key: cord-1046965-w0oi9tcw
authors: Ganneru, Brunda; Jogdand, Harsh; Daram, Vijaya Kumar; Das, Dipankar; Molugu, Narasimha Reddy; Prasad, Sai D.; Kannappa, Srinivas V.; Ella, Krishna M.; Ravikrishnan, Rajaram; Awasthi, Amit; Jose, Jomy; Rao, Panduranga; Kumar, Deepak; Ella, Raches; Abraham, Priya; Yadav, Pragya; Sapkal, Gajanan N.; Shete, Anita; Desphande, Gururaj Rao; Mohandas, Sreelekshmy; Basu, Atanu; Gupta, Nivedita; Vadrevu, Krishna Mohan
title: Th1 Skewed immune response of Whole Virion Inactivated SARS CoV 2 Vaccine and its safety evaluation
date: 2021-03-10
journal: iScience
DOI: 10.1016/j.isci.2021.102298
sha: af4b1086ad35e7e76320ce4b410ed79f8c30071a
doc_id: 1046965
cord_uid: w0oi9tcw

We report the development and evaluation of safety and immunogenicity of a whole virion inactivated (WVI) SARS-CoV-2 vaccine (BBV152), adjuvanted with aluminium 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 was 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 containing 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.

INTRODUCTION 47 study (RDT, In-vivo) , to evaluate that repeated administration of Algel-IMDG does not cause any 157 systemic toxicity or mortality. Mutagenicity assay performed with Algel-IMDG at various 158 concentrations revealed that there was no substantial increase in revertant colony numbers in any 159 of the tested strains at any dose level, in both the plate-incorporation and pre-incubation methods 160 in the presence or absence of metabolic activation (S9 mix) (Table S1) . Thus, the Algel-IMDG used 161 in the BBV152 A and B adjuvanted vaccine formulations were found to be non-mutagenic. Further, 162 maximum tolerated dose study performed with single dose of Algel-IMDG also revealed that the 163 Algel-IMDG was tolerated at the tested dose (20 µg agonist/animal) in mice and rats as 164 demonstrated by lack of erythema, edema, or any other macroscopic lesions at the site of injection 165 (Table S2) . 166

Moreover, repeated administration of (N+1 dose regimen) high dose of either Algel-IMDG alone 167 (30µg agonist/animal) in Swiss Albino mice and Wistar rats or high dose of adjuvanted vaccine 168 formulation (9µg Ag with30µg agonist /animal, which is more than HSD) in Wistar rats did not 169

show any clinical illness, change in body weight (Figure S1 ), or histopathological changes, except 170 inflammation at the site of injection ( Figure S3 ) and thus establishing the safety of both Algel-IMDG 171 and adjuvanted vaccine formulations at high dose. 172

Further, the safety of adjuvanted vaccine formulations (BBV152 A, B and C) at full HSD, were also 173 evaluated to be safe in three animal models (BALB/c mice, S. albino mice, and NZW rabbits), as 174 demonstrated by the repeated dose toxicity study with no mortality and with no changes in clinical 175 signs, body weight gain, body temperature, or feed consumption in any of the animals . 176

Representative data of body temperature as a parameter are shown in Table S3 . 177

Clinical pathological parameters such as haematology, clinical biochemistry, coagulation studies, and 178 urinalysis performed in repeated dose toxicity (RDT) studies, showed that the animals administered 179 either with adjuvanted vaccine candidates or adjuvants/antigen-alone were comparable to control 180 ( Figure S2, Table S4 ), except increased levels of Alpha 1-acid glycoprotein and neutrophils count on 181

Day 2 in adjuvant-alone or adjuvanted vaccine formulation groups. However, these values were 182 comparable to control on Day 21. This transient increase may be due to inflammation at the 183 injection site after administration of the first dose. These findings were further correlated with the 184 inflammatory reaction at the injection site observed microscopically, in the animals administered 185 with adjuvant-alone and adjuvanted vaccine with Algel and Algel-IMDG. This inflammation was 186 found to be slightly higher in animals that received Algel-IMDG than in animals which received Algel. 187 However, this inflammation reduced by day 28 (Figure S3 and S4) . Other than local reaction at the 188 site of injection, no other treatment-related microscopic findings observed in any of the animals 189 administered with antigen or adjuvant or adjuvanted vaccine formulations. Histopathological 190 examination of organs such as spleen, lungs, heart and lymph nodes etc., of all animal models 191 administered with antigen or adjuvant or adjuvanted vaccine formulations were normal ( Figure S5  192 and S6). 193

