IgG Antibody Seroprevalence Post Covishield Vaccination in Western Uttar Pradesh: A Hospital Based Study

Background: Covid-19, a zoonotic disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCov-2) has emerged as a worldwide infection and has been declared pandemic since March, 2020, by WHO. This has brought about tremendous burden on the health care system of not only the developing or the third world countries but also that of prosperous counties of the world. The recent Covid-19 pandemic has pushed the worldwide scientific and medical community to find a solution with the help of vaccines to control SARS-CoV-2 pandemic. An effective vaccine is one which leads to synthesis of IgG antibodies against SARS-CoV-2, thus aiding the control and decline of the pandemic. Aim: This study aims to evaluate the antibody titres post 1st dose and post 2nd dose Covishield vaccination and reveals the safety and efficacy of ChAdOx1 nCoV-19(Recombinant) Covishield vaccine. Methodology: The levels of IgG antibodies were estimated in 215 subjects (both normal subjects and Covid-19 positive subjects) using Enzyme Linked Immunosorbent Assay (ELISA) Technique. Original Research Article Kapoor et al.; JPRI, 33(43B): 236-247, 2021; Article no.JPRI.71486 237 Results: Two hundred & fifteen subjects from Teerthanker Mahaveer Hospital of Moradabad (western Uttar Pradesh) were enrolled for the study. The participants were divided into two different groups. Group I comprised of 215 subjects who received 1st dose of the Covishield vaccine. Group II comprised of 101 subjects who received both the doses of the ChAdOx1 nCoV-19(Recombinant) Covishield vaccine. Levels of IgG were analysed 28 days post 1st dose of ChAdOx1 nCoV19(Recombinant) Covishield vaccine and post 2nd dose of ChAdOx1 nCoV-19(Recombinant) Covishield vaccination. After testing serologically for neutralising IgG antibodies, the titre was found to be below the threshold level of 1.1 in 67.40% of the subjects in the study group 1, whereas 32.60% (n=70) were found to be in the seroprotective range (i.e IgG titre > 1.1). Out of the total 101 participants who took both the doses, 39 participants (38.6%) were found to be in the seroprotective range (i.e IgG titre > 1.1). Conclusion: The two doses of Covishield vaccination (4 weeks interval) given to subjects resulted in increase in IgG antibody titre (Neutralising Antibodies) against both spike protein and nucleocapsid protein after 1st dose and 2nd dose and that single dose may suffice for seroprotection in subjects with previous history of COVID-19 who had recovered from the disease.


