Immunogenicity and protective ecacy of BBV152: a whole virion inactivated SARS CoV-2 vaccine in the Syrian hamster model

The availability of a safe and effective vaccine would be the eventual measure to deal with SARS-CoV-2 threat. Here, we have developed and assessed the immunogenicity and protective ecacy of an inactivated SARS-CoV-2 vaccine (BBV152) in hamsters. Three dose vaccination regime with three formulations of BBV152 induced signicant titres of SARS-CoV-2 specic IgG and neutralizing antibodies. The formulation with imidazoquinoline adsorbed on alum adjuvant remarkably generated a quick and robust immune response. Th 1 biased immune response was demonstrated by the detection of IgG2 antibodies. Post-SARS-CoV-2 infection, vaccinated hamsters did not show any histopathological changes in the lungs. The protection of the hamsters 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 ndings conrm the immunogenic potential of BBV152 and further protection of hamsters challenged with SARS-CoV-2.


Introduction
Since the rst report in December 2019, severe acute respiratory syndrome coronavirus-2 (SARS-  has spread at an alarming rate and has infected more than 24 million people until August 31 st 2020 (World Health Organization, 2020). Globally, scienti c communities are actively engaged and trying to develop suitable vaccine candidates and speci c antiviral therapies against this virus. Vaccination is the most signi cant pharmaceutical intervention for the prevention of any infectious disease impacting the health of communities worldwide. Accelerated efforts are being taken for the development of a safe and Currently along with the conventional vaccine development platforms, advanced technologies are being used for the development of vaccines against SARS-CoV-2 like messenger RNA, DNA, viral vectors, recombinant subunit proteins, virus-like particles etc (Pandey et al., 2020). Despite the advances in vaccine design technologies, the development of an inactivated vaccine remains the most simple and relatively less expensive approach to produce a safe and effective vaccine. Inactivated vaccines have been used effectively to curb many infectious diseases in the past (Sanders et al., 2015). Recently, two inactivated SARS-CoV-2 vaccine candidates have shown promising results in preclinical trials (Gao et al., 2020, Wang et al., 2020). Among various small laboratory animal models, Syrian hamsters was found to be a suitable model for SARS-CoV-2 research, as the virus has been shown to replicate both in the upper and lower respiratory tract (Mohandas et al., 2020). The pulmonary pathology and lung viral load coincides with weight loss in hamsters during the rst week of infection , and can be used for the assessment of protective e cacy of vaccine candidates. This animal model has been successfully used to evaluate medical countermeasures for SARS-CoV-2 by multiple research groups (Luan et al., 2020, Imai et al., 2020. We have developed a whole virion inactivated vaccine candidate (BBV152) using β-propiolactone (BPL) inactivation method. The vaccine candidate along with aluminum hydroxide adjuvant alone or with aluminum hydroxide chemisorbed with imidazoquinoline was found to be immunogenic and safe in the preclinical studies on laboratory mice, rats and rabbits. Here, we report the immunization of Syrian hamsters with the vaccine candidate (BBV152) and evaluation of protective e cacy against SARS-CoV-2 by carrying out virus challenge experiment.