We report immunogenicity of three BBV152 formulations in BALB/c mice and New Zealand white 195 rabbits. BALB/c mice were administered either with full or 1/10 th or 1/20 th HSD, whereas Rabbits 196 were immunized with intended HSD. antigen-or adjuvants-alone at 1/20 th of the intended HSD (i.e., 1/20th of 3µg, 6µg, and 9 µg/mouse 201 or 0.15µg, 0.3 µg, and 0.45µg/mouse), to determine the optimal dose. ELISA titers (Figure 2A) and 202

NAb titers ( Figure 2B ) determined at various time points revealed that immune response elicited 203

against these adjuvanted vaccine formulations tested at three antigen concentrations elicited high 204 levels of binding and NAb titer (Figure 2A and B) . Antibody response determined on day 7 was found 205 to be less (10 2 titer) robust or negligible compared to Day 14 & Day 21 with a titer of 10 3 and 10 4 , 206 respectively. Notably, 3 and 6µg formulations induced high spike specific antibody titers compared 207 to 9µg group. Hence, adjuvanted formulation with high antigen dose (9µg) was eliminated in further 208 studies of the immunogenicity, whereas safety was evaluated in Wistar rats at this high dose. These 209 results also indicated that adjuvanted vaccine formulations either with Algel or Algel-IMDG elicited 210 high Spike (S1) specific antibody binding titers compared to antigen alone tested at all three 211 concentrations (Figure 2A) . This was further evaluated by administering BALB/c mice intramuscularly 212 with antigen at actual HSD (6µg Ag) either in the presence or absence of adjuvant (Algel-IMDG) and 213 compared with 1/10 th HSD of adjuvanted vaccine formulation (0.6µg Ag and Algel-IMDG). Immune 214 response elicited against adjuvanted vaccine formulation (BBV152B) was significantly (1 log) higher 215 than the antigen alone (6µg Ag), which is comparable with 1/10 th HSD of adjuvanted vaccine 216 formulation (1/10 th of BBV152B). These results suggest the dose sparing effect of Algel-IMDG ( with Algel and Algel-IMDG at 1/10 th human intended single dose (0.3, and 0.6 μg/dose with Algel or 221 J o u r n a l P r e -p r o o f 2D). Further, sera collected on Day 21 were analyzed by ELISA to determine S1, RBD, and N specific 223 binding titer ( Figure 2E ) and shown 100% seroconversion with S1, RBD & N protein. Analysis of 224 Plaque Reduction Neutralization Test (PRNT 90 ) , performed with individual mice sera, showed high 225

NAb's in all adjuvanted vaccines ( Figure 2F ). 226

We also compared different immunization dose schedules (Day 7 vs Day 14), where-in BALB/c mice 227 were administered intramuscularly with adjuvanted vaccine (full HSD), with one group receiving 2 nd 228 dose on 7-day and the other group on 14-day post initial immunization. Our results indicated 8-fold 229 increase in spike-protein specific antibody titer, when booster dose was given with 14 day interval as 230 compared to that given on 7-day ( Figure 2G) . Figure 2H) . Similarly, we also found 236 sustained NAb titers up to Day 98 ( Figure 2I) , which indicates the BBV152 vaccine candidates were 237 able to produce long-term immunity. 238

To assess the immunogenicity of adjuvanted vaccine formulations at full Human Single Dose (HSD, (3 240 and 6 μg antigen/dose), Rabbits (n=4) were immunized intramuscularly, on days 0, 7, and 14. Similar 241 to mice, immune response in rabbits also found to be time dependant and not all animals were 242 seroconverted on day 7 and showed less antibody binding titer (≥10 2 ). However, on day 21, we 243 found 100% seroconversion with spike specific antibody binding titer of ≥10 4 . All three formulations 244 (BBV152A, Band C) showed high binding antibody response ( Figure 3A) , with no statistically 245 significant difference. Similarly, PRNT 90 results showed high neutralizing antibody titers in all three 246 adjuvanted vaccine formulations on day 21 ( Figure 3B ). However, there is no significant difference 247 and similar results were also observed by MNT 50 titers ( Figure 3C ). Further, NAb titers determined by 248 MNT 50 were slightly higher or comparable with NAb titers of human convalescent sera collected 249 from recovered symptomatic COVID-19 patients ( Figure 3C) . 250

