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

Vaccine candidate preparation

The seed virus was adapted to a highly characterized GMP Vero cell platform, amplified to produce the master and working virus bank. The master virus bank was well characterized based on WHO Technical Report Series guidelines (WHO-TRS_978_Annex_3.pdf, 2013) , including identity, sterility, mycoplasma, virus titration, adventitious agents, hemadsorption etc. GMP production of virus bulk was performed in the bio-safety level-3 (BSL-3) facility using bioreactors.

Growth kinetics analysis revealed that the SARS-CoV-2 virus replicated to 7.0 log10 TCID₅₀ between 36 and 72 h (Figure 1A). The β-propiolactone was utilized for the inactivation of the virus by mixing the virus stock at 2-8°C. Inactivation kinetics was performed with varying conditions and concentrations, and samples were collected at various time points (between 0 and 24 h, at 4-h intervals) to evaluate the cytopathic effect. Three consecutive inactivation procedures (each lasting 24 h) were performed to ensure complete viral inactivation without affecting the antigen stability (Figures 1A and 1B). Purified and inactivated whole-virion antigen produced from three production batches was also characterized by western blot for its identity using anti-Spike (S1 & S2), anti-RBD, and anti-N protein (Figure 1B) antibodies. These results demonstrated that the final purified inactivated bulk of the vaccine candidate contains spike and nucleocapsid (N) protein. The uncleaved full-length spike was detected by the spike (S2) (Figure 1B first panel), spike (RBD) (Figure 1B second panel), and spike (S1) (Figure 1B third panel) antibodies. The spike (S2) antibody also detected the S2 subunit (Figure 1B first panel). The full-length spike (S), S2 fragment (S2), and nucleocapsid (N) protein bands were of expected molecular weight (Figure 1B). Transmission electron microscopy (TEM) analysis also showed that the inactivated and purified virus particles were intact, oval-shaped, and were accompanied by a crown-like structure representing the well-defined spike protein on the virus membrane (Figure 1C), and these data corroborate with electron micrograph of the live virus (Prasad et al., 2020).

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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.

Vaccine formulations with adjuvants and their stability

Inactivated whole-virion SARS CoV-2 (BBV152) vaccine candidates were formulated with two alum-based adjuvants: Algel (aluminum hydroxide gel) and Algel-IMDG, an imidazoquinoline class molecule (TLR7/TLR8 agonist, abbreviated as IMDG) chemisorbed onto aluminum hydroxide gel. Three vaccine formulations were prepared: first two contain 3 μg and 6 μg antigen with Algel-IMDG (BBV152A and BBV152B, respectively) and the third one contains 6μg antigen with Algel (BBV152C). To determine the stability of these formulations, Algel-IMDG vaccine formulations (BBV152A and BBV152B) were stored at 37°C and 2–8°C temperature for 7 days. These vaccine formulations were diluted 20 times (1/20^th human single dose (HSD), equivalent to 0.15 and 0.3 μg antigen) and administered in BALB/c mice intra-peritoneally to evaluate NAb titer by microneutralization test (MNT₅₀) at 7-day post-immunization. Our results demonstrated that the both vaccine formulations are relatively stable at 37°C for 7 days, as shown by equivalent NAb titer compared with formulation stored at real time (2–8°C) temperature (Figure 1D).

Safety evaluation of Algel-IMDG and adjuvanted vaccine formulations (BBV152)

Algel is a well-known adjuvant having been used in a large number of vaccines globally. Hence extensive safety evaluation was performed for the Algel-IMDG adjuvant alone along with three adjuvanted vaccine formulations (BBV152A, B, and C), as per the regulatory guidelines (WHO, 2013; OECD, 2020; CDSCO, 2019). Table 2 summarizes the key toxicology studies/tests performed and the observations thereof.

Safety of Algel-IMDG was evaluated by three experiments: (1) mutagenicity assay (in-vitro) to determine mutagenic potential of the Algel-IMDG; (2) maximum tolerated dose test (MTD, in-vivo), to ensure the human intended adjuvant dose is tolerable; and (3) repeated dose toxicity study (RDT, in-vivo), to evaluate that repeated administration of Algel-IMDG does not cause any systemic toxicity or mortality. Mutagenicity assay performed with Algel-IMDG at various concentrations revealed that there was no substantial increase in revertant colony numbers in any of the tested strains at any dose level, in both the plate-incorporation and pre-incubation methods in the presence or absence of metabolic activation (S9 mix) (Table S1). Thus, the Algel-IMDG used in the BBV152 A and B-adjuvanted vaccine formulations was found to be non-mutagenic. Further, maximum tolerated dose study performed with single dose of Algel-IMDG also revealed that the Algel-IMDG was tolerated at the tested dose (20 μg agonist/animal) in mice and rats as demonstrated by lack of erythema, edema, or any other macroscopic lesions at the site of injection (Table S2).

