Comparable Polyfunctionality of Ectromelia Virus- and Vaccinia Virus-Specific Murine T Cells despite Markedly DifferentIn VivoReplication and Pathogenicity

Abstract

Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunctional CD8 ؉ T-cell responses after immunization of humans. However, the degree to which the functional profile of T cells induced by VACV is similar to that generated during natural poxvirus infection remains unknown. In this study, we monitored virus-specific T-cell responses following the dermal infection of C57BL/6 mice with ECTV or VACV. Using polychromatic flow cytometry, we measured levels of degranulation, cytokine expression (gamma interferon [IFN-␥], tumor necrosis factor alpha [TNF-␣], and interleukin-2 [IL-2]), and the cytolytic mediator granzyme B. We observed that the functional capacities of T cells induced by VACV and ECTV were of a similar quality in spite of the markedly different replication abilities and pathogenic outcomes of these viruses. In general, a significant fraction (>50%) of all T-cell responses were positive for at least three functions both during acute infection and into the memory phase. In vivo killing assays revealed that CD8 ؉ T cells specific for both viruses were equally cytolytic (ϳ80% target cell lysis after 4 h), consistent with the similar levels of granzyme B and degranulation detected among these cells. Collectively, these data provide a mechanism to explain the ability of VACV to induce protective T-cell responses against pathogenic poxviruses in their natural hosts and provide further support for the use of VACV as a vaccine platform able to induce polyfunctional T cells.

Impact of Distinct Poxvirus Infections on the Specificities and Functionalities of CD4+T Cell Responses

“…In order to identify T CD4 ϩ with cytotoxic potential, we also assessed degranulation by measuring the surface expression of CD107a after stimulation (50). In agreement with our prior study (15), the average T CD4…”

“…ICS assay. ICS assays were performed as previously described (15). Briefly, bone marrow-derived dendritic cells (1 ϫ 10 6 ) generated using previously published methods (35) were either pulsed with peptides (3 g/ml) for 1 h in a 96-well plate or were infected with VACV WR (multiplicity of infection [MOI] of 5) for between 10 and 18 h before the addition of 1 ϫ 10 6 to 2 ϫ 10 6 splenocytes (pooled from two to five mice that were immunized with VACV WR for 10 days).…”

“…3A and C and Table 1). Eleven of these had been previously identified in the VACV screen, with two common epitopes (residues 46 to 60 of A18R [A18R 46 -60 ] and I1L [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] ) differing by a single, apparently neutral, amino acid (Table 2). Thus, three epitopes (I4L 632-646 , B13R 14 -28 , and E2L 426 -440 ) were novel (Table 3).…”

“…Furthermore, ECTV produces species-specific factors that allow for evasion and subversion of host responses and far greater replication in mice, as well as a greater antigen load, which will affect the levels of epitope display. Virulence factors and antigen load can also affect the cytokine milieu, which is markedly different in the two infections (15,(63)(64)(65)(66)(67) and which can strongly influence antigen-processing capabilities (68,69). Chief among the cytokines of interest is IFN-␥, which, in addition to influencing the expression of antigen-processing components (68)(69)(70)(71), drives upregulation of MHC-II (72).…”

“…Moreover, these poxviruses have distinct courses of infection after intradermal infection in mice. ECTV multiplies rapidly at the site of infection before disseminating into the lymphatics and bloodstream, where it leads to a systemic infection that affects both the liver and spleen (14,15), whereas VACV remains relatively localized after intradermal infection and does not lead to systemic infection (16). Importantly, because VACV is not a natural mouse pathogen, despite a high degree of homology with ECTV, the relevance of results from the widely studied VACV murine infection model may be limited.…”