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Summary Background BBV152 is a whole-virion inactivated SARS-CoV-2 vaccine (3 μg or 6 μg) formulated with a toll-like receptor 7/8 agonist molecule (IMDG) adsorbed to alum (Algel). We previously reported findings from a double-blind, multicentre, randomised, controlled phase 1 trial on the safety and immunogenicity of three different formulations of BBV152 (3 μg with Algel-IMDG, 6 μg with Algel-IMDG, or 6 μg with Algel) and one Algel-only control (no antigen), with the first dose administered on day 0 and the second dose on day 14. The 3 μg and 6 μg with Algel-IMDG formulations were selected for this phase 2 study. Herein, we report interim findings of the phase 2 trial on the immunogenicity and safety of BBV152, with the first dose administered on day 0 and the second dose on day 28. Methods We did a double-blind, randomised, multicentre, phase 2 clinical trial to evaluate the immunogenicity and safety of BBV152 in healthy adults and adolescents (aged 12–65 years) at nine hospitals in India. Participants with positive SARS-CoV-2 nucleic acid and serology tests were excluded. Participants were randomly assigned (1:1) to receive either 3 μg with Algel-IMDG or 6 μg with Algel-IMDG. Block randomisation was done by use of an interactive web response system. Participants, investigators, study coordinators, study-related personnel, and the sponsor were masked to treatment group allocation. Two intramuscular doses of vaccine were administered on day 0 and day 28. The primary outcome was SARS-CoV-2 wild-type neutralising antibody titres and seroconversion rates (defined as a post-vaccination titre that was at least four-fold higher than the baseline titre) at 4 weeks after the second dose (day 56), measured by use of the plaque-reduction neutralisation test (PRNT50) and the microneutralisation test (MNT50). The primary outcome was assessed in all participants who had received both doses of the vaccine. Cell-mediated responses were a secondary outcome and were assessed by T-helper-1 (Th1)/Th2 profiling at 2 weeks after the second dose (day 42). Safety was assessed in all participants who received at least one dose of the vaccine. In addition, we report immunogenicity results from a follow-up blood draw collected from phase 1 trial participants at 3 months after they received the second dose (day 104). This trial is registered at ClinicalTrials.gov , NCT04471519 . Findings Between Sept 5 and 12, 2020, 921 participants were screened, of whom 380 were enrolled and randomly assigned to the 3 μg with Algel-IMDG group (n=190) or 6 μg with Algel-IMDG group (n=190). Geometric mean titres (GMTs; PRNT50) at day 56 were significantly higher in the 6 μg with Algel-IMDG group (197·0 [95% CI 155·6–249·4]) than the 3 μg with Algel-IMDG group (100·9 [74·1–137·4]; p=0·0041). Seroconversion based on PRNT50 at day 56 was reported in 171 (92·9% [95% CI 88·2–96·2] of 184 participants in the 3 μg with Algel-IMDG group and 174 (98·3% [95·1–99·6]) of 177 participants in the 6 μg with Algel-IMDG group. GMTs (MNT50) at day 56 were 92·5 (95% CI 77·7–110·2) in the 3 μg with Algel-IMDG group and 160·1 (135·8–188·8) in the 6 μg with Algel-IMDG group. Seroconversion based on MNT50 at day 56 was reported in 162 (88·0% [95% CI 82·4–92·3]) of 184 participants in the 3 μg with Algel-IMDG group and 171 (96·6% [92·8–98·8]) of 177 participants in the 6 μg with Algel-IMDG group. The 3 μg with Algel-IMDG and 6 μg with Algel-IMDG formulations elicited T-cell responses that were biased to a Th1 phenotype at day 42. No significant difference in the proportion of participants who had a solicited local or systemic adverse reaction in the 3 μg with Algel-IMDG group (38 [20·0%; 95% CI 14·7–26·5] of 190) and the 6 μg with Algel-IMDG group (40 [21·1%; 15·5–27·5] of 190) was observed on days 0–7 and days 28–35; no serious adverse events were reported in the study. From the phase 1 trial, 3-month post-second-dose GMTs (MNT50) were 39·9 (95% CI 32·0–49·9) in the 3μg with Algel-IMDG group, 69·5 (53·7–89·9) in the 6 μg with Algel-IMDG group, 53·3 (40·1–71·0) in the 6 μg with Algel group, and 20·7 (14·5–29·5) in the Algel alone group. Interpretation In the phase 1 trial, BBV152 induced high neutralising antibody responses that remained elevated in all participants at 3 months after the second vaccination. In the phase 2 trial, BBV152 showed better reactogenicity and safety outcomes, and enhanced humoral and cell-mediated immune responses compared with the phase 1 trial. The 6 μg with Algel-IMDG formulation has been selected for the phase 3 efficacy trial. Funding Bharat Biotech International. Translation For the Hindi translation of the abstract see Supplementary Materials section.

