Antibody Development through SARS-CoV-2 Infection and Vaccination in India

Abstract

In this study, we examined the spread of SARS-CoV-2 infection and the dose-dependent efficacy of the COVID-19 vaccine Covishield (Oxford/AstraZeneca). During the period between December 2020 and February 2021, we tested the level of natural infection among individuals by estimating the reactivity of their sera towards SARS-CoV-2 spike (S) and nucleoprotein (N) proteins. The seropositivity of the population in different communities ranged from 17% to 51%, depending on their connectivity to the nearest metropolis Kolkata (population 14.85 million), the disease epicenter. We further found that while 90% of the people administered with two doses of Covishield developed antibody against SARS-CoV-2, only 55% developed antibody after one dose. Primarily those who had developed antibody through natural infection remained seropositive after the first vaccine dose in contrast to those with undetected infection. Our experimental findings not only contribute to a better understanding of COVID-19 epidemiology in India but also pave the way for an effective vaccination strategy involving the individual history of infection. OriginAl ArtiCle *Corresponding authors: Gourisankar Ghosh, Department of Chemistry & Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA 1Bio Bharati Life Science, EN 35, Sector V, Salt Lake, Kolkata, India 2Department of Chemistry & Biochemistry, University of California San Diego, La Jolla, CA, USA 3CSIR Indian Institute of Chemical Biology, 4 Raja Subodh Chandra Mullick Road, Jadavpur, Kolkata, India 4Sister Nivedita University, DG 1/2 New Town, Kolkata, India Check for updates 2020 [2]. COVID-19 began in Wuhan in China in late 2019 and spread all over the world by the middle of 2020. As of May 27, 2021, ~3.5 million people died of COVID-19 and an additional ~170 million people around the world were known to have been infected [3]. However, both the numbers might be higher in reality since many countries have underreported and infections and deaths [4] and a large number of asymptomatic infected people have never been tested for the infection. Coronaviruses belong to a large family of viruses. The family now has seven established human coronaviruses including SARS-CoV-2 [5]. The first two Human Coronaviruses (HCoV)-OC43 and -229E were isolated in the1960s [6,7]. These two viruses cause only mild respiratory problem and common cold. Four more human coronaviruses were found by 2012. Human coronaviruses HCoV-NL63 and HCoV-HKU1 are not pathogenic. But the other two -SARS coronavirus 1 (SARS-CoV), which emerged in November 2002, and MERS (Middle East Respiratory Syndrome) coronavirus (MERS-CoV), which emerged in 2012, [8,9] are highly pathogenic. Fortunately, they disappeared quickly. Finally, SARS-CoV-2 emerged in late 2019 [10,11]. The genome of the coronavirus is a single stranded Introduction COVID-19, the abbreviated form of ‘Coronavirus Disease-2019’, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1]. World health Organization declared COVID-19 as pandemic in March ISSN: 2474-3658 DOI: 10.23937/2474-3658/1510219 Ghosh et al. J Infect Dis Epidemiol 2021, 7:219 • Page 2 of 9 • ribonucleic acid (RNA) polymer [12,13]. The advantage of having an RNA genome is that some of the very essential proteins are generated just after the SARSCoV-2 enters the host (human) cell. The viral surface protein, Spike (S), attaches through its receptor-binding domain (RBD) to the host cell receptor for entry into the cell. Membrane (M), envelope (E) and nucleocapsid (N) are other major structural proteins. N protein is involved in the transcription and replication of viral RNA and packaging of the encapsidated genome into virions. Both N and S are highly immunogenic and are used for the diagnosis of SARS-CoV-2 infection [14]. Generation of circulating anti-viral antibody in the host after viral infection or virus specific vaccine administration is essential for blocking viral entry into host cells [15]. The detection of SARS-CoV-2-specific antibodies in the blood plasma of unvaccinated individuals is a measure of host infection by the virus [16]. Most COVID-19 vaccines use S protein as the target with the idea that antibody generated against the S protein will successfully block viral entry into the host cell [17-19]. Vaccine shortages have started to impact the global vaccination efforts [20]. Thus although vaccinating as many people as possible and as quickly as possible without allowing emergence of antibody evading variants is the best way out of the pandemic situation, efficacy of fractional vaccination of the previously infected individuals [21] is now being discussed as part of a protection plan [22,23]. However, such examination of efficacy has only been carried out in three industrialized nations (U.S.A., Italy, and Israel) with the mRNA vaccines (Pfizer-BioNTech and Moderna) [24-28]. In India, the first wave of infection and death