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1.  An ontology for major histocompatibility restriction 
MHC molecules are a highly diverse family of proteins that play a key role in cellular immune recognition. Over time, different techniques and terminologies have been developed to identify the specific type(s) of MHC molecule involved in a specific immune recognition context. No consistent nomenclature exists across different vertebrate species.
To correctly represent MHC related data in The Immune Epitope Database (IEDB), we built upon a previously established MHC ontology and created an ontology to represent MHC molecules as they relate to immunological experiments.
This ontology models MHC protein chains from 16 species, deals with different approaches used to identify MHC, such as direct sequencing verses serotyping, relates engineered MHC molecules to naturally occurring ones, connects genetic loci, alleles, protein chains and multi-chain proteins, and establishes evidence codes for MHC restriction. Where available, this work is based on existing ontologies from the OBO foundry.
Overall, representing MHC molecules provides a challenging and practically important test case for ontology building, and could serve as an example of how to integrate other ontology building efforts into web resources.
Electronic supplementary material
The online version of this article (doi:10.1186/s13326-016-0045-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4709943  PMID: 26759709
Major histocompatibility complex; Ontology; MHC; Immune epitope
2.  RSV-specific airway resident memory CD8+ T cells and differential disease severity after experimental human infection 
Nature Communications  2015;6:10224.
In animal models, resident memory CD8+ T (Trm) cells assist in respiratory virus elimination but their importance in man has not been determined. Here, using experimental human respiratory syncytial virus (RSV) infection, we investigate systemic and local virus-specific CD8+ T-cell responses in adult volunteers. Having defined the immunodominance hierarchy, we analyse phenotype and function longitudinally in blood and by serial bronchoscopy. Despite rapid clinical recovery, we note surprisingly extensive lower airway inflammation with persistent viral antigen and cellular infiltrates. Pulmonary virus-specific CD8+ T cells display a CD69+CD103+ Trm phenotype and accumulate to strikingly high frequencies into convalescence without continued proliferation. While these have a more highly differentiated phenotype, they express fewer cytotoxicity markers than in blood. Nevertheless, their abundance before infection correlates with reduced symptoms and viral load, implying that CD8+ Trm cells in the human lung can confer protection against severe respiratory viral disease when humoral immunity is overcome.
Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections. Here the authors analyse cellular immune responses of individuals experimentally infected with RSV and reveal the presence of high frequencies of virus- specific resident memory CD8+ T cells in the airway, which correlate with improved viral control.
PMCID: PMC4703893  PMID: 26687547
3.  CD4 T cells specific for a latency-associated γ-herpesvirus epitope are polyfunctional and cytotoxic1 
The oncogenic γ-herpesviruses EBV and KSHV are ubiquitous human pathogens that establish lifelong latent infections maintained by intermittent viral reactivation and reinfection. Effector CD4 T cells are critical for control of viral latency and in immune therapies for virus-associated tumors. Here we exploited γHV68 infection of mice to enhance our understanding of the CD4 T cell response during γ-herpesvirus infection. Using a consensus prediction approach, we identified 16 new CD4 epitope-specific responses that arise during lytic infection. An additional epitope encoded by the M2 protein induced uniquely latency-associated CD4 T cells, which were not detected at the peak of lytic infection but only during latency, and were not induced after infection with a latency-deficient virus. M2-specific CD4 T cells were selectively cytotoxic, produced multiple antiviral cytokines, and sustained IL-2 production. Identification of latency-associated cytolytic CD4 T cells will aid in dissecting mechanisms of CD4 immune control of γ-herpesvirus latency and the development of therapeutic approaches to control viral reactivation and pathology.
PMCID: PMC4301266  PMID: 25378595
4.  Immune Responses in Acute and Convalescent Patients with Mild, Moderate and Severe Disease during the 2009 Influenza Pandemic in Norway 
PLoS ONE  2015;10(11):e0143281.
