Remarkable immunogenicity and protective e cacy of BBV152, an inactivated SARS-CoV-2 vaccine in rhesus macaques


 The COVID-19 pandemic is a global health crisis that has severely affected mankind and posed a great challenge to the public health system of affected countries. The availability of a safe and effective vaccine is the need of the hour to overcome this crisis. Here, we have developed and assessed the protective efficacy and immunogenicity of an inactivated SARS-CoV-2 vaccine (BBV152) in rhesus macaques (Macaca mulata). Twenty macaques were divided into four groups of five animals each. One group was administered a placebo while three groups were immunized with three different vaccine candidates at 0 and 14 days. All the macaques were challenged with SARS-CoV-2 fourteen days after the second dose. The protective response was observed with increasing SARS-CoV-2 specific IgG and neutralizing antibody titers from 3rd-week post-immunization. Viral clearance was observed from bronchoalveolar lavage fluid, nasal swab, throat swab, and lung tissues at 7 days post-infection in the vaccinated groups. No evidence of pneumonia was observed by histopathological examination in vaccinated groups, unlike the placebo group which showed features of interstitial pneumonia and localization of viral antigen in the alveolar epithelium and macrophages by immunohistochemistry. Data from this study substantiate the immunogenicity of the vaccine candidates and BBV152 is being evaluated in Phase I clinical trials in India (NCT04471519).


Introduction
The pandemic of coronavirus disease 2019 (COVID- 19) has caused an unprecedented public health burden in several countries across the globe 1 . The spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is has infected more than 23 million people until August 2020 2 . With all the public health measures in place, including behavioural modi cations such as the use of masks, hand sanitization; pharmaceutical interventions such as antiviral drugs or safe and effective vaccines seem to be the only means of stopping this raging pandemic. With the release of the rst genome sequence of SARS-CoV-2 from China on 11 January 2020, the race against the virus and time had begun for the development of an effective COVID-19 vaccine. Multiple vaccine development platforms from traditional to next-generation approaches are being used by different research groups worldwide. Puri ed inactivated viruses have been traditionally used in vaccine development. These inactivated vaccines are safe and effective in the prevention of diseases i.e., rabies, polio, hepatitis A, and in uenza 3 . Here, we report the assessment of immunogenicity and protective e cacy of three formulations of a puri ed whole-virion inactivated SARS-CoV-2 (BBV152) vaccine candidate in the rhesus macaques.
Viral load in the nasal swab, throat swab and bronchoalveolar lavage uid Genomic RNA (gRNA) was detected from nasal swab (NS) specimens of all animals in the placebo group from 1 to 7 DPI. Viral clearance was observed in NS specimens of all the animals from the vaccinated group on 7 DPI. (Figure 2A). Subgenomic RNA (sgRNA) was detected in two of ve animals at 3 DPI and one of ve animals at 7 DPI of the placebo group. sgRNA was detected in the NS sample of only one animal of the vaccinated group IV on 5 DPI (Figure 2A).
Throat swab (TS) specimens of the placebo group were tested positive for gRNA at 1, 3, 5, and 7 DPI. Vaccinated groups had a detectable level of gRNA from 1 to 5 DPI with viral clearance on 7 DPI ( Figure  2B). sgRNA was not detected in TS specimens of animals from either group. Bronchoalveolar lavage (BAL) uid specimens of the animals from the placebo group were positive for gRNA from 1 to 7 DPI. In the vaccinated groups, gRNA was detected in BAL specimens until 3 DPI ( Figure 2C). sgRNA was detected in BAL specimens of four out of ve animals of the placebo group, while it was not detected in BAL specimens of vaccinated groups. Except for the placebo group, none of the vaccinated groups showed the presence of gRNA in lung lobes ( Figure 2D). The comparisons of viral copy numbers of the NS, TS, and the BAL uid samples of the vaccinated as compared to the placebo group were found to be statistically signi cant using the two-tailed Mann-Whitney test.
Viral load in the respiratory tract, lungs, and extra-pulmonary organs On 7 DPI, animals from all the groups were sacri ced and swab samples, BAL, and various organs were collected. The animals of the placebo group at 7 DPI showed bronchopneumonic patches and consolidation in the lungs at necropsy ( Figure 3A and 3B). In the placebo group, gRNA was detected in the trachea (3/5), nasopharyngeal mucosa (2/5), oropharyngeal mucosa (3/5) and nasal mucosa (1/5) specimens ( Figure 3C). Four out of ve animals had detectable gRNA and sgRNA in multiple lobes of the lungs ( Figure 3D). Lung specimens of all animals from the vaccinated groups were found negative for gRNA and sgRNA. In the placebo group, gRNA was detected in skin, ileum, colon, gall bladder, stomach, urinary bladder, and pancreas. Only one animal from group IV showed the presence of gRNA in ileum and caecum. Heart, liver, kidney, spleen, and brain were tested negative for gRNA in all animals.
Clinico-radiological analysis Weight loss, pyrexia, and worsening of SpO2 at room air, lethargy, reduced food and water intake, reduced self-grooming was observed in the placebo group and persisted till 7 DPI whereas these features resolved in the other group II and IV (Supplementary Table 1). The chest radiograph of the three animals in the placebo group showed in ltrates, bronchopneumonia, or lobar pneumonia which persisted till 7 DPI. Similar chest radiographic abnormalities were detected in two out of ve animals in group II and IV, but resolved by 5 DPI (Extended Data Figure 1A-D). No clinical or radiographic abnormalities were detected in group III animals.