INTRODUCTION
The pandemic caused by SARS-CoV-2, Corona virus has almost entirely shut down the worldwide movements and has restrained people from stepping to the outside world. Since, the virus has spread unanimously to the entire nations of the world; it has led to enormous loss of lives and economy. SARS-CoV-2 is believed to have originated in the year 2019 from Wuhan, China from bats and since then has been a pandemic affecting nearly 188 countries and 25 territories around the globe [1]. More than 175 million people have been infected with COVID-19 and above 3.7 million deaths have been recorded globally till date [2].
Initially, Corona virus SARS-CoV-2 causes moderate respiratory illness which usually culminates into Acute Respiratory Distress Syndrome(ARDS). Clinical outcomes may vary from asymptomatic infections to severe respiratory complications, Multi-organ Dysfunction Syndrome (MODS) and even death. Aged people above 60 years and those having co-morbidities like hypertension, heart diseases, lung diseases, Diabetes Mellitus, obesity and cancer, etc. have higher chances of progressing from mild to severe illness. The constitutional symptoms include fever, dry cough, loss of taste and smell, sore throat, nasal congestion, muscle and joint pains, etc. In severe COVID-19 disease, symptoms such as shortness of breath, loss of appetite, sleep disorders, anxiety and depression have been noted to varying degrees constituting the myriad occurrence of symptoms and signs in Covid-19 patients [3].
The novel corona viruses are structurally pleomorphic enveloped viruses. They have 4 structural proteins; Spike (S) protein, nucleocapsid (N) protein, envelope (E) and Membrane (M) proteins. Out of these proteins, S protein is responsible for attachment and penetration into hosts' cell via ACE receptors [4,5]. They have positive sense ssRNA genome with the N protein which forms the helical nucleocapsids. The genome is both capped and polyadenylated which prolongs the survival and exponential multiplication of the virion particle after entering the host cells [6].
Due to lack of confirmed and validated treatments for COVID-19, health professionals are providing optimal supportive care such as oxygen and advanced respiratory support such as mechanical ventilation for critical COVID-19 patients. Scientists globally are still working to develop definitive treatments for COVID-19. The current pandemic of COVID-19 has definitely challenged the scientific community worldwide to develop therapeutic measures and vaccines to prevent COVID-19 infections. Mankind never had more urgent task than creating broad immunity for Corona virus infection within the shortest span of time possible. This urgent need of COVID-19 vaccine throughout the world has led to the development of various vaccines. Any COVID-19 vaccine that evokes the production of neutralizing antibodies in the individuals is the primary target of all the COVID-19 vaccination programs [7]. The first mass vaccination program had been started in early December 2020 [8]. Covishield is a recombinant vaccine and is being manufactured using viral vector platform. A chimpanzee adenovirus-ChAdOx1 vector has been modified to carry the COVID-19 spike protein into the human cells [9]. Once the viral vector vaccine carrying the gene encoding S protein on the surface of SARS-CoV-2 is delivered inside the human cells, the inoculated gene is transcribed resulting in the synthesis of the S protein. This particular protein boosts the immune system of the individual by producing antibodies against specific S proteins.
The Covishield vaccination course consists of two separate doses of 0.5 ml each. The second dose needs to be administered between 4-6 weeks according to the latest factsheet of Serum Institute of India. The manufacturer of ChAdOx1 nCov-19 Corona virus vaccine, Serum Institute of India, documents the efficacy rate to be 81.3% [10].The actual effectiveness of these vaccines in terms of efficacy and control in the spread of the disease is still a subject of debate requiring further analysis, documentation and confirmation. The present study intends to analyze the antibody titres post 1st dose and post 2nd dose Covishield vaccination and to assess the safety and efficacy of ChAdOx1 nCov-19 (Recombinant) Covishield vaccine. In the present study, there will be an attempt to find out other common variables which might have an impact on performance in terms of efficacy, safety & immunogenicity of Covishield vaccine.

Study Design
This was a hospital based Descriptive Study.

Subject Selection and Sample Collection
Serum samples of 215 subjects of different age groups and their demographic, lifestyle habits, Covid-19 history, comorbidity history and drug history were collected to assess the IgG seroprevelance post Covishield vaccine inoculation in the Department of Medicine, Teerthanker Mahaveer Hospital (TMH), Moradabad, Western UP, India. The participants of age 19 years and above who had taken the Covishield vaccine dose were enrolled as a study sample [10]. Subjects who were administered with anti SARS-CoV-2 monoclonal antibodies or convalescent plasma (passive immunization) were excluded from the study.
Two different samples were taken from the enrolled subjects on two occasions, details of which are as follows; The following investigation was carried out in both the samples: IgG Antibodies using commercially available Enzyme Linked Immunosorbent Assay (ELISA) kit (DIAPRO).
A coating of spike antigens and recombinant nucleocapsid which are specific to COVID-19 were coated on the microplates. The sample which is diluted was applied with the solid phase with an aim to capture IgG by the antigens. After this washing was done and then in the second incubation process polyclonal specific anti h IgG antibodies labelled with horse radish peroxidase (HRP) were used to detect the bound antibodies. Optical signal was generated due to the enzyme being captured on the solid phase which acted on the mixture (substrate/chromogen) was used for calculation of cut off value and interpretation of results [11].
Interpretation of test results [11]  A cut-off value was decided and test results were calculated by using the following formula on the mean Optical density 450nm/620-630nm value of the Negative Control (NC):  NC + 0.250 = Cut-Off (Co)  In the follow-up of COVID-19 vaccination participants, following interpretation as indicated on manufacturer's test details were applied and interpreted.