Increased understanding of immune responses influencing clinical severity during pandemic influenza infection is important for improved treatment and vaccine development. In this study we recruited 46 adult patients during the 2009 influenza pandemic and characterized humoral and cellular immune responses. Those included were either acute hospitalized or convalescent patients with different disease severities (mild, moderate or severe). In general, protective antibody responses increased with enhanced disease severity. In the acute patients, we found higher levels of TNF-α single-producing CD4+T-cells in the severely ill as compared to patients with moderate disease. Stimulation of peripheral blood mononuclear cells (PBMC) from a subset of acute patients with peptide T-cell epitopes showed significantly lower frequencies of influenza specific CD8+ compared with CD4+ IFN-γ T-cells in acute patients. Both T-cell subsets were predominantly directed against the envelope antigens (HA and NA). However, in the convalescent patients we found high levels of both CD4+ and CD8+ T-cells directed against conserved core antigens (NP, PA, PB, and M). The results indicate that the antigen targets recognized by the T-cell subsets may vary according to the phase of infection. The apparent low levels of cross-reactive CD8+ T-cells recognizing internal antigens in acute hospitalized patients suggest an important role for this T-cell subset in protective immunity against influenza.
PMCID: PMC4659565  PMID: 26606759
5.  IL-10 producing intestinal macrophages prevent excessive anti-bacterial innate immunity by limiting IL-23 synthesis 
Nature communications  2015;6:7055.
Innate immune responses are regulated in the intestine to prevent excessive inflammation. Here we show that a subset of mouse colonic macrophages constitutively produce the anti-inflammatory cytokine IL-10. In mice infected with Citrobacter rodentium, a model for enteropathogenic Escherichia coli infection in humans, these macrophages are required to prevent intestinal pathology. IL-23 is significantly increased in infected mice with a myeloid cell-specific deletion of IL-10, and the addition of IL-10 reduces IL-23 production by intestinal macrophages. Furthermore, blockade of IL-23 leads to reduced mortality in the context of macrophage IL-10 deficiency. Transcriptome and other analyses indicate that IL-10-expressing macrophages receive an autocrine IL-10 signal. Interestingly, only transfer of the IL-10 positive macrophages could rescue IL-10 deficient infected mice. Therefore, these data indicate a pivotal role for intestinal macrophages that constitutively produce IL-10, in controlling excessive innate immune activation and preventing tissue damage after an acute bacterial infection.
PMCID: PMC4428691  PMID: 25959063
6.  Association Between Specific Timothy Grass Antigens and Changes in Thelper 1 and 2 Cell Responses Following Specific Immunotherapy 
Different populations of T cells are involved in the pathogenesis of allergic diseases.
We investigated changes in T-helper (Th) cell populations in patients with allergies following specific immunotherapy (SIT).
Peripheral blood mononuclear cells (PBMC) were isolated from patients with allergies who received specific immunotherapy (SIT) and those who did not (controls). We tested the ability of peptides from 93 Timothy grass (TG) proteins to induce T-cell responses (cytokine production). We used ELISPOT and staining assays for intracellular cytokines to measure production of interleukin (IL)4, IL5, IL13, interferon (IFN)γ, and IL10.
Compared with PBMC from controls, PBMC from patients who received SIT produced lower levels of Th2 cytokines upon incubation with several different TG peptides. These data were used to select 20 peptides to be tested an independent cohort of 20 patients with allergies who received SIT and 20 controls. We again observed a significant decrease in production of Th2 cytokines, and an increase in production of the Th1 cytokine IFNγ, in PBMC from the validation groups. These changes correlated with improved symptoms after SIT. Immunization with this selected pool of peptides (or their associated antigens) could protect a substantial proportion of the population from TG allergy.
We observed a significant decrease in production of Th2 cytokines by PBMC from patients who received SIT for TG allergy, compared with those who did not. These changes might be used to monitor response to therapy. The decrease occurred in response to antigens that elicit little (if any) immunoglobulin (Ig)E responses; these antigens might be developed for use in immunotherapy.
PMCID: PMC4253970  PMID: 25042980
T cells; specific immunotherapy; Timothy grass; cytokine
7.  Automatic Generation of Validated Specific Epitope Sets 
Journal of Immunology Research  2015;2015:763461.
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.
