Smallpox vaccine with integrated IL-15 demonstrates enhanced in vivo viral clearance in immunodeficient mice and confers long term protection against a lethal monkeypox challenge in cynomolgus monkeys

Abstract

Despite the eradication of smallpox, there is heightened concern that it could be reintroduced as a result of intentional release of Variola major virus through an act of bioterrorism. The live vaccine that was pivotal in the eradication of smallpox though considered a gold standard for its efficacy still retains sufficient residual virulence that can cause life-threatening sequelae especially in immune deficient individuals. Therefore, a safer smallpox vaccine that can match the efficacy of first generation vaccines is urgently needed. We previously reported that the integration of human IL-15 cytokine into the genome of Wyeth strain of vaccinia (Wyeth/IL-15), the same strain as the licensed vaccine, generates a vaccine with superior immunogenicity and efficacy in a mouse model. We now demonstrate that Wyeth/IL-15 is non-lethal to athymic nude mice when administered intravenously at a dose of 10(7) plaque forming units and it undergoes enhanced in vivo clearance in these immune deficient mice. Furthermore, a majority of cynomolgus monkeys vaccinated with vaccinia viruses with integrated IL-15, when challenged 3 years later with a lethal dose of monkeypox virus displayed milder clinical manifestations with complete recovery supporting the utility of Wyeth/IL-15 for contemporary populations as a safer and efficacious smallpox vaccine.

Fig. 1

In vivo biodistribution and clearance of Wyeth strain of vaccinia in immune competent BALB/c mice. Wyeth vaccinia derived from the Dryvax vaccine with the firefly luciferase gene integrated (Wyeth/Luc) or the firefly luciferase gene plus human IL-15 gene integrated (Wyeth/IL-15/Luc) into the hemagglutinin locus was injected intravenously (1 × 10⁷ pfu) in a volume of 50 μl via the tail vein to a group of 5 female BALB/c mice. Luciferase expression in mice was then sequentially visualized by luminescence imaging (IVIS 100 system from Xenogen, Caliper Life Sciences) starting 30 min post virus administration and continued every 12 h. Representative longitudinal images are shown. An integration time of 2 min was used for luminescence image acquisition.

Fig. 2

In vivo biodistribution and clearance of Wyeth strain of vaccinia in T cell-deficient nude mice. Wyeth vaccinia derived from the Dryvax vaccine with the firefly luciferase gene integrated (Wyeth/Luc) or the firefly luciferase gene plus human IL-15 gene integrated (Wyeth/IL-15/Luc) into the hemagglutinin locus was injected intravenously and luminescence was monitored longitudinally over a period of 3 weeks as described in the legend to Fig. 1. Asterisk in 13 days panel indicates the unavailability of the mouse due to death at that time point.

Fig. 3

Post vaccination skin lesions induced by the Wyeth strain of vaccinia or its derivatives with integrated cytokines. Each candidate smallpox vaccine, Wyeth, Wyeth/IL-2 and Wyeth/IL-15 was administered intradermally in a volume of 50 μl containing 1 × 10⁸ pfu to a shaved area between the scapulae. Lesions formed were photographed at the indicated time points after vaccination.

Fig. 4

Induction of vaccinia specific, interferon gamma producing CD8⁺ T cells. Fresh peripheral blood mononuclear cells (PBMC) isolated from vaccinated animals at indicated time points (3 × 10⁶cells) mixed with an equal number of irradiated (3000 rads) autologous PBMC that were infected with Wyeth vaccinia at a multiplicity of infection of 5 for 4 h prior to being irradiated. After 4 h of co-culture at 37 °C, Brefeldin A was added and incubation continued for further 8 h. Cells were then stained for surface CD3, CD8 and intracellular IFN gamma as described previously . Cells stained with appropriate antibodies were analyzed on a FACSCalibur instrument (Becton Dickinson) using FloJo software.