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 http://tools.iedb.org/immunogenicity/) 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.
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 http://tools.iedb.org/immunogenicity/). 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.
Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design.
Although there is an increased recognition of the role of T-cells in both dengue pathogenesis and protection, comprehensive analysis of T-cell activation during dengue infection is hampered by the small repertoire of known human dengue T-cell epitopes. Although dengue serotype 3 (DENV3) is responsible for numerous outbreaks worldwide, most of the known epitopes are from studies of dengue 2 serotype (DENV2). In this study, we identified novel DENV3 T-cell epitopes in HLA transgenic mice that were confirmed by HLA binding assays. A subset of these epitopes activated memory T-cells from subjects who were dengue IgG positive and primed naïve T-cells from dengue IgG negative individuals. Notably, some of HLA class II epitopes bearing highly conserved regions common to all four dengue serotypes could bind to multiple HLAs. We postulate that these highly conserved and HLA promiscuous T-helper epitopes can be important components of a dengue tetravalent vaccine.
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.
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).
Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.
HSV-1 is an endemic human herpesvirus worldwide that establishes a lifelong latent infection of neurons in the trigeminal ganglion (TG), allowing intermittent reactivation resulting in recurrent disease in some persons. Studies in HSV-1 models suggest a central role of TG-infiltrating virus-specific CD8 T-cells to control reactivation. In humans, however, the functional properties and fine specificity of intra-TG T-cell responses remain enigmatic. The current study used molecular, immunological and in situ analysis platforms on human cadaveric TG obtained within hours after death to characterize the local HSV-1 specific T-cell response in latently infected human TG in detail. We identified that CD4 and CD8 T-cells were juxtaposed to TG neurons and expressed host transcripts and proteins consistent with in situ antigen recognition and antiviral function. The intra-TG T-cell response, involving both CD4 and CD8 T-cells, was directed to a limited set of HSV-1 proteins per person, which was not limited to a specific kinetic or structural class of viral proteins. Collectively, the data indicate that the human TG is an immunocompetent environment for CD4 and CD8 T-cell recognition of diverse HSV-1 proteins expressed during latent infection and that the viral antigens identified herein are rational candidates for HSV-1 subunit vaccines.
Here, we evaluated the hypothesis that CD8+ T cell responses to caspase-cleaved antigens derived from effector T cells undergoing apoptosis, may contribute to multiple sclerosis (MS) immunopathology.
The percentage of autoreactive CD8+ T effector cells specific for various apoptotic T cell-associated self-epitopes (apoptotic epitopes) were detected in the peripheral blood and cerebrospinal fluid (CSF) by both enzyme-linked immunospot and dextramers of class I molecules complexed with relevant apoptotic epitopes. Moreover, the capacity of dextramer+ CD8+ T cells to produce interferon (IFN)-γ and/or interleukin (IL)-17 in response to the relevant apoptotic epitopes was evaluated by the intracellular cytokine staining. Cross-presentation assay of apoptotic T cells by dendritic cells was also evaluated ex vivo.
We found that polyfunctional (IFN-γ and/or IL-17 producing) autoreactive CD8+ T cells specific for apoptotic epitopes were represented in MS patients with frequencies significantly higher than in healthy donors. These autoreactive CD8+ T cells with a strong potential to produce IFN-γ or IL-17 in response to the relevant apoptotic epitopes were significantly accumulated in the CSF from the same patients. In addition, the frequencies of these autoreactive CD8+ T cells correlated with the disease disability. Cross-presentation assay revealed that caspase-cleaved cellular proteins are required to activate apoptotic epitope-specific CD8+ T cells ex vivo.
Taken together, these data indicate that apoptotic epitope-specific CD8+ T cells with strong inflammatory potential are recruited at the level of the inflammatory site, where they may be involved in MS immunopathology through the production of high levels of inflammatory cytokines.
Apoptosis; CD8+ T cells; Multiple sclerosis
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.
