Th1 skewed immune response of whole virion inactivated SARS CoV 2 vaccine and its safety evaluation

Abstract

We report the development and evaluation of safety and immunogenicity of a whole virion inactivated (WVI) SARS-CoV-2 vaccine (BBV152), adjuvanted with aluminum hydroxide gel (Algel), or TLR7/8 agonist chemisorbed Algel. We used a well-characterized SARS-CoV-2 strain and an established Vero cell platform to produce large-scale GMP-grade highly purified inactivated antigen. Product development and manufacturing process were carried out in a BSL-3 facility. Immunogenicity and safety were determined at two antigen concentrations (3μg and 6μg), with two different adjuvants, in mice, rats, and rabbits. Our results show that BBV152 vaccine formulations generated significantly high antigen-binding and neutralizing antibody titers (NAb), at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation contains TLR7/8 agonist adjuvant-induced Th1-biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2-specific IFN-γ⁺ CD4⁺ T lymphocyte response. Our results support further development for phase I/II clinical trials in humans.

Keywords: Immune Response; Immunology; Virology.

Graphical abstract

Figure 1

Characterization of inactivated SARS-CoV-2 and evaluation of the stability of BBV152 vaccine formulations (A) SARS-CoV-2 virus (Strain NIV-770-2020) growth kinetics and its cytopathic effect (CPE) before and after inactivation. (1) Line graph represents virus titer (10⁶–10⁷) measured by CCID₅₀ at every 3 h up to 48 h and after that every 12 h (24, 27, 30, 33, 36, 39, 42), (2) microscopic images represent cells with cytopathic effect (CPE) before inactivation and no CPE after inactivation, (3) image of Vero cell monolayer with no CPE observed from 16 to 36 h; (B) Western blot analysis of purified inactivated SARS-CoV-2 produced from three production batches; the antibody used for each panel is mentioned on the left side of the image; (C) Representative electron micrograph of purified inactivated SARS-CoV-2 candidate vaccine (BBV152) at a scale bar: 100 nm (right) and 200 nm (left); (D) Bar graph represents microneutralization antibody titer of day 14 individual sera (7 days after 2^nd dose) collected from mice vaccinated with 1/20^th dose of adjuvanted formulations (0.15 μg Ag with Algel-IMDG and 0.3 μg Ag with Algel-IMDG), after subjecting them for stability at 37°C for 7 days and compared with 2–8°C. Error bars represent median with 95% CI and the statistical analysis performed using one-sample T test shown not significant.

Figure 2

BBV152 vaccine induces high virus-specific antibody response in mice (A–I) Immune response elicited against antigens at three concentrations of antigen or adjuvanted vaccine formulations in BALB/c mice (n = 5, female) were represented. Animals were administered via IP route either with 1/20^th (Fig A & B) or 1/10^th (Fig D, E, & F) human single dose (HSD) or administered via intramuscular route with either full HSD or 1/10th dose (C, G, & H). S1-specific total IgG antibody binding titers measured using individual sera collected (A) at various time points (day 0, 7, 14, & 21) at 1/20^th dose; (D) on day 21 with 1/10^th dose; (C) on day 21 either with full HSD or 1/10^th dose; (G)on post-dose 1 (on day 7 or 14) and 2 (on day 14 or 28), when BALB/c mice were administered with BBV152B via IM route with different immunization schedule with an interval of 7 or 14 days, and (H) at various time points (day 21, 28, 42, 56, 84, and 98) at 1/10^th dose via IM route. (E) SARS-CoV-2 specific (S1, RBD, N and total inactivated antigen) antibody binding titers elicited against adjuvant vaccines (BBV152A, B & C) on day 21; neutralizing antibody titers performed by PRNT_90, using day 21 sera collected from BALB/c mice, when administered via IP route either with 1/20^th (B) or 1/10^th dose (F) or collected at various time points day 21, 28, 42, 56, 84, and 98 (I), when administered at 1/10^th dose via IM route. Antibody binding titers were performed by ELISA and neutralizing antibody titers by PRNT₉₀. Bar graphs representing data represented as mean ± SD (G), mean/mean ± SEM (A, H, & I) derived from individual mice sera data analysis. For the long-term immunogenicity study, sera from four animals per group were tested for ELISA and MNT₅₀ analyzed. Statistical analysis performed by (B) Wilcoxon rank test indicates significant difference between 0.3 μg antigen Algel and 0.3 μg antigen Algel-IMDG at p value < 0.05 and error bars indicate median with 95% CI, whereas in figure D, statistical analysis performed by Mann Whitney test showed significant difference at p value < 0.005 (∗∗) and <0.05 (∗), respectively. ns indicates not significant.

