The study was approved by the Institutional Project Review Committee, and Institutional Biosafety Committee, ICMR-National Institute of Virology (NIV), Pune. The study was recommended by the Institutional Animal Ethics Committee (Registration 43/GO/ReBi/SL/99/CPCSEA) and further approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi letter No. V11011 (13)/7/2020-CPCSEA-DADF dated 08.06.2020. The permission from the Office of Principal Chief Conservator of Forests (PCCF), Maharashtra state, and Central India was obtained for capturing of Rhesus Macaques from the wild. The research was conducted in compliance with the guidelines laid down by CPCSEA, Government of India16.
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 flasks and cell stacks using Dulbecco's Modified 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, clarified, and aliquoted. The virus was inactivated with ß-propiolactone at a ratio of 1:2000 – 1:4000 at 2-8°C for 24-32 h. It was further purified by column chromatography and concentrated using a tangential flow filtration system. A total of three vaccine formulations were prepared, namely BBV152A (3µg+alum+ imidazoquinoline), BBV152B (6µg+alum+ imidazoquinoline), and BBV152C (6µg+alum). Imidazoquinoline are TLR7 / TLR8 agonists, which are highly immunostimulatory in nature.
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 ad-libitum 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 fit for the study. Animals were screened for Kyasanur forest disease virus and SARS-CoV-2 and IgG antibodies and found to be negative17, 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 five 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 purified 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 106.5/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 fluid were collected on 0, 1, 3, 5, and 7 DPI. BAL fluid collection was performed using a flexible 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 fluid 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 fluid (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 earlier20. 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 confluent 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% CO2 for 5 days. Plates were stained with 1% amido black for an hour. Antibody titers were defined 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 Scientific, USA). Two hundred µl of each specimen (NS, TS, rectal swab, whole blood, BAL fluid, urine, CSF, and tissue homogenates) was used for RNA extraction using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, 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 earlier21, 22. Subgenomic RNA for the E gene was amplified using real-time RT-PCR from the above specimens using earlier published literature23.
Lymphocyte subset and cytokine/chemokine profile
To analyze phenotype and proportion of T helper, T cytotoxic and B cells anti-coagulated (0.1ml) whole blood was surface-stained with appropriate fluorochrome-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 40C 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 CO2 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 confirmed by real-time RT-PCR21.
Histopathological examination and Immunohistochemistry
Tissue sections from lungs were immersion-fixed in 10% neutral buffered formalin. Tissue processing and embedding were performed by techniques described earlier25. 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 antibody25, 17.
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 significant using the Kruskal-Wallis test were further assessed using the Mann-Whitney test. A group-wise comparison was performed to assess significance between the placebo and the other vaccinated groups using a two-tailed Mann-Whitney test. The p-values less than 0.05 were considered significant and are marked on the figures. Non-significant values are not depicted in the figures. 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 figures.
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All the data other than those presented in the article are provided in the form of supplementary files.