Histopathological examination and immunohistochemistry
Lung tissue showed mild to moderate interstitial pneumonia in the animals of the placebo group ( Table  2) characterized by thickening of alveolar septa, hyaline membrane formation, accumulation of edematous uid, and brin. Occasionally, certain foci of bronchioles showed necrosis and loss of epithelium with neutrophils and macrophage in ltration. Moderate to severe disease was present in 3 and mild disease in 2 animals of the placebo group with the involvement of four to six lung lobes. In group II, two animals had a single lobe of the lung affected (Table 3). Viral antigens were detected in the alveolar epithelium by immunohistochemistry (IHC) in the placebo group suggestive of SARS-CoV-2 infection.
These ndings indicate signi cant protection of the lungs from the vaccinated group as compared to the placebo group. Group II, III, and IV had signi cantly lower disease burden compared to the placebo group (  Table 3).
Virus isolation NS, TS, BAL uid, urine, stool, and lung tissue specimens (1 to 7 DPI) from placebo and vaccinated groups were processed for virus isolation. Cytopathic effect (CPE) was observed in TS and NS specimens of the placebo group on 1 and 3 DPI. BAL, urine, stool, and lungs specimens did not show CPE in any group. Two TS and one NS specimen of group II and IV respectively yielded virus isolation on 1 DPI.