Moreover, repeated administration of (N+1 dose regimen) high dose of either Algel-IMDG alone (30μg agonist/animal) in Swiss Albino mice and Wistar rats or high dose of adjuvanted vaccine formulation (9μg Ag with 30μg agonist/animal, which is more than HSD) in Wistar rats did not show any clinical illness, change in body weight (Figure S1), or histopathological changes, except inflammation at the site of injection (Figure S3) and thus established the safety of both Algel-IMDG and adjuvanted vaccine formulations at high dose.

Further, the safety of adjuvanted vaccine formulations (BBV152 A, B, and C) either at full HSD or 1/10^th or 1/20^th HSD was also evaluated to be safe in three animal models (BALB/c mice, S. albino mice, and NZW rabbits), as demonstrated by the repeated dose toxicity study with no mortality and with no changes in clinical signs, body weight gain, body temperature, or feed consumption in any of the animals. Representative data of body temperature as a parameter are shown in Table S3.

Clinical pathological parameters such as hematology, clinical biochemistry, coagulation studies, and urinalysis performed in repeated dose toxicity (RDT) studies showed that the animals administered with either adjuvanted vaccine candidates or adjuvants/antigen-alone were comparable with control (Figure S2, Table S4), except increased levels of Alpha 1- acid glycoprotein and neutrophils count on day 2 in adjuvant-alone or adjuvanted vaccine formulation groups. However, these values were comparable with control on day 21. This transient increase may be due to inflammation at the injection site after administration of the first dose. These findings were further correlated with the inflammatory reaction at the injection site observed microscopically, in the animals administered with adjuvant-alone and adjuvanted vaccine with Algel and Algel-IMDG. This inflammation was found to be slightly higher in animals that received Algel-IMDG than in animals that received Algel. However, this inflammation reduced by day 28 (Figures S3 and S4). Other than local reaction at the site of injection, no other treatment-related microscopic findings were observed in any of the animals administered with antigen or adjuvant or adjuvanted vaccine formulations. Histopathological examination of organs such as spleen, lungs, heart, and lymph nodes etc., of all animal models administered with antigen or adjuvant or adjuvanted vaccine formulations was normal (Figures S5 and S6).

Adjuvanted vaccine formulations (BBV152) induced high neutralization antibody titers

We report immunogenicity of three BBV152 formulations in BALB/c mice and New Zealand white rabbits. BALB/c mice were administered either with full or 1/10^th or 1/20^th HSD, whereas Rabbits were immunized with intended HSD. Table 2 summarizes the immunogenicity studies performed and the observations thereof.

Immunogenicity in BALB/c mice

Initially, BALB/c mice (n = 5/group, female) were administered with adjuvanted vaccine formulations, antigen or adjuvants alone at 1/20^thof the intended HSD (i.e., 1/20^th of 3 μg, 6 μg, and 9 μg/mouse or 0.15 μg, 0.3 μg, and 0.45 μg/mouse), to determine the optimal dose. ELISA titers (Figure 2A) and NAb titers (Figure 2B) determined at various time points revealed that immune response elicited against these adjuvanted vaccine formulations tested at three antigen concentrations elicited high levels of binding and NAb titer (Figures 2A and 2B). Antibody response determined on day 7 was found to be less (10²titer) robust or negligible compared with day 14 and day 21 with a titer of 10³ and 10⁴, respectively. Notably, 3 and 6 μg formulations induced high or similar spike-specific antibody titers compared with 9 μg group. Hence, adjuvanted formulation with high antigen dose (9 μg) was eliminated in further studies of the immunogenicity, whereas safety was evaluated in Wistar rats at this high dose. These results also indicated that adjuvanted vaccine formulations either with Algel or Algel-IMDG elicited high spike (S1)-specific antibody binding titers compared with antigen alone tested at all three concentrations (Figure 2A). This was further evaluated by administering BALB/c mice intramuscularly with antigen at actual HSD (6 μg Ag) either in the presence or in the absence of adjuvant (Algel-IMDG) and compared with 1/10^th HSD of adjuvanted vaccine formulation (0.6 μg Ag and Algel-IMDG). Immune response elicited against adjuvanted vaccine formulation (BBV152B) was significantly (1 log) higher than the antigen alone (6 μg Ag), which is comparable with 1/10^th HSD of adjuvanted vaccine formulation (1/10^th of BBV152B). These results suggest the dose-sparing effect of Algel-IMDG (Figure 2C).