Immunoglobulin subclasses (IgG1, IgG2a and IgG3) were analyzed on day 14 hyperimmunized 252 BALB/c mouse sera samples to evaluate the Th1/Th2 polarization. The average ratio of IgG2a/IgG1 or 253 ( Figure 4A ). Antigen alone showed Th1 biased response at three tested different concentrations with 255 an average Th1:Th2 index of 3, however, ELISA & PRNT 90 titers are less compared to Adjuvanted 256 vaccine formulations. Similarly, the same sera, when measured for interferon-γ (IFNγ) by ELISA,6µg 257

Algel-IMDG samples induced significantly higher responses of IFNγ compared to Algel ( Figure 4B) . 258

Additionally, expression of other cytokines such as Interleukin (IL)-2, IL-4, IL-6, Tumor Necrosis Factor 259 (TNF)-α, IL-17A, IL-10 including IFNγ, were noticeably higher in the 6µg Algel-IMDG when compared 260 to 6µg Algel ( Figure 4D ), especially on day 7 and 14 hyperimmunized sera. 261

To further evaluate Th1 skewed immune response induced by Algel-IMDG, intracellular staining was 262 performed using vaccinated mice splenocytes after stimulation with inactivated SARS-CoV-2 antigen 263 and determined IFNγ producing T lymphocytes. Interestingly, we found that the adjuvanted 264 formulation BBV152A (0.3µg Ag+Algel-IMDG) showed elevated levels of IFNγ producing CD4 cell 265 population, whereas, BBV152B formulation (0.6µg Ag+Algel-IMDG) induced comparable levels of 266

IFNγ producing CD4 + T cells to the BBV152C (0.6Ag+Algel) ( Figure 4C ). However, there is no 267 significant difference among the three groups. Similarly, BBV152A formulation (0.3µg Ag+Algel-268 IMDG) also induced higher IFNγ production in CD3 + and CD8 + T cells compared to other two 269 formulations. 270

To assess the effect of adjuvants (Algel or Algel-IMDG) on immune response and understanding the 271 critical role of IMDG in eliciting IFNα (which induces robust antibody and Th1 response), we 272 stimulated PBMCs from healthy volunteers with Adjuvants alone and adjuvanted vaccines for 36-273 72hrs at 2-fold dilutions of BBV152A, B and C and measured IFNα in the cell supernatant. We found 274 that Algel-IMDG containing TLR7/8 agonists alone stimulated IFN-α, but not the Algel. Additionally, 275

Adjuvanted vaccine formulations with Algel-IMDG induced elevated levels of IFN-α compared to 276

Algel formulation, demonstrating the enhanced activation of immune system by the Algel-IMDG 277 group compared to Algel ( Figure 4E) . Here, we report the development of a whole virion inactivated SARS-CoV-2 vaccine candidates 281 (BBV152A-C). The strain (NIV-2020-770) , which belongs to G clade used for this vaccine candidate is 282 pathogenic in humans, which is the current predominant circulating strain all over the world. It is 283 also observed that the D614G mutation increases viral neutralization (Sarkale et al., 2020) . This 284 strain also showed extensive genetic stability and appropriate growth characteristics and thus we 285 chose NIV-2020-770 strain for further vaccine development. show depot formation at the site of injection, which helps the antigen for slow release (Gupta and 292 Gupta, 2020) . The microscopic findings at the site of injection in the present study showed the 293 infiltration of macrophages and mononuclear cells indicates the activation of innate immunity. The 294 other adjuvant namely Algel-IMDG, containing TLR7/8 agonist, induced slightly higher reactogenicity. 295

