Nonclinical Testing of rBV AB PlChia Pastoris Vaccine

Before submission of the IND for rBV A/B (Pichia pastoris) vaccine, the proposed vaccine formulation was tested in GLP toxicity studies to establish its safety. Three studies were performed: repeat dose toxicity, repeat dose neurotoxicity, and repeat dose immunogenicity. The designs of these three studies were identical. Mice (CD1) received one (day 0), two (day 0 and day 28), or three (day 0, day 28, day 56) doses of vaccine formulated with or without 0.2% (w/v) Alhydrogel at serotype-specific antigen concentrations of 0, 1, 2, or 4 |g.

In each formulation, the concentration of the adjuvant and other excipients was the same as those intended for the Phase 1 clinical trial. For the repeat dose toxicity study, the animals were evaluated through clinical observation, body weight measurement, vaccination site reactogenicity, physical examination, food consumption, clinical pathology (hematology and serum chemistry), terminal organ weight, and macroscopic and microscopic examinations of tissues. For the repeat dose neuro-toxicity study, the animals were observed for behavioral, reflex, and physiological (elicited) changes after vaccination. For the repeat dose immunogenicity study, neutralizing antibody concentrations (NACs) in mice after each vaccination and in unvaccinated control mice were determined via mouse neutralization assays (MNAs).77 Identical assays and controls were used for the Phase 1 clinical trial to establish the immune responses to the vaccine antigens and to confirm that antibodies were present during the assessment of toxicity in the previously described studies. The results of the immunogenicity study are presented in Figure 4.5 and Figure 4.6. No vaccine-related toxicity was observed in any of these studies.

A fourth study of local reactogenicity in New Zealand White rabbits was performed at the request of the U.S. Food and Drug Administration (FDA). This species was chosen because the complete proposed 0.5 mL human dose could be

FIGURE 4.5 Serum NAC to BoNT/A. Groups of mice were vaccinated with 1, 2, or 4 |lg (of each antigen) of rBV A/B (P pastoris) with 0.2% (w/v) Alhydrogel (A) or without Alhydrogel (B) by intramuscular injection on days 0, 28, and 56. The serum NAC to BoNT/A was determined using the MNA: on day 28 in mice vaccinated on day 0 (one dose); on day 56 in mice vaccinated on days 0 and 28 (two doses); and on day 70 in mice vaccinated on days 0, 28, and 56 three doses). Untreated mice were euthanized and serum collected for determination of baseline NAC (0 |lg).

FIGURE 4.5 Serum NAC to BoNT/A. Groups of mice were vaccinated with 1, 2, or 4 |lg (of each antigen) of rBV A/B (P pastoris) with 0.2% (w/v) Alhydrogel (A) or without Alhydrogel (B) by intramuscular injection on days 0, 28, and 56. The serum NAC to BoNT/A was determined using the MNA: on day 28 in mice vaccinated on day 0 (one dose); on day 56 in mice vaccinated on days 0 and 28 (two doses); and on day 70 in mice vaccinated on days 0, 28, and 56 three doses). Untreated mice were euthanized and serum collected for determination of baseline NAC (0 |lg).

administered via intramuscular injection. The animals received two injections, one each on day 0 and day 28, using identical formulations proposed for the Phase 1 clinical trial of serotype-specific antigen dosage levels of 5, 10, or 20 |g. Both antigens were combined in a 1:1 ratio based on mass (|g). Therefore, each 0.5 mL dose of vaccine at each dosage level contained 10, 20, or 40 |g total protein. Injection sites were examined after each vaccination by the dermal Draize observation and scoring method, and histopathological examination of the site (skin and muscle) at

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