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Persistent Antibody Responses up to 18 Months after Mild SARS-CoV-2 Infection

Abstract Background Humoral immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may wane rapidly in persons recovered from mild coronavirus disease 2019 (COVID-19), but little is known about the longevity. Methods Serum samples were obtained 8, 12, and 18 months after infection from 20 patients with mild COVID-19. The binding activities of serum antibodies (IgA, IgG, and IgM) against SARS-CoV-2 antigens of the Wuhan-1 reference strain (wild-type) and the B.1.1.7, P.1, B.1.167.2, and B.1.1.529 variants were measured by enzyme-linked immunosorbent assays. Neutralizing antibody titers were measured using a cytopathic effect-based live virus neutralization assay. Results Serum IgA and IgG antibodies against spike or receptor-binding domain (RBD) protein of wild-type SARS-CoV-2 were detected for up to 18 months, and neutralizing antibodies persisted for 8 to 18 months after infection. However, any significant antibody responses against RBD proteins of SARS-CoV-2 variants were not observed, and median neutralizing antibody titers against the Delta variant at 8, 12, and 18 months were 8–11 fold lower than against wild-type viruses (P < .001). Conclusions Humoral immunity persisted for up to 18 months after SARS-CoV-2 infection in patients with mild COVID-19. Humoral immune activity against more recently circulating variants, however, was reduced in this population.


INTRODUCTION
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection induces humoral immune responses, including the production of neutralizing antibodies, even in patients with asymptomatic or mild coronavirus disease 2019 (COVID-19) [1]. Neutralizing antibodies play an essential role in virus clearance and have been considered a critical component of the immune system for protection against viral diseases. The persistence of these antibodies is, therefore, clinically meaningful. The initial humoral response is not as strong in patients with mild than with more severe COVID-19, raising concerns about waning immunity in patients with mild disease [2,3]. In addition, the duration of the humoral responses was found to vary more in patients with mild COVID-19 than in those with severe disease [4].
In response to the COVID-19 epidemic, the Korean government established non-hospital facilities called "community treatment centers (CTCs)" for the isolation of patients with mild diseases [5,6]. Individuals were admitted to the CTCs if they were alert, age < 65 years, had no or well-controlled underlying disease, and did not have dyspnea. Therefore, the CTCs provided a unique opportunity to study patients with mild COVID-19, including determinations of cross-sectional serologic responses 8 and 12 months after infection [7,8]. The present study comprehensively analyzed A c c e p t e d M a n u s c r i p t

Study Participants and Design
Patients were eligible for this study if they were previously healthy; were diagnosed with mild COVID-19, as confirmed by real-time reverse transcription-PCR assays; and had been isolated in a CTC operated by Seoul National University Hospital (SNUH) from March 5 to April 22, 2020 [5]. During their stay in a CTC, all patients were comprehensively evaluated twice daily by physicians and nurses using a video consultation system and were confirmed as not having shortness of breath, dyspnea on exertion, or abnormalities on chest radiography [6]. Asymptomatic infection was defined as those who had no symptoms for the duration of their CTC stay, and mild illness was defined as those who had COVID-19 symptoms but did not have shortness of breath, dyspnea, or abnormal chest imaging, according to the World Health Organization's definition of COVID-19 severity [9]. These patients did not receive specific treatments, such as antiviral agents, anti-SARS-CoV-2 monoclonal antibodies, or convalescent plasma.
Blood samples were prospectively collected 8, 12, and 18 months after SARS-CoV-2 infection, and serum was separated within 6 h after blood collection and stored at -80°C. At each time of blood sampling, infectious disease specialists interviewed the participants to obtain clinical information, including re-exposure to SARS-CoV-2, vaccination against SARS-CoV-2, and morbidity or medication potentially affecting immune responses. Serum samples were also collected from healthy volunteers who participated in a SARS-CoV-2 vaccination study [10].
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Ethical Approval
The Institutional Review Board of SNUH approved this study (IRB no. H-2009(IRB no. H- -168-1160, and all participants provided written informed consent in accordance with the Declaration of Helsinki. USA) and his-tagged SARS-CoV-2 proteins were purified according to the manufacture's instructions using Ni-NTA agarose resin (Thermo Fisher Scientific) affinity chromatography, as described [12]. A commercially available SARS-CoV-2 nucleocapsid recombinant protein was also used (Thermo Fisher Scientific, Waltham, MA, USA).  Anti-S1 (spike subunit) IgG antibodies were semi-quantitatively measured using a commercial ELISA kit (Euroimmun, Lübeck, Germany) according to the manufacturer's instructions. Optical density ratios (sample/calibrator) of ≥1.1, ≥0.8 to <1.1, and <0.8 were regarded as positive, borderline, and negative, respectively, according to the manufacturer's instructions.

