key: cord-0828831-xvlbtxx2 authors: Desphande, Gururaj Rao; Kaduskar, Ojas; Deshpande, Ketki; Bhatt, Vaishali; Yadav, Pragya; Gurav, Yogesh; Potdar, Varsha; Khutwad, Kirti; Vidhate, Shankar; Salunkhe, Asha; Patil, Chetan; Shingade, Snehal; Jarande, Kajal; Tilekar, Bipin; Salvi, Pavan; Patsuthe, Sudhir; Dange, Varsha; Kumar, Sudeep; Gurav, Shilpa; Chate, Sadhana; Abraham, Priya; Sapkal, Gajanan title: Longitudinal clinico-serological analysis of anti-nucleocapsid and anti-receptor binding domain of spike protein antibodies against SARS-CoV-2 date: 2021-09-17 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.09.024 sha: 13b496b1a8fef38ffbe2a4af620ca233b59fe42c doc_id: 828831 cord_uid: xvlbtxx2 Objectives Monitoring the antibody responses to SARS-CoV-2 infection and its correlation to clinical spectrum of disease is critical in understanding the disease progression and protection against re-infection. We assessed the nucleocapsid (N) and receptor-binding-domain of spike (SRBD) protein specific IgG and neutralizing antibody (NAb) responses in COVID-19 patients up to 8 months and its correlation with diverse disease spectrum. Methods During the first wave of SARS-CoV-2 pandemic, from 284 COVID-19 patients, 608 samples were collected up to 8 months post infection. The patients were categorized as asymptomatic, symptomatic and severe. The N and SRBD IgG and NAb titers were evaluated and correlated with clinical data. Results A steep increase in antigen specific antibody titers was observed till 40 days post onset of the disease (POD), followed by a partial decline till 240 days. Severe disease was associated with a stronger SRBD IgG response and higher NAb titers. The persistence of antibody response was observed in 76% against N, 80% against SRBD and 80% for NAbs of cases up to 8 months POD. Conclusion RBD and N protein specific IgG persisted till 240 days POD which correlated with NAb response, irrespective of individual`s symptomatic status indicating overall robust protection against re-infection. Keywords: SARS-CoV-2, nucleocapsid, receptor binding domain, neutralizing antibody response, IgG antibody response, 240 days. With over 221 million people infected across the globe (WHO-COVID- 19-global-data, n.d.) , the pandemic of COVID -19 is still a public health emergency and posing a significant threat to life. After a period of initial global decline in COVID-19 cases, the virus has strongly reemerged in many countries. During the first wave of SARS-CoV-2 infection in India, the maximum number of cases per day reached its peak during the months of September and October 2020, and subsequently declined until February 2021 followed by upsurge (second wave) of the COVID-19 cases in May 2021 (Worldometer, n.d.) . The clinical manifestations due to SARS-CoV-2 infection can vary from asymptomatic to mild infection to severe acute respiratory distress syndrome (ARDS) (Singhal, 2020) . Research indicated that about 40-45% of the SARS-CoV-2 cases are asymptomatic (Oran and Topol, 2020) and about 10% present with symptoms of severe disease such as increased respiratory rate, dyspnoea and low blood oxygen saturation (Brochot et al., 2020) . These varied clinical manifestations can be evaluated using various biochemical markers (Ciaccio and Agnello, 2020) (Pourbagheri-Sigaroodi et al., 2020) . The approved method for diagnosis of the infection is real time RT-PCR (Definitions, 2020) and the reported duration for the RT-PCR positivity is from 3 days prior to onset of symptoms up to 83 days post onset of disease (POD) (Walsh et al., 2020) . Since the viral detection period is so varied, understanding immune response is crucial. 6 Antibody response is one of the key factors for development of immunity and preventing reinfection. In our earlier study, we observed that the antibodies appear as early as 4 th day POD . However, the response to specific antigen may differ, due to the level of expression and immunogenicity and time. SARS-CoV-2 has four major structural proteins -spike (S), membrane (M), envelop (E) and nucleocapsid (N) (Kontou et al., 2020) . Of the four structural proteins, N and S proteins are the primary viral antigens responsible for eliciting antibody response . In India, vaccination drive has been started from 16 th January and as the waning of antibody is of major concern, it is pertinent to investigate the persistence of antibody response against COVID-19 virus (Brochot et al., 2020) (Choe et al., 2021a) (Choe et al., 2021b) (Dobaño et al., 2021) (Thangaraj et al., 2021) . Also, limited information regarding a combined analysis of SARS-CoV-2 antigen specific antibodies and neutralizing antibodies response over a longer period of