Erratum: T cells from patients with Parkinson's disease recognize α-synuclein peptides.

This corrects the article DOI: 10.1038/nature22815.

T cells from patients with Parkinson's disease recognize α-synuclein peptides.

Genetic studies have shown the association of Parkinson's disease with alleles of the major histocompatibility complex. Here we show that a defined set of peptides that are derived from α-synuclein, a protein aggregated in Parkinson's disease, act as antigenic epitopes displayed by these alleles and drive helper and cytotoxic T cell responses in patients with Parkinson's disease. These responses may explain the association of Parkinson's disease with specific major histocompatibility complex alleles.

Immunoproteomic analysis of house dust mite antigens reveals distinct classes of dominant T cell antigens according to function and serological reactivity.

BACKGROUND: House dust mite (HDM) allergens are a common cause of allergy and allergic asthma. A comprehensive analysis of proteins targeted by T cells, which are implicated in the development and regulation of allergic disease independent of their antibody reactivity, is still lacking. OBJECTIVE: To comprehensively analyse the HDM-derived protein targets of T cell responses in HDM-allergic individuals, and investigate their correlation with IgE/IgG responses and protein function. METHODS: Proteomic analysis (liquid chromatography-tandem mass spectrometry) of HDM extracts identified 90 distinct protein clusters, corresponding to 29 known allergens and 61 novel proteins. Peripheral blood mononuclear cells (PBMC) from 20 HDM-allergic individuals were stimulated with HDM extracts and assayed with a set of ~2500 peptides derived from these 90 protein clusters and predicted to bind the most common HLA class II types. 2D immunoblots were made in parallel to elucidate IgE and IgG reactivity, and putative function analyses were performed in silico according to Gene Ontology annotations. RESULTS: Analysis of T cell reactivity revealed a large number of T cell epitopes. Overall response magnitude and frequency was comparable for known and novel proteins, with 15 antigens (nine of which were novel) dominating the total T cell response. Most of the known allergens that were dominant at the T cell level were also IgE reactive, as expected, while few novel dominant T cell antigens were IgE reactive. Among known allergens, hydrolase activity and detectable IgE/IgG reactivity are strongly correlated, while no protein function correlates with immunogenicity of novel proteins. A total of 106 epitopes accounted for half of the total T cell response, underlining the heterogeneity of T cell responses to HDM allergens. CONCLUSIONS AND CLINICAL RELEVANCE: Herein, we define the T cell targets for both known allergens and novel proteins, which may inform future diagnostics and immunotherapeutics for allergy to HDM.

Immunodominance in allergic T-cell reactivity to Japanese cedar in different geographic cohorts.

BACKGROUND: Japanese cedar (JC) pollen is a common trigger for allergic rhinitis in Japan. Pollen proteins targeted by IgE, including Cry j 1 and Cry j 2, and isoflavone reductase (IFR) have been identified. OBJECTIVE: To compare antigen-specific IgE titers and T-cell responses to JC pollen-derived extract and peptides in cohorts with high and low pollen exposure. METHODS: Peripheral blood mononuclear cells from JC pollen allergic or nonallergic patients who have lived in Japan for at least 1 year and JC pollen allergic patients who have never been to Japan were tested for T-cell responses against JC pollen extract and peptide pools derived from Cry j 1, Cry j 2, or IFR. T-cell reactivity was assessed by interleukin 5 and interferon γ production by ELISPOT. RESULTS: JC pollen-specific T-cell reactivity and IgE titers were significantly higher in the allergic compared with the nonallergic Japanese cohort, which was also associated with different patterns of polysensitization. Interestingly, a significant overlap was observed in the hierarchy of the T-cell epitopes in the allergic Japanese cohort compared with the allergic non-Japanese cohort. In all 3 cohorts, T-cell reactivity was dominantly directed against peptides from the major allergens Cry j 1 and 2, with few T-cell responses detected against IFR. CONCLUSION: Our studies identify common denominators of T-cell reactivity in patient populations with different sensitization patterns, suggesting that generally applicable immunotherapeutic approaches might be developed irrespective of exposure modality.

The common equine class I molecule Eqca-1*00101 (ELA-A3.1) is characterized by narrow peptide binding and T cell epitope repertoires.

