 80% of the D8 protein sequence. Interestingly, LA5 binds with a high-affinity lock-and-key mechanism above this crevice with an unusually large antibody-antigen interface, burying 2,434 Å2 of protein surface.
PMCID: PMC3421696  PMID: 22623786
20.  Predicting Cell Types and Genetic Variations Contributing to Disease by Combining GWAS and Epigenetic Data 
PLoS ONE  2013;8(1):e54359.
Genome-wide association studies (GWASs) identify single nucleotide polymorphisms (SNPs) that are enriched in individuals suffering from a given disease. Most disease-associated SNPs fall into non-coding regions, so that it is not straightforward to infer phenotype or function; moreover, many SNPs are in tight genetic linkage, so that a SNP identified as associated with a particular disease may not itself be causal, but rather signify the presence of a linked SNP that is functionally relevant to disease pathogenesis. Here, we present an analysis method that takes advantage of the recent rapid accumulation of epigenomics data to address these problems for some SNPs. Using asthma as a prototypic example; we show that non-coding disease-associated SNPs are enriched in genomic regions that function as regulators of transcription, such as enhancers and promoters. Identifying enhancers based on the presence of the histone modification marks such as H3K4me1 in different cell types, we show that the location of enhancers is highly cell-type specific. We use these findings to predict which SNPs are likely to be directly contributing to disease based on their presence in regulatory regions, and in which cell types their effect is expected to be detectable. Moreover, we can also predict which cell types contribute to a disease based on overlap of the disease-associated SNPs with the locations of enhancers present in a given cell type. Finally, we suggest that it will be possible to re-analyze GWAS studies with much higher power by limiting the SNPs considered to those in coding or regulatory regions of cell types relevant to a given disease.
PMCID: PMC3559682  PMID: 23382893
21.  Positional Bias of MHC Class I Restricted T-Cell Epitopes in Viral Antigens Is Likely due to a Bias in Conservation 
PLoS Computational Biology  2013;9(1):e1002884.
The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from Defective Ribosomal Products (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.
Author Summary
To defend the host from an infection, the immune system continuously scans cell surfaces for foreign objects. Specifically, a virus inside a cell exploits the host to make copies of its proteins; viral proteins are broken up into peptide fragments; and the fragments are displayed on the infected cell's surface, thereby allowing detection and cell-killing. How these peptide fragments for cell-surface presentation are generated remains unknown. An understanding of this step will lead to rational design of vaccines and insights into tumor immunosurveillance and autoimmunity. One possible mechanism is that the peptide fragments come from defective proteins missing either the beginning or end regions, which may result in a bias. Here, we analyzed locations of a large set of known viral epitopes, peptide fragments recognized by the immune system, within their proteins. We find that all regions of proteins are represented well by the immune system. However, there is a statistically significant bias in the central regions of proteins, which correlate with a pattern of conservation spanning the length of viral proteins. Our results suggest a combined effect of conservation and enhancement of immune responses through repeated exposures in shaping the distribution of known viral epitopes.
PMCID: PMC3554532  PMID: 23357871
22.  Memory T Cells in Latent Mycobacterium tuberculosis Infection Are Directed against Three Antigenic Islands and Largely Contained in a CXCR3+CCR6+ Th1 Subset 
PLoS Pathogens  2013;9(1):e1003130.
An understanding of the immunological footprint of Mycobacterium tuberculosis (MTB) CD4 T cell recognition is still incomplete. Here we report that human Th1 cells specific for MTB are largely contained in a CXCR3+CCR6+ memory subset and highly focused on three broadly immunodominant antigenic islands, all related to bacterial secretion systems. Our results refute the notion that secreted antigens act as a decoy, since both secreted proteins and proteins comprising the secretion system itself are targeted by a fully functional T cell response. In addition, several novel T cell antigens were identified which can be of potential diagnostic use, or as vaccine antigens. These results underline the power of a truly unbiased, genome-wide, analysis of CD4 MTB recognition based on the combined use of epitope predictions, high throughput ELISPOT, and T cell libraries using PBMCs from individuals latently infected with MTB.
Author Summary
Mycobacterium tuberculosis is one of the most life-threatening pathogens of all time, having infected one-third of the present human population. There is an urgent need for both novel vaccines and diagnostic strategies. Here, we were able to identify the targets most dominantly recognized by latently infected individual that successfully contain infection. These targets are contained in three broadly genomic antigenic islands, all related to bacterial secretion systems and composed by several distinct ORFs. Thus, our results suggest that vaccination with one or few defined antigens will fail to replicate the response associated with natural immunity. Our analysis also pinpoints that the Th1 cells dominating the response are associated with novel and well-defined phenotypic markers, suggesting that the response is molded by unique MTB associated factors. This study demonstrates further that the approach combining peptide binding predictions with modern high throughput techniques is generally applicable to the study of immunity to other complex pathogens. Together, our data provide a new angle in the worldwide fight against M. tuberculosis and could be used for diagnostic or vaccine developments.
PMCID: PMC3554618  PMID: 23358848
23.  Reliable B Cell Epitope Predictions: Impacts of Method Development and Improved Benchmarking 
PLoS Computational Biology  2012;8(12):e1002829.