Intra-muscular injection induces a depot effect followed by the passive trafficking of Algel particles 296 via lymphatic flow from the interstitial space to the draining lymph nodes, as revealed by IFN-297 β/luciferase reporter mice (unpublished data from Dr. Sunil David, ViroVax, LLC, KS, USA). The lymph 298 node-targeting of Algel-IMDG ensures high adjuvant activity in the target organ (lymph nodes) by 299 enabling the induction of a strong, specific, adaptive immune response while minimizing systemic 300 exposure. Further, Algel-IMDG did not show mutagenicity in the five strains of Salmonella 301 typhimurium tested. The local reaction in the studies conducted was consistent with those available 302 in the literature for these adjuvants, which is a physiological reaction to activate immune system 303 rather than any adverse event Sellers et al., 2020 ) . circulating worldwide . Our potency results are quite favourably comparable with 314 those reported in the literature for similar COVID-19 vaccines . 315

Further, in our preclinical studies, we demonstrated that all the three inactivated whole virion SARS-316

CoV-2 vaccine candidates showed 100% seroconversion with high titers of antigen binding and 317 neutralizing antibody responses. Further, the adjuvanted IMDG formulation (BBV152B), showed 318 more than 10 times higher antibody response, compared to antigen-alone (Figure 2C) , thus Algel-319 IMDG formulation provides dose sparing effect. Moreover, these formulations induced immunity 320 that is biased towards Th1 mediated response, as demonstrated by the ratio between IgG2a and 321 IgG1 (greater than 1) ( Figure 4A) . Additionally, secretion of anti-viral cytokines such as IL-2, IL-4, IL-6, 322 IL-10, IL-17, TNF-alpha, IFNγ observed on days 7 and 14 (7 days after the 1 st and 2 nd dose) ( Figure 4D ) 323 andhigher induction of IFN-alpha ( Figure 4E ) in Algel-IMDG adjuvanted formulations might have 324 contributed to enhance activation of antigen-presenting cells, such as dendritic cells or 325 macrophages. However, mechanism of action of Algel-IMDG in the induction of Th1 biased response 326 is yet to be investigated. These results further supported by our Hamster and non-human primate 327 animal challenge study, wherein Algel-IMDG adjuvanted formulations provided early protection 328 compared to Algel formulation, with the significant reduction in the viral load ( Mohandas et al., 329 2021; Yadav et al., 2020 ) . It is also reported earlier that TLR recognition in innate cell population 330 drives early type I IFN production, thereby promotes viral clearance and the early production of 331 proinflammatory cytokines (Hijano et al., 2019;  Stephens and Varga, 2020) 332

Though major research is focussed on Spike as the target protein for SARS CoV-2 vaccine 333 development, there is some attention being paid towards Nucleocapsid protein as a target protein, 334 due its 90% amino acid homology and stability with fewer mutations over time (Dutta et al., 2020; 335 Grifoni et al., 2020) . Thus, it is predicted that vaccine strategies with conserved epitope regions 336 could generate cross protective immunity across Beta coronaviruses. Le Bert et al., 2020 showed the 337 presence T cell responses against the structural (nucleocapsid (N) protein) and non-structural (NSP7 338 and NSP13 of ORF1) regions of SARS-CoV-2 in individuals convalescing from coronavirus disease 339 2019 (COVID-19) (Duan et al., 2020; Le Bert et al., 2020) . 340

Further recent research findings based on bioinformatic analysis of epitope mapping revealed that 341 nucleocapsid protein composed of both T and B cell immunodominant epitopes (Chen et al., 342 2020; Sarkar et al., 2020 ) . Earlier, animal studies conducted using DNA vaccine against SARS CoV 343 shown that the nucleocapsid is able to produce enhanced antigen specific humoral and cellular 344 immune responses (Chunling et al., 2006; Zhao et al., 2005) . It is also to be noted that, though, the 345 J o u r n a l P r e -p r o o f earlier immunization studies performed in animal models against nucleocapsid protein reported to 346 cause pneumonia (Deming et al., 2006; Yasui et al., 2008) yet, there is no much established research 347 evidence is so far, on the pathogenicity of nucleoprotein in humans. 348

In conclusion, we believe that, the ability to induce Th1 skewed immune response and the presence 349 of conserved S and N protein in inactivated vaccine candidate formulated in Algel-IMDG would help 350 to combat other SARS CoV-2 variants. 351

In our findings, we also observed high binding titers with a 100% seroconversion towards S1, RBD, 352 and N protein. 

after Inactivation (i) Line graph represents Virus titer (10 6 -10 7 ) measured by CCID 50 at every 3 hours 387

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