Longitudinal observation of antibody responses after SARS-CoV-2 infection at 14 months


 A better understanding regarding antibody responses against SARS-CoV-2 after natural infection could provide valuable insights into the future implementation of vaccination policies. In this study, we aimed to assess the dynamics of IgG antibody titers in recovered COVID-19 patients over 14 months after mild and moderately severe infection using two FDA-approved immunoassays against SARS-CoV-2 Nucleocapsid protein (NCP)and anti-spike-receptor binding domain (S-RBD) through sequential serological tests at different time points. The demographics and clinical profile, that might be associated with the magnitude and longevity of antibody response were also analyzed. 
Our results suggest antibody persistency in 31 out of 32 (96.8%) subjects at 14 months post-infection. A significant positive association was observed between disease severity and anti-S-RBD IgG titers at 14 months. Patients who reported a loss of smell and taste during the clinical course of the disease also developed significantly better antibody titers. Patients who were seronegative for anti-NCP IgG (n=7) at 10 months, were found to be seropositive for anti-S-RBD IgG at 12,13 and 14 months emphasizing on higher false-negative rate for N protein-based antibody assays when compared with the anti-S-RBD assays.
This study calls for prioritizing vaccination for “naive” individuals (with no previous history of COVID-19 infection) and recovered but antibody-negative individuals instead of “vaccination for all”. This strategy would be helpful in low-resource settings and areas experiencing vaccine shortages by saving time, effort, and assets that could be sourced for the vulnerable populations.


Longitudinal observation of antibody responses after SARS-CoV-2 infection at 14 months Introduction
As the worldwide vaccination implementation programs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing Coronavirus disease 2019  are progressing in full swing, information regarding the kinetics and longevity of acquired immunity post-natural infection necessitates analysis as well as documentation. The SARS-CoV-2 shares approximately 79.5% genomic homology with SARS-CoV-1 with a similar receptor-binding domain (RBD) structure.
[1] Therefore, much understanding of the immunity offered post-SARS-CoV-2 infection is derived from previous experiences with SARS-CoV-1, where protective antibodies were found to persist for at least 2 years in addition to real-time emerging data. [2][3][4] A "robust adaptive immune response" with positive S-specific neutralizing antibodies (n Abs), memory B cells, and circulating follicular helper T cells have been demonstrated in recovered patients after SARS-CoV-2 infection. [5][6][7] In this study, we aimed to assess the dynamics of IgG antibody titers against SARS-CoV-2 in recovered COVID-19 patients over 14 months after mild and moderately-severe infection. The demographics and clinical profile, that might be associated with the magnitude and longevity of antibody response was also analyzed. To our knowledge, the current study provides the longest follow-up reported in the literature till date.

Patient cohort
A monocentric pilot observational study, that longitudinally analyzed the presence of antibodies against SARS-CoV-2 was conducted in patients based in Umbria region, Italy who had tested positive for SARS-CoV-2 in March 2020 by Reverse Transcriptasequantitative Polymerase Chain Reaction (RT-qPCR). The RT-qPCR tests were performed by the Local health regulatory authorities according to the national guidelines and standard operating protocols. The patients were managed as per the set protocols by treating the doctor, prescribing home isolation for mild and moderate cases, hospitalization for cases with increased severity. On recovery, all subjects were informed about the seroprevalence study and were invited for voluntary participation. After written informed consent, serological samples were collected and antibody titers were analyzed clinical history along with symptoms and treatment undertaken using a standardized questionnaire. They were then invited for voluntary follow up periodically for sequential serum sample antibody assessment. The study participants did not receive any compensation or any other benefit, but were informed individually about their antibody status.