Virus-specific CD8+ T cells play an important role in controlling HIV/SIV replication. These T cells recognize intracellular pathogen-derived peptides displayed on the cell surface by individual MHC class I molecules. In the SIV-infected rhesus macaque model, five Mamu class I alleles have been thoroughly characterized with regard to peptide binding, and a sixth was shown to be uninvolved. In this study, we describe the peptide binding of Mamu-A1*007:01 (formerly Mamu-A*07), an allele present in roughly 5.08% of Indian-origin rhesus macaques (n=63 of 1240). We determined a preliminary binding motif by eluting and sequencing endogenously bound ligands. Subsequently, we used a positional scanning combinatorial library and panels of single amino acid substitution analogs to further characterize peptide binding of this allele and derive a quantitative motif. Using this motif, we selected and tested 200 peptides derived from SIVmac239 for their capacity to bind Mamu-A1*007:01, 33 were found to bind with an affinity of 500nM or better. We then used PBMC from SIV-infected or vaccinated but uninfected, A1*007:01-positive rhesus macaques in IFN-γ Elispot assays to screen the peptides for T cell reactivity. In all, eleven of the peptides elicited IFN-γ+ T cell responses. Six represent novel A1*007:01-restricted epitopes. Furthermore, both Sanger and ultra-deep pyrosequencing demonstrated the accumulation of amino acid substitutions within four of these six regions, suggestive of selective pressure on the virus by antigen-specific CD8+ T cells. Thus, it appears that Mamu-A1*007:01 presents SIV-derived peptides to antigen-specific CD8+ T cells and is part of the immune response to SIVmac239.
SIV; MHC; Macaque; Epitope; Escape
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.
Malaria; Vaccine; Circumsporozoite protein; ELISpot; Flow cytometry; NetMHC; Epitope mapping; Class I restriction; Localization
Type 1 diabetes is an autoimmune disease characterized by T cell responses to beta cell antigens, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for beta cell-specific CD8+ T cells in the pathogenic process. As CD8+ T cells specific for beta cell antigens are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human β2-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify beta cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8+ T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified here have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.
Rhesus and pigtail macaques have proven to be valuable animal models for several important human diseases, including HIV, where they exhibit similar pathology and disease progression. Because rhesus macaques have been extensively characterized in terms of their major histocompatibility complex (MHC) class I alleles, their demand has soared, making them increasingly difficult to obtain for research purposes. This problem has been exacerbated by a continued export ban in place since 1978. Pigtail macaques represent a potential alternative animal model. However, because their MHC class I alleles have not been characterized in detail, their use has been hindered. To address this, in the present study, we have characterized the peptide binding specificity of the pigtail macaque class I allele Mane-A1*082:01 (formerly known as Mane A*0301), representative of the second most common MHC class I antigen detected across several cohorts. The motif was defined on the basis of binding studies utilizing purified MHC protein and panels of single amino acid substitution analog peptides, as well as sequences of peptide ligands eluted from Mane- A1*082:01. Based on these analyses, Mane-A1*082:01 was found to recognize a motif with H in position 2 and the aromatic residues F and Y, or the hydrophobic/aliphatic residue M, at the C-terminus. Finally, analysis of the binding of a combinatorial peptide library allowed the generation of a detailed quantitative motif that proved effective in the prediction of a set of high-affinity binders derived from chimeric SIV/HIV, an important model virus for studying HIV infection in humans.
MHC; Macaques; Peptide binding motif; T cell epitope; Class I
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.
Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross reactivity against widely divergent circulating strains. Rational approaches for sequence selection to maximize immunogenicity and minimize genetic distance across circulating strains may enhance vaccine induction of optimal cytotoxic T cell responses. We assessed T cell recognition of potential hepatitis C virus vaccine sequences generated using three rational approaches: 1) combining epitopes with predicted tight binding to the major histocompatibility complex (MHC), 2) consensus sequence (most common amino acid at each position), and 3) representative ancestral sequence that had been derived using Bayesian phylogenetic tools. No correlation was seen between peptide MHC binding affinity and frequency of recognition as measured by an interferon-gamma T cell response in human leukocyte antigen-matched HCV infected individuals. Peptides encoding representative, consensus, and natural variant sequences were then tested for the capacity to expand CD8 T cell populations and to elicit cross-reactive CD8 T cell responses. CD8+ T cells expanded with representative sequence HCV generally more broadly and robustly recognized highly diverse circulating HCV strains than T cell expanded with either consensus sequence or naturally occurring sequence variants. These data support the use of representative sequence in HCV vaccine design.