reached a peak around September-October, 2020 [3]. In February, cases reached to its lowest level but a much stronger second wave largely caused by a mutant (B.1.617.2) that has already been detected worldwide is currently sweeping through the country. Although mass vaccination started in the country in late February, only about 14% of the population has received the vaccine so far. In this study, we examined the severity of COVID-19 in the Indian population and evaluated the seropositivity of individuals after both natural infection and one or two doses of AstraZeneca’s Adenovirus Vaccine Covishield. We noted that the disease severity in a certain population was controlled at least partly by its socioeconomic status. We also observed a positive correlation between natural infection and development of circulating antibody. While the first dose of vaccine helped maintain seropositivity in previously SARS-Cov-2 infected individuals, the second dose of vaccine was critical for development of antibody in those people with undetected SARS Cov 2 infection. Overall, these results provide a basis for modifications of policies in determining the dosage requirement of the Covishield vaccine. Materials & Methods Cloning, expression, and purification of N protein N gene (Nucleocapsid protein gene) was synthesized and cloned in pUC 57 (Ampr) followed by subcloning in BioBharati pHIS-TEV vector (Cat# V0020) between Bam HI and Xho I restriction sites. Then E. coli (Rosetta DE3) cells were transformed with pHis TEV vector containing histidine tagged N gene. Cells were grown in LB media containing ampicillin (100 μg/ml) and chloramphenicol (17 μg/ml) at 37 °C till the light scattering value at 600 nm reached 0.6. Then the culture was induced with 0.5 mM IPTG at 37 °C for 4 hours. The cells were centrifuged and the pellet was resuspended in Buffer A [25 mM Tris-Cl (pH 7.5), 300 mM NaCl, 0.5% Triton-X 100, 5% Glycerol, 2 M Urea] and sonicated. The suspension was centrifuged at 12500 rpm for 30 minutes at 4 °C. Supernatant was loaded onto 1 ml Ni-NTA agarose beads pre-equilibrated with Buffer A at 4 °C. Flow through was discarded. Beads were washed 3 times with 10 column volume of Buffer A + 20 mM Imidazole. Protein was eluted using Buffer A containing 250 mM imidazole. Collected fractions were analyzed using 12.5% SDS PAGE. Purified protein concentration was estimated and stored as 50% glycerol stocks at -80 °C. S-RBD was obtained from a commercial source (Genscript, Catalog #Z03514-100) and as a gift from Dr. Gene Tan of J. Craig Venter Institute (La Jolla, USA). Preparation of protein A/G conjugated to HRP Horseradish peroxidase (HRP) was first dissolved in water and activated with 0.25 M sodium periodate at room temperature for 30 minutes followed by purification by gel filtration in Sephadex G-25 column. Purified activated HRP was then conjugated to protein A/G at 4:1 molar ratio in the presence of 0.5 M carbonate buffer, pH 9.6 for 16 h at 4 °C. Next, Protein AG-HRP was dialyzed against 0.1 M PBS, pH 7.5. The dialyzed Protein AG-HRP was then concentrated and mixed with BSAGlycerol mix so that the final concentration of protein is 1 mg/ml. Goat anti human IgG/A/M antibody HRP conjugated was obtained from Invitrogen (Catalog # A18847). Blood sample collection Roughly two drops of blood were from the fingertip of each subject by inserting a sharp needle. The age range of all subjects, both males and females, was from 20 to 80 yr with nearly 50% with age below 45 yr. Samples were incubated overnight at 4 °C followed by centrifugation to separate bold sera. Sera were preserved frozen at -20 °C until ELISA. Enzyme linked immunosorbent assay (ELISA) ELISA was performed following the method published [29] with some modifications using a test kit developed in house. Briefly, 100 ng of S-RBD was ISSN: 2474-3658 DOI: 10.23937/2474-3658/1510219 Ghosh et al. J Infect Dis Epidemiol 2021, 7:219 • Page 3 of 9 • deviation and n is sample number). Then table was used to determine the p value range from the t-statistic. Samples from different communities were compared to that of Kolkata (Figure 2) using unpaired two-sample one-tailed tt est in Microsoft Excel. Samples from the same set of individuals obtained at two different times (Figure 3C, Figure 3D and Supplementary Figure 2) were compared using paired two-sample one tailed t-test in Microsoft Excel. Results obtained with protein A/G and IgG were compared using paired two sample two tailed t-test in Microsoft Excel. Samples were analysed for the distribution plot with Graph Pad Prism version 8.3.1 software. Ethical considerations An Institutional Review Board (IRB) on Human Subjects was constituted following the guidelines of the Indian Council for Medical Research (ICMR). The IRB read and discussed the research proposal, and provided certificate of approval for the research conducted in this study. Samples were collected following the protocol approved by the IRB. Samples were collected from do