Evaluation of a ricin vaccine candidate (RVEc) for human toxicity using an in vitro vascular leak assay.

To protect against ricin intoxication, a genetically derived ricin A chain vaccine candidate (RVEc) was developed lacking the toxic N-glycosidase activity (Olson et al., 2004). The vaccine protects animals against an aerosolized ricin holotoxin (RT) challenge (Carra et al., 2007). In the current study, the RVEc vaccine was evaluated for its interaction and effect on human endothelial cells. RVEc was tested in an in vitro cellular-based bioassay, consisting of primary human endothelial cells cultured on collagen-coated inserts, to which concentrations of the vaccine candidate (0.6, 2, 2.5 or 9 µM) were added. RVEc showed no signs of adverse activity on the cells (e.g., cytotoxicty activity) as measured by changes in trans-endothelial electrical resistance (TEER). In contrast, ricin toxin (RT) cytotoxicity was observed at all concentrations tested. Under light microscopy, no cytotoxicity was visible at 24h with 0.6 or 9 µM of RVEc. However, cytotoxicity was observed for RT and to a lesser degree for RTA. Flow cytometric analysis showed binding of RT, slight binding of RTA, and no binding of the RVEc vaccine to endothelial cells. The presence of RTB as a contaminant contributing to the cytotoxicity in the RTA preparation was ruled out by a RTB-specific ELISA. In addition, RTA at 9 µM produced a cytotoxic activity that could not be explained exclusively by the presence of azide in the RTA buffer. In the current study, the model demonstrated no discernable adverse events of the RVEc vaccine on human endothelial cells, when compared to the toxicity caused by holotoxin or native RTA preparations.

An alternative approach to combination vaccines: intradermal administration of isolated components for control of anthrax, botulism, plague and staphylococcal toxic shock.

BACKGROUND: Combination vaccines reduce the total number of injections required for each component administered separately and generally provide the same level of disease protection. Yet, physical, chemical, and biological interactions between vaccine components are often detrimental to vaccine safety or efficacy. METHODS: As a possible alternative to combination vaccines, we used specially designed microneedles to inject rhesus macaques with four separate recombinant protein vaccines for anthrax, botulism, plague and staphylococcal toxic shock next to each other just below the surface of the skin, thus avoiding potentially incompatible vaccine mixtures. RESULTS: The intradermally-administered vaccines retained potent antibody responses and were well- tolerated by rhesus macaques. Based on tracking of the adjuvant, the vaccines were transported from the dermis to draining lymph nodes by antigen-presenting cells. Vaccinated primates were completely protected from an otherwise lethal aerosol challenge by Bacillus anthracis spores, botulinum neurotoxin A, or staphylococcal enterotoxin B. CONCLUSION: Our results demonstrated that the physical separation of vaccines both in the syringe and at the site of administration did not adversely affect the biological activity of each component.The vaccination method we describe may be scalable to include a greater number of antigens, while avoiding the physical and chemical incompatibilities encountered by combining multiple vaccines together in one product.

Improved formulation of a recombinant ricin A-chain vaccine increases its stability and effective antigenicity.

Ricin is a potent toxin associated with bioterrorism for which no vaccine or specific countermeasures are currently available. A stable, non-toxic and immunogenic recombinant ricin A-chain vaccine (RTA 1-33/44-198) has been developed by protein engineering. We identified optimal formulation conditions for this vaccine under which it remained stable and potent in storage for up to 18 months, and resisted multiple rounds of freeze-thawing without stabilizing co-solvents. Reformulation from phosphate buffer to succinate buffer increased adherence of the protein to aluminum hydroxide adjuvant from 15 to 91%, with a concomitant increase of nearly threefold in effective antigenicity in a mouse model. Using Fourier-transform infrared spectroscopy, we examined the secondary structure of the protein while it was adhered to aluminum hydroxide. Adjuvant adsorption produced only a small apparent change in secondary structure, while significantly stabilizing the protein to thermal denaturation. The vaccine therefore may be safely stored in the presence of adjuvant. Our results suggest that optimization of adherence of a protein antigen to aluminum adjuvant can be a useful route to increasing both stability and effectiveness, and support a role for a "depot effect" of adjuvant.