Summary Background To mitigate the effects of COVID-19, a vaccine is urgently needed. BBV152 is a whole-virion inactivated SARS-CoV-2 vaccine formulated with a toll-like receptor 7/8 agonist molecule adsorbed to alum (Algel-IMDG) or alum (Algel). Methods We did a double-blind, multicentre, randomised, controlled phase 1 trial to assess the safety and immunogenicity of BBV152 at 11 hospitals across India. Healthy adults aged 18–55 years who were deemed healthy by the investigator were eligible. Individuals with positive SARS-CoV-2 nucleic acid and/or serology tests were excluded. Participants were randomly assigned to receive either one of three vaccine formulations (3 μg with Algel-IMDG, 6 μg with Algel-IMDG, or 6 μg with Algel) or an Algel only control vaccine group. Block randomisation was done with a web response platform. Participants and investigators were masked to treatment group allocation. Two intramuscular doses of vaccines were administered on day 0 (the day of randomisation) and day 14. Primary outcomes were solicited local and systemic reactogenicity events at 2 h and 7 days after vaccination and throughout the full study duration, including serious adverse events. Secondary outcome was seroconversion (at least four-fold increase from baseline) based on wild-type virus neutralisation. Cell-mediated responses were evaluated by intracellular staining and ELISpot. The trial is registered at ClinicalTrials.gov (NCT04471519). Findings Between July 13 and 30, 2020, 827 participants were screened, of whom 375 were enrolled. Among the enrolled participants, 100 each were randomly assigned to the three vaccine groups, and 75 were randomly assigned to the control group (Algel only). After both doses, solicited local and systemic adverse reactions were reported by 17 (17%; 95% CI 10·5–26·1) participants in the 3 μg with Algel-IMDG group, 21 (21%; 13·8–30·5) in the 6 μg with Algel-IMDG group, 14 (14%; 8·1–22·7) in the 6 μg with Algel group, and ten (10%; 6·9–23·6) in the Algel-only group. The most common solicited adverse events were injection site pain (17 [5%] of 375 participants), headache (13 [3%]), fatigue (11 [3%]), fever (nine [2%]), and nausea or vomiting (seven [2%]). All solicited adverse events were mild (43 [69%] of 62) or moderate (19 [31%]) and were more frequent after the first dose. One serious adverse event of viral pneumonitis was reported in the 6 μg with Algel group, unrelated to the vaccine. Seroconversion rates (%) were 87·9, 91·9, and 82·8 in the 3 μg with Algel-IMDG, 6 μg with Algel-IMDG, and 6 μg with Algel groups, respectively. CD4+ and CD8+ T-cell responses were detected in a subset of 16 participants from both Algel-IMDG groups. Interpretation BBV152 led to tolerable safety outcomes and enhanced immune responses. Both Algel-IMDG formulations were selected for phase 2 immunogenicity trials. Further efficacy trials are warranted. Funding Bharat Biotech International.

