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

A Significant Fraction of Epitope Sequences Are Conserved in S-OIV.

The overall goal of the analysis presented in this section was to establish whether the epitopes described in the literature and presumably associated with pre-existing immunity in the general population, would be conserved in S-OIV sequences.

At the time of this writing, the epitope information available in the IEDB related to influenza A encompassed information from 594 references (journal articles and direct submissions), describing 3,724 distinct molecular structures (linear and discontinuous peptides) derived from influenza A that were tested experimentally for interaction with immune receptors. This roughly doubled the amount of information on influenza epitopes that was available in 2006 (7), emphasizing the important contribution and greatly enhanced throughput of recent influenza epitope mapping efforts, which were stepped up since the emergence of H5N1 avian flu.

We focused our analysis on influenza A epitope mapping experiments that have the most relevance for human immunity and considered only those epitopes that are present in the sequences of recently circulating strains of H1N1 (i.e., isolates from 1988 to 2008). We considered an epitope relevant if it showed recognition by antibodies or by T cells in the context of human MHC and if the epitopes were mapped in the context of whole influenza organisms or proteins. As B-cell epitopes are infrequently identified in humans (7), B-cell epitopes mapped in the context of any host organism were included. We think that this is justified, as studies have shown substantial overlap for B-cell epitopes defined in different species, presumably because the structural constraints associated with their recognition is similar across species (11).

We distinguished between two primary categories of epitopes: (i) those recognized by B cells/antibodies, and (ii) those recognized by T cells. The latter were further categorized into those recognized by CD8⁺ T cells in the context of major histocompatibility complex (MHC) class I molecules and those recognized by CD4⁺ T cells in the context of MHC class II. If multiple epitopes in the same category had sequences that were nested within each other, a representative epitope was chosen that was associated with the highest specific immune response. Full details of the specific IEDB queries are given in Materials and Methods. As shown in Table 1, a total of 26 B-cell epitopes were found in recently circulating H1N1 strains, whereas 139 CD4⁺ and 78 CD8⁺ T-cell epitopes were found.

Next, we asked how many of these experimentally defined epitopes, for which we would expect some pre-existing immunity, are totally conserved in the emerging S-OIV strains. Human S-OIV sequences were retrieved from isolates originating in Mexico, the United States, and elsewhere, as described in Materials and Methods. As shown in Table 1, a substantial number of epitopes present in recent seasonal H1N1 strains are also found to be 100% conserved in S-OIV sequences. Notably, whereas only ≈31% of the B-cell epitopes are conserved in S-OIV strains, up to 41% and 69% of the CD4⁺ and CD8⁺ T-cell epitopes, respectively, are conserved. The complete list of individual epitopes is provided in Table S1.

We also assessed whether seasonal influenza viruses of the H3N2 subtype contained additional epitopes conserved in S-OIV strains. A repeat of the analysis above showed that this was not the case, as all epitopes that are conserved between the H3N2 subtype and as S-OIV are also shared with seasonal H1N1 isolates. Overall, these data demonstrated that a sizeable fraction of epitopes derived from H1N1 seasonal flu strains and described in the literature is conserved in S-OIV sequences. This raised the possibility that some level of immunity against S-OIV sequences might exist in the general population.

Distribution of Conserved Epitopes in Different Influenza Proteins.

The proteins hemagglutinin (HA) and neuraminidase (NA), which make up the majority of the viral surface, are more variable than other influenza proteins. Therefore, it can be expected that epitopes from those proteins are less likely to be conserved across strains than others. As shown in Table 2, this is indeed the case. For T-cell epitopes, only five of 43 (12%) HA and NA epitopes are conserved, as opposed to 106 of 174 (61%) epitopes derived from other proteins. Similarly, at the level of B-cell epitopes, only a single HA/NA epitope among six is conserved (17%), whereas seven of 20 of the remaining epitopes in other proteins are conserved (35%). This shows that for both B-cell and T-cell epitopes, fewer epitopes are conserved in the virion surface antigens.

For B-cell responses, neutralizing epitopes are primarily located on the virion surface proteins HA and NA. That means that the majority of B-cell epitopes conserved in S-OIV are unlikely to be neutralizing. For example, the NP protein contains 9 H1N1 B-cell epitopes, four of which are conserved in S-OIV sequences. However, to be targets of directly neutralizing antibodies, epitopes need to be exposed on the virion surface. Therefore, although epitopes in the NP protein are often targeted by serological immune responses, they are not protective as they are thought to be inaccessible from the surface (12).

