Vaccinia virus is the central component of the smallpox vaccine, used in the only successful eradication of an infectious disease (reviewed in [55
]). Concerns about the potential use of smallpox virus as a biological weapon, combined with a high morbidity rate and contraindications to immunization with vaccinia virus in some segments of the population, and recent outbreaks of monkeypox and other related poxviruses [56
], all highlight the need for a new generation of smallpox vaccines [58
]. For these reasons, new strains of vaccinia virus have been developed [60
] and subunit vaccines for smallpox are in development [11
]. However, advancement in this field is hampered by the lack of well-characterized CD4+
T cell epitopes required for the induction of long-lasting cellular and humoral immune responses.
We approached the challenge of identifying CD4+
T cell epitopes in vaccinia using an algorithm that combines independent assessments of MHC–peptide affinity [23
] and propensity for MHC-mediated antigen presentation [33
]. These two algorithms each proved highly predictive in an evaluation of 18 well-characterized HLA-DR1-restricted immunodominant epitopes. The combination approach was extremely effective in identifying vaccinia-derived CD4+
T cell epitopes. Of 36 potential epitopes tested, we observed IFN-γ responses to 25 peptide sequences, with strong responses in multiple donors observed for ten peptides. By comparison, in a recent study >2,000 peptides were screened to identify only 14 epitopes [20
]. The 25 peptides for which we observed T cell responses were derived from 23 proteins (A28L and D11L had two epitopes each). These proteins represent a mixture of early, late, and intermediate proteins, and include proteins present in the virion as well as proteins expressed only in infected cells ().
Peptides 301 and 334, which were recognized by all of our vaccinia-immune donors, are derived from proteins F17R and A10L, respectively, proteins that have been reported to be among the very most abundant proteins in the intracellular mature virion particles [62
]. A CD4+
T cell response to a different region of protein A10L was recently reported [18
]. Peptides 342 (D11L), 343 (D11L), 344 (A24R), and 302 (D1R) are all derived from enzymes involved in nucleic acid metabolism, and which are also highly represented in vaccinia virions. Ten other epitopes also derive from proteins present in vaccinia virions (IMV), but are reported to be at lower abundance [62
]. We used a crude lysate of infected cells as a source of vaccinia virus antigens for in vitro amplification of TCLs, and so we expected that our experimental protocol would allow identification of T cell responses directed against proteins present in viral particle and also against proteins not present in the virus but which are expressed in infected cells. Six peptides (305, 319, 328, 332, 337, and 338) derive from proteins not reported to be present in vaccinia virions. Peptide 332, derived from putative protein F16L, is recognized by all the vaccinia-exposed donors, but is not present in purified virions [62
]. Two peptides, 337 and 338, are derived from the immunomodulatory and virulence factors A44L (hydroxysteroid dehydrogenase) [65
] and B16R (IL-1 β inhibitor) [66
], and also are not expected to be present in the virion. Interestingly, we observed preferential responses to these epitopes in the infected as compared to vaccinated donors. However, inter-individual variation in the T cell responses to vaccinia virus as reported by Jing et al. could also explain these observations [18
]. T cell responses to peptides 306, 308, 314, and 327 were only evident after a second in vitro expansion. These peptides belong to the proteins I8R, F1L, I7L, and A48R, respectively. Only F1L, containing 306, and I7L, containing 314, are present in the virion. DR-restricted CD4+
T cell responses to proteins I8R and A48R have been reported to other regions of these proteins [18
Envelope proteins A27L, A33R, B5R, and L1R have attracted attention as possible subunit vaccine candidates because antibodies against these proteins correlate with protection against viral challenge in animal models [12
]. We tested two peptides derived from L1R (312 and 317), but did not observe responses in our donors. Both A33R and B5R have at least one peptide with very favorable HLA-DR1 binding prediction and antigen presentation scores that, however, fell just outside the range that we tested, and both of these proteins as well as L1R have additional peptides with scores in a slightly more generous region. Additional testing will be required to determine whether these high-scoring sequences do elicit the CD4+
T cell responses observed in these proteins. A27L does not contain potential epitopes that score highly by this algorithm. Although responses to A27L, A33R, B5R, and L1R were not observed among the set of peptides that we tested, we did identify T cell epitopes (peptides 322, 325, and 338) from three other proteins expected to be present on the virion membrane, A17L (p21 membrane protein), A28L (IMV membrane protein required for membrane fusion), and F16L (IL-1 β inhibitor). A17L is reported to be present in the inner of the two membranes of the IMV particles, with the N-terminus of the protein protruding to the surface of the particle, and antibodies raised against the exposed fraction of the protein neutralize vaccinia virus [67
]. These proteins might be considered as possible candidates for inclusion in subunit vaccine development efforts. Finally, antibody responses have been reported for protein I1L, containing peptide 345, and protein A10L, containing peptide 334 [9
]. However, there is currently no information about the possible role of these antibodies in immunity to vaccinia.
Only very recently have CD4+
T cell epitopes been identified in vaccinia virus [17
]. Jing et al. [18
] reported human T cell responses to 35 vaccinia proteins, and Moutaftsi et al. [20
] reported murine T cell responses to 13 vaccinia proteins. In this study, we detected in total human T cell responses to 28 peptide sequences from 24 different proteins, including the peptides with low prediction scores. T cell responses defined in this study and the ones reported by Jing et al. [18
] have in common only the response to three proteins, with no overlap in the actual peptide epitopes recognized. Comparison of the human T cell responses reported in our study and the murine responses reported by Moutaftsi et al. reveals a partial overlap in the T cell response to peptide 328, and a complete overlap in the response to peptides 325 and 345 in the proteins A20R, A28L, and I1L, respectively. Tang et al. [17
] reported human responses to three A27L epitopes in T cells isolated from blood samples obtained after 1 month or 3 years of vaccination; no epitopes from this protein were identified in our study or in the studies of Jing et al. [18
] or Moutaftsi et al. [20
]. Finally, using the same set of peptides tested here, Mitra-Kaushik et al. [19
] reported cytotoxic CD4+
T cell to D1R (MVA302) and A24R (MVA341) peptides, for which we observed robust IFN-γ responses. Whether the difference between these reports reflect individual (“private”) patterns of immunodominance, differences between the TCL and assay protocols used by the different groups, or incomplete sampling of a broad response to many different antigens, remains to be established by further work.
An important factor to consider in the characterization of T cell epitopes is the sequence conservation, since viral variation potentially can evade T cell immunity. Current vaccines induce substantial cross-protection between poxvirus family members. The 35 peptide sequences presented in this study are highly conserved among vaccinia strains, with only peptides 301 and 310 exhibiting variation within the core epitope or the immediately flanking residues (). The epitope sequences also are conserved within the larger poxvirus family (). Peptides 301, 332, 334, 335, 344, and 345, which each were recognized by strong responses in at least two of the donors in our study, are conserved in at least three poxvirus, including the human pathogens variola (all peptides) and monkeypox (all but 332). CD4+ T cells recognizing three of these peptides (332, 334, and 345) were observed in PBMCs from a vaccinated donor, indicating that cells with these specificities make up a significant part of the long-lasting memory pool elicited by immunization with vaccinia virus.
Our identification of highly conserved CD4+ T cell epitopes derived from vaccinia virus and recognized by donors of multiple MHC haplotypes could aid efforts to track cellular immunity induced by next-generation smallpox vaccines and could contribute to selection of candidate proteins for inclusion in potential subunit vaccine approaches. The epitope identification algorithm described here appears to be a significant improvement over current approaches, and could find application in prediction of class II MHC-restricted T cell responses to other large-genome viral and bacterial pathogens.