(T1430-06-31) Development of a Lyophilized Single Vial Adjuvanted Sub-unit Vaccine for Bed Side Reconstitution

Purpose: Vaccine adjuvants such as those made using an oil-in-water compositions [e.g., MF59 (Novartis) and Adjuvant System 03 (AS03) (GSK Vaccines)] show potent clinical immunogenicity when combined with sub-unit antigens. However, these adjuvants require bedside mixing with antigen and cold chain for storage and shipping. Single vial liquid vaccines containing both antigen and adjuvant have shown reduced immunogenicity of the vaccine as a result of unwanted hydrophobic interactions of the antigen with adjuvant. To address these challenges, the goal of this study was to develop a single vial dried vaccine for reconstitution, containing both antigen and adjuvant. The immunogenicity of reconstituted single vial vaccine was compared with liquid vaccine using cytomegalovirus (CMV) pentamer antigen as a model antigen.

Methods: In previous work we have shown that emulsion adjuvants can be formulated using an alternative self-emulsification process. In this study we used an optimized self-emulsified adjuvant (SE-AS44) and developed a lyophilization cycle to create a dried adjuvant with and without antigen. The lyophilized vaccine was checked for changes in size and PDI using DLS, and antigen integrity using SDS-PAGE gels and compared with controls. The formulations were characterized for pH, osmolarity, size, PDI, squalene and alpha-tocopherol content and endotoxin before immunization. These vaccines were evaluated in 6-8 week old female C57BL/6 mice with three immunizations (each 3 weeks apart). Sera from non-terminal bleed were analyzed for neutralizing antibody titers and IgG antibody titers. Neutralizing antibody responses were determined from serum three weeks post each immunization using the retinal pigment epithelial cell line (ARPE -19) and TB40 (a CMV strain).  IgG antibody titers were determined from serum three weeks post immunization 2 and 3 using sandwich ELISA (enzyme linked immunosorbent assay). T-cell responses were analyzed 4 weeks post 3^rd immunization by intracellular cytokine staining (ICS) of in vitro CMV stimulated spleenocytes. Spleens from individual mouse were processed to form single cell suspensions. The cytokine responses were analyzed using flow cytometry and appropriate gating strategies.

Results: CMV in post-lyophilized vaccine has shown structural integrity when compared to CMV only and pre-lyophilized vaccine on gel. The size distribution for post-lyophilized vaccine shows slight increase in size but similar PDI. Collectively, the results showed feasibility of single vial lyophilized emulsion adjuvanted CMV vaccine. Neutralizing antibody responses and anti-CMV pentamer IgG titers showed that both lyophilized and liquid vaccine groups showed no significant difference when compared with each other and with AS03. ICS of in vitro CMV stimulated spleenocytes were analyzed for CD4+ and CD8+ responses. The results showed a dominant Th0/Th2 response with adjuvanted groups with CMV pentamer. The trend was similar humoral immune response where adjuvanted groups showed higher overall frequencies of antigen-specific CD4+ T cells than unadjuvanted CMV pentamer alone

Conclusion: The results demonstrated a proof-of-concept for single vial adjuvanted lyophilized vaccine that maintained its potency in vivo as compared to bed-side mixed liquid vaccine. We have also demonstrated that self-emulsification process shows equivalence in animal models for humoral and cellular responses compared to the current complex manufacturing processes.

References: Lodaya, R.N., et al., Formulation Design, Optimization and In Vivo Evaluations of an α-Tocopherol Containing Self-Emulsified Adjuvant System using Inactivated Influenza Vaccine. 2019. 316: p. 12-21.
Shah, R.R., et al., The development of self-emulsifying oil-in-water emulsion adjuvant and an evaluation of the impact of droplet size on performance. 2015. 104(4): p. 1352-1361.

Acknowledgments: Ethics Statement: All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee by the ethical review process at the institution where the work was performed. All studies were executed in compliance with provisions of the USDA Animal Welfare Act, the Public Health Service Policy on Humane Care and Use of Laboratory Animals and the U.S. Interagency Research Animal Committee Principles for the Utilization and Care of Research Animals.

Funding: This study was funded by GlaxoSmithKline Vaccines Biologicals SA which was involved in all stages of the study conduct and analysis.

nAb titers in serum obtained three weeks post 1st, 2nd and 3rd immunization, against CMV TB40 strain of virus.



Anti-CMV pentamer IgG antibody titers in serum obtained three weeks post 2nd and 3rd immunization.



Antigen specific CD4+ T cells using ICS assay.