A single HA B-cell epitope is conserved in S-OIV. To determine its location in the protein, we mapped the epitope onto the three-dimensional structure of a homologous protein of the S-OIV HA trimer complex for the precursor HA (Fig. 1 A and B) and onto a homology model of the cleaved HA (Fig. 1 C and D), as the epitope is located at the N-terminal of the fusion peptide. In the precursor, the epitope is solvent accessible, with an accessible surface area (ASA) of 632 Ų, which is similar to the surface area of interfaces in known structures of antibody–peptide complexes. In the cleaved HA, the epitope is partially buried in the cavity of HA, into which the fusion peptide inserts after precursor cleavage, with an ASA of 294 Ų. The location of this epitope in the conserved HA2 glycopeptide is atypical, as most neutralizing antibodies target HA1, and it is unclear how frequent pre-existing immune responses are against this peptide. At the same time, this epitope was mapped to a monoclonal antibody that inhibited cell fusion in vitro and provided protection from challenge in vivo (13). Taken together, these data suggest that although the one B-cell epitope that is conserved might be partially exposed in some HA isoforms, the vast majority of known B-cell epitopes are not conserved in S-OIV and are unlikely to be relevant for protection.

Experimental Demonstration of Memory T-Cell Response Recognizing H1N1 Influenza Sequences.

The analysis presented above suggests that a significant level of T-cell immunity might pre-exist in the general population against epitope sequences that are found totally conserved in S-OIV. To experimentally address this point, we synthesized four groups of peptide epitopes. One group corresponded to the CD4⁺ T-cell epitopes totally conserved in S-OIV sequences; the second group consisted of CD8⁺ T-cell epitopes conserved in S-OIV; and two additional groups of peptides consisted of CD4⁺ and CD8⁺ T-cell epitopes not conserved in S-OIV sequences. These four separate peptide pools were tested with peripheral blood mononuclear cells (PBMCs) from normal blood donors for induction of interferon (IFN)–γ secretion in direct ex vivo ELISPOT assays. PBMC from anonymous blood donors from the San Diego region were banked in the context of an independent study of influenza responses, and collected at least 1 year before the onset of the current S-OIV pandemic.

We found that, on average, responses significantly greater than zero were detected against the pool of epitopes conserved in S-OIV sequences, both in terms of CD4⁺ and CD8⁺ T-cell responses (P < 0.01 for all epitopes sets, one-tailed single sample t test) (Fig. 2). The responses to the conserved S-OIV epitope pools were similar in magnitude to the responses observed with their nonconserved counterparts, and the observed difference was not statistically significant (average of 40 spot-forming cells [SFC]/10⁶ cells for conserved and 41 SFC for nonconserved CD8+ T-cell epitopes (P = 0.94, two-tailed paired t test), and 51 versus 74 SFC/10⁶ for CD4⁺ T-cell conserved and nonconserved, respectively (P = 0.14, two-tailed paired t test). Information on age and sex was available only for half of the 20 donors (Table S2); thus investigating correlations of responses with these parameters was not possible.

For selected donors, we sought to further document that the responding cells were of the memory phenotype. Accordingly, intracellular cytokine and cell-surface staining assays were used to characterize the IFN-γ responses against CD4⁺ and CD8⁺ T-cell epitopes conserved in S-OIV. Specifically, CD4⁺ or CD8⁺ T cells producing IFN-γ were examined for the expression of various memory markers. We found that the responding CD4⁺IFN-γ⁺ T cells, stimulated with the epitope pool conserved in S-OIV, were CD45RA^medCD62L^loCCR7⁻ (Fig. 3). Likewise, the CD8⁺IFN-γ⁺ T cells stimulated with the epitope pool conserved in S-OIV were also CD45RA^medCD62L^loCCR7⁻. Overall, these data show that both CD4⁺ and CD8⁺ T-cell memory responses against S-OIV epitopes can be detected directly ex vivo, and that the magnitude of responses against epitopes conserved in S-OIV is at least comparable to that of other epitopes in recently circulating influenza strains.

Comparison of Literature-Defined Epitope Content for Seasonal Influenza and S-OIV at the Individual Isolate Level.

Next, we asked how the level of pre-existing immunity for an average S-OIV isolate compares to that of an average seasonal H1N1 influenza isolate. For 559 seasonal H1N1 flu isolates from 2000 to 2008, we determined the number of epitopes present in each isolate that were also present in H1N1 seasonal flu isolates of the preceding 20 years. As before, such epitopes are considered likely targets of pre-existing immune responses. Table 3 lists the median number of epitopes on a per-virus basis as 16 B cell, 93 CD4⁺, and 66 CD8⁺ T-cell epitopes. These numbers were used as a baseline against which we compared the number of epitopes conserved in S-OIV isolates, again on a per-virus basis. Interestingly, no variability in the epitope sequences within different S-OIV isolates was detected. Thus, the number of conserved epitopes in any S-OIV isolate is 8, 57, and 54 for B-cell, CD4⁺, and CD8⁺ T-cell epitopes, respectively. Thus, it would be predicted that the levels of pre-existing immunity against S-OIV, albeit reduced in comparison to the levels directed against other H1N1 strains, would still be significant, particularly in the case of T-cell epitopes, in general, and CD8⁺ T-cell epitopes, in particular.

