Development of a highly efficacious vaccinia-based dual vaccine against smallpox and anthrax, two important bioterror entities

Abstract

Bioterrorism poses a daunting challenge to global security and public health in the 21st century. Variola major virus, the etiological agent of smallpox, and Bacillus anthracis, the bacterial pathogen responsible for anthrax, remain at the apex of potential pathogens that could be used in a bioterror attack to inflict mass casualties. Although licensed vaccines are available for both smallpox and anthrax, because of inadequacies associated with each of these vaccines, serious concerns remain as to the deployability of these vaccines, especially in the aftermath of a bioterror attack involving these pathogens. We have developed a single vaccine (Wyeth/IL-15/PA) using the licensed Wyeth smallpox vaccine strain that is efficacious against both smallpox and anthrax due to the integration of immune-enhancing cytokine IL-15 and the protective antigen (PA) of B. anthracis into the Wyeth vaccinia virus. Integration of IL-15 renders Wyeth vaccinia avirulent in immunodeficient mice and enhances anti-vaccinia immune responses. Wyeth/IL-15/PA conferred sterile protection against a lethal challenge of B. anthracis Ames strain spores in rabbits. A single dose of Wyeth/IL-15/PA protected 33% of the vaccinated A/J mice against a lethal spore challenge 72 h later whereas a single dose of licensed anthrax vaccine protected only 10%. Our dual vaccine Wyeth/IL-15/PA remedies the inadequacies associated with the licensed vaccines, and the inherent ability of Wyeth vaccinia virus to be lyophilized without loss of potency makes it cold-chain independent, thus simplifying the logistics of storage, stockpiling, and field delivery in the event of a bioterror attack involving smallpox or anthrax.

Fig. 1.

The expression of glycosylated B. anthracis PA by recombinant vaccinia viruses with an integrated IL-15 gene. BHK-21 cells were infected with Wyeth/IL-15/PA and MVA/IL-15/PA for 48 h, and the infected BHK-21 cell lysates were subjected to SDS/PAGE on a 10% acrylamide gel. Western blot analyses were performed using a polyclonal goat PA-specific antibody (List Biological Laboratories). The glycosylation of vaccinia virus-expressed PA polypeptide in BHK21 cells was assessed by the treatment of vaccinia-infected cell lysates with PNGase F enzyme (New England Biolabs) before SDS/PAGE. The positions of molecular weight markers are on shown on left.

Fig. 2.

Assessment of PA-specific antibodies in A/J mice vaccinated with vaccinia-based dual vaccines. Mice were vaccinated twice with Wyeth/IL-15/PA, MVA/IL-15/PA (1 × 10⁷ pfu/mouse), or Biothrax/AVA (25 μL/mouse) s.c. 4 wk apart, and 3 wk after the second dose of vaccine, animals were bled. Serial dilutions of pooled sera from each group (n = 6) vaccinated with respective vaccinia recombinant vaccines or Biothrax/AVA were then analyzed by a PA-specific ELISA to determine the levels of PA-specific antibody induced by vaccination.

Fig. 3.

Long-term persistence and Ig subclass specificities of PA-specific antibodies induced by vaccinia-based dual vaccines. Groups of A/J mice (n = 6) were vaccinated with two doses of either MVA/IL-15/PA (1 × 10⁷ pfu/mouse) or Biothrax/AVA (25 μL/mouse) s.c. 4 wk apart, and their serum samples were collected 6 mo after the second vaccination. Pooled serum samples from each group were then tested in a PA-specific ELISA to determine the total anti-PA IgG levels as well as anti-PA IgG_2a and anti-PA IgG_2b levels using either total or subclass-specific anti-mouse IgG secondary antibodies in the ELISA.

Fig. 4.

Superior induction kinetics of PA-specific antibodies after vaccination with vaccinia-based dual vaccines. A group of A/J mice (n = 6) were given a single dose of MVA/IL-15/PA vaccine (1 × 10⁷ pfu/mouse) or Biothrax/AVA (25 μL/mouse). Animals were bled longitudinally at indicated time points following vaccination, and serial dilutions of pooled sera from each group were tested in a PA-specific ELISA to determine the kinetics of PA-specific antibody induction and to quantitate the levels of such PA-specfic antibodies in their sera. Prebleeds taken from each group before vaccination served as a baseline control.

Fig. 5.

Induction kinetics of vaccinia-neutralizing antibodies following vaccination with vaccinia-based dual vaccines. Mice were vaccinated with a single dose of MVA/IL-15/PA vaccine (1 × 10⁷ pfu/mouse), and longitudinal serum samples were collected as described in the legend to Fig. 4. The induction kinetics of vaccinia-neutralizing antibodies and the quantitation of such antibodies were determined using a plaque reduction assay as described in Materials and Methods. Prebleeds taken from each group before vaccination served as a baseline control.