Animals-human; T cells; Infections-viral; Processes-vaccination
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.
MHC; HLA class I; HLA class II; Peptide binding; T cell epitopes
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, http://www.iedb.org) 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.
CD8+ T cells (TCD8) confer protective immunity against many infectious diseases, suggesting that microbial TCD8 determinants are promising vaccine targets. Nevertheless, current T cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection — information that is critical for the rational design of TCD8-targeted vaccines. We employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a complex pathogen, vaccinia virus (VACV), that are presented by the most frequent representatives of four major HLA class I supertypes. Immunologic characterization revealed that many previously unidentified VACV determinants were recognized by smallpox-vaccinated human peripheral blood cells in a variegated manner. Many such determinants were recognized by HLA class I–transgenic mouse immune TCD8 too and elicited protective TCD8 immunity against lethal intranasal VACV infection. Notably, efficient processing and stable presentation of immune determinants as well as the availability of naive TCD8 precursors were sufficient to drive a multifunctional, protective TCD8 response. Our approach uses fundamental insights into T cell epitope processing and presentation to define targets of protective TCD8 immunity within human pathogens that have complex proteomes, suggesting that this approach has general applicability in vaccine sciences.
There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121–129) and IL9 (ILDAHSLYL, N165–173), were defined. VT9- and IL9-specific CD8+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.
The antigens recognized by individual CD8+ T cells are small peptides bound to major histocompatibility complex (MHC) class I molecules. The CD8+ T cell response to a virus is restricted to several peptides, and the magnitudes of the effector as well as memory phases of the response to the individual peptides are generally hierarchical. The peptide eliciting a stronger response is called immunodominant (ID), and those with smaller-magnitude responses are termed subdominant (SD). The relative importance of ID and SD determinants in protective immunity remains to be fully elucidated. We previously showed that multispecific memory CD8+ T cells can protect susceptible mice from mousepox, an acute lethal viral disease. It remained unknown, however, whether CD8+ T cells specific for single ID or SD peptides could be protective. Here, we demonstrate that immunization with dendritic cells pulsed with ID and some but not all SD peptides induces memory CD8+ T cells that are fully capable of protecting susceptible mice from mousepox. Additionally, while natural killer (NK) cells are essential for the natural resistance of nonimmune C57BL/6 (B6) to mousepox, we show that memory CD8+ T cells of single specificity also protect B6 mice depleted of NK cells. This suggests it is feasible to produce effective antiviral CD8+ T cell vaccines using single CD8+ T cell determinants and that NK cells are no longer essential when memory CD8+ T cells are present.
The yellow fever vaccines (YF-17D-204 and 17DD) are considered to be among the safest vaccines and the presence of neutralizing antibodies is correlated with protection, although other immune effector mechanisms are known to be involved. T-cell responses are known to play an important role modulating antibody production and the killing of infected cells. However, little is known about the repertoire of T-cell responses elicited by the YF-17DD vaccine in humans. In this report, a library of 653 partially overlapping 15-mer peptides covering the envelope (Env) and nonstructural (NS) proteins 1 to 5 of the vaccine was utilized to perform a comprehensive analysis of the virus-specific CD4+ and CD8+ T-cell responses. The T-cell responses were screened ex-vivo by IFN-γ ELISPOT assays using blood samples from 220 YF-17DD vaccinees collected two months to four years after immunization. Each peptide was tested in 75 to 208 separate individuals of the cohort. The screening identified sixteen immunodominant antigens that elicited activation of circulating memory T-cells in 10% to 33% of the individuals. Biochemical in-vitro binding assays and immunogenetic and immunogenicity studies indicated that each of the sixteen immunogenic 15-mer peptides contained two or more partially overlapping epitopes that could bind with high affinity to molecules of different HLAs. The prevalence of the immunogenicity of a peptide in the cohort was correlated with the diversity of HLA-II alleles that they could bind. These findings suggest that overlapping of HLA binding motifs within a peptide enhances its T-cell immunogenicity and the prevalence of the response in the population. In summary, the results suggests that in addition to factors of the innate immunity, “promiscuous” T-cell antigens might contribute to the high efficacy of the yellow fever vaccines.