We report the development and evaluation of safety and immunogenicity of a whole virion inactivated SARS-COV-2 vaccine (BBV152), adjuvanted with aluminium hydroxide gel (Algel), or a novel TLR7/8 agonist chemisorbed Algel. We used a well-characterizedSARS-CoV-2 strain and an established vero cell platform to produce large-scale GMP grade highly purified inactivated antigen, BBV152. Product development and manufacturing were carried out in a BSL-3 facility. Immunogenicity was determined at two antigen concentrations (3µgand 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, at both concentrations, in all three species with excellent safety profiles. The inactivated vaccine formulation containing TLR7/8 agonist adjuvant-induced Th1 biased antibody responses with elevated IgG2a/IgG1 ratio and increased levels of SARS-CoV-2 specific IFN-Υ+ CD4 T lymphocyte response. Our results support further development for Phase I/II clinical trials in humans. Funding: This work was supported and funded by Bharat Biotech International Limited and the Indian Council of Medical Research. Conflict of Interest: All authors have no personal financial or non-financial interests to disclose. Ethical Approval: All animal experiments were performed after obtaining necessary approvals from the Institutional Animal Ethics Committee (IAEC). Maintenance (housing and care) of all animals (mice, rats & rabbits) involved in the study was adhered to guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, 2003). All studies were performed following both national and international guidelines in compliance with OECD (Organization for Economic Co-operation and Development) Principles of Good Laboratory Practice (1997)(“OECD Guidelines for Testing of Chemicals, Section 4, No. 471: ‘Bacterial Reverse Mutation Test’, adopted July 21st, 1997,” n.d.).

Background: BBV152 is a whole-virion inactivated SARS-CoV-2 vaccine (3 μg or 6 μg) formulated with a Toll-Like Receptor 7/8 agonist molecule adsorbed to alum (Algel-IMDG) or 6 μg with alum (Algel). Methods: We conducted a double-blind, randomised, multicentre, controlled phase 1 clinical trial to evaluate the safety and immunogenicity of BBV152. A total of 375 healthy adults Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation were randomised equally to receive three vaccine formulations (n=100 each) prepared with 3 μg with Algel-IMDG, 6 μg with Algel-IMDG, and 6 μg with Algel, and an Algel only control arm (n=75). Two intramuscular doses of vaccines were administered (twoweeks apart). Primary outcomes were reactogenicity and safety. Secondary outcome was seroconversion (≥4-fold above baseline) based on wild-type virus neutralisation. Cell-mediated responses were evaluated by intracellular staining and ELISpot. Findings: Among 827 participants screened between July 13 and July 30, 2020, 375 participants were randomised. After both doses, the proportion (95%CI) of solicited local and systemic adverse reactions were 17% (10·5, 26·1), 21% (13·8, 30·5), 14% (8·1, 22·7), and 10% (6·9, 23·6) in the 3 μg with Algel-IMDG, 6 μg with Algel-IMDG, 6 μg with Algel, and control groups, respectively . The most common solicited adverse events were injection site pain, headache, and fatigue. All solicited adverse events were mild (43, 69·3%) or moderate (19, 30·7%) and were more frequent after the first dose. One serious adverse event was reported in the 6 μg with Algel group, unrelated to the vaccine. Seroconversion rates (%) were 87·9, 91·9, and 82·8 in the 3 μg with AlgelIMDG, 6 μg with Algel-IMDG and 6 μg with Algel groups, respectively. CD4 + and CD8 + T cell responses were detected in a subset from both Algel-IMDG groups. Interpretations: BBV152 led to tolerable safety outcomes and enhanced immune responses. Both Algel-IMDG formulations were selected for phase 2 immunogenicity trials. Further efficacy trials are warranted. Trial Registration Number: Clinicaltrials.gov : NCT04471519 Funding: This work was supported and funded by Bharat Biotech International Limited. Conflict of Interest: This work was supported and funded by Bharat Biotech International Limited. All authors are employees of one of the above-mentioned organisations, with no stock options or incentives. Co-author- K.E is the Chairman and Managing Director of Bharat Biotech. Ethical Approval: The trial was conducted across 11 sites in 9 states in India. The trial was approved by the National Regulatory Authority (India) and the respective Ethics Committees and was conducted in compliance with all International Council for Harmonization 114 (ICH) Good Clinical Practice guidelines.

