Stabilization of a recombinant ricin toxin A subunit vaccine through lyophilization

Abstract

Lyophilization was used to prepare dry, glassy solid vaccine formulations of recombinant ricin toxin A-chain containing suspensions of colloidal aluminum hydroxide adjuvant. Four lyophilized formulations were prepared by using combinations of rapid or slow cooling during lyophilization and one of two buffers, histidine or ammonium acetate. Trehalose was used as the stabilizing excipient. Aggregation of the colloidal aluminum hydroxide suspension was reduced in formulations processed with a rapid cooling rate. Aluminum hydroxide particle size distributions, glass transition temperatures, water contents, and immunogenicities of lyophilized vaccines were independent of incubation time at 40°C for up to 15 weeks. Mice immunized with reconstituted ricin toxin subunit A (RTA) vaccines produced RTA-specific antibodies and toxin-neutralizing antibodies (TNA) regardless of the length of high temperature vaccine storage or the degree of aluminum adjuvant aggregation that occurred during lyophilization. In murine studies, lyophilized formulations of vaccines conferred protection against exposure to lethal doses of ricin, even after the lyophilized formulations had been stored at 40°C for 4 weeks. A corresponding liquid formulation of vaccine stored at 40°C elicited RTA-specific antibody titers but failed to confer immunity during a ricin challenge.

Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection

“…This formulation of RiVax is biochemically stable at 40°C for at least 1 y (21). When used in mice, it remained fully immunogenic and protective (21).…”

“…Although ricin holotoxin is stable, isolated RTA is unstable in an aqueous milieu, rapidly forming macromolecular aggregates after disruption of tertiary structure (22). To stabilize RiVax on the surface of alum for potential long-term storage and thermostability, we developed a process to lyophilize the adjuvant and protein complex in the presence of glassifying excipients to preserve its conformation (21).…”

“…The current vaccine is an alum-adsorbed vaccine, but lyophilized with a glassifying excipient and reconstituted with water immediately before use (21). Lyophilization results in long-term stability that will be important for stockpiling.…”

“…Passive protection studies in mice with RT-neutralizing MAbs have suggested that most of the protective antibodies recognize conformational determinants (19,20). We have recently developed a lyophilized thermostable adjuvant formulation of RiVax that appears to retain its tertiary structure and, hence, its conformational epitopes (21). In the Significance Ricin toxin (RT) is a CDC-designated select agent that can be dispersed as an aerosol.…”

Recent advances in the development of vaccines against ricin

“…17 Because this formulation of RiVax was effective in mice after many months of incubation at 40 C, it is presumed that the adjuvant-bound antigen was not grossly destabilized. Any unfolding of the antigen that may have taken place had occurred during the aqueous phase processing.…”