Figure 3

BBV152 induces robust neutralizing antibody response in rabbits New Zealand white rabbits (n = 4) were administered intramuscularly on days 0, 7, and 14 with full HSD. SARS-CoV-2-specific antibody titers were measured by ELISA. NAb tires were measured by PRNT₉₀ and MNT₅₀. Data Points represent median/mean of individual animal data. (A) S1-specific Ab binding titer of sera collected at various time points (day 0, 7, 14, and 21) (B) PRNT₉₀ neutralizing antibody titers of day 21 sera; error bars indicate median with 95% CI, and statistical analysis performed using Wilcoxon signed rank test found no significant difference among the three adjuvanted vaccine groups. (C) MNT₅₀ neutralizing antibody titers of sera collected at various time points (day 0, 7, 14, and 21) along neutralizing antibody titer (MNT₅₀) with human convalescent sera (HCS) from recovered COVID-19 patients (n = 15). Samples were collected between 21 and 65 days of virological confirmation. Error bars indicate mean with 95% CI.

Figure 4

BBV152 induces a robust virus-specific T cell response Panel of figures represent cell-mediated response data generated either from vaccinated sera or from splenocytes collected from BALB/c mice or cell culture supernatant collected, after stimulation of unvaccinated huPBMCs (ex-vivo). BALB/c mice (n = 10) were vaccinated with 1/10th HSD of adjuvanted vaccine formulations (BBV 152 A, B, and C) via the IP route. (A) Th1:Th2 index generated using the formulas IgG2a/IgG1 or (IgG2a + IgG3)/IgG1, from endpoint antibody (immunoglobulin subclasses: IgG1, IgG2a, and IgG3) titer analysis measured by ELISA, using sera collected from day 21 (7 days post-third dose), from BALB/c mice, administered with 1/20th HSD of adjuvanted vaccine formulations (BBV 152 A, B, and C) via the IP route. Endpoint titer of respective immunoglobulin sub classes obtained from PBS/Algel/Algel-IMDG were taken as baseline. Error bars represent mean ± SEM. (B) Secreted IFN gamma levels estimated by ELISA, on day 21 sera (7 days post-third dose). Statistical analysis done by Mann Whitney test showed significant difference at p value < 0.05, between 0.6 μg antigen Algel and 0.6 μg Algel-IMDG. (C) Percent of CD3⁺ (Left) or CD4⁺ (Middle) or CD8⁺ (right) T lymphocytes producing IFN gamma measured by intracellular staining assay using the splenocytes of individual BALB/c mice administered with 1/10^th HSD. Error bars indicate mean ± SEM. (D) Cytokine profile measured on various time points using vaccinated BALB/c mice sera, when administered with adjuvanted vaccine formulations (1/20^th HSD via IP route). Left—BBV152C-antigen 6μg + Algel. Right—BBV152B-antigen 6μg + Algel-IMDG. Error bars indicate mean ± SDE. IFNα levels measured by ELISA from culture supernatant, when stimulated healthy PBMCs with Algel or Algel-IMDG or adjuvanted vaccine formulations (BBV152A, B, and C)). Two-fold serial dilutions of the human intended dose of adjuvanted vaccine formulations were used. Corresponding antigen or adjuvant alone concentration were also maintained simultaneously as controls. Error bars indicate mean ± SD of triplicate values.