PMCID: PMC4629045  PMID: 26568965
8.  Transcriptional profile of TB antigen-specific T cells reveals novel multifunctional features1 
In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4+ T cells as important contributors. Here we show that TB-specific CD4+ T cells have a characteristic chemokine expression signature (CCR6+CXCR3+CCR4−), and that the overall number of these cells is significantly increased in LTBI donors compared to healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6+CXCR3+CCR4− cells and find significant differences to conventional Th1, Th17 and Th2 cells, but no major changes between healthy and LTBI donors. CCR6+CXCR3+CCR4− cells display linage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6+CXCR3+CCR4− cells reveals characteristics important for controlling latent TB infections.
PMCID: PMC4157075  PMID: 25092889
9.  Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer 
PLoS Pathogens  2015;11(9):e1005148.
Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.
Author Summary
Before the eradication of smallpox (variola virus) from nature, hundreds of million of people succumbed to the infection. The discovery of vaccinia virus (VACV), the active ingredient of the smallpox vaccine, ultimately led to the eradiation of smallpox from the human population. Vaccination with VACV leads to a strong antibody response that protects against variola virus. As the protective antibodies recognize viral proteins that are highly similar in sequence between the different orthopox strains, such as A33 used in this study, several antibodies have the capacity to neutralize a larger breath of orthopx viruses. In this study we have identified an anti-A33 antibody from a larger panel that exhibits a unique binding mode to A33. This antibody, A27D7, is also resistant to single amino acid changes throughout the protein and binds to engineered A33 variants that mimic ectromelia and orthopox A33 in the antibody-binding site. As the antibody further protects against ectromelia infection of mice, this antibody appears to be a potent orthopox cross-species protective antibody with therapeutic potential.
PMCID: PMC4556652  PMID: 26325270
10.  In vivo RNA interference screens identify regulators of antiviral CD4+ and CD8+ T cell differentiation 
Immunity  2014;41(2):325-338.
Classical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time-consuming. Here we developed a pooled approach to screen 30–100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4+ and CD8+ T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper-1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTL). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL, and enhanced Tfh and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies.
PMCID: PMC4160313  PMID: 25148027
11.  The Human CD8+ T Cell Responses Induced by a Live Attenuated Tetravalent Dengue Vaccine Are Directed against Highly Conserved Epitopes 
Journal of Virology  2014;89(1):120-128.
The incidence of infection with any of the four dengue virus serotypes (DENV1 to -4) has increased dramatically in the last few decades, and the lack of a treatment or vaccine has contributed to significant morbidity and mortality worldwide. A recent comprehensive analysis of the human T cell response against wild-type DENV suggested an human lymphocyte antigen (HLA)-linked protective role for CD8+ T cells. We have collected one-unit blood donations from study participants receiving the monovalent or tetravalent live attenuated DENV vaccine (DLAV), developed by the U.S. National Institutes of Health. Peripheral blood mononuclear cells from these donors were screened in gamma interferon enzyme-linked immunosorbent spot assays with pools of predicted, HLA-matched, class I binding peptides covering the entire DENV proteome. Here, we characterize for the first time CD8+ T cell responses after live attenuated dengue vaccination and show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue infection. Interestingly, whereas broad responses to structural and nonstructural (NS) proteins were observed after monovalent vaccination, T cell responses following tetravalent vaccination were, dramatically, focused toward the highly conserved NS proteins. Epitopes were highly conserved in a vast variety of field isolates and able to elicit multifunctional T cell responses. Detailed knowledge of the T cell response will contribute to the identification of robust correlates of protection in natural immunity and following vaccination against DENV.
IMPORTANCE The development of effective vaccination strategies against dengue virus (DENV) infection and clinically significant disease is a task of high global public health value and significance, while also being a challenge of significant complexity. A recent efficacy trial of the most advanced dengue vaccine candidate, demonstrated only partial protection against all four DENV serotypes, despite three subsequent immunizations and detection of measurable neutralizing antibodies to each serotype in most subjects. These results challenge the hypothesis that seroconversion is the only reliable correlate of protection. Here, we show that CD8+ T cell responses in vaccinees were readily detectable and comparable to natural dengue virus infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection in natural immunity and vaccination against DENV.
PMCID: PMC4301095  PMID: 25320311
12.  Acyclovir Has Low but Detectable Influence on HLA-B*57:01 Specificity without Inducing Hypersensitivity 
PLoS ONE  2015;10(5):e0124878.
