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Pre-clinical assessment of safety and immunogenicity of LT adjuvanted intranasal flu vaccines by means of ad hoc toxicological and immunogenicity studies in different animal species

Pre-clinical studies of immunogenicity, immune-adjuvanticity and toxicology of the different components of the vaccine, as well of the final formulations of the vaccine to be used in the clinical trial, have been performed.

TOXICOLOGICAL STUDIES:Following assessment of the immunogenicity of the candidate vaccines in Balb/c mice using formulations containing the pandemic strain H5N3 (7,5mcg/mouse) and LTK63 (3 mcg/mouse) and based on the results of the preliminary stability study, four vaccines were formulated in GLP conditions for Toxicology Studies. The toxicological vaccine lots have been produced using only one influenza virus strain, H3N2, instead of the 3 different strains that will be present in the clinical lots.

In two separate studies in mice and in rabbits, no toxicological effects were found on upper and lower respiratory tract after single or repeated administration of the vaccine preparation with either LT mutant up to 150 days. Due to the reported possibility of retrograde transport of the molecules through the olfactory nerves, a particular attention was given to possible neurological effects after immunization.

However, no abnormalities were observed in the olfactory bulbs, olfactory nerves, cervical spinal cord, brain (coronal sections at three levels to include cerebrum, cerebellum, and brain stem), and meninges. Furthermore, immuno-histochemical studies with a sensitive anti-LT monoclonal antibody, capable of detecting trace amounts of LT on Y-1 cells in vitro, failed to detect any amount of LT in the olfactory nerves and in the brain of mice after intranasal treatment. STABILITY STUDIES. Stability studies for toxicological lots were performed at two different temperatures (+2-8 °C and +36-38°C).

Additionally, a set of analytical techniques were developed and validated in order to evaluate quality and stability of the candidate vaccine lots. Based on the data collected during the stability studies of the toxicological lots, it was concluded that the formulations are stable up to 6 months at 2-8°C and 2 weeks at 36-38.

IMMUNOGENITY STUDIES. Vaccine lots used for the Toxicology Study were also analyzed, against the appropriate controls, to evaluate their immunogenicity in Balb/c mice. In addition, the immunogenicity of intra nasal vaccines formulations, prepared with and without SMBV particles, and containing all 3 FLU Ag’s was evaluated. The results showed that, in mice, LT mutants (LTK63 and LTR72) and SMBV, administered intranasally, have a synergistic effect to enhance serum and mucosal antibody responses to the three flu antigens.

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53100 SIENA
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