T-cell responses are considered to be very important; however, the role of T-cell responses in vaccine mediated immunity is still controversial. One reason may be that most studies of human T-cell responses are focused on a few epitopes. We still lack a systematic view of the repertoire of peptides presented by the different HLA class I and II molecules and how the peptides presented by the different HLAs interact within the host to develop T-cell responses. Here we present a study of the T-cell responses against the YF-17DD vaccine in the context of a cohort of 220 volunteers and observed that the most prevalent T-cell responses are targeted at peptides that bind to multiple types of HLA molecules. Based on these results we postulate that promiscuous T-cell epitopes might have a critical role in the development of adaptive immunity. These results may have broader implications for other pathogens, since the yellow fever vaccine is currently being developed as a vaccine vector for other diseases. Therefore, these epitopes might have a functionally cooperative role in boosting specific neutralizing antibody responses. In addition, we propose that promiscuous T-cell antigens may be better immunogens for vaccine development; however more studies are necessary.
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.
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.
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.
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.
Ability of CD8+ T cells to act as cytolytic effectors and produce IFN-γ was shown to mediate resistance to Toxoplasma gondii in murine models due to recognition of peptides restricted by murine MHC Class I molecules. However, no T. gondii specific HLA-B07 restricted peptides were proven protective against T gondii. Recently, two T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA720–28 (LPQFATAAT) and GRA327–35 (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702, and elicited IFN-γ from PBMCs of seropositive HLA-B*0702 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ producing T cells were identified by ELISPOT and proliferation assays utilizing splenic T lymphocytes from HLA transgenic mice. When HLA-B*0702 mice were immunized with one of the epitopes identified, GRA720–28 in conjunction with a universal CD4+ T cell epitope (PADRE) and adjuvants (CD4+ T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam2Cys for CD8+ T cells), this immunization induced CD8+ T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates feasibility of bioinformatics followed by an empirical approach based on HLA binding to test this biological activity for identifying protective HLA-B*0702 restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.
Toxoplasma gondii; HLA-B07 epitopes; PADRE; adjuvant; vaccine
Here we analyzed the molecular targets associated with myasthenia gravis (MG) immune responses, enabled by an immune epitope database (IEDB) inventory of approximately 600 MG-related epitopes derived from 175 references. The vast majority of epitopes were derived from the α-subunit of human AChR suggesting that other MG-associated autoantigens should be investigated further. Human α-AChR was mostly characterized in humans, whereas reactivity primarily to T. californica AChR was examined in animal models. While the fine specificity of T-cell response was similar in the two systems, substantial antibody reactivity to the C-terminus was detected in the nonhuman system, but not in humans. Further analysis showed that the reactivity of nonhuman hosts to the C-terminus was eliminated when data were restricted to hosts tested in the context of autoimmune disease (spontaneous or induced), demonstrating that the epitopes recognized in humans and animals were shared when disease was present. Finally, we provided data subsets relevant to particular applications, including those associated with HLA typing or restriction, sets of epitopes recognized by monoclonal antibodies, and epitopes associated with modulation of immunity or disease. In conclusion, this analysis highlights gaps, differences, and similarities in the epitope repertoires of humans and animal models.
The Immune Epitope Database and Analysis Resource (IEDB, http://www.iedb.org) 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.
epitope conservation; epitope predictions; vaccine design; Major Histocompatibility Complex