Immune mediated adverse drug reactions (IM-ADRs) remain a significant source of patient morbidity that have more recently been shown to be associated with specific class I and/or II human leukocyte antigen (HLA) alleles. Abacavir-induced hypersensitivity syndrome is a CD8+ T cell dependent IM-ADR that is exclusively mediated by HLA-B*57:01. We and others have previously shown that abacavir can occupy the floor of the peptide binding groove of HLA-B*57:01 molecules, increasing the affinity of certain self peptides resulting in an altered peptide-binding repertoire. Here, we have identified another drug, acyclovir, which appears to act in a similar fashion. As with abacavir, acyclovir showed a dose dependent increase in affinity for peptides with valine and isoleucine at their C-terminus. In agreement with the binding studies, HLA-B*57:01 peptide-elution studies performed in the presence of acyclovir revealed an increased number of endogenously bound peptides with a C-terminal isoleucine. Accordingly, we have hypothesized that acyclovir acts by the same mechanism as abacavir, although our data also suggest the overall effect is much smaller: the largest changes of peptide affinity for acyclovir were 2-5 fold, whereas for abacavir this effect was as much as 1000-fold. Unlike abacavir, acyclovir is not known to cause IM-ADRs. We conclude that the modest effect of acyclovir on HLA binding affinity in contrast to the large effect of abacavir is insufficient to trigger a hypersensitivity syndrome. We further support this by functional in vitro studies where acyclovir, unlike abacavir, was unable to produce an increase in IFN-γ upon expansion of HLA-B*57:01+ PBMCs from healthy donors. Using abacavir and acyclovir as examples we therefore propose an in vitro pre-clinical screening strategy, whereby thresholds can be applied to MHC-peptide binding assays to determine the likelihood that a drug could cause a clinically relevant IM-ADR.
PMCID: PMC4449000  PMID: 26024233
13.  Analysis of Human RSV Immunity at the Molecular Level: Learning from the Past and Present 
PLoS ONE  2015;10(5):e0127108.
Human RSV is one of the most prevalent viral pathogens of early childhood for which no vaccine is available. Herein we provide an analysis of RSV epitope data to examine its application to vaccine design and development. Our objective was to provide an overview of antigenic coverage, identify critical antibody and T cell determinants, and then analyze the cumulative RSV epitope data from the standpoint of functional responses using a combinational approach to characterize antigenic structure and epitope location. A review of the cumulative data revealed, not surprisingly, that the vast majority of epitopes have been defined for the two major surface antigens, F and G. Antibody and T cell determinants have been reported from multiple hosts, including those from human subjects following natural infection, however human data represent a minority of the data. A structural analysis of the major surface antigen, F, showed that the majority of epitopes defined for functional antibodies (neutralizing and/or protective) were either shown to bind pre-F or to be accessible in both pre- and post-F forms. This finding may have has implications for on-going vaccine design and development. These interpretations are in agreement with previous work and can be applied in the larger context of functional epitopes on the F protein. It is our hope that this work will provide the basis for further RSV-specific epitope discovery and investigation into the nature of antigen conformation in immunogenicity.
PMCID: PMC4441423  PMID: 26001197
14.  Immunodominance Changes as a Function of the Infecting Dengue Virus Serotype and Primary versus Secondary Infection 
Journal of Virology  2014;88(19):11383-11394.
Dengue virus (DENV) is the causative agent of dengue fever (DF). This disease can be caused by any of four DENV serotypes (DENV1 to -4) which share 67 to 75% sequence homology with one another. The effect of subsequent infections with different serotypes on the T cell repertoire is not fully understood. We utilized mice transgenic for human leukocyte antigens (HLA) lacking the alpha/beta interferon (IFN-α/β) receptor to study responses to heterologous DENV infection. First, we defined the primary T cell response to DENV3 in the context of a wide range of HLA molecules. The primary DENV3 immune response recognized epitopes derived from all 10 DENV proteins, with a significant fraction of the response specific for structural proteins. This is in contrast to primary DENV2 infection, in which structural proteins are a minor component of the response, suggesting differential antigen immunodominance as a function of the infecting serotype. We next investigated the effect of secondary heterologous DENV infection on the T cell repertoire. In the case of both DENV2/3 and DENV3/2 heterologous infections, recognition of conserved/cross-reactive epitopes was either constant or expanded compared to that in homologous infection. Furthermore, in heterologous infection, previous infection with a different serotype impaired the development of responses directed to serotype-specific but not conserved epitopes. Thus, a detrimental effect of previous heterotypic responses might not be due to dysfunctional and weakly cross-reactive epitopes dominating the response. Rather, responses to the original serotype might limit the magnitude of responses directed against epitopes that are either cross-reactive to or specific for the most recently infecting serotype.
