Deliverables
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 Ags 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.
The LTK63 adjuvanted influenza vaccines formulations, with and without delivery system, for intranasal administration, have been developed. Clinical grade lots the recombinant proteins LT-K63 and LT-R72 were produced.
Following a change in the study plans only LTK63 was used in intranasal formulations. Formulation studies were performed to devise a suitable way of preparing clinical vaccines containing the adjuvant as well as three influenza antigens. Flu antigens were of three following strains (H5N3, B, A/H3N2).Five different lots of vaccine (four nasal and 1 intramuscular) were formulated and extensively tested for immunogenicity and stability over time in different conditions.
Three of the nasal formulations contained the biopolymer SMBV (the Supramolecular Biovector), a new delivery system.
The clinical lots produced for the Phase I trial were the following:
- 3 µg/dose LTK63 + SMBV + HA (3X 7.5 mcg);
- 10 µg/dose LTK63 + SMBV + 3HA (3X 7.5 mcg);
- 30 µg/dose LTK63 + SMBV + 3HA (3X 7.5 mcg);
- 30 µg/dose LTK63 + 3HA (3X 7.5 mcg) ;
- IN placebo;
- IM adjuvanted flu vaccine (active control)Vaccine preparations for human use were packaged and labelled according to the study protocol and shipped to the clinical site in Leicester, UK.
The phase I clinical trial was performed in Leicester, involving recruitment, immunization and monitoring of 84 volunteers. A particular effort was placed on careful monitoring for the possible occurrence of side effects.
No patient was lost to follow-up, and no relevant side effects were noticed after intranasal or intramuscular immunisation.
Furthermore, samples of blood, nasal secretions and crevicular fluid were collected and delivered to partners K. Mills, J. Holmgren and M. Zambon for analysis. A special arrangement had to be set to deliver samples by air courier to K. Mills, performing studies of cell-mediated immunity, which required same-day delivery.Ad hoc individual case report forms (CRFs) were filled by the investigators and sent to Chiron Vaccines data management center in Amsterdam, The Netherlands, in order to be entered into the clinical database. Data were subsequently fully analysed and reported in a clinical study report.
The clinical trial performed in this period provided a number of relevant information: 1.A nasal trivalent influenza vaccine containing the non-toxic LT mutant LTK63 is very well tolerated and is able to induce a systemic immune response against influenza B and, to a lesser extent, against H3N2 antigens.2.A dose-dependent mucosal immune response against all the flu antigens included in the vaccine was observed in the nose after nasal immunization.3.No systemic immune response could be detected against the pandemic antigen H5N3 after nasal immunization. Furthermore, the systemic immune response against the other antigens was observed only at the highest dose of LT mutant. Therefore, a higher dose of adjuvant, a mutant with stronger adjuvant activity (such as LTR72) or/and a different schedule of administration could be necessary for optimal immunization against these antigens. In particular, studies of cell-mediated immunity suggested that a longer interval could be needed between the first and the second immunization, to allow the development of immunologic memory particularly against the pandemic strain.
Two additional findings of great practical interest were found in the study:4.
The systemic immune response against H5N3 observed in the control group immunized by the intramuscular route, was similar to the one previously observed using a monovalent vaccine. Therefore the pandemic antigen can be formulated in a trivalent vaccine without loss of activity.5.A marked increase in nasal specific IgG against the three flu antigens was observed after parenteral immunization. Thus, local immunoglobulins might participate in the protective effect of the intramuscular vaccine, in addition to the systemic immune response.
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