Melika Haghighi - Advanced Therapy Medicinal Product (ATMP) Department, Breast Cancer Research Center, Motamed Cancer Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Akbar Khorasani - Department of FMD Vaccine Production, Razi Vaccine & Serum Research Institute, Agricultural Research, Education & Extension Organization (AREEO), Karaj, Iran
Pegah Karimi - Advanced Therapy Medicinal Product (ATMP) Department, Breast Cancer Research Center, Motamed Cancer Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran
Mehdi Mahdavi - Advanced Therapy Medicinal Product (ATMP) Department, Breast Cancer Research Center, Motamed Cancer Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran

Objective(s): SARS-CoV-۲, emerging as a major threat to public health, has to be controlled through vaccination. Naloxone (NLX), an opioid receptor antagonist, demonstrated its adjuvant activity for microbial vaccines. In this study, inactivated SARS-CoV-۲ was developed in the Alum/NLX adjuvant to increase the potency of the inactivated SARS-CoV-۲ vaccine. Materials and Methods: BALB/c mice were immunized on days ۰ and ۱۴ with inactivated SARS-CoV-۲-Alum, -Alum + NLX ۳ mg/kg, -Alum + NLX ۱۰ mg/kg, and -Freund adjuvant, as well as PBS. IFN-γ and IL-۴ cytokines and Granzyme-B release were assessed with ELISA. In addition, specific total IgG, IgG۱/IgG۲a isotypes, and ratio as well as anti-RBD IgG responses were assessed with an optimized ELISA. Results: SARS-CoV-۲-Alum-NLX۱۰ group showed a significant increase in the IFN-γ cytokine response versus SARS-CoV-۲-Alum, SARS-CoV-۲-Alum-NLX۳, and PBS groups. The SARS-CoV-۲-Alum-NLX۳ group exhibited a significant decrease in IL-۴ cytokine versus SARS-CoV-۲-Alum. The mice immunized with SARS-CoV-۲-Alum-NLX۱۰ showed a significant increase in CTL activity versus SARS-CoV-۲-Alum and PBS. In addition, mice immunized with SARS-CoV-۲-Alum-NLX۳, SARS-CoV-۲-Alum-NLX۱۰ and SARS-CoV-۲-Freund demonstrated an increase in IgG response, as compared with SARS-CoV-۲-Alum and PBS group. Furthermore, all formulations of SARS-CoV-۲ vaccines could induce both IgG۱ and IgG۲a isotypes. But, the IgG۲a/IgG۱ ratio in SARS-CoV-۲-Freund and SARS-CoV-۲-Alum-NLX۱۰ revealed an increase as compared with that of the SARS-CoV-۲-Alum group. Anti-RBD IgG response in the SARS-CoV-۲-Alum-NLX۱۰ group showed a significant increase as compared with the Alum-based vaccine. Conclusion: Formulation of inactivated SARS-CoV-۲ virus in NLX/alum adjuvant improved the potency of humoral and, especially, cellular responses.

Alum Adjuvant, Immune responses, Inactivated SARS-CoV-۲ - virus, Naloxone, Vaccine formulation