time, its correlation with clinical findings and disease severity is hindering our understanding of the roles of humoral immunity in COVID-19 protection. Furthermore, the kinetics of antibodies against SARS-CoV-2 is of great importance after the introduction of the new vaccines as it will be useful in development of therapeutic and preventive modalities for halting the pandemic. Here, we report immune responses of COVID-19 patients against N and SRBD proteins up to eight months POD. Also the levels of N and SRBD specific IgG were correlated with the plaque reduction neutralization (PRN) assay, hemoglobin (Hb), total Red blood cell (RBC) count, total white blood cell (WBC) count and platelet count in asymptomatic, symptomatic and severe symptomatic patients. The study was approved by Institutional Ethics Committee of ICMR -National Institute of Virology. In this study, a total of 608 serum/plasma samples were collected from 284 COVID-19 patients between April 2020 to February 2021 during the first wave of SARS CoV-2 from designated COVID-19 hospitals in Pune Municipal Corporation (PMC) and Pimpri-Chinchwad Municipal Corporation (PCMC), Maharashtra, India. RT-PCR done from the throat/ nasal swabs collected from a subset of these patients revealed that the circulating SARS-CoV-2 strains were from the G (D614G) clade (Potdar et al., 2020) (Potdar et al., 2021) . After the discharge of the patients, follow up blood samples were collected till a maximum period of eight months from the onset date of illness at PMC/PCMC clinic by the trained staff. None of the recovered patients were re-infected within the 8 months period of follow up. Among qRT-PCR confirmed positive cases, the participants were classified into three categories based on their clinical symptoms. Asymptomatic cases were patients who did not develop any symptoms throughout the course of the disease. Symptomatic cases were patients with fever, fatigue, body ache, diarrhoea, abdominal pain, dyspnoea and respiratory symptoms like runny nose, cough, sore throat, and nasal discharge. Whereas, patients with any of the following 3 criteria with or without the above symptoms were included in severe cases: Respiratory distress with breathing difficulty (≥30 breaths/min) or Oxygen saturation with ≤90% at rest or chest imaging with >50% obvious lesions (Ministry of Health and Family Welfare., 2020) (Xiang et al., 2020) . The study included 208 symptomatic, 60 asymptomatic and 16 severe symptomatic patients. Of the 284 patients, 132 patients provided multiple samples (56 patients provided 2, 23 patients provided 3, 19 patients provided 4, 21 patients provided 5, 3 patients provided 6, 6 patients provided 7, 3 patients provided 8 and 1 patient provided 10 samples) while 152 patients provided one sample. Case histories of all the patients were documented from hospitals. Hematological parameters like Hb, platelet 8 count, total RBC count, total and differential WBC counts were recorded for 125 of the 284 patients. Anti-SARS-CoV-2 SRBD protein IgG in human serum specimens was tested by coating the recombinant SRBD protein on to the microtitre wells followed by post-coating procedures. Serum samples were diluted in the ratio of 1:50 with sample diluent. Fifty microlitres each of the diluted samples, positive and negative controls were added to respective wells. ELISA plate was incubated at 37 o C for 1 hour followed by washing with wash buffer 5 times. Fifty microlitres of ready to use anti-human IgG HRP was added to each well and was incubated at 37 o C for 30minutes. After washing, 100μL of liquid 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate added and incubated at room temperature in dark for 10 minutes. The reaction was stopped by adding 100μL stop solution (1N H 2 SO 4 ) after 10 minutes. The absorbance was measured at 450nm. If OD value of sample tested exceeds 0.2 and sample OD/ negative control OD (sample ratio) >2.7, the sample was considered positive (Supplementary Figure 1 ). As described for S-RBD ELISA, similar procedures and criteria were followed for dilution of the samples, testing protocol and interpretation for recombinant N protein ELISA (Supplementary Figure 1) . Total 298 samples were tested for PRNT (subset of samples tested for N and SRBD protein specific IgG by ELISAs); performed as described elsewhere . Descriptive statistics were calculated for continuous variables, counts and percentages for categorical variables. Mann-Whitney U-tests and Kruskal-Wallis test were performed to 9 compare the differences between groups. Pearson's correlation was drawn to evaluate correlation between two methods. Geometric mean titers were calculated for ELISA ratios and NAb titers. The