Maya F. Kotturi

Pre-existing immunity against swine-origin H1N1 influenza viruses in the general human population

A major concern about the ongoing swine-origin H1N1 influenza virus (S-OIV) outbreak is that the virus may be so different from seasonal H1N1 that little immune protection exists in the human population. In this study, we examined the molecular basis for pre-existing immunity against S-OIV, namely the recognition of viral immune epitopes by T cells or B cells/antibodies that have been previously primed by circulating influenza strains. Using data from the Immune Epitope Database, we found that only 31% (8/26) of B-cell epitopes present in recently circulating H1N1 strains are conserved in the S-OIV, with only 17% (1/6) conserved in the hemagglutinin (HA) and neuraminidase (NA) surface proteins. In contrast, 69% (54/78) of the epitopes recognized by CD8+ T cells are completely invariant. We further demonstrate experimentally that some memory T-cell immunity against S-OIV is present in the adult population and that such memory is of similar magnitude as the pre-existing memory against seasonal H1N1 influenza. Because protection from infection is antibody mediated, a new vaccine based on the specific S-OIV HA and NA proteins is likely to be required to prevent infection. However, T cells are known to blunt disease severity. Therefore, the conservation of a large fraction of T-cell epitopes suggests that the severity of an S-OIV infection, as far as it is determined by susceptibility of the virus to immune attack, would not differ much from that of seasonal flu. These results are consistent with reports about disease incidence, severity, and mortality rates associated with human S-OIV.

A Protective Role for Dengue Virus-Specific CD8+ T Cells

Infection with one of the four serotypes of dengue virus (DENV1–4) can result in a range of clinical manifestations in humans, from dengue fever to the more serious dengue hemorrhagic fever/dengue shock syndrome. Although T cells have been implicated in the immunopathogenesis of secondary infections with heterologous DENV serotypes, the role of T cells in protection against DENV is unknown. In this study, we used a mouse-passaged DENV2 strain, S221, to investigate the role of CD8+ T cells in the immune response to primary DENV infection. S221 did not replicate well in wild-type mice, but did induce a CD8+ T cell response, whereas viral replication and a robust CD8+ T cell response were observed after infection of IFN-α/βR−/− mice. Depletion of CD8+ T cells from IFN-α/βR−/− mice before infection resulted in significantly higher viral loads compared with undepleted mice. Mapping the specificity of the CD8+ T cell response led to the identification of 12 epitopes derived from 6 of the 10 DENV proteins, with a similar immunodominance hierarchy observed in wild-type and IFN-α/βR−/− mice. DENV-specific CD8+ T cells produced IFN-γ, TNF-α, expressed cell surface CD107a, and exhibited cytotoxic activity in vivo. Finally, immunization with four of the immunodominant CD8+ T cell epitopes enhanced viral clearance. Collectively, our results reveal an important role for CD8+ T cells in the host defense against DENV and demonstrate that the anti-DENV CD8+ T cell response can be enhanced by immunization, providing rationale for designing DENV-specific vaccines that induce cell-mediated immunity.

Naive Precursor Frequencies and MHC Binding Rather Than the Degree of Epitope Diversity Shape CD8+ T Cell Immunodominance

The primary CD8+ T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8+ T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2Kb, resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F1 hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8+ T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33–41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8+ T cell responses.

Molecular Determinants of T Cell Epitope Recognition to the Common Timothy Grass Allergen