Summary The availability of a safe and effective vaccine would be the eventual measure to deal with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) threat. Here, we have assessed the immunogenicity and protective efficacy of inactivated SARS-CoV-2 vaccine candidates BBV152A, BBV152B, and BBV152C in Syrian hamsters. Three dose vaccination regimes with vaccine candidates induced significant titers of SARS-CoV-2-specific IgG and neutralizing antibodies. BBV152A and BBV152B vaccine candidates remarkably generated a quick and robust immune response. Post-SARS-CoV-2 infection, vaccinated hamsters did not show any histopathological changes in the lungs. The protection of the hamster was evident by the rapid clearance of the virus from lower respiratory tract, reduced virus load in upper respiratory tract, absence of lung pathology, and robust humoral immune response. These findings confirm the immunogenic potential of the vaccine candidates and further protection of hamsters challenged with SARS-CoV-2. Of the three candidates, BBV152A showed the better response., Graphical Abstract, Highlights • Vaccine candidates, BBV152 induced potent humoral immune response in Syrian hamsters • Early viral clearance from lower respiratory tract in vaccinated hamsters • BBV152 vaccine candidates protected Syrian hamsters from pneumonia, Biological Sciences; Immunity; Immunology; Viral Microbiology; Virology

The clinical evaluation of the MDCK-based H1N1 pandemic influenza vaccine HNVAC in adults aged 18–65 years is reported. In the Phase I randomized, double-blind, placebo-controlled, single-centre study, 160 subjects were parallelly assigned 3:1 to vaccine:placebo groups (n = 60:20) with both the aluminium hydroxide adjuvanted and non-adjuvanted vaccine formulations. A single dose of both the formulations containing 15 μg of haemagglutinin protein showed minimal adverse reactions, the most common of which were pain at injection site (11.67%) and fever (10.00%). Both formulations produced 74–81% seroprotection (SRP: titre of ≥40), 67–70% seroconversion (SRC: four-fold increase in titres between days 0 and 21), and a four-fold increase in geometric mean titres (GMT). Aluminium hydroxide did not have a significant effect either on immunogenicity or on reactogenicity. Nevertheless, based on its recognized positive effects on the stability and immunogenicity of many vaccines, and its marginal benefit in both pre-clinical and Phase I studies of HNVAC, alum adjuvanted HNVAC was further tested in a staggered Phase II/III randomized, double-blind, placebo-controlled, multi-centre study of 200 and 195 subjects, respectively, parallelly assigned 4:1 to adjuvanted vaccine and placebo groups. In these studies, the most common adverse reactions were pain at injection site (6.88% and 5.77% in Stage 1 and Stage 2, respectively) and fever (7.50% and 7.05%, respectively), and a single dose resulted in 87–90% SRP, 85–86% SRC, and a nearly six-fold increase in GMT, meeting or exceeding licensing criteria. It is concluded that HNVAC is safe and immunogenic to adults of 18–65 years.