The interaction between antibodies and antigens is one of the most important immune system mechanisms for clearing infectious organisms from the host. Antibodies bind to antigens at sites referred to as B-cell epitopes. Identification of the exact location of B-cell epitopes is essential in several biomedical applications such as; rational vaccine design, development of disease diagnostics and immunotherapeutics. However, experimental mapping of epitopes is resource intensive making in silico methods an appealing complementary approach. To date, the reported performance of methods for in silico mapping of B-cell epitopes has been moderate. Several issues regarding the evaluation data sets may however have led to the performance values being underestimated: Rarely, all potential epitopes have been mapped on an antigen, and antibodies are generally raised against the antigen in a given biological context not against the antigen monomer. Improper dealing with these aspects leads to many artificial false positive predictions and hence to incorrect low performance values. To demonstrate the impact of proper benchmark definitions, we here present an updated version of the DiscoTope method incorporating a novel spatial neighborhood definition and half-sphere exposure as surface measure. Compared to other state-of-the-art prediction methods, Discotope-2.0 displayed improved performance both in cross-validation and in independent evaluations. Using DiscoTope-2.0, we assessed the impact on performance when using proper benchmark definitions. For 13 proteins in the training data set where sufficient biological information was available to make a proper benchmark redefinition, the average AUC performance was improved from 0.791 to 0.824. Similarly, the average AUC performance on an independent evaluation data set improved from 0.712 to 0.727. Our results thus demonstrate that given proper benchmark definitions, B-cell epitope prediction methods achieve highly significant predictive performances suggesting these tools to be a powerful asset in rational epitope discovery. The updated version of DiscoTope is available at
Author Summary
The human immune system has an incredible ability to fight pathogens (bacterial, fungal and viral infections). One of the most important immune system events involved in clearing infectious organisms is the interaction between the antibodies and antigens (molecules such as proteins from the pathogenic organism). Antibodies bind to antigens at sites known as B-cell epitopes. Hence, identification of areas on the surface antigens capable of binding to antibodies (also known as B-cell epitopes) may aid the development of various immune related applications (e.g. vaccines and immunotherapeutic). However, experimental identification of B-cell epitopes is a resource intensive task, thereby making computer-aided methods an appealing complementary approach. Previously reported performances of methods for B cell epitope predictive have been moderate. Here, we present an updated version of the B-cell epitope prediction method; DiscoTope, that on the basis of a protein structure and epitope propensity scores predicts residues likely to be involved in B-cell epitopes. We demonstrate that the low performances to some extent can be explained by poorly defined benchmarks, and that inclusion of additional biological information greatly enhances the predictive performance. This suggests that, given proper benchmark definitions, state-of-the-art B cell epitope prediction methods perform significantly better than generally assumed.
PMCID: PMC3531324  PMID: 23300419
24.  Applications for T-cell epitope queries and tools in the Immune Epitope Database and Analysis Resource 
Journal of immunological methods  2010;374(1-2):62-69.
The Immune Epitope Database and Analysis Resource (IEDB, hosts a continuously growing set of immune epitope data curated from the literature, as well as data submitted directly by experimental scientists. In addition, the IEDB hosts a collection of prediction tools for both MHC class I and II restricted T-cell epitopes that are regularly updated. In this review, we provide an overview of T-cell epitope data and prediction tools provided by the IEDB. We then illustrate effective use of these resources to support experimental studies. We focus on two applications, namely identification of conserved epitopes in novel strains of a previously studied pathogen, and prediction of novel T-cell epitopes to facilitate vaccine design. We address common questions and concerns faced by users, and identify patterns of usage that have proven successful.
PMCID: PMC3041860  PMID: 21047510
epitope conservation; epitope predictions; vaccine design; Major Histocompatibility Complex
25.  Insights into HLA restricted T cell responses in a novel mouse model of dengue virus infection point towards new implications for vaccine design1 
The frequency of dengue virus (DENV) infection has increased dramatically in the last few decades, and the lack of a vaccine has led to significant morbidity and mortality worldwide. To date, a convenient murine system to study human T cell responses to DENV has not been available. Mice transgenic for human leukocyte antigens (HLA) are widely used to model human immune responses and it has been shown that mouse-passaged DENV is able to replicate to significant levels in IFN-α/βR−/− mice. To cover a wide range of HLA phenotypes, we backcrossed IFN-α/βR−/− mice with HLA A*0201, A*0101, A*1101, B*0702 and DRB1*0101 transgenic mice. A DENV proteome-wide screen identified a total of 42 epitopes across all HLA-transgenic IFN-α/βR−/− strains tested. In contrast only 8 of these elicited responses in the corresponding IFN-α/βR+/+ mice. We were able to identify T cell epitopes from 9 out of the 10 DENV proteins. However, the majority of responses were derived from the highly conserved nonstructural proteins NS3 and NS5. The relevance of this model is further demonstrated by the fact that most of the epitopes identified in our murine system are also recognized by PBMC from DENV exposed human donors, and a dominance of HLA B*0702 restricted responses has been detected in both systems. Our results provide new insights into HLA-restricted T cell responses against DENV, and we herein describe a novel murine model, which allows the investigation of T cell-mediated immune mechanisms relevant to vaccine design.
PMCID: PMC3186824  PMID: 21918184

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