(W1330-01-05) Extrapolating the Use of Microfluidic Chips to Formulate Reproducible and Effective Nanoemulsions for Subunit Vaccines: A Paradigm Shift in Manufacturing Emulsion Adjuvants

Purpose: Vaccine adjuvants such as those made using an oil-in-water compositions [e.g., MF59 (Novartis) and Adjuvant System 03 (AS03) (GSK Vaccines)] have been used in the clinic to improve immunogenicity when combined with sub-unit antigens. Current process of making these oil-in-water nanoemulsions by microfluidizer is an expensive, complex, and high-shear process. Nanoemulsions have been prepared by other low-energy processes like phase inversion temperature and self-emulsification. In the past, squalene oil emulsion (self-emulsifying adjuvant 160 (SEA160)) has been prepared by the self-emulsification process with similar particle size and immunogenicity to MF59. However, industrial scalability of the self-emulsification process remains a challenge. Simpler processes like a microfluidic chip involving a pump-driven scalable formulation technique, currently used to formulate lipid nanoparticles (LNPs), can be used to make these nanoemulsions. We have shown that the extrapolation of the microfluidic chip-based method for manufacturing these nanoemulsions could prove beneficial in making the process simpler and scalable. Additionally, we have shown reproducibility in the process and non-inferiority in animal models.

Methods: In previous work, we have shown that emulsion adjuvants can be formulated using an alternative self-emulsification process. In this study, we optimized the process using Precision Nanosystems Nanoassemblr Ignite™ and NxGen™ microfluidic chip to make emulsion adjuvants. We used a one parameter at a time approach to optimize the process and used SEA160 as our control formulation. Antigen integrity was determined using ovalbumin as model antigen and run on SDS-PAGE gels and compared with controls. The formulations were characterized for size and PDI using DLS. Optimized and stable formulations were characterized for pH, osmolarity, size, PDI, squalene content and endotoxin and tested for immune responses in suitable animal model. Five-eight-week-old CB6F1 female mice were immunized three times (each three weeks apart) with adjuvant and Cytomegalovirus glycoprotein B (CMV gB) as the antigen. 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 (Total and subclass) were determined from serum three weeks post immunization 2 and 3 using Luminex.

Results: Microfluidic chip process for making emulsions was optimized to yield formulation with similar characteristics of SEA160 (sizes around 160 nm and PdI < 0.2). The resultant microfluidic chip formulation (MC160) were found to be stable for up to four weeks. Ovalbumin mixed with these formulations showed structural integrity when compared with controls on SDS-PAGE gels. Neutralizing antibody responses and antigen specific total IgG titers showed that both SEA160 and MC160 groups showed no significant difference when compared with each other. Moreover, both groups showed an adjuvant effect when compared with unadjuvanted CMV gB. MC160 and SEA160 both the groups showed higher CMV gB specific IgG subclass1 titers (compared to unadjuvanted CMV gB) which is a marker of Th2 induced responses.

Conclusion: The results demonstrated a proof-of-concept for using microfluidic chip platform to make emulsion adjuvants. Additionally, we demonstrated non-inferiority in animal models for humoral responses compared to SEA160.

References: 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.
Lodaya, Rushit N, Amey P Kanitkar, Asma Ashraf, Douty Bamba, Mansoor M Amiji, and Derek T O’Hagan, A Self-Emulsified Adjuvant System Containing the Immune Potentiator Alpha Tocopherol Induces Higher Neutralizing Antibody Responses Than a Squalene-Only Emulsion When Evaluated with a Recombinant Cytomegalovirus (CMV) Pentamer Antigen in Mice. Pharmaceutics, 2023. 15(1): p. 238.

Acknowledgements: 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.

Neutralizing antibody titers in serum obtained three weeks post 1st, 2nd, and 3rd immunization, against CMV TB40 strain of virus. Each bar represents geometric mean titers (GMT) with 95% confidence interval (CI) from n=10 animals per group. Statistics were performed on titers post-log transformation using one-way ANOVA followed by Tukey’s test to compare all groups with each other. Differences are marked on the graph. All comparisons marked are for 3wp3 titers; where, ns = not significant, *= p˂0.05, **= p˂0.01, ***= p˂0.001 and ****= p˂0.0001

Total IgG antibody titers in serum obtained three weeks post 2nd and 3rd immunization, against CMV gB. Each bar represents geometric mean titers (GMT) with 95% confidence interval (CI) from n=10 animals per group. Statistics were performed on titers post-log transformation using one-way ANOVA followed by Tukey’s test to compare all groups with each other. Differences are marked on the graph. All comparisons marked are for 3wp3 titers; where, ns = not significant, *= p˂0.05, **= p˂0.01, ***= p˂0.001 and ****= p˂0.0001.

IgG subclass antibody titers (A: IgG1, B: IgG2a and C: IgG2b) in serum obtained three weeks post 3rd immunization, against CMV gB. Each bar represents geometric mean titers (GMT) with 95% confidence interval (CI) from n=10 animals per group. Statistical analyses were performed on titers post-log transformation using one-way ANOVA followed by Tukey’s test to compare all groups with each other. Differences are marked on the graph; where, ns = not significant, *= p˂0.05, **= p˂0.01, ***= p˂0.001 and ****= p˂0.0001