Patient selection
From May 2020 to January 2021, anti-NCP antibodies developed against of SARS-CoV-2 were analysed using the MAGLUMI® 2019-nCoV lgM/lgG CLIA assay through sequential serum samples. We treated time as a factor and defined six different time points (TPs); (T0-T5). The first blood sample was collected in the month of May 2020, 2 months after the month of infection-March and was defined as T0. Consecutive serological samples were analysed at different TPs; three months (T1), five months (T2), seven months (T3), eight months (T4) and ten months (T5) post infection in June, August, October, November of 2020 and January 2021 respectively. At this point, a more specific immunoassay; MAGLUMI® SARS-CoV-2 S-RBD IgG CLIA was adopted for future assesments.
From late February 2021, an additional n=12 patients (8 female and 4 male), who met the eligibility criteria for participation, were enrolled in the study and added to the original cohort (n=30). These patients (n=12), similar to the original cohort, had a history of testing positive for SARS-CoV-2 by RT-qPCR in March 2020, updating the sample size to n=42.
Since the legal provisions adopted by the Italian Ministry of Health advised mandatory vaccination for all Healthcare Workers, irrespective of previous disease status, n=10 patients (4 female and 6 male) were gradually vaccinated from mid-March 2021 and hence excluded from the original cohort, making the revised final sample size as n=32. The study design, study findings and the temporal distribution of sequential serological sampling time points are described in Figure 1. The study group was divided into two groups at each TP based on disease severity; Mild and Moderately-Severe and the antibody assessments were done accordingly. [9] In brief, antibodies against NCP were analyzed at T0-T5 (over 10 months post infection; March 2020-January 2021) in n=30 patients followed by analysis of antibodies against Spike-RBD from T6-T8 (over 12-14 months post infection) in n=32 patients. The blood samples were collected after informed consent by the patients and with the approval of the ethics committee of the Associazione Naso Sano (Document number ANS-2020/001) at an accredited lab (Laboratory of Nuclear Lipid BioPathology, CRABION, Perugia, Italy). Data collection and analysis was masked from main principal investigator, who was also a part of the study sample in order to avoid observer bias. [10,11] The study was conducted in accordance with the Declaration of Helsinki and national and institutional standards. The STROBE statement checklist can be found in Supplementary Table S.

Analytical system used
As per the Specifications, anti-SARS-CoV-2 S-RBD IgG assay had a sensitivity 100% with CI [99.9%-100.0%] at ≥15 days post symptom onset and specificity of 99.6%; CI [98.7%-100.0%]. High concentration samples were diluted automatically by analyzers and the recommended dilution was 1:9 with the diluent in kit. The sample, buffer and magnetic microbeads coated with S-RBD recombinant antigen were mixed thoroughly and incubated, forming immune-complexes. After precipitation, decanting of supernatant, and performing a wash cycle, ABEI labeled with anti-human IgG antibody was added, and incubated to form complexes. Again after precipitation in a magnetic field, decanting of supernatant, and performing another wash cycle, the Starter 1+2 were added to initiate a chemiluminescent reaction. The light signal was measured by a photomultiplier as relative light units (RLUs), which is proportional to the concentration of SARS-CoV-2 S-RBD IgG presented in the sample. The measurements and interpretation of results were made according to the manufacturer's instructions. The analyzer automatically calculates the concentration in each sample by means of a calibration curve which is generated by a 2point calibration master curve procedure. The results were expressed in AU/mL. A result less than 1.00 AU/mL (<1.00 AU/mL) was considered to be non-reactive while a result greater than or equal to 1.00 AU/mL (≥1.00 AU/mL) was considered to be reactive. [12]

STATISTICAL ANALYSIS
The descriptive statistics for the main characteristics of the study group were expressed as Median, [1st -3rd] quartile for continuous variables and as absolute frequency (column percentage) for the categorical variables. The normal distribution of data was tested by One sample Kolmogorov-Smirnov test. The p-values resulted from Mann Whitney U test, Friedman Test, Pearson's Chi-squared test (for cell frequency n≥5) and Fisher's exact test (for cell frequency n<5). Statistical significance was defined for p < 0.05. All analyses and data plotting were performed using SPSS Version 22.

Study group characteristics during infection and at 14 months post infection
N abs against S-RBD of SARS-CoV-2 were analyzed for n=32 at 14 months post infection.
Of these, n=21 (66%) were females and n=11 (34%) were males. The disease severity was rated as mild and moderately-severe in n=19 (59.3%) and n=13 (40.7%), respectively. It was noted that n=18 (56.2%) declared one or more comorbidities such as asthma/seasonal allergies, diabetes, hypertension or cardiovascular diseases. Of these, n=5 (38.5%) subjects of the moderately-severe category, had a cardiovascular disease which was a significant finding (p.=0.006). In terms of clinical symptoms experienced, the subjects in the moderately-severe group (n=13) had significant shortness of breath (n=10, p=0.029) , fatigue (n=12, p=0.011) and headache (n=8, p=0.046). The baseline clinical, demographic features and disease characteristics of the study subjects at the time of infection (March 2020) are reported in Table 1. The main characteristics were expressed as Median (q2) with First and Third quartiles i.e., (q1-q3) for continuous variables and as absolute frequency and column percentage for binary variables. The p-values resulted from Mann Whitney U test, Pearson's Chi-squared test (for cell frequencies n≥5) and Fisher's exact test (for cell frequencies n<5). Table 2 reports the baseline clinical, demographic features and disease characteristics of subjects followed up at 14 months with respect to disease severity.

