Ethical statement:
The study was approved by the Institutional Animal Ethics Committee and further approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi letter number V-11011(13)/7/2020-CPCSEA-DADF.
Virus strains
Virus propagation and titration:
SARS-CoV-2 (NIV-2020-770) strain was isolated from throat/nasal swab specimen of COVID-19 positive patient in Vero CCL-81 cells at the maximum containment facility of ICMR-NIV, Pune 13.
SARS-CoV-2 stock was prepared by inoculating the known titer of virus in three passages in Vero CCL-81 cells. Cytopathic effect was first observed on second post-infection day (PID) and harvested on third PID. Virus titrations were performed in Vero CCL-81 cells using tissue culture infectious dose 50% (TCID50) assay. Virus titre (TCID50/ml) was calculated by the Reed-Muench method and found to be 106.5 TCID50/ml.
Antigen preparation
Gamma inactivation of the virus: Gamma irradiation of the virus stock was performed using Co-60 source (24 kGy) of GC-5000 Gamma chamber (BRIT, Mumbai). This irradiated stock was again inoculated in Vero CCL-81 twice and observed for five days to confirm the complete inactivation of the virus (Elliot et al., 1982)14.
Concentration of gamma-inactivated antigen: Gamma irradiated SARS-CoV-2-infected tissue culture fluid was concentrated using 30 kDa filters (Pall, Germany) and further passed through 0.2 μm filters, aliquoted and stored at −80°C. Concentrated viral antigen was also aliquoted in 1 and 2 ml volumes in frosted glass bottles and further lyophilized. The lyophilized vials were stored at −20°C to be used as a source of whole virus antigen
Equine Immunization:
Ten 4–10 years old, healthy equines (160–200 kg in weight) that had no detectable antibodies against SARS-CoV-2, were chosen for primary immunization at Biologicals E. Ltd (Bio E). The equines were numbered HK1 to HK10, and the same numbers were used to represent the plasma obtained from individual equines accordingly. Equines were inoculated with inactivated SARS-CoV-2/VeroCCL81/P-4 antigen subcutaneously along with Freund’s adjuvant. After completion of initial immunization, the equines were test bled and plasma samples were collected and anti-SARS-CoV-2 IgG was tested via ELISA and plaque reduction neutralization assay (PRNT).
Enzyme-linked immunosorbent assay (ELISA)
Specificity of antibodies raised in equines were evaluated by sandwich ELISA using inactivated SARS-CoV-2 antigen (Sapkal et al., 2020)15. Briefly, 96-well polystyrene microtitre ELISA plates (Nunc, Thermo Fisher Scientific, USA) were coated with inactivated SARS-CoV-2 antigen (1:10 diluted, 100 µl/well) in 1x Phosphate-Buffered Saline (PBS) (pH 7.2), overnight at 4°C and then were blocked with a 1% BSA in 1x PBS for one hour at 37°C. The plates were washed three times with 1x PBST, pH 7.4 with 0.1 % tween-20 (PBST). To the coated plate, 100 µl of 1:100 diluted equine plasma samples were added and incubated at 37°C for one hour. After each step, the plate was washed five times using 1x PBST. Following this, 100 µl/well of anti-horse IgG horseradish peroxidase (HRP, Sigma-Aldrich, USA) (1:16000) diluted in 1x PBST and added; and plates were incubated for one hour at 37⁰C. Further, 100 µl of 3, 3’, 5, 5’-tetramethylbenzidine (TMB, Cellbiosis) substrate was added and incubated for 10 min. The reaction was stopped by adding 100ul of 1N sulphuric acid (H2SO4), and the absorbance values were measured at 450 nm using an ELISA reader (Thermo Fischer scientific, USA). Normal horse plasma (non-immunized) was used as negative control and pooled plasma of 10 immunized animals been used as positive control. The cut-off for the assay was defined as mean of negative control optical density plus three standard deviation (3SD).
Further to identify the binding efficacy of the antibodies, multiple dilutions of pooled plasma collected from the equines and the purified bulk preparations produced from the plasma were tested in ELISA. For negative control, normal horse serum (non-immunized) was added. The bound antibodies were then probed with anti-horse IgG-HRP conjugate. The end point titre was defined as the reciprocal of the highest dilution of the sample that gives result above the cutoff16.
Plaque reduction neutralization test (PRNT): PRNT was performed as described by Deshpande et al., (2020)17. Briefly, four-fold serial dilutions of heat inactivated (56°C for 1 h) horse plasma samples were 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 one hour, each virus-diluted plasma 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 one hour, overlay medium (2% CMC with 2% FBS in 2× MEM) was added to the cell monolayer, and the plate was further incubated at 37°C in 5% CO2 for 4 days. Plaques were observed and the plates were stained with 1% amido black for an hour. Antibody titres were defined as the highest plasma dilution that resulted in >90 per cent (PRNT90) reduction in the number of plaques.
Purification of SARS-CoV-2 F(ab’)2 equine immunoglobulin
Plasma collected from the 10 equines was pooled to produce a plasma pool and was used to produce multiple batches of purified F(ab’)2 fragments as per established manufacturing technology at BioE at pilot scale. In the first step, the plasma pool was diluted and the pH adjusted to 3-3.5 and enzyme pepsin was added to initiate IgG digestion. During the process, the pH and temperature was controlled to ensure complete digestion. After the completion of the enzymatic reaction, the pH of the solution was raised and heated till 55oC to inactivate residual pepsin as well as any equine viruses potentially present in the plasma. After heat inactivation, caprylic acids were added to precipitate contaminating plasma proteins such as albumin. The precipitated proteins were removed from the solution via cloth filtration. The filtered solution containing F(ab’)2 fragments was then further purified using ultrafiltration-diafiltration and formulated into a glycine-sodium chloride buffer. The formulated bulk was filtered through a 0.2 micron filter to produce Purified Bulk F(ab’)2 fragment immunoglobulins.
Two to three batches of purified bulk were pooled and mixed with the glycine-sodium chloride buffer and then sterile filtered to produce final bulks. The final bulk preparations were again sterile filtered in an on-line manner on an automated vial filling line and filled into 2R glass vials stopper with rubber bungs and sealed with flip-off aluminium caps to produce final lot product suitable for clinical evaluation. Non-reducing SDS-PAGE gels, using the buffer system described by Laemmli (1970)18, were used to monitor the digestion process.
Overall scheme of immunization strategy and equine hyperimmune globlulin production has been represented in Figure-1.
Quality control assessment of the purified equine anti-SARS-CoV-2 Immunoglobulin.
Physical and biochemical properties of equine anti SARS-CoV-2 final bulk and final lot Immunoglobulin was determined as per the standard guidelines and in-house specifications for clinical product characterization19,20,21,22 (Supplementary data).
Data Analysis
The data analysis were performed with IBM SPSS statistics 20 (NY, USA) and GraphPad Prism 8 (San Diego, CA, USA). The neutralizing endpoint as the reduction in the number of plaque count by 90 per cent (PRNT90) was calculated by probit analysis. ELISA results (OD) were plotted as mean OD for each sample.