Poxvirus CD8+ T-cell determinants and cross-reactivity in BALB/c mice

Abstract

Mouse models of orthopoxvirus disease provide great promise for probing basic questions regarding host responses to this group of pathogens, which includes the causative agents of monkeypox and smallpox. However, some essential tools for their study that are taken for granted with other mouse models are not available for these viruses. Here we map and characterize the initial CD8+ T-cell determinants for poxviruses in H-2d-haplotype mice. CD8+ T cells recognizing these three determinants make up around 40% of the total responses to vaccinia virus during and after resolution of infection. We then use these determinants to test if predicted conservation across orthopoxvirus species matches experimental observation and find an unexpectedly cross-reactive variant peptide encoded by ectromelia (mousepox) virus.

FIG. 1.

Characterization of H-2^d-restricted VACV T_CD8+ determinants. Mice were infected with 10⁶ PFU VACV-WR i.p., and 7 days later, splenocytes were used in ICS assays. (A) Antigenic potency of synthetic peptides. Peptides at the indicated concentrations were used as stimulators for ICS (IFN-γ) assays. Data are expressed as percentages of maximum stimulation and are representative of two or more experiments. (B) Anti-TNF and anti-IFN-γ expression in VACV determinant-specific T_CD8+. TNF was used in addition to IFN-γ in ICS. Plots show CD8⁺ gated events and are representative of several experiments. Note that TNF⁺, IFN-γ⁻ events are similar in negative controls and for all VACV peptides.

FIG. 2.

T_CD8+ responses to VACV determinants in the context of the total anti-VACV response. Mice were immunized i.p. with 10⁶ PFU of VACV-WR, and T_CD8+ responses of splenocytes were measured by ICS 7 days (A) and 12 weeks (B) later. (Left) Graphs show the percentages of T_CD8+ that produce IFN-γ in ex vivo stimulations with peptides indicated (name of gene shown). (Right) Graphs compare a summation of the peptide data with similar data from the same splenocytes stimulated with VACV-infected P815 cells. Data are means and standard errors of the means from groups of four mice.

FIG. 3.

Cross-reactivity between T_CD8+ elicited by VACV-WR and variant peptides from other orthopoxviruses. In all panels, VACV-WR peptides are indicated by protein name; variants are F2(G) (SPGAAGYDL), F2(NH) (SNHAAGYDL), and E3(I) (VGPSNSPIF). (A) Mice were infected with the orthopoxvirus shown in the top left of each graph: VACV-WR and MVA, 10⁶ and 10⁸ PFU, respectively, i.p.; CPXV, 10⁵ PFU i.p.; and ECTV-TK⁻, 2 × 10⁷ PFU s.c. in the rear leg shank. Splenic T_CD8+ responses to peptides were measured by ICS after 7 days (VACV strains) and 8 days (CPXV and ECTV). Data are percentages of T_CD8+ that produce IFN-γ in ex vivo stimulations with peptides. Means and standard errors of the means for groups of four or five mice are plotted and are representative of repeated experiments. (B and C) A T_CD8+ line was derived from VACV-WR immune splenocytes by five restimulations with SPYAAGYDL in vitro and tested for cross-reactivity with variant peptides at various concentrations by ICS (B) and to peptides and virus-infected cells by cytotoxicity assay (C). (B, left) Percent T_CD8+ that produce IFN-γ after a short stimulation with the peptide shown (circle, SPYAAGYDL; square, SPGAAGYDL; triangle, SNHAAGYDL); (B, right) the same data presented as percentages of the maximum response. Panel C shows percent specific lysis at the E:T ratios indicated for this T_CD8+ line against ECTV-infected (ECTV, unfilled triangle) or VACV-WR-infected (VACV, unfilled circle) P815 cells or peptide-pulsed P815 as indicated above the graph (symbols as in panel B).