Here we describe a detailed quantitative peptide-binding motif for the common equine leukocyte antigen (ELA) class I allele Eqca-1*00101, present in roughly 25 % of Thoroughbred horses. We determined a preliminary binding motif by sequencing endogenously bound ligands. Subsequently, a positional scanning combinatorial library (PSCL) was used to further characterize binding specificity and derive a quantitative motif involving aspartic acid in position 2 and hydrophobic residues at the C-terminus. Using this motif, we selected and tested 9- and 10-mer peptides derived from the equine herpesvirus type 1 (EHV-1) proteome for their capacity to bind Eqca-1*00101. PSCL predictions were very efficient, with an receiver operating characteristic (ROC) curve performance of 0.877, and 87 peptides derived from 40 different EHV-1 proteins were identified with affinities of 500 nM or higher. Quantitative analysis revealed that Eqca-1*00101 has a narrow peptide-binding repertoire, in comparison to those of most human, non-human primate, and mouse class I alleles. Peripheral blood mononuclear cells from six EHV-1-infected, or vaccinated but uninfected, Eqca-1*00101-positive horses were used in IFN-γ enzyme-linked immunospot (ELISPOT) assays. When we screened the 87 Eqca-1*00101-binding peptides for T cell reactivity, only one Eqca-1*00101 epitope, derived from the intermediate-early protein ICP4, was identified. Thus, despite its common occurrence in several horse breeds, Eqca-1*00101 is associated with a narrow binding repertoire and a similarly narrow T cell response to an important equine viral pathogen. Intriguingly, these features are shared with other human and macaque major histocompatibility complex (MHC) molecules with a similar specificity for D in position 2 or 3 in their main anchor motif.

Development and validation of a broad scheme for prediction of HLA class II restricted T cell epitopes.

Computational prediction of HLA class II restricted T cell epitopes has great significance in many immunological studies including vaccine discovery. In recent years, prediction of HLA class II binding has improved significantly but a strategy to globally predict the most dominant epitopes has not been rigorously defined. Using human immunogenicity data associated with sets of 15-mer peptides overlapping by 10 residues spanning over 30 different allergens and bacterial antigens, and HLA class II binding prediction tools from the Immune Epitope Database and Analysis Resource (IEDB), we optimized a strategy to predict the top epitopes recognized by human populations. The most effective strategy was to select peptides based on predicted median binding percentiles for a set of seven DRB1 and DRB3/4/5 alleles. These results were validated with predictions on a blind set of 15 new allergens and bacterial antigens. We found that the top 21% predicted peptides (based on the predicted binding to seven DRB1 and DRB3/4/5 alleles) were required to capture 50% of the immune response. This corresponded to an IEDB consensus percentile rank of 20.0, which could be used as a universal prediction threshold. Utilizing actual binding data (as opposed to predicted binding data) did not appreciably change the efficacy of global predictions, suggesting that the imperfect predictive capacity is not due to poor algorithm performance, but intrinsic limitations of HLA class II epitope prediction schema based on HLA binding in genetically diverse human populations.

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.

Discovering naturally processed antigenic determinants that confer protective T cell immunity.

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.

Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals.

T cells play an important role in the pathogenesis of allergic diseases. However, the proteins considered as potential immunogens of allergenic T-cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is a well-studied inhaled allergen for which major IgE-reactive allergens have also been shown to trigger T helper 2 (Th2) responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE reactivity. A TG pollen extract was analyzed by 2D gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry of selected protein spots in combination with de novo sequencing of the whole TG pollen transcriptome identified 93 previously undescribed proteins for further study, 64 of which were not targeted by IgE. Predicted MHC binding peptides from the previoulsy undescribed TG proteins were screened for T-cell reactivity in peripheral blood mononuclear cells from allergic donors. Strong IL-5 production was detected in response to peptides from several of the previously undescribed proteins, most of which were not targeted by IgE. Responses against the dominant undescribed epitopes were associated with the memory T-cell subset and could even be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic, proteomic, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.

Measurement of MHC/peptide interactions by gel filtration or monoclonal antibody capture.

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.

A strategy to determine HLA class II restriction broadly covering the DR, DP, and DQ allelic variants most commonly expressed in the general population.

Classic ways to determine MHC restriction involve inhibition with locus-specific antibodies and antigen presentation assays with panels of cell lines matched or mismatched at the various loci of interest. However, these determinations are often complicated by T cell epitope degeneracy and promiscuity. We describe a selection of 46 HLA DR, DQ, and DP specificities that provide worldwide population (phenotypic) coverage of almost 90 % at each locus, and account for over 66 % of all genes at each locus. This panel afforded coverage of at least four HLA class II alleles in over 95 % of the individuals in four study populations of diverse ethnicity from the USA and South Africa. Next, a panel of single HLA class II-transfected cell lines, corresponding to these 46 allelic variants was assembled, consisting of lines previously developed and 15 novel lines generated for the present study. The novel lines were validated by assessing their HLA class II expression by FACS analysis, the in vitro peptide binding activity of HLA molecules purified from the cell lines, and their antigen presenting capacity to T cell lines of known restriction. We also show that these HLA class II-transfected cell lines can be used to rapidly and unambiguously determine HLA restriction of epitopes recognized by an individual donor in a single experiment. This panel of lines will enable high throughput determination of HLA restriction, enabling better characterization of HLA class II-restricted T cell responses and facilitating the development of HLA tetrameric staining reagents.