We investigated the molecular determinants of allergen-derived T cell epitopes in humans utilizing the Phleum pratense (Timothy grass) allergens (Phl p). PBMCs from allergic individuals were tested in ELISPOT assays with overlapping peptides spanning known Phl p allergens. A total of 43 distinct antigenic regions were recognized, illustrating the large breadth of grass-specific T cell epitopes. Th2 cytokines (as represented by IL-5) were predominant, whereas IFN-γ, IL-10, and IL-17 were detected less frequently. Responses from specific immunotherapy treatment individuals were weaker and less consistent, yet similar in epitope specificity and cytokine pattern to allergic donors, whereas nonallergic individuals were essentially nonreactive. Despite the large breadth of recognition, nine dominant antigenic regions were defined, each recognized by multiple donors, accounting for 51% of the total response. Multiple HLA molecules and loci restricted the dominant regions, and the immunodominant epitopes could be predicted using bioinformatic algorithms specific for 23 common HLA-DR, DP, and DQ molecules. Immunodominance was also apparent at the Phl p Ag level. It was found that 52, 19, and 14% of the total response was directed to Phl p 5, 1, and 3, respectively. Interestingly, little or no correlation between Phl p-specific IgE levels and T cell responses was found. Thus, certain intrinsic features of the allergen protein might influence immunogenicity at the level of T cell reactivity. Consistent with this notion, different Phl p Ags were associated with distinct patterns of IL-5, IFN-γ, IL-10, and IL-17 production.

The CD8+ T-Cell Response to Lymphocytic Choriomeningitis Virus Involves the L Antigen: Uncovering New Tricks for an Old Virus

ABSTRACT CD8+ T-cell responses control lymphocytic choriomeningitis virus (LCMV) infection in H-2b mice. Although antigen-specific responses against LCMV infection are well studied, we found that a significant fraction of the CD8+ CD44hi T-cell response to LCMV in H-2b mice was not accounted for by known epitopes. We screened peptides predicted to bind major histocompatibility complex class I and overlapping 15-mer peptides spanning the complete LCMV proteome for gamma interferon (IFN-γ) induction from CD8+ T cells derived from LCMV-infected H-2b mice. We identified 19 novel epitopes. Together with the 9 previously known, these epitopes account for the total CD8+ CD44hi response. Thus, bystander T-cell activation does not contribute appreciably to the CD8+ CD44hi pool. Strikingly, 15 of the 19 new epitopes were derived from the viral L polymerase, which, until now, was not recognized as a target of the cellular response induced by LCMV infection. The L epitopes induced significant levels of in vivo cytotoxicity and conferred protection against LCMV challenge. Interestingly, protection from viral challenge was best correlated with the cytolytic potential of CD8+ T cells, whereas IFN-γ production and peptide avidity appear to play a lesser role. Taken together, these findings illustrate that the LCMV-specific CD8+ T-cell response is more complex than previously appreciated.

Identification of broad binding class I HLA supertype epitopes to provide universal coverage of influenza A virus.

Influenza virus remains a significant health concern, with current circulating strains that affect millions each year plus the threat of newly emerging strains, such as swine-origin H1N1 and avian H5N1. Our hypothesis is that influenza-derived HLA-class I-restricted epitopes can be identified for use as a reagent to monitor and quantitate human CD8(+) T-cell responses and for vaccine development to induce protective cellular immunity. Protein sequences from influenza A virus strains currently in circulation, agents of past pandemics and zoonotic infections of man were evaluated for sequences predicted to bind to alleles representative of the most frequent HLA-A and -B (class I) types worldwide. Peptides that bound several different HLA molecules and were conserved among diverse influenza subtypes were tested for their capacity to recall influenza-specific immune responses using human donor PBMC. Accordingly, 28 different epitopes antigenic for human donor PBMC were identified and 25 were 100% conserved in the newly emerged swine-origin H1N1 strain. The epitope set defined herein should provide a reagent applicable to quantitate CD8(+) T cell human responses irrespective of influenza subtype and HLA composition of the responding population. In addition, these epitopes may be suitable for vaccine applications directed at the induction of cellular immunity.

A multivalent and cross-protective vaccine strategy against arenaviruses associated with human disease.

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8+ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8+ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8+ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.

