Development of smallpox vaccine candidates with integrated interleukin-15 that demonstrate superior immunogenicity, efficacy, and safety in mice

Abstract

The potential use of variola virus, the etiological agent of smallpox, as a bioterror agent has heightened the interest in the reinitiation of smallpox vaccination. However, the currently licensed Dryvax vaccine, despite its documented efficacy in eradicating smallpox, is not optimal for the vaccination of contemporary populations with large numbers of individuals with immunodeficiencies because of severe adverse effects that can occur in such individuals. Therefore, the development of safer smallpox vaccines that can match the immunogenicity and efficacy of Dryvax for the vaccination of contemporary populations remains a priority. Using the Wyeth strain of vaccinia virus derived from the Dryvax vaccine, we generated a recombinant Wyeth interleukin-15 (IL-15) with integrated IL-15, a cytokine with potent immunostimulatory functions. The integration of IL-15 into the Wyeth strain resulted in a >1,000-fold reduction in lethality of vaccinated athymic nude mice and induced severalfold-higher cellular and humoral immune responses in wild-type mice that persisted longer than those induced by the parental Wyeth strain. The superior efficacy of Wyeth IL-15 was further demonstrated by the ability of vaccinated mice to fully survive a lethal intranasal challenge of virulent vaccinia virus even 10 months after vaccination, whereas all mice vaccinated with parental Wyeth strain succumbed. By integrating IL-15 into modified vaccinia virus Ankara (MVA), a virus currently under consideration as a substitute for the Dryvax vaccine, we developed a second vaccine candidate (MVA IL-15) with greater immunogenicity and efficacy than Dryvax. Thus, Wyeth IL-15 and MVA IL-15 viruses hold promise as more-efficacious and safe alternatives to the Dryvax vaccine.

FIG. 1.

IL-15-integrated vaccine viruses display augmented CD8⁺ and CD4⁺ T-cell responses. Animals were immunized subcutaneously with 2 × 10⁶ PFU of the respective virus. At the indicated time points, three to four immunized animals were sacrificed and their splenocytes harvested and pooled. Stimulator cells were prepared from syngeneic splenocytes infected with the Wyeth strain of vaccinia virus at an MOI of 5 for 6 h, followed by γ-irradiation. Responder cells were stained for intracellular IFN-γ according to the vendor's protocol (Pharmingen). Panel A depicts vaccinia virus-specific CD8⁺ T cells, and panel B depicts vaccinia virus-specific CD4⁺ T cells. Data are the means ± standard errors for two separate experiments that showed similar results.

FIG. 2.

IL-15-integrated vaccines induce CD8⁺ CTLs with enhanced lytic activity and prolonged memory. Animals were immunized subcutaneously with 2 × 10⁶ PFU of the indicated vaccine candidate. Twelve days (A), 1 month (B), 5 months (C), and 10 months (D) after immunization, three to four mice were sacrificed from each group, their splenocytes were harvested and pooled, and CD8⁺ T cells were isolated. CD8⁺ T cells were then stimulated in vitro for 1 week with syngeneic splenocytes infected with the Wyeth strain of vaccinia virus at an MOI of 5 and subjected to γ-irradiation. P815 cells infected with the Wyeth strain (filled circles) served as target cells, while uninfected P815 cells (open circles) served as controls in a 5-hour ⁵¹Cr release assay. The x axis represents the effector-to-target cell ratio. Two separate experiments showed similar results, and the data presented are the means ± standard errors for a triplicate treatment in one experiment.

FIG. 3.

A host previously vaccinated with a replicating vaccinia virus can be effectively boosted with replication-deficient MVA to induce cellular immune responses. Animals were first vaccinated with 2 × 10⁶ PFU of WR vaccinia virus and 14 months later boosted with an identical dose of MVA or MVA-IL-15 virus subcutaneously. Nine days after the boost, splenocytes were harvested and pooled from three animals in each group. Pooled splenocytes were then stimulated with Wyeth strain-infected (5 MOI), irradiated syngeneic spleen cells. After in vitro stimulation, the responder cells were stained for surface CD4⁺ and CD8⁺ expression, followed by intracellular cytokine staining for IFN-γ (IFN-g) and TNF-α (TNF-a). Spleen cells from unvaccinated animals (control group) and animals that did not receive a booster vaccination were also included in the comparative analysis. The data shown are representative of two independent experiments.

FIG. 4.

Rapid induction of vaccinia virus-neutralizing antibodies in vaccinated animals augmented by coexpression of IL-15. (A) Animals were vaccinated with 2 × 10⁶ PFU of the indicated vaccine agent subcutaneously, and 12 days later, blood was collected by retro-orbital bleeding and sera were separated. Within groups, sera were pooled (from five animals) and serially diluted sera were mixed with 150 PFU of WR vaccinia virus for plaque reduction neutralization. (B) Animals were vaccinated with Wyeth or Wyeth-IL-15 (2 × 10⁶ PFU), and 14 months later, blood was collected and sera were separated. Serial dilutions (dil) of pooled sera from each group (five animals per group) were tested for the presence of vaccinia virus-neutralizing antibodies by a plaque reduction assay. The data shown are representative of three independent experiments. VAC, vaccinia virus.

FIG. 5.

IL-15-integrated vaccinia virus induces higher neutralizing antibody levels even in animals with preexisting vaccinia virus-neutralizing antibodies. Animals were first vaccinated with MVA (2 × 10⁶ PFU) and boosted 10 weeks later with MVA or MVA-IL-15 (MVA15). Six months after the boosting serum, vaccinia virus-neutralizing antibody levels were determined in revaccinated animals by a plaque reduction assay, using serial dilutions (dil) of pooled sera from each group of five animals. The data shown are representative of three independent experiments. VAC, vaccinia virus.

FIG. 6.

Animals vaccinated with IL-15-integrated vaccinia viruses better tolerate a lethal intranasal vaccinia virus challenge. Four groups of mice, five animals per group, were vaccinated with equivalent doses (2 × 10⁶) of the respective vaccine agents. (A) Vaccinated animals were challenged 30 days after vaccination. (B) Vaccinated animals were also challenged 10 months after vaccination. For intranasal challenge of vaccinated animals, 1 × 10⁶ PFU of the WR strain of vaccinia virus was used. The body weights of individual animals were measured daily after intranasal challenge. Age-matched unimmunized mice were included in the challenge experiments to serve as controls. Two separate experiments showed similar weight loss patterns.