Discussion
A safe and effective vaccine is the need of the hour to overcome the COVID-19 pandemic. In the global race for the development of vaccines, few research groups have reported the preclinical studies of viral vector vaccines (ChAdOx1 nCoV-19 and Ad-26.COV.2.S) 4, 5 , mRNA vaccine (mRNA-1273) 6 , DNA vaccine (INO-4800) 7 and inactivated vaccines (PiCoVacc and BBIBP-CorV) 8,9 . Phase I/II clinical trial are either completed or on-going for these vaccines. Here, we report the two-dose vaccination regimen of inactivated SARS-CoV-2 vaccine candidates found to induce a strong immune response and protection of animals from the infection of SARS-CoV-2.
Neutralizing antibodies and IgG responses were observed from 3 rd -week post-immunization in vaccinated groups. IgG titer rose in an increasing pattern with the highest response in group III. The presence of gRNA in NS was observed in the placebo group until 7 DPI. Vaccinated groups had no detectable gRNA in NS on 7 DPI indicating the ability of vaccine candidates to limit upper respiratory tract viral replication, which is a key factor determining the virus transmission. gRNA and sgRNA were not detected in the BAL uid from 5 DPI suggesting that vaccination hindered virus replication and enabled faster clearance from lower airway protecting the animals. gRNA was detected in multiple organs at necropsy in the placebo group, whereas it was found to be cleared in the vaccinated groups. An elevated level of IL-6 is highly consistent in COVID-19 disease and lymphopenia may be associated with a high level of IL-6 10, 11 . Our experimental data showed an elevated level of IL-6 in the placebo group compared to vaccinate groups (II and III) on 1 DPI, suggestive of protection against the SARS-CoV-2 infection. The transient lymphopenia in the placebo group could be suggestive of suboptimal cellular immunity in the placebo group or may be due to the recruitment of lymphocytes to the in amed respiratory tract 12,13 . Furthermore, lymphopenia is speci cally CD8+ T cells biased 12,14 , this possibly explains the elevated proportion of CD4+ T cells in the placebo group 12 . IL-5, a Th 2 cytokine, is associated with eosinophilia, and evidence of eosinophils for antiviral activity is documented 15. Our study demonstrated an elevated level of IL-5 in vaccinated group II compared to the placebo group. IL-8, a key chemokine, responsible for the recruitment of neutrophils and other immune cells was substantially high in vaccinated group III indicative of infection.
Organ viral load, interstitial pneumonia, and detection of viral antigen by IHC in lung tissue strengthen the evidence of SARS CoV-2 induced pulmonary disease in placebo animals. group III (3 µg, Adjuvant-A) animals had no clinical/radiological evidence of disease activity demonstrating signi cant protection from SARS-CoV-2. These ndings correlate well with protection from disease demonstrated by serological, gross pathological, and HPE ndings.
Altogether this study demonstrates that a two-dose vaccination regimen using 3µg dose of the vaccine candidate with adjuvant B induce a signi cant immune response and provide effective protection in animals challenged with SARS-CoV-2. Data from this study substantiate the immunogenicity of BBV152 which is being evaluated in Phase I clinical trials in India (NCT04471519). Generation of vaccine Bharat Biotech has established biosafety level 3 (BSL3) manufacturing facilities with a highly characterized and safe vero cell manufacturing platform that were readily deployed towards process development and large-scale manufacturing. Vero CCL-81 cells were initially grown in tissue culture asks and cell stacks using Dulbecco's Modi ed Eagle Medium (DMEM) (Sigma-Aldrich, India) containing 5-10% newborn calf serum (NBCS). Virus propagation was done in bioreactors at the temperature of 36 ± 1°C and was harvested at 36-72 h post-infection and supernatants were collected, clari ed, and aliquoted.

