Production of BBV154. The master and working virus banks of the BBV154 vaccine and the HEK cells were prepared in a GMP facility. Virus and cell banks were characterized for identity, safety and purity according to ICH guidelines. Infection of the HEK cells with the BBV154 produced a cytopathic effect (CPE) which included rounding of cells and lysis. Growth kinetics of the BBV154 in HEK cells was carried out with five different multiplicities of infection (MOI) ranging from 0.25 to 3 MOI. Based on the results of ChAd genome copies (Fig. 1a) and infectious titer (data not shown) estimations of the growth kinetics sample, an optimum MOI of 1 and a harvest time between 60–72 hours were selected. The downstream purification cascade as mentioned in the Methods section was followed to obtain a purified and formulated BBV154 vaccine candidate (Fig. 1b).
Characterization of BBV154. The BBV154 vaccine candidate was tested for the integrity of the expression cassette by conventional PCR. The primers flanking the expression cassette were used to amplify an expected product of 5889 base pairs (bp) from three different batches. A PCR product of 2069 bp was obtained with the ChAd vector control (Fig. 1c).
The spike expression by BBV154 was accessed by infection of HEK cells and the cell lysates were subjected to western blotting with antibody against the receptor binding domain (RBD) of spike protein (Fig. 1d). The RBD antibody detected the full-length spike in the cell lysates derived from BBV154 infected cells (Fig. 1d, lanes 2–4) and the spike protein size corresponds to that of the positive control (Fig. 1d, lane 5). The spike expression of BBV154 was also demonstrated by immunocytochemistry, spike expression products being visualized as distinct spots (Fig. 1e and 1f).
In order to test for the presence of replication competent adenovirus (RCA), BBV154 was passaged in A549 cells that do not have complement E1. As expected, CPE was not observed even after three consecutive passages indicating absence of RCA. To exclude the possibility of inhibition of the RCA in the BBV154 sample, BBV154 spiked with wild type Ad5 (1 or 10 TCID) displayed CPE with an amplification between 109 to 1010 TCID50 (Table 1).
Table 1
Test for detection of RCA in the BBV154 sample
Sample Details
|
Number of passages in A549 cells
|
TCID50 assay performed on
|
Virus titers after three passages (TCID50)
|
BBV154 sample alone*
|
3
|
A549 cells
|
No CPE
|
BBV154 sample spiked with 1 TCID of Ad5
|
1010
|
BBV154 sample spiked with 10 TCID of Ad5
|
1010.35
|
Ad5 alone 10 TCID
|
1011
|
BBV154 sample alone
|
HEK cells
|
No CPE
|
* Hexon copies: 10.58 log copies/ mL; Spike copies: 10.18 log copies/ mL; virus particle by A260: 2.82 x 1012 virus particle/ mL. |
Safety evaluation of BBV154 formulation. An extensive safety evaluation for the vaccine candidate BBV154 formulation was performed as per the regulatory guidelines20,21. Safety of BBV154 was assessed by repeated dose toxicity studies conducted in both rodent (Swiss albino mice, BALB/c mice and Wistar rats) and non-rodent (New Zealand White rabbits) species, after the administration of N + 1 dose regimen. No mortality, systemic toxicity or clinical signs was observed in any of the animal models throughout the tested experimental period. Body weight gain (Supplementary Fig. 1), food consumption and body temperature of the animals were within the normal range. Clinical chemistry parameters such as hematology, clinical biochemistry, urinalysis, coagulation analysis and acute phase protein values did not indicate any variation in the vaccine-treated animals from placebo-treated animals even at the highest dose tested (5 x 1011 VP/animal). Further, the coagulation factors leading to thrombosis or thrombocytopenia, platelet count, prothrombin time and activated partial thromboplastin time (APTT) were well within the normal range and comparable with the concurrent control group and were unaffected by BBV 154 (Supplementary Tables 2 & 3). The skin around the nose did not show any local reactogenicity such as erythema, edema or eschar formation on gross observation. Absolute and relative (to body weight) organ weights were comparable to placebo animals. Further extensive histopathological evaluation, following Registry of Industrial Toxicology Animal-data (RITA) guidance, of three levels of nasal cavity (Supplementary Fig. 2) and related organs such as larynx, trachea, lungs or associated lymph nodes (Supplementary Fig. 3) did not reveal any abnormalities in microscopic observations.