Chronic Lymphocytic Choriomeningitis Virus Infection Actively Down-Regulates CD4+ T Cell Responses Directed against a Broad Range of Epitopes

Activation of CD4+ T cells helps establish and sustain CD8+ T cell responses and is required for the effective clearance of acute infection. CD4-deficient mice are unable to control persistent infection and CD4+ T cells are usually defective in chronic and persistent infections. We investigated the question of how persistent infection impacted pre-existing lymphocytic choriomeningitis virus (LCMV)-specific CD4+ T cell responses. We identified class II-restricted epitopes from the entire set of open reading frames from LCMV Armstrong in BALB/c mice (H-2d) acutely infected with LCMV Armstrong. Of nine epitopes identified, six were restricted by I-Ad, one by I-Ed and two were dually restricted by both I-Ad and I-Ed molecules. Additional experiments revealed that CD4+ T cell responses specific for these epitopes were not generated following infection with the immunosuppressive clone 13 strain of LCMV. Most importantly, in peptide-immunized mice, established CD4+ T cell responses to these LCMV CD4 epitopes as well as nonviral, OVA-specific responses were actively suppressed following infection with LCMV clone 13 and were undetectable within 12 days after infection, suggesting an active inhibition of established helper responses. To address this dysfunction, we performed transfer experiments using both the Smarta and OT-II systems. OT-II cells were not detected after clone 13 infection, indicating physical deletion, while Smarta cells proliferated but were unable to produce IFN-γ, suggesting impairment of the production of this cytokine. Thus, multiple mechanisms may be involved in the impairment of helper responses in the setting of early persistent infection.

A detailed analysis of the murine TAP transporter substrate specificity.

Background The transporter associated with antigen processing (TAP) supplies cytosolic peptides into the endoplasmic reticulum for binding to major histocompatibility complex (MHC) class I molecules. Its specificity therefore influences the repertoire of peptides presented by MHC molecules. Compared to human TAP, murine TAPs binding specificity has not been characterized as well, even though murine systems are widely used for basic studies of antigen processing and presentation. Methodology/Principal Findings We performed a detailed experimental analysis of murine TAP binding specificity by measuring the binding affinities of 323 peptides. Based on this experimental data, a computational model of murine TAP specificity was constructed. The model was compared to previously generated data on human and murine TAP specificities. In addition, the murine TAP specificities for known epitopes and random peptides were predicted and compared to assess the impact of murine TAP selectivity on epitope selection. Conclusions/Significance Comparisons to a previously constructed model of human TAP specificity confirms the well-established differences for peptide substrates with positively charged C-termini. In addition these comparisons show that several residues at the N-terminus of peptides which strongly influence binding to human TAP showed little effect on binding to murine TAP, and that the overall influence of the aminoterminal residues on peptide affinity for murine TAP is much lower than for the human transporter. Murine TAP also partly prefers different hydrophobic amino acids than human TAP in the carboxyterminal position. These species-dependent differences in specificity determined in vitro are shown to correlate with the epitope repertoire recognized in vivo. The quantitative model of binding specificity of murine TAP developed herein should be useful for interpreting epitope mapping and immunogenicity data obtained in humanized mouse models.

Of mice and humans: how good are HLA transgenic mice as a model of human immune responses?

BackgroundPrevious studies have defined vaccinia virus (VACV)-derived T cell epitopes in VACV-infected human leukocyte antigen-A*0201 (HLA-A2.1) transgenic (Tg) mice and A2.1-positive human Dryvax vaccinees. A total of 14 epitopes were detected in humans and 16 epitopes in A2.1 Tg mice; however, only two epitopes were independently reported in both systems. This limited overlap raised questions about the suitability of using HLA Tg mice as a model system to map human T cell responses to a complex viral pathogen. The present study was designed to investigate this issue in more detail.ResultsRe-screening the panel of 28 A2.1-restricted epitopes in additional human vaccinees and in A2.1 Tg mice revealed that out of the 28 identified epitopes, 13 were detectable in both systems, corresponding to a 46% concordance rate. Interestingly, the magnitude of responses in Tg mice against epitopes originally identified in humans is lower than for epitopes originally detected in mice. Likewise, responses in humans against epitopes originally detected in Tg mice are of lower magnitude.ConclusionThese data suggest that differences in immunodominance patterns might explain the incomplete response overlap, and that with limitations; HLA Tg mice represent a relevant and suitable model system to study immune responses against complex pathogens.