analysis was performed on GraphPad Prism 9. Cox proportional hazards was evaluated for the association between neutrophil to lymphocyte ratio and antibody responses and Receiver Operating Characteristic (ROC) curves for SRBD and N IgG ELISAs against PRNT were plotted on IBM SPSS 26. For total of 284 patients, the median age of the study population was 38 years with interquartile range (IQR) of 26-51, of which (158) 55.6% of them were males and (126) 44.3% were females. About 21.12% and 78.88% patients were asymptomatic and symptomatic with mild to severe symptoms respectively. Of the 224 symptomatic patients, 7.14% patients presented with severe symptoms. Majority of the older males (62.50%) showed severe symptoms. All the hematological parameters were recorded for 125 patients. Further analyses revealed that patients with severe disease condition had higher neutrophil (78.4%) and lower lymphocyte counts (15.4%). The most common symptoms observed were cold/cough/sore throat/nasal discharge (72.76%), fever (66.96%), body ache (28.57%) and breathing problems / Dyspnoea (14.73%), while comparatively less common were fatigue (11.16%) and diarrhoea (4.55%)( Table 1 ). The SARS-CoV-2 N and SRBD protein-specific IgG antibody response (n = 608) and NAb activities (n = 298) were investigated and correlated in acute (0- female (SRBD ratio = 10.06; N ratio = 7.89) patients as well (data not shown). Although there was a slight variance between the protein-specific IgG immune response mounted when comparing individuals on either side of age 50 years. Patients above the age of 50 years (SRBD ratio = 11.97; N ratio = 8.63) showed a slightly higher antibody levels as compared to those below the age of 50 years (SRBD ratio = 9.56; N ratio = 7.32). Considering neutrophil to lymphocyte ratio (NLR) is linked to innate immunity (Zhang et al., 2020) and is an early warning signal of severe COVID-19 (Xia et al., 2020) , we analyzed NLR data against IgG response among asymptomatic, symptomatic and severe symptomatic patients (n = 125). Both N and SRBD IgG ratios were high with increased NLR in severe symptomatic patients (Figure: 5A, 5B) . Some symptomatic patients exhibited high NLR but the cumulative NLR of this category was within range. Also, the IgG ratios for both N and SRBD for symptomatic patients were not associated with increase in the NLR. We then performed a time-dependent covariate Cox regression analysis of antibody responses (adjusted for sex and stratified for NLR at the time of sampling) on subsequent sampling In SARS-CoV-2 infection, waning of immunity and probability of re-infection is a major concern and there are reports affirming that antibody titers decline more quickly in asymptomatic or mild symptomatic cases than severe cases (Yamayoshi et al., 2021) . Studies showing longevity of antibody response in SARS CoV-2 in a large sample size are very scarce with contrasting results (Hartley et al., 2020) (Choe et al., 2021b) . Here, we provide a comprehensive analysis of antibody dynamics and persistence of antibody response by 13 evaluating protein specific IgG levels in 284 individuals with varied disease severity up to 240 days POD. The antibodies against SRBD IgG appeared slightly earlier and remained substantially persistent than the N protein specific IgG throughout the course of the study (240 POD). Notably, the antibody levels tend to decrease with increased interval between days post onset of symptoms until they reached a constant value. Although seropositivity reaches its maximum by week 4, average relative IgG titers against both N and SRBD antigens continue to increase till week 6 followed by a steady decline in average relative titers during weeks 7- It is worth mentioning that various factors may predict an enhanced initial antibody response against SARS-CoV-2 and the persistence of antibodies over time (Terpos et al., 2021) . Of the measured SARS-CoV-2 antibodies, the IgG response against the SRBD domain was associated with NAbs (r = 0.79) independent of other factors such as sex or age. This indicated that SRBD specific antibodies in the patient sera meant improved patient survival rate supporting the concept that these antibodies are a major contributor to the protective effect of humoral immunity in COVID-19. This finding may have implications in the anticipated protection against re-infection over time. Additionally, the SRBD specific IgG response showed comparatively higher average levels of antibody ratios in patients with severe disease, than the asymptomatic and symptomatic cases. A previous study reported that the severe disease was associated with more robust serological responses including early seroconversion (