Statistical Analysis
The data collected was tabulated and statistical calculations were done using SPSS version 23. Frequency distribution of variables was determined and presented in tabular and graphical form. Association of lifestyle habits, COVID-19 status and adverse effects post vaccination with seroprevalence was done by chi-square test. The p value < 0.05 was considered statistically significant.

DISCUSSION
COVID-19, a zoonotic disease caused by Severe Acute respiratory Syndrome Coronovius-2 (SARS-Cov-2) has emerged as a worldwide infection and has been declared pandemic since March, 2020, by WHO [12]. This has brought about tremendous burden on the health care systems of not only the developing or the third world countries but also that of affluent counties of the world. Nonetheless, economic activities have been jeopardized to a great extent all over the world [13]. WHO has since then, emphasized on curtailing the progression of COVID-19 pandemic by advocating judicious use of the existing drugs, discovery and use of newer drugs and providing a definitive approach to eradicate the disease by vaccine development [14]. This might also interfere with the detoxification process, by interfering with the functions of mixed function oxidases, hydroxylase system or cytochrome P450 enzyme system of the cells [17].
On the other hand, in non tobacco users (n=198), 34.34% were found to be in the seroprotective range, reinforcing the negative influence of tobacco usage in eliciting immune response. Apparent difference was found in seroprevalence of IgG neutralizing antibodies with alcohol consumption but, no firm grounds could be established regarding the effect of alcohol consumption on immune response of the participants [18]. Coexisting morbidities, although numerically had shown to negatively influence the generation of protective secondary immune response, but the results obtained are statistically not significant (p>0.05). Further, drug intake and supplement intake have shown apparently more individuals to be in serologically non protective zone but, statistically the results obtained were not significant, (p= 0.81) and (p=0.35) respectively. This further demands the determination of the nature of the drug and/or supplement consumed by the participants.
In table 4, COVID-19 status has proved to be a strong predictor of increased future immune response. 78.26% (n=18) were seroprotective with the production of significant amount of IgG antibodies as compared to Covid non-sufferers where 26.70% (n=5) were in seroprotective range (p=.001). The mechanisms may be manifold and far more complex than assumed, as described earlier in this section.
Out of 215 participants perceiving adverse effects of any kind like fever, malaise, myalgia, headache, etc were 154, out of which 36.36% (n=56) were found to have high IgG antibody titre in comparison to participants those not experiencing any adverse/side effects were 61 participants, of which only 22.9% (n=14) were in seroprotective range. This possibly indicates that occurrence of minor transient adverse effects are nothing but the indication that the immune response is being stimulated and active immune response is coming into play to build up of long term immune response [19].
As shown in table 4, Anaphylactoid reactions were seen in 46 candidates (21.40%). Out of whom, 39.13% (n=18) had protective IgG antibody titre and those not suffering from any kind of anaphylactoid reactions, 31% (n=52) were in the protective range [20]. Thus, statistically anaphylactoid reactions did not seem to play any role in building up of protective antibody titre of immune response (p=0.29).
Group II result and outcomes: Study participants having received both the shots of ChAdOx1 nCov-19 (Recombinant) Covishield Vaccine (n=101) were considered to have completed the entire vaccination regimen and were grouped in the Group II. Table 5 depicts the demographic profile of the individuals included in the 2 nd group which are almost comparable to that of the 1 st group except that the compliance of male participants 61.4% (n=62) was better for 2 nd dose of ChAdOx1 nCov-19 (Recombinant) Covishield vaccine which is pointing towards some sociodemographic factor in this geographical area in particular, resulting in hesitancy or abstinence for 2nd dose of vaccine amongst female counterparts. This emphasizes the need for spreading more awareness among the female participants for completion of vaccination protocol.
Participants those complying for the inoculation of 2 nd shot of ChAdOx1 nCov-19 (Recombinant) Covishield vaccine were also found to have difference in IgG seroprevalence i.e, immunogenicity in terms of COVID-19 history. Out of the total 101 participants who were compliant to take 1 st as well 2 nd doses, 38.6% participants (n=39) were found to be in the seroprotective range after 28 days of 2 nd shot of vaccine. 16 participants from 2 nd group were having the history of positivity for COVID-19 history out of which 75% (n=12) were having seroprotective range of IgG antibodies. Form the 2 nd group, 85 participants who had never suffered from COVID-19, about 32% (n=27) participants were found to have protective neutralizing IgG antibodies.
In accordance to the findings obtained from group I and group II outcomes, it can be inferred that in future the duration and frequency of the booster doses can be deduced from the statistical results thus, obtained.

