Protection against Anthrax Lethal Toxin Challenge by Genetic Immunization with a Plasmid Encoding the Lethal Factor Protein

Abstract

The ability of genetic vaccination to protect against a lethal challenge of anthrax toxin was evaluated. BALB/c mice were immunized via gene gun inoculation with eucaryotic expression vector plasmids encoding either a fragment of the protective antigen (PA) or a fragment of lethal factor (LF). Plasmid pCLF4 contains the N-terminal region (amino acids [aa] 10 to 254) of Bacillus anthracis LF cloned into the pCI expression plasmid. Plasmid pCPA contains a biologically active portion (aa 175 to 764) of B. anthracis PA cloned into the pCI expression vector. One-micrometer-diameter gold particles were coated with plasmid pCLF4 or pCPA or a 1:1 mixture of both and injected into mice via gene gun (1 g of plasmid DNA/injection) three times at 2-week intervals. Sera were collected and analyzed for antibody titer as well as antibody isotype. Significantly, titers of antibody to both PA and LF from mice immunized with the combination of pCPA and pCLF4 were four to five times greater than titers from mice immunized with either gene alone. Two weeks following the third and final plasmid DNA boost, all mice were challenged with 5 50% lethal doses of lethal toxin (PA plus LF) injected intravenously into the tail vein. All mice immunized with pCLF4, pCPA, or the combination of both survived the challenge, whereas all unimmunized mice did not survive. These results demonstrate that DNA-based immunization alone can provide protection against a lethal toxin challenge and that DNA immunization against the LF antigen alone provides complete protection.

A dually active anthrax vaccine that confers protection against both bacilli and toxins

“…These findings indicate that anti-PA IgGs elicited by DAAVs or PA have the same functional potency to neutralize PA. To directly verify the effectiveness of DAAVs in protection against anthrax, we challenged immunized mice with anthrax lethal toxin. Ten days after the third immunization, each mouse was given a mixture of 48 g of PA and 20 g of LF through its tail vein, corresponding to about 4 times the LD 50 of lethal toxin (19). Without exception, control mice immunized with PBS or PGA died within 24 h (Fig.…”

“…Two weeks after the third dose of vaccination, each mouse was challenged through its tail vein with a mixture of 48 g of PA and 20 g of LF (List Biological Laboratories, Campbell, CA), the equivalent of approximately 4 times the LD 50 of lethal toxin in mice (19). Without exception, unprotected mice died within 24 h. Protected mice were monitored closely for at least 2 weeks.…”

Multicomponent anthrax toxin display and delivery using bacteriophage T4

“…The tripartite anthrax toxin is comprised of three interacting partners, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). Although PA has been the principal target for numerous vaccine development strategies [16,17], inclusion of LF and EF could elicit additional protective immune responses [18,19], which may be necessary to develop a more robust next generation anthrax vaccine. We fused multiple anthrax toxins and their domains to Hoc and assembled them in vitro on phage T4 in various combinations.…”

Serum IgG antibody response to the protective antigen (PA) of Bacillus anthracis induced by anthrax vaccine adsorbed (AVA) among U.S. military personnel

“…AVA is produced from the culture supernatant of a mutant strain of B. anthracis, V63340 77/-NP1-R that expresses mostly PA and does not induce antibodies to the LF or the EF [9]. Patients convalescent from anthrax have antibodies to all three components of anthrax toxin [10].…”