Immunogenicity of single dose vaccination of BBV154. Immunogenicity of BBV154 candidate vaccine was evaluated in both inbred (BALB/c) and outbred (Swiss albino) mice. Humoral and cellular immune responses were assessed in mice two-weeks post vaccination with either one-tenth, one quarter or half (1x1010, 2.5x1010 or 5x1010 VP respectively) of a human single dose (HSD). Single dose vaccination of BBV154 elicited systemic and mucosal immune response against SARS-CoV-2 spike as early as 14 days post vaccination (Supplementary Fig. 4a). On day 21 after vaccination, significant spike-specific systemic IgG and IgA responses were detected in most of the vaccinated animals, with comparable spike-specific IgG immune responses in BALB/c and Swiss Albino sera (Supplementary Fig. 4b). Mice vaccinated with 5 × 1010 VP had higher spike-specific antibody titers than those given 1×1010 VP, demonstrating a dose-dependent response (Fig. 2b & 2c). Additionally, substantial increases in serum IgG and IgA levels were observed on day 56 compared with day 21. Spike-specific antibodies were persistent and still measurable on day 70, ten weeks after vaccination, demonstrating durable immunity. IN immunization is known to stimulate mucosal IgA antibodies, providing a first line of defence against respiratory pathogens, and IN vaccination with BBV154 induced S1-specific IgG and IgA responses in the bronchoalveolar lavage (BAL) fluids (Fig. 2e). As with the systemic IgG and IgA, dose-dependent pulmonary antibody responses were observed.
Vaccine-induced neutralizing antibody responses against SARS-CoV-2 NIV-2020-770 (containing the D614G mutation)22 were assessed using a live virus microneutralization test (MNT50). Consistent with the spike-specific antibody responses, immune sera from BBV154 vaccinated mice neutralized SARS-CoV-2. Responses were dose-dependent: mice vaccinated with 5×1010 VP had higher neutralizing antibody titers (GMT = 264), than those given 2.5×1010 VP (GMT = 76) which in turn were higher than those given 1×1010 VP (GMT = 45) (Fig. 2e). Notably, these neutralizing antibody GMTs remained unchanged even after 56 days post vaccination. Further, the assessment of SARS-CoV-2 neutralizing antibodies in bronchoalveolar lavage (BAL) fluid of BBV154 immunized mice also displayed SARS-CoV-2 neutralizing antibodies in dose-dependent manner (Fig. 2f). As expected, neither serum nor BAL fluid from placebo-treated mice exhibited any SARS-CoV-2 neutralizing activity. Further, the level of the neutralizing antibody response was well-correlated with S1-specific systemic IgG levels quantified in individual animals (Pearson r2 = 0.7504, Fig. 2g), signifying that robust antibody responses to spike protein were allied with generation of potentially protective neutralizing antibodies.
Having observed a robust antibody response in vaccinated mice, we next examined the cell mediated immune (CMI) response activated via IN immunization. Ex vivo re-stimulation of splenocytes of vaccinated animals with S1 protein resulted in a significant induction of Th1 associated IFN-γ or TNF-α cytokines (Fig. 2h). Moreover, T-cells from the BBV154 immune animals produced low levels of IL-10 and IL-4 when compared with T-cells from the placebo-treated mice. This indicates that IN vaccination of BBV154 did not initiate a Th2 response but rather induced the expected antiviral T-cell responses.
Repeated-dose IN immunization of BBV154 elicits anti-spike humoral response. The immunogenicity and tolerability of clinical batch samples of BBV154 vaccine were evaluated in mice, Wistar rats and New Zealand White rabbits with a full human dose (N + 1) regimen. Repeated doses of different concentrations of candidate vaccine (5x109 VP [low-dose], 5x1010 VP [medium-dose], or 5x1011 VP [high-dose] per animal) were administered IN on days 0, 21 and 28. Serum samples were collected 21 days post-primary or pre-prime booster immunization and spike-specific IgG and IgA responses were evaluated by ELISA. Mice, rats and rabbits immunized with high- and medium-doses of vaccine elicited significantly higher IgG and IgA responses against purified S1 antigen than in the low-dose group (Fig. 3b-3e). Consistent with the spike binding antibody response, BBV154 vaccination elicited substantial increases in the SARS-CoV-2 specific neutralizing antibodies in mice, rats and rabbits (Fig. 3f & 3g). Notably, boosting enhanced serum neutralization activity two- to four-fold in high-dose group (5x1011 VP) animals, with insignificant increases in rabbit immune sera (Table 2), whereas no neutralizing antibodies were detected in sera from placebo-treated animals after primary immunization or boosting.