CONCLUSIONS
Thus, in this study the mainstay of study areas were the assessment of efficacy and safety ChAdOx1 nCoV-19(Recombinant) Covishield vaccine. Efficacy of the vaccination may be deduced from the fact that there is a significant increase of IgG antibodies titre (Neutralizing Antibodies) against S protein and N protein both after 1st dose and 2nd dose of vaccination (8 weeks). However, earlier increase in protective IgG antibody titre was seen in COVID-19 positive patients indicating that in such individuals a single dose might suffice and act as a booster as the active immunity has already been acquired by naturally acquired corona virus/incidental infection and that, it has already evoked the activation of memory B and T cells which on receiving the 2nd dose generates secondary immune response. • Trials for monoclonal IgG neutralizing antibodies can also be done for contacts and frontline workers/healthcare professionals as prophylaxis to limit the transmission of viruses among the caretakers of COVID-19 patients.

Future Implications
• As evidenced by our study findings, lifestyle modifications may be emphasized to the patients to get better immune response post vaccination regimen.
• Another mainstay application of the research findings of this study may at least in part, help to decide the duration and frequency of booster dose(s) of ChAdOx1 nCov- 19 (Recombinant) Covishield Vaccine in future.

Limitations of the study
• Findings and outcomes of the present study may be confirmed on a larger size to increase the generability and external validation. • Study on adolescents and children which couldn't be made possible in this study needs to be done for wholesome assessment across all age groups.
• Segregation of study groups on the basis of type of comorbidities, drugs and supplements needs to be done for enhancing the capability of this study across varied medical scenario.
• Long follow up couldn't be done in this study which needs to be done for further validation of research outcomes.

SUMMARY
Various scientists and researchers across different regions of the world have advocated the administration of vaccination to permanently get rid of the pandemic caused by SARS-CoV2. Inspired by the current evidences, an attempt has been made in the study to assess the immunogenicity, safety and variables association with current proposed ChAdOx1 nCov-19 (Recombinant) Covishield vaccine regimen in India. This study has been done specifically on a cohort of North Indian population of Western UP reporting to our tertiary care hospital, who had consented in writing for their immunoglobulin levels in blood to be assayed quantitatively. Results after dosage 1 ( post 28 days) and after dosage 2 ( post 28 days of dose I ) were analyzed extensively and at large the vaccine has been found to be effective, safe, well tolerated and protective in statistically significant number of participants. Furthermore, it has also been deduced from the findings obtained that the constraints or burden on the health care systems might be reduced at least in Covid positive patients ( i.e., RT PCR positive-6 months prior ) by administering only a single dose of vaccine, as single dose was seen to be sufficient to generate appreciable immune response in these category of participants.
Similar findings may be extrapolated on larger population to increase the generalizability of the study outcomes by extending a similar kind of study on children and adolescents. This approach might increase the accountability and applicability of the findings of the study across all strata of age groups of Indian population.

DISCLAIMER
Authors have declared that the products used for this research are commonly and predominantly used in our area of research. We do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by the institute.

CONSENT
Enrolled participants had signed a duly filled consent form stating the sampling and usage of clinical data.

ETHICAL APPROVAL
The present study was conducted under the guidelines laid down by given Institutional Ethical Committee.