Relatively little is known about the specificity of human T-cell responses to Y. pestis
. This is important in a number of ways: (i) to gain a better understanding of subversion of immunity in the pathogenesis of plague, (ii) to design epitope vaccines, and (iii) from a perspective of fundamental immunology, to contribute to our understanding of the interface between bacterial infection and host T-cell immunity. Because of the logistical problems inherent in identifying Y. pestis
immune human subjects, we have here undertaken complete analysis HLA-DR1-restricted CD4 T-cell epitopes of Caf1 of Y. pestis
by multiple approaches. We used mice transgenic for HLA DRB1*0101 in the absence of endogenous mouse MHC class II so that all T-cell responses detected were DR1 restricted and epitopes could be identified in the absence of any competing MHC class II products. Vaccination with Caf1 protects mice against plague effectively (35
) but induces relatively poor T-cell responses in vivo
) (Fig. ). It is therefore of considerable interest to dissect the basis of Caf1 immune recognition in more detail. We show here that the HLA-DR-restricted CD4 T-cell response is dominated by recognition of a single Caf1 epitope and that this response is likely to have broad-ranging applicability to human populations since the epitope binds with varying affinity to all of the HLA-DR alleles tested.
The single immunodominant epitope of Caf1 was identified within peptide 15 (Caf1141-160), overlapping with the sequence in peptide 14 (Caf1131-150) and demonstrated by multiple approaches. Peptide 15 was the only peptide to recall T-cell responses consistently in Caf1-immunized mice measured both as proliferation and as IFN-γ-secreting cell frequency. The presence of an epitope shared by peptides 14 and 15 was confirmed by proliferation responses and IFN-γ-secreting cell frequency in peptide-immunized mice, and both peptides 14 and 15 were also recognized by the majority of T-cell hybridomas generated from Caf1-immunized mice. However, lymph node T cells taken directly ex vivo from Caf1-immunized mice recognized peptide 15 but not 14, possibly showing a dominance of a particular fine specificity for peptide 15 in the in vivo T-cell repertoire. In addition, it was shown that peptide 15 was among the five peptides that bound DR1 with high affinity and was the only peptide from the mature Caf1 protein predicted as an epitope by consensus of the combined algorithms of the IEDB analysis resource.
Peptides 7, 10, 11, 12, 13, and 16 also stimulated IFN-γ-producing cells, although not recalling proliferation responses in Caf1-immunized mice, suggesting additional immunodominant or subdominant epitopes in these peptides. Of these, only peptides 11 and 16 induced IFN-γ-producing cells in peptide-immunized mice. The presence of an epitope on peptide 16 was confirmed by the specificity of one of the T-cell hybridomas generated from Caf1-immunized mice. The epitope on peptide 16 is distinct from that on peptide 14/15 because neither the peptide 16-specific T-cell hybridoma nor peptide 16-immunized mice recognized peptide 15. Peptide 16 also bound DR1 with intermediate affinity and was one of the higher-scoring peptides by epitope prediction. The epitope on peptide 11 was also confirmed in proliferation and IFN-γ assays following peptide immunization, although this peptide bound HLA-DR with low affinity.
All five peptides that bound DR1 elicited a T-cell response in at least one of the functional assays. However, several peptides (peptides 4 and 6 to 14), recognized by T cells in one or more of the assays, did not show detectable binding to HLA-DR1, suggesting that strong binding to a particular MHC class II allele is not a prerequisite for a functional helper T-cell epitope. Additional peptides (peptides 4, 5, 8, and 9) were shown to include epitopes by peptide immunization using both proliferation and IFN-γ assays. The lack of strong responses to these peptides in Caf1-immunized mice identify them as cryptic epitopes (30
An unexpected outcome of the present study was the identification of one or more DR1-restricted epitopes in the signal sequence of Caf1. All 20 amino acids of peptide 1 and the first 11 amino acids of peptide 2 represent the signal sequence of Caf1 (17
). T-cell responses to peptide 1 in Caf1-immunized mice were not detected (Fig. ) and would not be expected because the mature rCaf1 protein lacks the signal sequence. However, a marginal response in lymph node proliferation assay was detected in one mouse immunized with peptide 1 (Fig. ) and in separate experiments cells from one peptide-immunized mouse responded by IFN-γ production (Fig. ). Peptide 1 also bound with high affinity to DR1, as well as DR7 and DR15 (Table ), and scored highest in epitope prediction analysis. Similar results were obtained for peptide 2. Thus, the data suggest the presence of at least one CD4 T-cell epitope within the signal sequence by several approaches. In addition, we have infected DR1-transgenic mice with Y. pestis
and measured T-cell responses as secreted IFN-γ upon in vitro
recall to the Caf1 peptide set, and some mice responded to peptides 1 and 2 (data not shown), suggesting that after cleavage from Caf1, the signal sequence is available for antigen presentation during infection. It remains to be determined whether the location of CD4 T-cell epitopes within the signal sequence of proteins from bacteria is a general phenomenon.
Although the functional mapping of T-cell epitopes was confined to HLA-DR1, we also examined the binding of all 16 Caf1 peptides to seven different, common HLA-DR alleles. Remarkably 15 of the 16 Caf1 peptides bound with high or intermediate affinity to one or more of the DR alleles tested, including alleles of the same (DR1, DR4, DR7, and DR11) or different HLA-DR supertypes (10
). In particular, peptides 2, 14, and 15 demonstrated the greatest promiscuous binding properties, each binding to five of the seven alleles. Of the remaining peptides, three bound to three alleles, five bound to two alleles, and three bound to a single allele, and peptide 9 was the only peptide that bound none of the DR alleles. Epitopes that bind multiple HLA alleles are termed promiscuous or universal (32
) and would be particularly valuable for use in epitope-defined vaccines. In addition, we have previously mapped HLA-DQ8-restricted CD4 T-cell epitopes of Caf1, as well as the immunodominant mouse H-2d
- and H-2b
-restricted epitopes of Caf1, including one within peptide 15 (Table ) (31
In conclusion, this is the first report identifying HLA-restricted CD4 T-cell epitopes of a major plague vaccine antigen. In particular, we have shown that the T-cell response after Caf1 vaccination is focused on a single immunodominant epitope with properties of a promiscuous HLA class II binder.