Protection against lethal vaccinia virus challenge in HLA-A2 transgenic mice by immunization with a single CD8+ T-cell peptide epitope of vaccinia and variola viruses

Abstract

CD8(+) T lymphocytes have been shown to be involved in controlling poxvirus infection, but no protective cytotoxic T-lymphocyte (CTL) epitopes are defined for variola virus, the causative agent of smallpox, or for vaccinia virus. Of several peptides in vaccinia virus predicted to bind HLA-A2.1, three, VETFsm(498-506), A26L(6-14), and HRP2(74-82), were found to bind HLA-A2.1. Splenocytes from HLA-A2.1 transgenic mice immunized with vaccinia virus responded only to HRP2(74-82) at 1 week and to all three epitopes by ex vivo enzyme-linked immunosorbent spot (ELISPOT) assay at 4 weeks postimmunization. To determine if these epitopes could elicit a protective CD8(+) T-cell response, we challenged peptide-immunized HLA-A2.1 transgenic mice intranasally with a lethal dose of the WR strain of vaccinia virus. HRP2(74-82) peptide-immunized mice recovered from infection, while naïve mice died. Depletion of CD8(+) T cells eliminated protection. Protection of HHD-2 mice, lacking mouse class I major histocompatibility complex molecules, implicates CTLs restricted by human HLA-A2.1 as mediators of protection. These results suggest that HRP2(74-82), which is shared between vaccinia and variola viruses, may be a CD8(+) T-cell epitope of vaccinia virus that will provide cross-protection against smallpox in HLA-A2.1-positive individuals, representing almost half the population.

FIG. 1.

Binding of vaccinia virus peptide to HLA-A2. The 9-mer peptides were tested for binding by the standard T2 binding assay. HLA-A2 expression was measured with the antibody BB7.2 and compared with expression on T2 cells unpulsed with peptide. Each data point represents a single peptide concentration on the titration curve. This result was reproduced in three independent experiments.

FIG. 2.

Ex vivo ELISPOT on splenocytes from vaccinia virus-infected HLA-A2 transgenic mice. Mice from the AAD strain of HLA-A2 transgenic mice were injected i.p. with the replication-competent vaccinia virus vSC8. Spleens were removed at 1 week of infection or 4 to 6 weeks postinfection (1 month) and screened ex vivo by ELISPOT for IFN-γ-secreting T lymphocytes recognizing each of the three predicted vaccinia virus epitopes on peptide-pulsed C1R.AAD targets. The data are representative averages from the AAD strain of mice in three or four independent experiments with three to nine mice in each experiment. Error bars are based on standard errors of the means.

FIG. 3.

Natural processing and recognition of HRP2(74-82) in vaccinia virus-infected C1R.AAD targets. HLA-A2 transgenic mice were immunized twice s.c. in the base of the tail with the vaccinia virus peptide HRP2(74-82). Spleens were removed and restimulated twice in vitro on HRP2(74-82)-pulsed antigen-presenting cells and then screened by ELISPOT for IFN-γ-secreting T lymphocytes recognizing either peptide-pulsed or vaccinia virus (vSC8)-infected C1R.AAD targets. [Error bars represent standard errors of the means; P = 0.02 for recognition of virally infected versus uninfected cells by T cells raised on HRP2(74-82) peptide.]

FIG. 4.

(A) Survival of wild-type vaccinia virus challenge. HLA-A2 transgenic mice (strain A2Kb, five or six mice per group) were immunized once i.p. with vSC8 vaccinia virus or three times s.c. with peptide HRP2(74-82) or A26L(6-14) or left unimmunized. Some of the immunized mice were depleted of CD8⁺ T cells. One month after the third immunization with HRP2(74-82) or A26L(6-14), the mice were challenged intranasally with a lethal amount (10⁶ PFU) of wild-type vaccinia virus (strain WR). Survival was monitored out to 14 days postchallenge (where day 0 is the day of challenge) [P = 0.004 for unimmunized versus HRP2(74-82)-, vSC8-, or A26L(6-14)-immunized mice; P = 0.40 for CD8-depleted versus unimmunized mice; P = 0.02 for CD8-depleted versus HRP2(74-82)-immunized mice]. The results shown are from an experiment representative of two with similar results. (B) Disease course following vaccinia virus challenge. Disease course was tracked by monitoring mouse weight loss daily over the same 14-day period as survival. Mice that lost more than 25% of body weight were sacrificed according to NIH guidelines. Numbers represent pooled average weights of 11 mice [HRP2(74-82)-immunized group], 4 mice [A26L(6-14)-immunized group and CD8-depleted group], 7 mice (vSC8-immunized group), and 9 mice (unimmunized group), from two independent experiments [P = 0.008 for unimmunized mice versus HRP2(74-82)-immunized mice, P = 0.02 for vSC8-immunized mice versus unimmunized mice, P = 0.03 for A26L(6-14)-immunized mice versus unimmunized mice, P = 0.001 for CD8-depleted mice versus HRP2(74-82)-immunized mice, and P = 0.45 for unimmunized mice versus CD8-depleted mice]. Error bars represent standard errors of the means. Mean weights were not plotted beyond the point at which most or all of the mice in the group were dead. The results shown are from an experiment representative of two with similar results.

FIG. 5.

(A) Survival of HHD-2 mice following vaccinia virus challenge. HHD-2 mice, which have only human HLA-A2 and no mouse class I MHC molecules, were immunized three times s.c. with either HRP2(74-82) (five mice) or A26L(6-14) (three mice) or left unimmunized (four mice). Two weeks after the second peptide immunization, mice were challenged intranasally with 10⁶ PFU of wild-type vaccinia virus (strain WR). Survival was monitored to 14 days postchallenge (where day 0 is the day of challenge) [P = 0.01 for HRP2(74-82)-immunized mice versus unimmunized mice]. The results shown are from an experiment representative of two with similar results. (B) Disease course in HHD-2 mice following vaccinia virus challenge. As with A2Kb mice, weight loss was monitored in the HHD-2 mice over the same 14-day period as survival. Mice that lost more than 25% of body weight were sacrificed according to NIH guidelines. Numbers represent average weights of three to five mice in each group (as in panel A) (or average weight of surviving mice) [P = 0.005 for HRP2(74-82)-immunized mice versus unimmunized mice]. Error bars represent standard errors of the means. Mean weights were not plotted beyond the point at which most or all of the mice in the group were dead. The results shown are from an experiment representative of two with similar results.

FIG. 6.

Immunodominance of response to HRP2(74-82) in peptide-immunized versus whole-virus-immunized mice. (A) Average precursor frequency of HRP2(74-82) peptide-specific CD8⁺ T cells as measured by IFN-γ ELISPOT in pre- and post-virus-challenge mice immunized with either peptide or whole virus. (B) Relative dominance of HRP2(74-82) epitope following virus challenge in each group of immunized mice. Percent immunodominance is defined as [spots per million on HRP2(74-82)-pulsed targets/spots per million on whole-virus-infected targets] × 100. In both panels, error bars represent the standard errors of the means. (For both expansion pre- to postchallenge in panel A and relative dominance in panel B, P = 0.01.)