Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies

Abstract

Generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for human immunodeficiency virus type 1 (HIV-1) and other pathogens. Virus-specific CD8(+) T-lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8(+) T-lymphocyte responses may prove to be critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Here we show that epitope modification strategies can alter CD8(+) T-lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing simian immunodeficiency virus Gag lacking the dominant D(b)-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D(b)-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes.

FIG. 1.

Construction of the Gag dAL11 DNA vaccine. (A) The Gag dAL11 DNA vaccine was produced by mutating the position 5 anchor residue of the AL11 epitope from asparagine (N) to alanine (A). (B) Expression of Gag WT and Gag dAL11 DNA vaccines was assessed in transiently transfected 293 cells by enzyme-linked immunosorbent assay. (C) AL11-specific CD8⁺ T-lymphocyte responses were evaluated by D^b/AL11 tetramer binding assays of C57BL/6 mice immunized with Gag WT and Gag dAL11 DNA vaccines. Error bars indicate standard errors.

FIG. 2.

Cellular immune responses elicited by Gag WT and Gag dAL11 DNA vaccines. Groups of C57BL/6 mice (n = 12/group) were immunized i.m. at week 0 and week 4 with 50 μg Gag WT or Gag dAL11 DNA vaccine. AL11- and KV9-specific CD8⁺ T-lymphocyte responses were assessed by IFN-γ ICS assays. (A) Representative mice at week 2. (B) Mean responses in both groups at multiple time points following immunization. (C) IFN-γ ELISPOT assays in response to a pool of overlapping Gag peptides, the CD4⁺ T-lymphocyte epitope DD13, and the CD8⁺ T-lymphocyte epitopes AL11 and KV9 at week 4 and week 8. SFC, spot-forming cells. Error bars indicate standard errors.

FIG. 3.

Durability of epitope-specific cellular immune responses. Groups of C57BL/6 mice (n = 4/group) were immunized i.m. once with 50 μg Gag WT or Gag dAL11 DNA vaccine. AL11- and KV9-specific CD8⁺ T-lymphocyte responses (A) and DD13-specific CD4⁺ T-lymphocyte responses (B) were assessed by IFN-γ ICS assays 6 months after immunization. Error bars indicate standard errors.

FIG. 4.

Anatomic separation of Gag WT and Gag dAL11 DNA vaccines. Groups of C57BL/6 mice (n = 8/group) were immunized once i.m. with the following DNA vaccine regimens: 50 μg Gag WT (half the dose in each leg), 50 μg Gag dAL11 (half the dose in each leg), 25 μg Gag WT and 25 μg Gag dAL11 mixed together in the same syringe (half the dose in each leg), or 25 μg Gag WT (left leg) and 25 μg Gag dAL11 (right leg). AL11- and KV9-specific CD8⁺ T-lymphocyte responses were assessed by IFN-γ ICS assays at week 2 following immunization. Error bars indicate standard errors.

FIG. 5.

Immunogenicities and protective efficacies of heterologous prime-boost regimens. Groups of C57BL/6 mice (n = 8/group) were immunized i.m. at week 0 with 50 μg Gag WT or Gag dAL11 DNA vaccine and boosted at week 4 with 10⁶ vp of rAd5 expressing Gag WT. Control mice received a sham plasmid and an empty rAd5 vector. (A) AL11- and KV9-specific CD8⁺ T-lymphocyte responses by IFN-γ ICS assays 2 weeks after the boost immunization at week 6. (B) Mice were challenged i.p. at week 10 with 5 × 10⁶ PFU of vaccinia virus-Gag, and vaccinia virus titers in ovaries were assessed on day 6 after challenge. Virus titers in both vaccinated groups (*) were significantly lower than those in the sham control group (P < 0.001). Virus titers were lower in mice primed with the Gag dAL11 DNA vaccine than in mice primed with the Gag WT DNA vaccine (P < 0.05). Error bars indicate standard errors.