Study design and experiments on rhesus macaques
Rhesus macaques (Macaca mulatta) were housed in individual cages at the animal facility of ICMR-NIV, Pune. The animals were maintained on commercial pelleted feed, fruits, vegetables, and adlibitum potable drinking water with a 12h/12h dark/light cycle. All the animals were clinically evaluated for skin/systemic disorders, hemoglobin, total leukocyte count, differential leukocyte count, platelet count, packed cell volume, biochemical parameters (AST, ALT, bilirubin, serum proteins, alkaline phosphatase, LDH, BUN, creatinine, cholesterol, triglycerides, sodium, potassium, glucose), abdominal ultrasonography, chest X-ray, tuberculin test and were found t for the study. Animals were screened for Kyasanur forest disease virus and SARS-CoV-2 and IgG antibodies and found to be negative 17,18 . Biomedic data systems temperature transponder was implanted in the interscapular region subcutaneously for monitoring of body temperature during the study.
Twenty adult animals aged 3 -12 years were divided into 4 groups of ve animals (3 M, 2 F) each viz. the placebo (group I), group II, III, and IV. The placebo group was administered Phosphate buffer saline (PBS), group II, III, and IV were immunized with formulations of puri ed inactivated SARS-CoV-2 vaccine candidate 6μg+Adjuvant-A(BBV152C), 3μg+Adjuvant-B (BBV152A), and 6μg+Adjuvant-B (BBV152B) respectively. Animals were administered with two doses of vaccine/placebo on days 0 and 14 respectively intramuscularly in the deltoid region. Blood samples were collected on 0, 12, 19, 26, and 28 days for assessing the anti-SARS IgG antibody and NAb titers ( Figure 1A).
After completion of twenty eight-days of immunization, animals were shifted to animal biosafety level-4 facility. Animals were challenged with 1 ml of SARS-CoV-2 (P-3, NIV-2020770, TCID50 10 6.5 /ml) 19 intratracheally and 0.25 ml in each nostril. Animals were monitored twice daily and clinical scoring was performed based on parameters as listed in Supplementary Table 3. Clinical examination was done on 0, 1, 3, 5, and 7 DPI along with body temperature, body weight, pulse rate, and oxygen saturation at room air (Supplementary Table 3). NS, TS, rectal swab, chest X-ray, blood specimens, and BAL uid were collected on 0, 1, 3, 5, and 7 DPI. BAL uid collection was performed using a exible pediatric bronchoscope (Pentax Medical India Private Limited) under general anesthesia. The bronchoscope was inserted into the trachea and was guided through bronchus past the 3rd bifurcation; 5 ml of normal saline was instilled and aspirated from the lower/middle lobes of the lungs bilaterally. On 7 DPI, a detailed bronchoscopy and BAL uid collection from lobes of the lungs bilaterally were performed.
During necropsy, the following organs; brain, nasal mucosa, tonsil, nasopharynx, oropharynx, cervical lymph node, trachea, lungs, mediastinal lymph node, heart, spleen, liver, kidneys, urinary bladder, gastrointestinal tract and skin along with underlying deltoid muscle from the immunization site and cerebrospinal uid (CSF) were collected.
Enzyme-linked immunosorbent assay for detection of anti-SARS-CoV-2 IgG antibody Immunoplates (Maxisorp, Nunc) were coated with 100 μL/well of SARS CoV-2 antigen overnight at 4°C in the carbonate buffer. Subsequently, wells were blocked with liquid plate sealer (CANDOR Bioscience GmbH, Germany) for two hours at room temperature (25-30°C). One hundred μL of diluted rhesus macaque serum samples (1:100 in 1% bovine serum albumin in 1×PBS containing 0.1% Tween (PBST) were added to duplicate wells and incubated at 37°C for one hour. To each well, 100 μL of a 1:15000 dilution of goat anti-monkey IgG peroxidase-conjugated antibodies (Jackson ImmunoResearch, USA) was added wells and incubated at 37°C for one hour. The plates were washed 5 times with a wash buffer (1×PBST) post all incubations. One hundred microliters of (3,3'5,5'-Tetramethylbenzidine (TMB) substrate was added and incubated for 10 min. The reaction was stopped by 1N H2SO4 and absorbance was measured at 450 nm18. The sample was considered positive when the P/N ratio was more than 1.5. and optical density values with the SARS-CoV-2 antigen was above 0.2.

Plaque reduction neutralization test
The plaque reduction neutralization test (PRNT) was performed as described earlier 20 . A four-fold serial dilution of rhesus macaque's serum samples was mixed with an equal amount of virus suspension containing 50-60 plaque-forming units (PFU) in 0.1 ml. After incubating the mixtures at 37°C for 1 h, each virus-diluted serum sample (0.1 ml) was inoculated onto one well of a 24-well tissue culture plate containing a con uent monolayer of Vero CCL-81 cells. After incubating the plate at 37°C for 60 min, an overlay medium consisting of 2% carboxymethyl cellulose (CMC) with 2% fetal calf serum (FCS) in 2× MEM was added to the cell monolayer, and the plate was further incubated at 37°C in 5% CO 2 for 5 days.
Plates were stained with 1% amido black for an hour. Antibody titers were de ned as the highest serum dilution that resulted in >50 (PRNT50) reduction in the number of plaques.
Detection of SARS-CoV-2 genomic and subgenomic viral RNA The organs harvested during necropsy were uniformly weighed and homogenized (Tissue homogenizer, Qiagen, Germany) using 1 ml of sterile MEM (GIBCO, Thermo Fisher Scienti c, USA). Two hundred µl of each specimen (NS, TS, rectal swab, whole blood, BAL uid, urine, CSF, and tissue homogenates) was used for RNA extraction using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scienti c, USA). SARS-CoV-2 real-time RT-PCR was performed and a standard curve was plotted using in vitro transcribed RNA for E gene (gRNA) as described earlier 21,22 . Subgenomic RNA for the E gene was ampli ed using real-time RT-PCR from the above specimens using earlier published literature 23 .