Neutralizing Antibody Live Virus Assay
Neutralization assays based on cytopathic effect (CPE) were performed as previously described [13]. Briefly, 50 μL serum samples diluted from 2-to 4,096-fold were added to 50 μL of cell culture medium containing 50 times the tissue culture infective dose of the WT virus without the D614G mutation (BetaCoV/Korea/SNU01/2020) [14] or the Delta variant virus (isolated from SNUH, GenBank accession no.MZ853946) in each of a 96-well plate, and the plates were incubated at 37 °C for 2 h in CO2 5% vol/vol. Virus antibody mix was added to these wells, and the plates were incubated at 37°C for 5 days. CPE was assessed after 5 days by microscopic examination. The highest dilution of each serum sample that inhibited SARS-CoV-2 activity was defined as the neutralizing antibody titer. Each assay was performed in duplicate with negative control samples.
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Graphs and Statistical Analysis
Data were analyzed and were visualized using GraphPad Prism ver. 9.2.0 software (San Diego, CA, USA). Immune responses were reported as geometric mean titers (GMT) with 95% confidence intervals (CIs) for neutralizing antibody titers at each timepoint. Differences between GMTs at various timepoint differences were analyzed by Wilcoxon matched-pairs signed-ranks test with two-sided P values < .05 considered statistically significant.

DISCUSSION
Recent reports on COVID-19 vaccines with strong efficacy provide hopes for pandemic control [15,16], and many nations are pursuing the rollout of SARS-CoV-2 vaccination as an exit strategy from unprecedented social distancing [4]. However, achieving herd immunity among the population depends critically on the duration of protective immunity resulting from natural infection and vaccination. Therefore, determining the longevity of humoral immunity against SARS-CoV-2 among individuals who have recovered from COVID-19 is essential for predicting herd immunity. The present study describes binding and neutralizing antibody responses against WT and variant viruses for up to 18 months after infection in individuals who have recovered from mild COVID-19, a group more susceptible to waning immunity than those who have recovered from more severe COVID-19.
A c c e p t e d M a n u s c r i p t Early reports have shown that the antibody titers of patients with mild SARS-CoV-2 infection declined more rapidly than those reported for patients infected with SARS-CoV-1 [17], and neutralizing antibodies acquired upon natural infection could disappear within 3 months [18]. Antibody titers in patients recovering from Middle East respiratory syndrome (MERS) were found to decrease rapidly during the first 6 months after infection, but to decrease less steeply between 6 months and 1 year after infection and to be maintained over a longer period [19]. Antibody kinetics profiles in patients recovering from COVID-19 were similar to those in patients recovering from MERS [20], with humoral immunity against SARS-CoV-2 remaining relatively stable for up to 8-12 months [21][22][23][24].
This study found that antibody titers against spike and RBD WT proteins persisted for 18 months in this patient cohort without significant change. These results indicate that patients recovering from COVID-19 have long-lived plasma cells producing anti-spike and RBD antibodies, as reported previously [25]. Although mucosal IgA level was not directly measured in the present study, the persistence of the serum IgA response to S protein suggests that these antibodies contribute to viral clearance in mucosal membranes for up to 18 months after infection [26]. The present study also found that neutralizing antibody activities against SARS-CoV-2 persisted for up to 18 months. This finding is consistent with recent reports that humoral immunity to SARS-CoV-2 is maintained for at least 1 year after infection, and that primary SARS-CoV-2 infection decreased the risk of subsequent infection by 77-83% for at least 12 months [27]. Our results suggest that humoral immunity and protection from reinfection with the original virus causing the primary infection may persist for over 18 months, even after mild infection. M a n u s c r i p t Serum antibodies in our cohort were generated by infection with SARS-CoV-2 WT, but the current circulating SARS-CoV-2 variants of concern have several mutations in their spike and RBD proteins, resulting in high transmissibility and potential immune escape [28]. Thus, we tested the binding activities of serum antibodies in these patients against the RBD proteins of the SARS-CoV-2 Alpha, Gamma, Delta, and Omicron variants. These serum samples rarely showed significant antibody responses against the RBD proteins of the SARS-CoV-2 variants and barely neutralized the Delta variant, implying that these patients are susceptible to reinfection by these variant viruses.
Although these SARS-CoV-2 variants of concern have been reported to be less susceptible to neutralization by serum from vaccinated individuals [29], the effectiveness of two-dose AstraZeneca and Pfizer vaccines have been reported to be 60% and 88%, respectively [30]. Furthermore, although pre-vaccination sera from previously infected persons rarely neutralized the Beta variant in vitro, a single dose of vaccine boosted neutralizing antibody titers against all variants by up to 1000-fold [31]. Vaccination of persons previously diagnosed with COVID-19 was found to increase anti-RBD IgG to levels that strongly neutralize the Alpha and Beta variants regardless of pre-vaccine IgG levels and vaccine type [32]. These results suggest that vaccination augments immune responses, even to variants to which infected individuals had not been previously exposed. Indeed, a recent study showed that unvaccinated individuals were 2.34 times likely to be reinfected with COVID-19 than those who were fully vaccinated after initial SARS-CoV-2 infection [33]. These findings, together with our results, indicate that persons who have recovered from mild SARS-CoV-2 infection should be offered the COVID-19 vaccine.
A c c e p t e d M a n u s c r i p t The limitations of the present study include relatively small sample size and the predominance of healthy young individuals, reducing the ability to generalize from our results. However, the substantial heterogeneity in antibody responses among individuals with mild SARS-CoV-2 infection [34] suggests that results obtained from our study cohort, with relatively homogenous age and disease severity, may help to determine the longevity of immune responses to SARS-CoV-2 infection.
In conclusion, humoral immunity in persons who had recovered from mild COVID-19 was found to persist for up to 18 months after SARS-CoV-2 infection.
Humoral immune activity against recently circulating viral variants was significantly blunted, however, underscoring the need for vaccination of this population. M a n u s c r i p t  A c c e p t e d M a n u s c r i p t