Optimization of SARS-CoV-2 challenge dose in Syrian hamsters
For assessment of the SARS-CoV-2 challenge dose after immunization a 10 fold serial dilutions of 10 5.5 Median Tissue Culture Infectious Dose (TCID50) were used. One hundred microliter from each of the 5 dilutions (10 5.5 , 10 4.4 ,10 3.5 , 10 2.5 , and 10 1.5 ) was inoculated intranasally in 5 groups of 6 hamsters each. On 3 day post infection (DPI), 3 hamsters each from all the 5 groups were sacri ced and the lung viral titres were measured. Virus load was found to be similar in all the hamsters by real time RT-PCR irrespective of the virus dilutions inoculated. Further to con rm this nding virus titration was performed on the same lung samples and TCID50 titre ranging between 10 5.48 to 10 5.58 was observed in all samples on 3DPI ( Figure S1). Similarly on day 14, the remaining hamsters were sacri ced and lung samples were found to show a titre of 10 2.5 TCID 50/ ml in all the animals, indicating that viral inoculum could induce disease in all hamsters ( Figure S1). On histopathological analysis, lungs samples of the hamsters inoculated with the 10 5.5 TCID50 and 10 4.5 TCID50 dilutions collected on 3DPI showed mild in ammatory changes indicating beginning of pneumonia, whereas other groups showed minimal or no changes. On 14 DPI, the lung pathological changes were found minimal in all groups indicating recovery from infection. Even though the lung viral titres in all groups were similar irrespective of the dose of virus inoculums, the lung pathological changes indicated the rapid induction of pneumonic changes in 10 5.5 and 10 4.5 dilutions. Hence, a virus dilution dose of 10 5.5 TCID50 was used for virus challenge.
Clinical observations during BBV152 immunization period and post SARS-CoV-2 challenge We immunized four groups of 6-8 week old Syrian hamsters (9 hamsters in each group), with phosphate buffered saline (group I), 6µg of BBV152 with Algel1 (group II), 3µg of BBV152 with Algel2 (group III) and 6µg of BBV152 with Algel2 (group IV) ( Figure 1). All the hamsters received two booster doses on day 14 and day 35. Rectal temperature remained within the normal range and no clinical signs were observed in all the groups throughout the immunization period. The body weight increased until 7 weeks postimmunization ( Figure 2A), but following SARS-CoV-2 infection on day 50, decrease in body weight was observed in all the groups ( Figure 2B). However, the percentage decrease in vaccinated groups were lesser compared to the group I. The decrease in body weight of group III hamsters was less as compared to other groups ( Figure 2). Inactivated whole virion vaccine candidates induced speci c IgG /neutralizing antibody and Th 1 biased immune response Anti-SARS-CoV-2 IgG antibody response was detected by 3 weeks in 8/9 hamsters of group IV with an average OD of 0.62, 8/9 hamsters in group III with an average OD of 0.42 and in 2/9 hamsters (average OD = 0.285) of group II. On day 48, IgG antibody response was found to be increasing in the vaccinated groups with an average OD of 1.32 in group IV (9/9 hamsters), 1.2 in group III (9/9 hamsters), 0.55 in group II (9/9 hamsters) ( Figure 3A, C). All the animals in group I remained negative for IgG antibody during immunization period whereas post virus challenge, 2/3 hamsters showed IgG positivity by 7 DPI IgG antibody response in vaccinated hamsters was further characterized to determine the IgG subclass pro les. On sub-typing IgG2 was detected in all the IgG antibody positive samples whereas it was negative for IgG1 during immunization and post-infection phase ( Figure 3E). All three formulations of vaccine candidates signi cantly induced IgG2 with an increasing trend post-infection indicating a Th 1 biased immune response (Figure 3 A-E).
Neutralizing antibody (NAb) started appearing in the immunized groups at 3rd week of immunization and increased till 7 th week with highest titre (mean = 28810) in group III ( Figure 3F). After virus infection the highest titre of NAb (mean = 85623) was seen in group III animals on 15 DPI. Group I did not show NAb response during immunization phase and after virus infection till 15 DPI ( Figure 3G).
Detection of SARS-CoV-2 genomic RNA (gRNA) in swabs/ organ samples post virus challenge SARS-CoV-2 viral genome copy number in throat swabs (TS) of group I were signi cantly high compared to other groups on 3, 5 and 7 DPI ( Figure 4A). The viral gRNA in group I persisted till 10 DPI, whereas it was cleared in all the vaccinated groups by 5 DPI. Higher copy numbers of viral gRNA were detected in the nasal washings of group I ( Figure 4B). Lungs ( Figure 5A), Nasal turbinates ( Figure 5B) and trachea ( Figure 5C) of group I showed higher viral gRNA copy number compared to other groups on 3 and 7 DPI. Trachea was cleared of viral gRNA by 7 DPI in all the groups. Complete gRNA clearance was observed from lungs of group III and IV on 7 DPI and from group II by 15 DPI. Nasal turbinates viral gRNA persisted in all the groups till 15 DPI, but with lower copy numbers in vaccinated groups compared to group I. No viral subgenomic (sg) RNA was detected in TS, nasal wash, nasal turbinate or trachea of animals of vaccinated groups. However viral sgRNA was detected in lungs (3/3), trachea (1/3) and nasal turbinate (1/3) in group I animals on 3 DPI. The spleen, kidney and small intestine of hamsters of group I showed viral gRNA positivity on 3 DPI ( Figure 5D).