IMPORTANCE DENV transmission occurs in more than 100 countries and is an increasing public health problem in tropical and subtropical regions. At present, no effective antiviral therapy or licensed vaccine exists, and treatment is largely supportive in nature. Disease can be caused by any of the four DENV serotypes (DENV1 to -4), which share a high degree of sequence homology with one another. In this study, we have addressed the question of how the T cell repertoire changes as a function of infections with different serotypes and of subsequent heterologous secondary infections. This is of particular interest in the field of dengue viruses, in which secondary infections with different DENV serotypes increase the risk of severe disease. Our results on the evolution of the immune response after primary and secondary infections provide new insights into HLA-restricted T cell responses against DENV relevant for the design of a vaccine against DENV.
PMCID: PMC4178794  PMID: 25056881
15.  Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site of Vulnerability in L1 Protein 
Journal of Virology  2014;88(19):11339-11355.
Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been included in multicomponent DNA or protein vaccines against orthopoxviruses. To further understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1 monoclonal antibodies (MAbs), which clustered into 3 distinct epitope groups. While two groups of anti-L1 failed to neutralize, one group of 3 MAbs potently neutralized VACV in an isotype- and complement-independent manner. This is in contrast to neutralizing antibodies against major VACV envelope proteins, such as H3, D8, or A27, which failed to completely neutralize VACV unless the antibodies are of complement-fixing isotypes and complement is present. Compared to nonneutralizing anti-L1 MAbs, the neutralization antibodies bound to the recombinant L1 protein with a significantly higher affinity and also could bind to virions. By using a variety of techniques, including the isolation of neutralization escape mutants, hydrogen/deuterium exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly conserved among antibodies recognizing the epitope. These antibodies, however, had divergent light-chain and heavy-chain CDR3 sequences. Our study demonstrates that the conformational L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent group of antibodies.
IMPORTANCE Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in human history, but it presents a level of risk that has become unacceptable for the current population. Studying the immune protection mechanism of smallpox vaccine is important for understanding the basic principle of successful vaccines and the development of next-generation, safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group of highly potent murine neutralizing antibodies. The analysis of antibody-antigen complex structure and the sequences of the antibody genes shed light on how these potent neutralizing antibodies are elicited from immunized mice.
PMCID: PMC4178804  PMID: 25031354
16.  Visual and functional demonstration of growing Bax-induced pores in mitochondrial outer membranes 
Molecular Biology of the Cell  2015;26(2):339-349.
We visualized Bax-induced pores in outer membrane vesicles (OMVs) using cryo-electron microscopy and monitored dextran release from these vesicles by flow cytometry. The data argue that Bax promotes mitochondrial outer membrane permeabilization by inducing the formation of large, solitary, and growing pores through a mechanism involving membrane-curvature stress.
Bax induces mitochondrial outer membrane permeabilization (MOMP), a critical step in apoptosis in which proteins are released into the cytoplasm. To resolve aspects of the mechanism, we used cryo-electron microscopy (cryo-EM) to visualize Bax-induced pores in purified mitochondrial outer membranes (MOMs). We observed solitary pores that exhibited negative curvature at their edges. Over time, the pores grew to ∼100–160 nm in diameter after 60–90 min, with some pores measuring more than 300 nm. We confirmed these results using flow cytometry, which we used to monitor the release of fluorescent dextrans from isolated MOM vesicles. The dextran molecules were released gradually, in a manner constrained by pore size. However, the release rates were consistent over a range of dextran sizes (10–500 kDa). We concluded that the pores were not static but widened dramatically to release molecules of different sizes. Taken together, the data from cryo-EM and flow cytometry argue that Bax promotes MOMP by inducing the formation of large, growing pores through a mechanism involving membrane-curvature stress.