Table 2
SARS-CoV-2 neutralizing antibody responses in serum following single or double intranasal administration of candidate vaccine BBV154
Animal Model
|
Dose (VP/animal)
|
Neutralizing Antibody Titers (MNT50)
(mean)
|
Prime
|
Booster
|
BALB/c
|
Placebo
|
7.6
|
6.7
|
5x109
|
7.8
|
45.8
|
5x1010
|
76.6
|
120.9
|
5x1011
|
264.8
|
454.1
|
Swiss Albino
|
Placebo
|
8.6
|
7.2
|
5x109
|
13.93
|
111.87
|
5x1010
|
91.25
|
359.5
|
5x1011
|
325.15
|
741.11
|
Wistar Rats
|
Placebo
|
5.66
|
7.44
|
5x109
|
17.3
|
16.7
|
5x1010
|
158.7
|
128.25
|
5x1011
|
278.3
|
545.2
|
New Zealand Rabbit*
|
Placebo
|
5.6
|
5.6
|
5x109
|
5.6
|
5.6
|
5x1010
|
12.36
|
9.46
|
5x1011
|
23.9
|
26.5
|
Each group consisting of 10–12 animals; * Consisting of 4–6 animals |
We then assessed the levels of anti-vector (ChAd36)-specific neutralizing antibodies in vaccinated animals. In line with a previous study23, most of the animals did not produce the ChAd36 neutralizing antibodies, only 3 out of 12 vaccinated rat sera appearing to have low titers of anti-ChAd36 antibodies (Fig. 4a). However, the immune sera derived from the same animals displayed significantly higher levels of SARS-CoV-2 virus neutralization activity compared with pre-immune sera (Fig. 4b). Absent or insignificant titers of vector (ChAd36)-specific neutralizing antibodies following three doses of BBV154 implies that IN administration may offer an advantage for repeat vaccination using adenovirus-vectored vaccines.
BBV154 immunogenicity in the young and aged hamsters. Aging is accompanied by changes in the immune system, particularly in adaptive immune responses which are diminished in the aged individuals24. We explored age-dependent differences in the immunogenicity of BBV154 candidate vaccine in young (9–11 weeks) or aged (28–36 weeks) Syrian Hamsters. Consistent with earlier preclinical studies by Bricker et al18, following vaccination significant spike-specific systemic and mucosal IgM and IgG responses were detected in most of the vaccinated hamsters. Further, BBV154-immunized young animals showed a moderate but significant increase in levels of spike-specific IgG compared with older animals (Fig. 5b), whereas both young and old animals presented comparable mucosal IgG responses (Fig. 5c). BBV154 vaccination in young and old animals elicited a lower IgG1 antibody response than IgG2, indicating Th1 phenotype, with IgG2a/IgG1 ratios greater than 1 (Fig. 5d). Due to non-availability of commercial Anti-hamster-IgA secondary antibodies, IgA responses were not analysed. However, we would anticipate finding the IgA response in hamsters to be similar to other animals that were vaccinated with BBV154. As with spike-specific binding antibodies, BBV154 immune sera from young animals had much higher levels of SARS-CoV-2 neutralizing activity than immune sera from old animals (Fig. 5e). Levels of neutralizing antibodies in saliva were found to be comparable in young and old animals (Fig. 5f).
Systemic prime-intranasal boost strategy augments BBV154 vaccine efficacy. Effective vaccination strategies need not be restricted to one route of administration alone; several vaccine studies have demonstrated that memory cells primed by parenteral vaccination can be “pulled” into mucosal sites by successive mucosal vaccination25− 27. To test this, a group of naive BALB/c mice were primed by IM vaccination with BBV154 on day 0 followed by an IN booster on day 28, and the immune responses were compared with mice which received two IN BBV154 vaccinations. The IM prime-IN booster vaccination induced similar levels of S1-specific IgG and IgA and slightly increased neutralizing antibody titers compared with the IN-prime and IN-boost mice (Fig. 6b-d). To test this further, a heterologous vaccination study was conducted in rabbits; groups of rabbits were primed IM with COVAXIN® (Whole-Virion Inactivated vaccine) followed by IN BBV154 booster and the resulting immune responses were compared with homologous COVAXIN IM priming and boosting. The BBV154 IN booster in COVAXIN-primed rabbits elicited significantly high levels of spike-specific IgG and IgA titers, compared with the homologous COVAXIN/COVAXIN immune model (Fig. 6f-6i). Further, COVAXIN/BBV154 heterologous immune sera showed two-fold higher SARS-CoV-2 neutralizing titers than immune sera derived from rabbits given the COVAXIN/COVAXIN homologous regimen (Fig. 6j and 6k). Emergence of SARS-CoV-2 VOC has raised concerns about the breadth and durability of neutralising antibody responses28. To address this, heterologous immune sera was subjected SARS-CoV-2 variants inhibition studies. Heterologous boosting with the BBV154 vaccine followed by COVAXIN priming showed an IC50 (binding inhibition by 50%) of 584.7; 305.5 and 5.75.44 against B.1, Delta, and omicron, respectively (Fig. 6l).