Role of co-morbidities on antibody titers
It was observed that the subjects with one or more comorbidities (n=18) with respect to disease severity, developed a better antibody titer at 14 months as compared to the group (n=14) without any comorbidity. The p value was significant. (p=0.033). A study by Huang et al. also found that diabetes was associated with higher IgG levels. [13] This could also mean that subjects with a more severe disease due to one or more comorbidities had a better antibody titer at 14 months. Similar findings were observed by studies by Gudbjartsson  al. [14][15][16][17] However, larger studies are needed to draw stronger conclusions regarding these associations.

Role of Loss of smell and taste on antibody titers
It was observed that the subjects who experienced loss of smell and taste during infection, with respect to disease severity, developed a better antibody titer at 14 months. (p=0.043 and p=0.031 respectively). Although similar p values of significance were observed for lower antibody titers developed at 14 months in healthcare workers as compared to nonhealthcare workers (p=0.023), a generalized comment is not justified. A significant p value in such a situation could be due to n=6 subjects in the healthcare workers group and n=26 for non-healthcare workers resulting in bias.  Figure 2] In terms of disease severity, it was observed that in both the groups, the antibody titers developed at different Time periods (TPs) were discrete and statistically significant ( p value <0.001 for the mild group and the Moderately-Severe group). The results are described in Table 3 [Table 3] Since the data repeats over a period of time, multiple comparison test; i.e The Friedman's Two-way ANOVA test was applied. [ Figure 3] In order to highlight the significance of the TPs with respect to each other, pairwise multiple comparison tests; i.e Two/More related samples test was applied.

Role of disease severity on antibody titers
Median neutralization titer (MNT) was calculated for both disease severity groups. It was observed that the subjects of the moderately-severe group developed a higher median antibody tire (14.78 AU/ml) at 14 months when compared to the mild group (5.55 AU/ml) but the dispersion or variation was higher for the Moderately-Severe group, indicating a larger degree of variability in development of antibodies. The box plot shows the Median line at the 25th percentile in both the cases indicating that the data is positively skewed i.e., some values are towards the higher end. The subjects in the mild group show less variability in terms of developing antibody titers and have a smaller median while subjects of the Modereately-Severe group exhibit larger variation and also has a higher median.

DISCUSSION
The SARS-CoV-2 is an enveloped virus with four structural proteins: spike (S) protein, membrane (M) protein, envelope (E) protein and nucleocapsid (N) protein. Among these four structural proteins, the S and N proteins are the main immunogens. The S protein is a major protective antigen that elicits highly potent neutralizing antibodies (nAbs) and plays an essential role in viral attachment, fusion, entry, and transmission. The S protein comprises of an N-terminal Sl subunit responsible for virus-receptor binding and a Cterminal S2 subunit responsible for virus-cell membrane fusion. The S1 sub-unit is further divided into an N-terminal domain (NTD) and a receptor-binding domain (RBD). The RBD within Sl interacts directly with host receptors, human angiotensin converting enzyme 2 (hACE2). [18] The immunity against any infectious disease is comprised of two arms: innate immunity and adaptive or acquired immunity. Each of these arms contain humoral (B cells) and the cell mediated (T cell) immune elements.
Antibodies are synthesized and secreted by plasma cells that are derived from the B cells of the immune system and can be used as a correlate of immunity. Antibody tests also known as serological tests, detect the presence of antibodies against a particular diseasecausing agent in the blood, to evaluate the immune response against it. Antibodies can come in different varieties known as isotypes or classes which differ in their biological properties and ability to deal with different antigens and are called Immunoglobulins.
Immunoglobulin (IgM) eliminates pathogens in the early stages of B Cell mediated immunity and is a marker of active infection, while immunoglobulin G (IgG) provides long lasting antibody-mediated immunity and is the only antibody capable of crossing the placenta to give passive immunity to the fetus. Neutralizing antibody titers (and total Spike antibody titers) have a positive correlation with COVID-19 disease severity in large cohort studies. [7,11,23]. This was also observed in our study findings. The subjects in the moderately-severe group developed a higher Median Neutralisation Titre (14.78 AU/ml , p=0.003) at 14 months when compared to the Mild group (5.55 AU/ml , p<0.001). [ Table 4] This result was statistically significant.
However, the relationship between the neutralizing antibodies, T follicular helper cells (Tfh cells), and COVID-19 disease severity appears to be complex. A higher neutralizing antibody titer is associated with severe disease and potentially "extrafollicular B cell responses" [23] whereas the SARS-CoV-2-specific Tfh cells are associated differently.
Moreover, antibodies could act like a useful surrogate marker of CD4 + T cell responses in many infections, since antibody assays are much easier to perform and more sensitive in small blood volumes when compared to antigen-specific T cell assays. [24] A recent study by Abu-Raddad LJ et al in Qatar, assessed the cumulative risk as well as incidence rate of SARS-CoV-2 reinfection in a nationwide cohort of 43,044 antibodypositive individuals. This study with a follow period of up to 35 weeks, demonstrated and confirmed through viral genome sequencing that SARS-CoV-2 reinfection occurs, but "only rarely" with a cumulative risk of ~2 per 1000 persons and reinfection incidence rate of <1 per 10,000 person weeks as compared to the complement cohort of 149,923 antibody-negative persons with a much higher cumulative risk of re-infection (~31 per 1000 persons after 46 weeks of follow-up) and estimated incidence rate of infection (~14 per 10,000 person-weeks. ). The estimated efficacy of natural infection against reinfection was 95%. Moreover, this study showed no evidence of waning protective immunity against reinfection in this cohort for over 7 months. [25] An important point that needs to be highlighted in our study is zero cases of re-infection despite the fact that the Umbria region has been experiencing multiple waves with mutant strains. [11]