Virus titration
Lungs, nasal turbinate and TS samples of group I showed an average titre of 10 6 , 10 5.5 and 10 4 TCID50/ml respectively on 3 DPI. In the vaccinated groups III and IV, nasal turbinates showed an average titre of 10 5 and 10 4 TCID50/ml on 3 DPI, whereas the TS and lungs titre was found considerably less (10 2.5 TCID50/ml) compared to group I. In contrast group II did not show live virus titre in any of the specimens on 3, 7 and 15 DPI. On 7 DPI only nasal turbinates of group I showed virus titre whereas vaccinated groups were negative. This correlates with the decreasing trend of gRNA and sgRNA in immunized groups after 3 DPI.

Pathological and Immuno-histochemistry ndings in lungs post virus inoculation
The lungs of the vaccinated groups appeared normal on 3, 7 and 15 DPI ( Figure 6B, 6C and 6D) on gross pathology whereas on 7 DPI the lungs of group I showed diffuse areas of consolidation and congestion ( Figure 6A). On histopathological examination, lung sections from group I animals showed congestion, haemorrhages, exudations in the alveoli, mononuclear cell in ltration in the alveolar interstitium and pneumocyte hyperplasia on 3 and 7 DPI ( Figure 6E, 6F). Occasionally, loss of bronchiolar epithelium was also observed. By 15 DPI, bro-elastic proliferation with collagen deposition at alveolar epithelial lining were observed in the lungs of group I ( Figure 6G

Materials Availability
This study did not generate new unique reagents.

Data and Code Availability Statement
The published article includes all datasets generated or analysed during this study.

Immunization of hamsters
Thirty-six, 6-8 week old, female Syrian hamsters were divided into four groups, viz., Group I, II, III, and IV of 9 hamsters each. The hamsters were housed in individually ventilated cages with ad libitum food and water. Group I was administered with phosphate-buffered saline (PBS), group II with 6µg of vaccine candidate along with Algel 1, group III with 3µg of vaccine candidate with Algel 2, and group 4 with 3µg of vaccine candidate with Algel 2. Animals of each group were immunized with 0.1 ml of PBS/vaccine formulations intramuscularly in the left hind leg under iso urane anaesthesia 0, 14, and 35 days. Post immunization hamsters were observed daily for clinical signs and injection site reaction. Rectal temperature was monitored every 24 hours for 3 days post-immunization and weekly thereafter. Body weight was measured every alternate day for the rst week and weekly thereafter. The hamsters were bled on day 12, 21, and 48 post-immunization to check for antibody response.

Challenge study in hamsters
The immunized hamsters were challenged with 0.1 ml of 10 5.5 TCID50 SARS-CoV-2 virus intranasally on the eighth-week post-immunization (day 50) in the containment facility of ICMR-National Institute of Virology, Pune under iso urane anaesthesia. Throat swabs were collected in 1 ml virus transport media on every alternate day post-inoculation for viral load estimation. Three hamsters from each group were euthanized on 3, 7 and 15 DPI to collect throat swab, nasal wash, rectal swab, blood and organ samples for viral RNA estimation, titration, histopathology, and immunological analysis.

