Therapeutic efficacy of equine botulism antitoxin in Rhesus macaques.

BACKGROUND: There are currently no licensed vaccines available for prevention of botulism in humans. The vaccination is not desirable due to expanding therapeutic indications of botulinum toxins. The only available specific treatment for botulism is antitoxin to remove circulating toxin, thus, preventing further neuronal damage. BAT® (Botulism Antitoxin Heptavalent (A, B, C, D, E, F, G)-(Equine)) has been developed and its therapeutic efficacy evaluated against botulinum neurotoxin serotype A (BoNT/A) in Rhesus macaques. METHODS AND FINDINGS: In a post-exposure prophylaxis (PEP) study, animals were exposed to 4x LD50/kg of BoNT/A and administered intravenously with either BAT (1x or 0.1x scaled human dose), or placebo at 4 hours post-exposure. The animals were monitored for 14 days. For the therapeutic intervention studies, animals were exposed to a 1.7x LD50/kg of BoNT/A and treated intravenously with either placebo or BAT at a 1x scaled human dose at the onset of clinical signs. Animals were monitored on an hourly basis for 14 or 21 days. In the PEP study, all animals tolerated equine based antitoxin without any adverse clinical signs. A 100% survival was observed in groups treated with the BAT compared to 0% survival in those treated with the placebo (p<0.001, Fisher's exact test). BAT antitoxin prevented the development of signs of neurotoxicity of botulinum toxin. In a therapeutic study, treatment with the BAT at scaled 1x human dose after the onset of clinical signs significantly enhanced survival compared to the placebo (46.6% vs. 0%, p<0.0001, Fisher's exact test). Additionally, treatment with the BAT delayed the progression of signs (muscular weakness, respiratory distress, oral/nasal discharge) of toxin intoxication and reduced the severity of the disease. CONCLUSIONS: A single dose of BAT, when administered to symptomatic monkeys, resulted in a statistically significant survival benefit compared to the placebo. Additionally, BAT completely protected monkeys from the clinical signs of intoxication and subsequent death when administered as PEP treatment. These data in part supported the licensure of BAT under the Animal Rule in the United States by the Food and Drug Administration.

Recombinant C fragment of botulinum neurotoxin B serotype (rBoNTB (HC)) immune response and protection in the rhesus monkey.

Botulinum neurotoxin B (BoNTB) is a distinct protein subtype of a family of neurotoxins with the potential for use in biological warfare or terrorist attacks. This study is one in a series evaluating the immunogenicity and protective effects of recombinant vaccines against the different subtypes of botulinum toxin. The recombinant subunit vaccines encoding the C fragment portion ( approximately 50 kDa) of the toxins are produced in the yeast, Pichia pastoris. In this study, groups of rhesus monkeys were vaccinated with three doses (1 and 5microg per dose) of rBoNTB(H(c)) vaccine. Total and neutralizing antibody titers were determined at various times during and postvaccination. Two groups of vaccinated monkeys plus non-vaccinated controls were actively challenged with B toxin by aerosol exposure. All monkeys receiving vaccine were protected from the toxin and no clinical signs of disease were observed, while controls displaying classic signs of botulism succumbed to the toxin challenge. Two additional groups of monkeys receiving the same vaccine regiment as the first two groups had significant levels of circulating neutralizing antibody titers up to 24 months postvaccination. This non-human primate study demonstrated the short- and long-term immunity afforded by the rBoNTB(H(c)) vaccine.

Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity.

At this time there are no vaccines or therapeutics to protect against staphylococcal enterotoxin B (SEB) exposure. Here, we report vaccine efficacy of an attenuated SEB in a nonhuman primate model following lethal aerosol challenge and identify several biomarkers of protective immunity. Initial in vitro results indicated that the mutation of key amino acid residues in the major histocompatibility complex (MHC) class II binding sites of SEB produced a nontoxic form of SEB, which had little to no detectable binding to MHC class II molecules, and lacked T-cell stimulatory activities. When examined in a mouse model, we found that the attenuated SEB retained antigenic structures and elicited protective immune responses against wild-type SEB challenge. Subsequently, a vaccine regimen against SEB in a nonhuman primate model was partially optimized, and investigations of immune biomarkers as indicators of protection were performed. SEB-naïve rhesus monkeys were vaccinated two or three times with 5 or 20 microg of the attenuated SEB and challenged by aerosol with wild-type SEB toxin. Unlike exposure to the native toxin, the vaccine did not trigger the release of inflammatory cytokines (TNF alpha, IL6, or IFN gamma). All rhesus monkeys that developed anti-SEB serum titers > or = 10(4) and elicited high levels of neutralizing antibody survived the aerosol challenge. These findings suggest that the attenuated SEB is fully protective against aerosolized toxin when administered to unprimed subjects. Moreover, experiments presented in this study identified various biomarkers that showed substantial promise as correlates of immunity and surrogate endpoints for assessing in vivo biological responses in primates, and possibly in humans, to vaccines against SEs.

Correlation of body temperature with protection against staphylococcal enterotoxin B exposure and use in determining vaccine dose-schedule.

The immunoprotective potential of a recombinant vaccine against the incapacitating effect of aerosolized staphylococcal enterotoxin B (SEB) in nonhuman primates is reported. SEB belongs to a family of structurally related superantigens responsible for serious, life threatening pathologies. Injecting the recombinant SEB vaccine did not induce temperature elevation in rhesus monkeys, a classical symptom of toxic-shock syndrome. No temperature elevation was noted following injection with control tetanus toxoid. In addition to 100% survival, we observed a clear correlation between vaccine dose and mitigation of temperature elevation after a lethal SEB aerosol challenge. We conclude that the recombinant SEB vaccine is non-pyrogenic and that monitoring changes in body temperature is an important biomarker of toxic shock in a primate animal model.

Protection against bacterial superantigen staphylococcal enterotoxin B by passive vaccination.

We investigated the ability of two overlapping fragments of staphylococcal enterotoxin B (SEB), which encompass the whole toxin, to induce protection and also examined if passive transfer of chicken anti-SEB antibodies raised against the holotoxin could protect rhesus monkeys against aerosolized SEB. Although both fragments of SEB were highly immunogenic, the fragments failed to protect mice whether they were injected separately or injected together. Passive transfer of antibody generated in chickens (immunoglobulin Y [IgY]) against the whole toxin suppressed cytokine responses and was protective in mice. All rhesus monkeys treated with the IgY specific for SEB up to 4 h after challenge survived lethal SEB aerosol exposure. These findings suggest that large fragments of SEB may not be ideal for productive vaccination, but passive transfer of SEB-specific antibodies protects nonhuman primates against lethal aerosol challenge. Thus, antibodies raised in chickens against the holotoxin may have potential therapeutic value within a therapeutic window of opportunity after SEB encounter.