key: cord-0758355-ny00gjqp
authors: Alkharaan, Hassan; Bayati, Shaghayegh; Hellström, Cecilia; Aleman, Soo; Olsson, Annika; Lindahl, Karin; Bogdanovic, Gordana; Healy, Katie; Tsilingaridis, Georgios; De Palma, Patricia; Hober, Sophia; Månberg, Anna; Nilsson, Peter; Pin, Elisa; Sällberg Chen, Margaret
title: Persisting Salivary IgG against SARS-CoV-2 at 9 Months After Mild COVID-19: A Complementary Approach to Population Surveys
date: 2021-05-12
journal: J Infect Dis
DOI: 10.1093/infdis/jiab256
sha: 359e6c035bd81f0cd1709a10ea07f80a37ce6105
doc_id: 758355
cord_uid: ny00gjqp

BACKGROUND: Declining humoral immunity in COVID-19 patients and the possibility of reinfection have raised concern. Mucosal immunity, particularly salivary antibodies, may be short-lived although long-term studies are lacking. METHODS: Using a multiplex bead-based array platform, we investigated antibodies specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in 256 saliva samples from convalescent patients 1-9 months after symptomatic COVID-19 (n=74, Cohort 1), undiagnosed individuals with self-reported questionnaires (n=147, Cohort 2), and individuals sampled pre-pandemic time (n= 35, Cohort 3). RESULTS: Salivary IgG antibody responses in Cohort 1 (mainly mild COVID-19) were detectable up to nine months post-recovery, with high correlations between spike and nucleocapsid specificity. At nine months, IgG remained in both blood and saliva in majority of patients. Salivary IgA was rarely detected at this timepoint. In Cohort 2, salivary IgG and IgA responses were significantly associated with a recent history of COVID-19 like symptoms. Salivary IgG also tolerated temperature and detergent pre-treatments. CONCLUSIONS: Unlike SARS-CoV-2 salivary IgA that appeared short-lived, the specific IgG in saliva appeared stable even after mild COVID-19 as noted for blood serology. This non-invasive saliva-based SARS-CoV-2 antibody test with home self-collection may therefore serve as a complementary alternative to conventional blood serology.

M a n u s c r i p t 4

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbroke in an abrupt fashion after its initial identification in Wuhan, China, in late December 2019 (1), and obligated the World Health Organization to declare a global health emergency which escalated the concern to a pandemic situation in March 2020. As of March 2021, SARS-CoV-2 has caused over 114 million cases of coronavirus disease and up to 2.5 million global deaths (2). The human adaptive immune system plays a key role in eliminating and memorizing pathogens by launching a cascade of activities that activate B and T lymphocytes. B lymphocytes produce antibodies that recognize and neutralize SARS-CoV-2 and protect against reinfection (3) (4) (5) . IgG, IgA, and IgM antibodies are activated against SARS-CoV-2 and detected in the circulating blood of >90% of infected individuals from the 11-13 day post-symptom onset (PSO) (6) (7) (8) . A recent study showed that circulating antibodies post-SARS-CoV-2 infection can persist for up to eight months (9) , while other studies have shown that this immunological memory persists for a certain period followed by a slight decline, especially in asymptomatic infected individuals (10) (11) (12) (13) (14) .

Oral and nasal cavities are considered the main gate for SARS-CoV-2 virus entry, and saliva secretory antibodies may be the first immunity arm that combat the infection through virus recognition. Salivary antibodies to SARS-CoV-2 can be detected early after symptom onset and persist for at least three months post-infection (8, 10, 12) . Hence, saliva sampling could be a sensible and non-invasive way to indicate SARS-CoV-2 exposure. Similar to the previous SARS-CoV and MERS-CoV viruses, the spike protein (S) of SARS-CoV-2 recognizes the angiotensin-converting enzyme 2 (ACE2) receptor and uses it to enter host cells (15) (16) (17) . Although antibodies play an important role in virus clearance (11, 18) , differential features of anti-SARS-CoV-2 antibodies negatively impacting disease severity, especially those related to complement deposition and systemic inflammation is also described (19) . Understanding the dynamics and durability of antibody memory to SARS-CoV-2 is an instrumental step to manage the pandemic and may be useful in deploying A c c e p t e d M a n u s c r i p t 5 vaccination strategies. As the mucosal immunity is known to be short-lived, the durability of SARS-CoV-2 specific antibodies in saliva could be limited. Whether they permit detection 3-4 months after infection (8, 10) is of great interest.

