OBJECTIVE: The present study aimed to evaluate the immunogenicity of BA.2 variant receptor binding domain (RBD) recombinant protein formulated with CpG 1826 plus alum dual adjuvant. METHODS: The BA.2 variant RBD (residues 308-548) fusing TT-P ₂ epitope was obtained from prokaryotic expression system, purification technology and dialysis renaturation, which was designated as Sot protein. The soluble Sot protein formulated with CpG 1826 plus alum dual adjuvant was designated as Sot/CA subunit vaccine and then the BALB/c mice were intramuscularly administrated with two doses of the Sot/CA subunit vaccine at 14-day interval (day 0 and 14). On day 28, the number of effector T lymphocytes secreting IFN-γ and IL-4 in mice spleen were determined by enzyme-linked immunospot (ELISpot) assay. The serum IgG, IgG1 and IgG2a antibodies were examined by enzyme-linked immunosorbent assay (ELISA). In addition, the level of neutralizing antibodies (NAbs) induced by Sot/CA subunit vaccine was also evaluated by the microneutralization assay. RESULTS: The high-purity soluble Sot protein with antigenicity was successfully obtained by the prokaryotic expression, protein purification and dialysis renaturation. The Sot/CA subunit vaccine induced a high level of IgG antibodies and NAbs, which were of cross-neutralizing activity against SARS-CoV-2 BA.2 and XBB.1.5 variants. Meanwhile, Sot/CA subunit vaccine also induced a high level of effector T lymphocytes secreting IFN-γ (635.00 ± 17.62) and IL-4 (279.20 ± 13.10), respectively. Combined with a decreased IgG1/IgG2a ratio in the serum, which indicating Sot/CA subunit vaccine induced a Th1-type predominant immune response. CONCLUSION The Sot protein formulated with CpG 1826 plus alum dual adjuvant showed that the excellent cellular and humoral immunogenicity, which provided a scientific basis for the development of BA.2 variant subunit vaccines and references for the adjuvant application of subunit vaccines.
Animals ; COVID-19 Vaccines/immunology* ; Alum Compounds/pharmacology* ; Mice, Inbred BALB C ; Vaccines, Subunit/immunology* ; Mice ; SARS-CoV-2/immunology* ; Oligodeoxyribonucleotides/administration & dosage* ; Female ; Adjuvants, Immunologic ; COVID-19/immunology* ; Antibodies, Viral/blood* ; Immunogenicity, Vaccine ; Spike Glycoprotein, Coronavirus/immunology* ; Antibodies, Neutralizing/blood* ; Adjuvants, Vaccine ; Immunoglobulin G/blood*

OBJECTIVE: This study aimed to investigate the effects of Montanide ISA-720 and Naloxone (NLX) in Hepatitis B surface antigen (HBsAg) vaccine formulation on cytokine and long-lasting antibody responses. METHODS: First, the HBsAg was formulated in Montanide ISA-720 adjuvant and Naloxone at 5 and 10 mg/kg. The experimental mice were immunized three times at a 2-week interval, and then IL-4, IL-2, TNF-α, and IFN-γ cytokines; long-lasting IgG antibody responses 220 days after the last shot; and IgG1/IgG2a isotypes were assessed by ELISA. RESULTS: The HBsAg-Alum group exhibited the highest IL-4 cytokine response among the experimental groups, whereas NLX in HBsAg-MON720 vaccine formulation did not affect cytokine responses. In addition, NLX in Alum-based vaccine suppressed IL-4 cytokine response and increased the IL-2/IL-4 cytokine ratio. Moreover, HBsAg-MON720 was more potent than HBsAg-Alum in the induction of antibody responses, and NLX in Alum- and MON720-based vaccines induced long-lasting antibody responses. CONCLUSION NLX in Alum-based vaccine decreased IL-4 cytokine response, increased IL-2/IL-4 cytokine ratio, and improved long-lasting humoral immune responses in both vaccine formulations. Therefore, the adjuvant activity of NLX in the vaccine formulation depends on the type of adjuvant and the nature of the antigen in the vaccine formulation.
Adjuvants, Immunologic/pharmacology* ; Alum Compounds ; Animals ; Cytokines ; Hepatitis B Surface Antigens ; Hepatitis B Vaccines ; Immunity, Humoral ; Immunoglobulin G ; Interleukin-2 ; Interleukin-4 ; Mice ; Mice, Inbred BALB C ; Mineral Oil ; Naloxone/pharmacology* ; Tumor Necrosis Factor-alpha