Adoption of anti-S-RBD immunoassay
The S1 subunit has low evolutionary protein homologies within the coronavirus family suggesting less cross-reactivities among the endemic coronaviruses, but the N proteinbased antibody assays exhibit a higher false-negative rate compared with the S1 subunit, making the anti-S-RBD assays more specific. Although the Nucleocapsid and Spike IgG titers are highly correlated [24], spike is the target of SARS-CoV-2 neutralizing antibodies, and the RBD of Spike is the target of >90% of neutralizing antibodies in COVID-19 cases [23,[26][27][28], with some neutralizing antibodies instead targeting the N-terminal domain (NTD). [29] Therefore, after following up the cohort for 10 months for anti NCP antibodies, a more specific assay was adopted that detected the presence of antibodies against the S-RBD. It was observed in our study the subjects , n=7 who were seronegative for anti-NCP antibodies at T5 , were found to be seropositive at T6, T7 and T8 for anti-S-RBD emphasizing on the fact that N protein-based antibody assays exhibit a higher falsenegative rate when compared with the anti-S-RBD assays. [ Figure 4]

Antibody seropositivity in the cohort
In this study, at T0, 2 months after the initial infection , 24 out of 30 (80%) subjects were positive for SARS-CoV-2 NCP IgG antibodies, followed by a slight dip with 20 out of 30 Our results are in line with previous studies showing a similar longevity and pattern of anti-SARS-CoV-2 Ab responses, with Ab levels reaching a peak at 23 days following symptom onset and being maintained for at least 4 months, [11,14,[30][31][32][33][34][35][36] yet contradictory to others, in which a low prevalence and rapid decay (within 3 months) of anti-SARS-CoV-2 Abs in COVID-19 patients with either mild or severe disease were observed. [37,38]

Average Antibody titers [Anti-NCP IgG and Anti-S-RBD IgG] developed at 14 months
The estimated marginal mean or average of antibody titers (in AU/ml) developed over different TPs such as T0-T5 and T6-T8 with respect to two different immunological assays against NCP and Spike protein respectively. It can be observed that the average of Anti-NCP IgG antibody titers tends to show a decreasing trend across T0-T5, whereas Anti-S-RBD IgG shows a better average of antibody titers, in terms of magnitude (seropositivity) across T6-T8.
In Conclusion, our study findings are consistent with recent studies reporting robust antibody persistency suggesting that induced SARS-CoV-2 immunity through natural infection, might be very efficacious against re-infection (>90%) and could persist for more than six months. Our study followed-up patients up to 14 months demonstrating presence of anti-S-RBD IgG in 96.8% of recovered COVID-19 subjects.
In such a scenario, prioritizing vaccination for "naive" individuals ( with no previous history of COVID-19 infection) and recovered but antibody-negative individuals would be helpful by saving time, effort and resources that could be used for the vulnerable populations.

Lower titers
Higher titers COLOR KEY FOR N=7 PATIENTS