Lymphocyte subset and cytokine/chemokine pro le
To analyze phenotype and proportion of T helper, T cytotoxic and B cells anti-coagulated (0.1ml) whole blood was surface-stained with appropriate uorochrome-conjugated antibodies along with their corresponding isotype controls. Two sets of sample tubes were prepared, one for T cell (CD3-FITC, CD8-PE, CD4-APC) and another for B cell (CD45-PerCP, CD3-FITC, CD20-PE). After incubation for 30 min at 4 0 C in dark, 2 ml of RBC lysing buffer were added to each tube, vortexed, and incubated at room temperature for 12 min. Two milliliters of washing solution was added to each tube. The sample tubes were centrifuged at 200 x g for 5 min. and the supernatant was carefully aspirated out. The cell pellets were suspended in 500μl wash buffer and vortexed. Υ, α Υ,α Virus isolation from clinical/necropsy specimens One hundred microliters of each specimen were inoculated onto 24-well Vero CCL-81 cell monolayers maintained in MEM (Gibco, UK), and incubated for 1 h at 37°C with rocking every 10 min. Subsequently, the media was removed and cells were washed with 1× PBS. Media with 2% FBS was added to each well and was incubated in a CO 2 incubator at 37°C for 5 days. The culture plate was examined daily for CPE using an inverted microscope (Nikon, Eclipse Ti, Japan) 19 . The cell culture supernatant from the wells showing CPE was further con rmed by real-time RT-PCR 21 .

Histopathological examination and Immunohistochemistry
Tissue sections from lungs were immersion-xed in 10% neutral buffered formalin. Tissue processing and embedding were performed by techniques described earlier 25 . Four micrometers thick tissue sections were used for hematoxylin and eosin staining. Anti-SARS-CoV-2 immunoreactivity in the tissues was assessed using mouse polyclonal serum. For IHC, mouse polyclonal serum was used as the primary antibody (1:500) and anti-mouse HRP antibody was used as a secondary antibody 25,17 .

Statistical analysis
Clinical, virological, hematological, biochemical, and immunological data were analyzed using GraphPad Prism software version 8.4.3 (GraphPad, San Diego, California) and Stata 14 software. (StataCorp LLC, USA). The clinical, virological, and serological data for the different groups were initially compared using the non-parametric Kruskal-Wallis test. The groups that were signi cant using the Kruskal-Wallis test were further assessed using the Mann-Whitney test. A group-wise comparison was performed to assess signi cance between the placebo and the other vaccinated groups using a two-tailed Mann-Whitney test.
The p-values less than 0.05 were considered signi cant and are marked on the gures. Non-signi cant values are not depicted in the gures. The log10 plots below the detection limits are depicted as log10 (1) for the illustration purpose. The detection limits are marked with the dotted lines on the respective gures.

Reporting summary
The nature research reporting summary provided with this paper contains the information on research design.

Data availability
All the data other than those presented in the article are provided in the form of supplementary les. Figure 1 Experimental summary and humoral response in vaccinated animals (A) Experiment summary of the work ow. Two doses of three different inactivated SARS-CoV-2 vaccine formulations were administered to the three groups of animals. Two doses of placebo were administered to the fourth group of the animals, which were the controls in the study. All the animals were challenged 14 days after the second dose. Samples were collected at different time point pre-challenge and post-infection (B) Anti-SARS-CoV-2

Figures
IgG response during a two-dose vaccine regime for four groups of animals observed from 1st to 4th week of immunization. (C) Anti-SARS-CoV-2 IgG response at post-infection (0, 1, 3, 5, and 7 DPI) for four groups of animals.. (D) Anti-SARS-CoV-2 IgG titers in animals at 7 DPI. (E) NAb titers in animals from 1st to 4th week of immunization. (F) NAb titers in animals at 0, 1, 3, 5, and 7 DPI. The statistical signi cance was assessed using the Kruskal-Wallis test followed by the two-tailed Mann-Whitney test between two groups; p-values of less than 0.05 were considered to be statistically signi cant. The dotted line in gure D indicates the limit of detection of the assay.  of respiratory tract tissues, (D) lungs tissue and (E) extrapulmonary organs at 7 DPI. The statistical signi cance was assessed using the Kruskal-Wallis test followed by the two-tailed Mann-Whitney test between the two groups; p-values less than 0.05 were considered to be statistically signi cant. The dotted lines indicate the limit of detection of the assay.