To test this hypothesis, we compared the magnitude of responses detected in the ELISPOT assay for IFN-γ release against the peptide pools corresponding to CD4⁺ and CD8⁺ T-cell epitopes conserved in S-OIV with peptide pools corresponding to CD4⁺ and CD8⁺ T-cell epitopes found in 2008 seasonal influenza. For this comparison, we used PBMC from blood donations obtained in 2007. The results obtained are shown in Fig. 4.

We found that significant CD4⁺ and CD8⁺ T-cell responses were detected to epitopes conserved in both S-OIV and 2008 seasonal influenza (P < 0.05 for all epitope sets, one-tailed single-sample t test). As predicted, the response to the epitopes in 2008 seasonal influenza was higher than the response to the S-OIV epitopes, in terms of both CD4⁺ and CD8⁺ T cells (17 versus 28 SFC/10⁶ for CD4⁺ T cells, and 44 versus 51 SFC/10⁶ for CD8⁺ T cells in response S-OIV and seasonal influenza epitopes, respectively); however, these differences did not reach statistical significance (P = 0.25 for CD4⁺ responses and P = 0.13 for CD8+ responses, according to a two-tailed paired t test). Overall, the level of pre-existing immunity was higher for CD8⁺ T cells compared with CD4⁺ T cells, as this likely reflects a higher level of conservation of the S-OIV CD8⁺ T-cell epitopes with recently circulating 2008 seasonal influenza.

Querying for Epitopes with Human Relevance in the IEDB.

Epitopes derived from influenza A were retrieved from the IEDB, as described in the SI Text.

Querying for Epitopes Conserved in Circulating H1N1.

The epitopes in the human-relevant set described above were searched against H3N2 and H1N1 sequences from circulating strains between 1988 and 2008, as described in the SI Text.

Querying for Epitopes Conserved in S-OIV (H1N1 2009).

The set of circulating H1N1 epitopes were searched against all S-OIV sequences (H1N1, 2009) and the conservation of epitopes was calculated in the same manner as above.

Querying for Influenza Sequences.

Circulating H1N1 and H3N2 influenza sequences were obtained from the NCBI Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). For details on the queries, see the SI Text.

At the time this analysis was performed, the NCBI database did not contain sequences from Mexican isolates. These were retrieved from the GISAID database. Table S3 lists the isolates for which sequences were obtained. All Mexican isolates were submitted by the Centers for Disease Control and Prevention (Atlanta, GA, Rebecca Garten).

Homology Modeling, Image Generation, and ASA Calculation.

The homology model for Fig. 1 was generated using the SWISS-MODEL homology-modeling server (26), as described in the SI Text.

PBMC Isolation.

PBMC were isolated from heparinized blood by gradient centrifugation with a Histopaque-1077 (Sigma-Aldrich, St. Louis, MO), suspended in fetal bovine serum (FBS) containing 10% dimethyl sulfoxide, and cryopreserved in liquid nitrogen.

Ex vivo IFN-γ ELISPOT.

IFN-γ ELISPOT assays were performed as described in ref. 27. In brief, 2 × 10⁵ PBMC (CD8⁺ assays) or enriched CD4⁺ T cells were incubated with pools of peptides (1 μg/ml per peptide). CD4⁺ T cells were positively enriched by incubating PBMC with anti-CD8 microbeads (Miltenyi Biotech, Auburn, CA) for 15 min at 4 °C. After washing, PBMC were resuspended in magnetic cell sorting (MACS) buffer and passed through a magnetized LD column (Miltenyi Biotech). Enriched CD4⁺ T cells were collected by washing the column three times with MACS buffer. After 20 h incubation with the peptide pools at 37 °C, plates were developed, and responses calculated as described in ref. 27.

Intracellular IFN-γ Staining.

PBMC were stimulated with pools of peptides (1 μg/ml per peptide). After 2 h, brefeldin A was added, and PBMC were cultured for an additional 6 h with the peptide pools. After incubation, cells were stained for cell surface antigens CD4, CD8, CD45RA, CD62L, and CCR7. Cells were then fixed, permeabilized, and stained for intracellular IFN-γ using a Cytofix/Cytoperm kit according to manufacturer's directions (BD Biosciences). After washing, samples were resuspended in phosphate-buffered saline, and data were acquired on an LSRII flow cytometer (BD Biosciences). The frequency of CD4⁺ and CD8⁺ T cells responding to each peptide pool was quantified by determining the total number of gated CD4⁺ or CD8⁺ and IFN-γ⁺ T cells using FlowJo software (Tree Star, San Carlos, CA).