PMCID: PMC4294680  PMID: 25411335
17.  Abacavir-Reactive Memory T Cells Are Present in Drug Naïve Individuals 
PLoS ONE  2015;10(2):e0117160.
Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.
To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.
Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.
We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.
PMCID: PMC4326126  PMID: 25674793
18.  Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility 
Nature immunology  2014;15(8):777-788.
A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.
PMCID: PMC4140783  PMID: 24997565
19.  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
20.  Murine Anti-vaccinia Virus D8 Antibodies Target Different Epitopes and Differ in Their Ability to Block D8 Binding to CS-E 
PLoS Pathogens  2014;10(12):e1004495.
The IMV envelope protein D8 is an adhesion molecule and a major immunodominant antigen of vaccinia virus (VACV). Here we identified the optimal D8 ligand to be chondroitin sulfate E (CS-E). CS-E is characterized by a disaccharide moiety with two sulfated hydroxyl groups at positions 4′ and 6′ of GalNAc. To study the role of antibodies in preventing D8 adhesion to CS-E, we have used a panel of murine monoclonal antibodies, and tested their ability to compete with CS-E for D8 binding. Among four antibody specificity groups, MAbs of one group (group IV) fully abrogated CS-E binding, while MAbs of a second group (group III) displayed widely varying levels of CS-E blocking. Using EM, we identified the binding site for each antibody specificity group on D8. Recombinant D8 forms a hexameric arrangement, mediated by self-association of a small C-terminal domain of D8. We propose a model in which D8 oligomerization on the IMV would allow VACV to adhere to heterogeneous population of CS, including CS-C and potentially CS-A, while overall increasing binding efficiency to CS-E.
Author Summary
Vaccinia virus (VACV) is an orthopox virus and considered the gold standard of vaccines as it was used to eradicate smallpox from the human population. Inoculation with VACV leads to a strong B cell immune response and the production of potent antibodies that simultaneously target several envelope proteins of the virus. Among those viral proteins, D8 is an adhesion molecule that binds chondroitin sulfate, a glycosaminoglycan, on the host cell surface. Here, we identified chondroitin sulfate E (CS-E), as the preferred ligand for D8 and assessed the role of a panel of anti-D8 antibodies in preventing D8 binding to CS-E. We further mapped the binding site of each antibody on the D8 surface to reveal the targeted epitopes. Finally, using several truncated D8 constructs, we identified that the C-terminal domain of D8 that is not involved in CS-E binding is in fact involved in oligomerization of native D8 in vitro and likely, also on the virion, as a means of increasing binding affinity to increase viral adhesion to CS on the host cell.
PMCID: PMC4256255  PMID: 25474621
21.  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
22.  The immune epitope database (IEDB) 3.0 
Nucleic Acids Research  2014;43(Database issue):D405-D412.
The IEDB,, contains information on immune epitopes—the molecular targets of adaptive immune responses—curated from the published literature and submitted by National Institutes of Health funded epitope discovery efforts. From 2004 to 2012 the IEDB curation of journal articles published since 1960 has caught up to the present day, with >95% of relevant published literature manually curated amounting to more than 15 000 journal articles and more than 704 000 experiments to date. The revised curation target since 2012 has been to make recent research findings quickly available in the IEDB and thereby ensure that it continues to be an up-to-date resource. Having gathered a comprehensive dataset in the IEDB, a complete redesign of the query and reporting interface has been performed in the IEDB 3.0 release to improve how end users can access this information in an intuitive and biologically accurate manner. We here present this most recent release of the IEDB and describe the user testing procedures as well as the use of external ontologies that have enabled it.
PMCID: PMC4384014  PMID: 25300482
23.  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
24.  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
25.  CLO: The cell line ontology 
Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions.
Construction and content
Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms.
Utility and discussion
The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development.
PMCID: PMC4387853  PMID: 25852852
Cell line; Cell line cell; Immortal cell line cell; Mortal cell line cell; Cell line cell culturing; Anatomy

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