Several limitations of the use of embryonated eggs and the threat of pandemics have highlighted the need for other platforms for the production of influenza vaccines. We report the indigenous development and pre-clinical testing of an MDCK-based H1N1 pandemic influenza vaccine HNVAC from India. The cell bank and virus seed were characterized extensively. The cells were characterized by PCR, electron microscopy, and karyotyping, and found to be of female canine epithelial origin. The virus was confirmed by neutralization, haemagglutination inhibition, neuraminidase inhibition, and PCR and nucleotide sequencing. Adventitious agent testing was performed by both in vitro and in vivo studies. The in vitro studies included culturing, haemadsorption, haemagglutination, PCR and RT-PCR, whereas in vivo studies included passage in embryonated eggs and in laboratory animals. Both cell bank and virus seed were free of adventitious agents. MDCK cell lysates as well as cellular DNA did not produce tumours in newborn or adult laboratory animals. The bioprocess parameters were standardized to recover antigen with minimal levels of process-related impurities. The vaccine bulk was tested for the presence of specific antigen, and quantified by single radial immunodiffusion. Finally, non-adjuvanted and aluminium hydroxide adjuvanted vaccine formulations were found to be safe in preclinical toxicity studies in mice, rats, guinea pigs and rabbits, and immunogenic in mice and rabbits. This is the first and only cell culture-based influenza vaccine platform developed in any developing country.

Adjuvants in vaccines are immune stimulants that play an important role in the induction of effective and appropriate immune responses to vaccine component. In search of a potent vaccine adjuvant, the water-soluble biopolymeric fraction BOS 2000 from Boswellia serrata was evaluated for desired activity. We investigated the ability of BOS 2000 to enhance HBsAg specific immune responses. The effect was determined in the form of protective anti-HBsAg titers, neutralizing antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation by using MTT assay, Th1 (IFN-gamma and TNF-alpha) and Th2 (IL-4) cytokines as well as T-lymphocyte subsets (CD4/CD8) and intracellular cytokines (IFN-gamma/IL-4), these responses were highest in BOS 2000 immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of adjuvant by 18.1-fold in comparison to alum, total IgG and its subtypes (IgG1 and IgG2a) antibodies titer in serum was significantly enhanced. Analysis of HBsAg specific cytokines revealed that alum was associated with a predominantly IL-4 response. In contrast, BOS 2000 was associated with production of both IFN-gamma and IL-4. We conclude that BOS 2000 is a potent enhancer of antigen-specific Th1 and Th2 immune responses in comparison to alum with Th2 limitation and is a promising adjuvant for vaccine applications.

Alum has been in use since long as an adjuvant for vaccines. However, its use as a vaccine adjuvant offers limitation in supporting cell mediated response. Therefore, a new plant based product RLJ-NE-299A from Picrorhiza kurroa reported for its immunostimulatory activity, has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with alum, antigen-specific immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with RLJ-NE-299A and alum in mice. The adjuvant RLJ-NE-299A up-regulated remarkably the expression of Th1 cytokines IL-2, IL-12, IFN-gamma, TNF alpha and Th2 cytokine IL-4 in lymph node cell cultures after 2 weeks of primary immunization with HBsAg. Further, the levels of both immunoglobulins IgG2a (Th1) and IgG1 (Th2) subtypes increased profoundly in blood sera of mice immunized with HBsAg/RLJ-NE-299A. The results indicated that RLJ-NE-299A has strong potential to increase both cell mediated and humoral immune responses and is capable of sustaining the total antigen-specific antibody response. Besides, the RLJ-NE-299A provides a signal to gear up both CD4 helper cells (Th1 and Th2) and CD8 cells populations, which may have important implications for vaccination against hepatitis B virus. Variable doses of RLJ-NE-299A (0.312-40 microg) containing vaccine antigen (HBsAg) were well tolerated with optimum T cell response at 2.5 microg/ml. Not only this, the adjuvant was also able to induce cellular immune responses to HBsAg as evidenced by Th1 and Th2 cytokines upregulation, which enabled mice to overcome the unresponsiveness to antigen HBsAg encountered with alum-adjuvanted vaccine in otherwise non-responding mice population. The study presents evidence that the HPLC standardized fraction RLJ-NE-299A, is an adjuvant of choice over alum in improving and maintaining the improved immune status against HBsAg, and may also prove useful adjuvant candidate with other vaccine antigens, too.