The identification of potentially pathogenic and therapeutic epitopes from common human allergens.

OBJECTIVES: To outline the processes involved in large-scale T-cell epitope identification from common allergens and illustrate their relevance to development of allergy specific immunotherapy. DATA SOURCES: A set of studies recently published by our laboratory illustrating high-throughput identification of allergen specific T-cell epitopes. STUDY SELECTION: T-cell responses contribute both directly and indirectly to allergy-related disease. However, the molecular targets (epitopes) recognized by allergen-specific T cells are largely undefined. We review several different studies in the last 2 years that identified novel T-cell epitopes from a panel of 32 different allergen sources. RESULTS: Allergen-specific T-cell responses are highly heterogeneous. Epitopes prevalently recognized in allergic patients are often capable of binding to multiple HLA class II molecules. This feature can be used to predict these promiscuous epitopes by bioinformatic predictions. This approach was validated in the Timothy grass system and then applied to a panel of 31 other allergen sources. CONCLUSION: T-cell epitopes for common allergens have been identified, and a general method to identify epitopes from additional allergens has been validated. Characterization of epitopes for common allergens might enable new diagnostics and immunotherapy regimens. These data will also allow the study of T-cell responses in different patient populations and throughout disease progression.

T cell responses to known allergen proteins are differently polarized and account for a variable fraction of total response to allergen extracts.

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 to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which 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-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas 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 specific immunotherapy treatment or varying disease severity (asthma or rhinitis).

Analysis of T cell responses to the major allergens from German cockroach: epitope specificity and relationship to IgE production.

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. 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 more than 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, whereas 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 copresented immunomodulators. In comparing Ab 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 cell-B cell help might support development of IgE responses. Finally, specific immunotherapy 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.

Drug hypersensitivity caused by alteration of the MHC-presented self-peptide repertoire.

Idiosyncratic adverse drug reactions are unpredictable, dose-independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggest that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkages between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations found has been for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. Abacavir adverse reactions were recently shown to be driven by drug-specific activation of cytokine-producing, cytotoxic CD8(+) T cells that required HLA-B*57:01 molecules for their function; however, the mechanism by which abacavir induces this pathologic T-cell response remains unclear. Here we show that abacavir can bind within the F pocket of the peptide-binding groove of HLA-B*57:01, thereby altering its specificity. This provides an explanation for HLA-linked idiosyncratic adverse drug reactions, namely that drugs can alter the repertoire of self-peptides presented to T cells, thus causing the equivalent of an alloreactive T-cell response. Indeed, we identified specific self-peptides that are presented only in the presence of abacavir and that were recognized by T cells of hypersensitive patients. The assays that we have established can be applied to test additional compounds with suspected HLA-linked hypersensitivities in vitro. Where successful, these assays could speed up the discovery and mechanistic understanding of HLA-linked hypersensitivities, and guide the development of safer drugs.

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-gamma, 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-gamma, IL-10, and IL-17 production.

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage.

BACKGROUND: Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4+ T cells are needed for optimal CD8+ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4+ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4+ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy. RESULTS: By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4+ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4+ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4+ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies. CONCLUSIONS: The identification of CD4+ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.

Definition of epitopes and antigens recognized by vaccinia specific immune responses: their conservation in variola virus sequences, and use as a model system to study complex pathogens.

In the last few years, a wealth of information has become available relating to the targets of vaccinia virus (VACV)-specific CD4(+) T cell, CD8(+) T cell and antibody responses. Due to the large size of its genome, encoding more than 200 different proteins, VACV represents a useful model system to study immunity to complex pathogens. Our data demonstrate that both cellular and humoral responses target a large number of antigens and epitopes. This broad spectrum of targets is detected in both mice and humans. CD4(+) T cell responses target late and structural antigens, while CD8(+) T cells preferentially recognize early antigens. While both CD4(+) and CD8(+) T cell responses target different types of antigens, the antigens recognized by T(H) cells are highly correlated with those recognized by antibody responses. We further show that protein abundance and antibody recognition can be used to predict antigens recognized by CD4(+) T cell responses, while early expression at the mRNA level predicts antigens targeted by CD8(+) T cells. Finally, we find that the vast majority of VACV epitopes are conserved in variola virus (VARV), thus suggesting that the epitopes defined herein also have relevance for the efficacy of VACV as a smallpox vaccine.

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.

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.