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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.

Further, to assess the immunogenicity and safety of clinical batch samples, BALB/c mice (n = 10/group, 5 male and 5 female) were vaccinated via IP route with three adjuvanted vaccine formulations with Algel and Algel-IMDG at 1/10^th human intended single dose (0.3 and 0.6 μg/dose with Algel or Algel-IMDG). All adjuvanted vaccine formulations elicited antigen-specific binding antibodies (Figure 2D). Further, sera collected on day 21 were analyzed by ELISA to determine S1, RBD, and N-specific binding titer (Figure 2E) and showed 100% seroconversion with S1, RBD, and N protein. Analysis of plaque reduction neutralization test (PRNT₉₀), performed with individual mice sera, showed high NAbs in all adjuvanted vaccines (Figure 2F).

We also compared different immunization dose schedules (day 7 versus day 14), wherein BALB/c mice were administered intramuscularly with adjuvanted vaccine (full HSD), with one group receiving second dose on day 7 and the other group on day 14 after initial immunization. Our results indicated 8-fold increase in spike-protein-specific antibody titer, when booster dose was given with 14-day interval as compared with that given on day 7 (Figure 2G).

In addition, to demonstrate long-lived immune response, BALB/c mice (n = 8/group, 4 male and 4 female) were vaccinated intramuscularly with three adjuvanted vaccine formulations (1/10^th HSD of BBV152A, B, and C) on day 0, 7, and 14 and evaluated antibody titer up to 12 weeks after last dose. These results revealed that the spike-specific antibodies reached peak level on day 28, and the antibody titers were sustained up to day 98, i.e., 12 weeks after last dose (Figure 2H). Similarly, we also found sustained NAb titers up to day 98 (Figure 2I), which indicates the BBV152 vaccine candidates were able to produce long-term immunity.

Immunogenicity in New Zealand white rabbits

To assess the immunogenicity of adjuvanted vaccine formulations at full human single dose (HSD, 3 and 6 μg antigen/dose), rabbits (n = 4) were immunized intramuscularly on days 0, 7, and 14. Similar to mice, immune response in rabbits was also found to be time dependent, and not all animals were seroconverted on day 7 and showed less antibody binding titer (≥10²). However, on day 21, we found 100% seroconversion with spike-specific antibody binding titer of greater than or equal to 10⁴. All three formulations (BBV152A, B, and C) showed high binding antibody response (Figure 3A), with no statistically significant difference. Similarly, PRNT₉₀ results showed high neutralizing antibody titers in all three adjuvanted vaccine formulations on day 21 (Figure 3B). However, there is no significant difference, and similar results were also observed by MNT₅₀ titers (Figure 3C). Further, NAb titers determined by MNT₅₀ were slightly higher or comparable with NAb titers of human convalescent sera collected from recovered symptomatic COVID-19 patients (Figure 3C).

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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.

Limitations of the study

Long-term protective efficacy of these vaccine candidates, cross-neutralization with other SARS CoV 2 variants, and mechanism of action of Algel-IMDG in inducing cell-mediated responses need to be evaluated further.

We thank Ms. Tejaswi Mandala, Ms. Spandana Sure, Dr. Ramulu Chintala, Dr. Rakesh Chandra Meka, Dr. Mohammad Feraz Ahsan, Dr. Balasankara Reddy Kaipa, BBIL, and Dr. Zaiham Rizvi, THSTI for their technical support. We greatly acknowledge the support provided by Dr. Vinay Aileni, in the alignment of manuscript. Our special thanks to Dr. Sunil David (ViroVax, LLC, KS, USA) for giving us adjuvant samples for evaluation in the initial development phase of the project. This vaccine candidate could not have been developed without the efforts of Bharat Biotech's manufacturing and quality control teams. All authors would like to express their gratitude for all the frontline health care workers during this pandemic.