Method details
Inactivated SARS-CoV-2 whole virion vaccine (BBV152) SARS-CoV-2 strain (NIV-2020-770) isolated at ICMR-NIV, Pune was propagated in Vero CCL81 cells and harvested on observation of cytopathic effect in the cells. At BBIL BPL (Ferak, Germany) was added to the virus harvest following ltration and stabilization of the harvest using a buffer. The mixture was kept at 2-8˚C with continuous stirring for 24 hrs and was further hydrolysed by incubating at 37˚C for 2hrs. Column chromatography was used for further puri cation and the process intermediate was concentrated to prepare the whole virion vaccine. Two different antigen concentrations (3 µg and 6 µg) and 2 adjuvants namely Algel 1 (Alum) and Algel 2 (TLR 7/8 (imidazoquinoline) agonist adsorbed alum) in combinations were used for the study. The vaccine formulations evaluated in the study were 6 µg antigen with Algel1, 3 µg with Algel 2, and 6 µg with Algel 2.

Enzyme-linked Immunosorbent Assay
Ninety-six well microtitre plates were coated with 1:10 diluted inactivated SARS-CoV-2 antigen with carbonate buffer (pH 9.5) overnight at 4 °C. Subsequently, wells were blocked with liquid plate sealer (CANDOR Bioscience GmbH, Germany) for two hours at room temperature (25-30°C). The wells were washed 5 times with phosphate-buffered saline with 0.05% Tween 20 (PBS-T) and were incubated at 37°C for one hour with 100μl of diluted hamster serum samples (1: 100). Negative control was added to each plate. After 5 washes with PBS-T, anti-hamster IgG antibodies 1:3000 (Thermoscienti c, USA) were added and incubated for 1 hour at 37°C. Following 5 washes with PBS-T, 100 μl of substrate, 3',3'5,5'tetramethylbenzidine (TMB) was added to each well. The colour reactions were developed for 10 minutes and after termination, absorbance was measured at 450 nm. Serum IgG titres were determined by testing serial 10-fold dilutions of each sample, starting from 1:100 dilution. Titre values were determined as the highest dilution at which the optical density was more than 0.2 and positive/negative (P/N) ratio above 1.5.
For antibody sub-typing, plates were coated with antigen and blocked as described earlier. Hamster serum, 1:100 diluted in 1% bovine serum albumin in 1× PBST was added to each well and incubated for 1 hour at 37°C. After washing the plates 5 times with 1× PBST wells were probed with biotinylated anti-Syrian hamster IgG1 /IgG2 antibodies diluted at 1:10000 (BD biosciences, USA) and incubated for 1 hour at 37°C. After washing, the plates were incubated with Streptavidin-horseradish peroxidase 1:8000 (Thermo-scienti c, USA) for 30 minutes at 37°C. The reaction was read as described earlier.

Plaque Reduction Neutralization test
The assay was performed as described by Gururaj et al., 2020. Brie y, a four-fold serial dilution of hamster serum samples was mixed with an equal amount of virus suspension and incubated at 37°C for 1 hour. Further 0.1 ml of the mixture was inoculated in a 24-well tissue culture plate containing a con uent monolayer of Vero CCL-81 cells. The plate was incubated at 37°C for 60 min and overlay medium (2% carboxymethyl cellulose with 2% FBS in 2X MEM) was added to the cell monolayer, which was further incubated at 37°C in 5% CO2 incubator for 4-5 days and PRNT 50 titres were calculated as described earlier.

Cytokine analysis
The serum cytokine levels (TNF-α, IFN-γ, IL-4, IL-6, IL-10 and IL-12) were assessed in hamsters post challenge at 3, 7, and 15 days. An ELISA based commercial assay (Immunotag, USA) was used for the hamster speci c cytokine quantitation. For this, plates pre-coated with hamster speci c cytokine antibody were used and a streptavidin based HRP system was used for detection and the absorbance was measured at 450 nm.

Histopathology and immunohistochemistry
Lungs samples collected during necropsy were xed in 10% neutral buffered formalin. The tissues were processed by routine histopathological techniques for hematoxylin and eosin staining. Duplicate sections were taken for immunohistochemical evaluation. An in-house developed anti-SARS-CoV-2 mouse polyclonal serum was used as the primary antibody for detection. The tissue sections were rehydrated and antigen retrieval was performed using 0.3% hydrogen peroxide in methanol. The slides were incubated with 1: 500 dilution of primary antibody for an hour and an anti-mouse HRP antibody (     in the scatter plot. 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. 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.