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.

Improved stability of a protein vaccine through elimination of a partially unfolded state.

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1-33/44-198 or 1-198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule-like state at 45 degrees C, rRTA 1-33/44-198 and 1-198 are significantly more thermostable, remaining completely folded at temperatures up to 53 degrees C and 51 degrees C, respectively. Deleting both an exposed loop region (amino acids 34-43) and the C-terminal domain (199-267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.

Comparative efficacy and immunogenicity of Q fever chloroform:methanol residue (CMR) and phase I cellular (Q-Vax) vaccines in cynomolgus monkeys challenged by aerosol.

Preliminary evidence gathered in rodents and livestock suggested that a phase I chloroform:methanol residue (CMR) extracted vaccine was safe and efficacious in protecting these animals from challenge with the obligate phagolysosomal pathogen (Coxiella burnetii). Prior to the initiation of phase II studies in human volunteers, we compared, in non-human primates (Macaca fascicularis), the efficacy of CMR vaccine with Q-Vax, a licensed cellular Australian Q fever vaccine that has been demonstrated to provide complete protection in human volunteers. Vaccine efficacy was assessed by evaluating thoracic radiographs and the presence of fever and bacteremia in monkeys challenged by aerosol with Coxiella burnetii. Changes in blood chemistries, hematology, behavior and pulmonary function were also examined. CMR, whether administered in single 30 or 100 microg doses or two 30 microg subcutaneous doses, gave equivalent protection in vaccine recipients as a single 30 microg dose of Q-Vax. In addition, vaccination resulted in significant, although temporary, increases in specific antibody titers against C. burnetii phases I and II antigens. The C. burnetii CMR vaccine may be an efficacious alternative to cellular Q fever vaccines in humans.

Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge.

Mucosal (oral) immunization of mice with carrier-delivered ricin toxoid (RT) vaccine was accomplished by one long (7 weeks) or two short (4 weeks) immunization schedules. For the long and short immunization schedule two lots of vaccine were administered prepared with the same procedure but at different occasions. The long schedule consisted of a total of seven doses of 50 microg of vaccine in microencapsulated (lot #108) or aqueous form administered on days 1, 2, 3, 28, 29, 30 and 49. With the short schedule a total of seven or six doses of 25 microg (lot #111) were administered on days 1, 2, 3, 14, 15, 16 and 30, or on 1, 2, 14, 15, 30, 31 and 32, respectively. Mice immunized orally with the long schedule, 50 microg of RT vaccine incorporated into poly-DL-lactide-co-glycolyde (DL-PLG) microspheres (MS) produced serum IgG, IgG2a and IgA ELISA antibodies. All mice immunized with RT in DL-PLG MS (RT-MS) were protected against a lethal ricin aerosol challenge. In contrast, with the same schedule and with the same dose, the aqueous vaccine (RT) failed to stimulate IgG, IgG2a and IgA antibodies, and these mice were not protected against an aerosol ricin toxin challenge. With the shorter immunization scheme, seven doses of 25 microg RT-MS stimulated a significant, though reduced, protection with the microencapsulated, but not with the aqueous vaccine. When the first and second 3-day cycles of the short immunization schedule was reduced to two doses, and the 3-day cycle was administered at the end of the schedule, neither RT-MS nor RT stimulated protection against the challenge. These results indicated that successful oral immunization with RT-MS depended on both the dose and the schedule, consisting of three consecutive days of administration in two cycles, 4 weeks apart. Altering this schedule and the dose, resulted in a reduced protection or no protection at all. Furthermore, under the conditions of this study, the advantage of the microencapsulated RT vaccine over the aqueous vaccine for effective oral immunization was well demonstrated.