In this study, we exploited a highly sensitive and specific multiplex SARS-CoV-2 serology platform previously validated for seroprevalence studies (20) to investigate SARS-CoV-2 antibodies in saliva. Samples from individuals 1) with a diagnosis of mild COVID-19 in the convalescent phase; 2) 1-9 months after diagnosis of COVID-19, and 3) with or without a history of COVID-19 symptoms (undiagnosed), were analysed and compared to prepandemic samples. Our data indicates that spike-specific IgG reactivity is detectable in saliva in the vast majority of patients at 1-9 months post-infection. This result was similar to those detected by blood serology performed in a clinical diagnostic laboratory. The IgA reactivity on the other hand was short-lived in saliva, detectable only during the first three months. Moreover, IgG and IgA reactivity to both spike and nucleocapsid significantly correlated with a history of COVID-19 like symptoms in undiagnosed individuals.

We applied a bead-based serology assay to detect IgG and IgA to SARS-CoV-2 proteins in saliva samples to evaluate its performance. The assay method is originally developed for detection of SARS-CoV-2 specific IgG in serum and plasma (20) where it showed 99.7% sensitivity and 100% specificity, and no cross-reaction when testing samples positive for other Coronaviruses. Salivary antibody responses to three different SARS-CoV-2 antigens (two spike and one nucleocapsid proteins) were first tested. The antigens´ performance in classifying positive and negative samples was evaluated for the single antigens as well as for antigens combined in panels. Best performing representations of spike and nucleocapsid were chosen in subsequent assessments.

A c c e p t e d M a n u s c r i p t 6

The study was approved by the human ethical authority (dnr 2020-01702, 2020-06381) and complied with the declaration of Helsinki. All participants were recruited after signing informed consent forms. Saliva samples (total n=256) were collected and arranged in the based on their health condition in the three months prior to sampling.

Expectorated unstimulated whole saliva was used throughout this study. All samples were self-collected using standardized instructions and sample tubes provided by this study.

Samples were processed and stored at -80°C within 24 h. Salivary stability tests were performed on sample subgroups to evaluate antibody reactivity following viral inactivation with either 1% Triton X-100 for 1 h at room temperature (RT), or heat-treatment at 56°C for 30 min (19) . Eighteen antibody-positive from Cohort 1 and, then antibody-negative samples from Cohort 2 were included in the comparison. Incubation at RT for one to three days was also tested in five samples to simulate the standard circumstances of the mailed-in saliva self-collection procedure. Saliva samples from convalescent patients (Cohort 1) were collected on same day as venous blood during a COVID-19 follow-up examination at department of Infectious Diseases, Karolinska University Hospital. 

The proteins were produced as follows: 1) Spike glycoprotein (Spike-f) in a soluble trimeric form stabilized in its prefusion-conformation, was expressed in HEK293 cells and purified using a C-terminal Strep II tag; 2) Spike S1 domain was expressed in CHO cells and purified using a C-terminal HPC4-tag; 3) Nucleocapsid C-terminal (NC-C) chain, were both expressed in E. coli and purified using a C-terminal His-tag (21, 22) .

The analysis of salivary antibodies was performed as previously described (20) 

Statistics and visualization of the multiplex bead array generated data were performed using 

The assay performance was evaluated by comparing the ability of each of the three antigens included in the assay to classify convalescent samples (Cohort 1, n=74) and pre-pandemic samples (Cohort 3, n=35), of which 12 samples from Cohort 3 were used to set the assay cut-offs. Among the three antigens, spike glycoprotein (Spike-f) and C-terminal fragment of the nucleocapsid (NC-C) showed the best performance in classifying SARS-CoV-2 convalescent samples from the pre-pandemic samples. Spike-f showed 88% sensitivity and A c c e p t e d M a n u s c r i p t 10 100% specificity, with one negative control sample reaching intensity signal at the cut off level. NC-C showed 66% sensitivity and 100% specificity (Table 1) . Here, we also evaluated the assay performance for all combined antigen panels of 2 and 3 antigens, considering a sample as positive when reactive to both antigens in a panel-of-two antigens and to at least two out of three antigens in a panel ( Table 1) . As noted, the best performance was reached by the Spike-f, S1, NC-C triple combination, showing 72% sensitivity and 100% specificity.

On the other hand, the IgA reactivities were identified only in a minority of cases, with higher prevalence of reactivity to Spike-f (17%) in this cohort (Table S1 ). It should be noted that larger sample sets are needed in order to establish and validate these sensitivity and specificity levels.

As shown in Table 2 , Cohort 1 mainly comprised patients who have had mild COVID-19 and were grouped according to duration after confirmed diagnosis. Some were hospitalized for isolation, but none received oxygen treatment or required ventilation-related treatment. All individuals were free from respiratory symptoms at the nine month follow-up but some residual symptoms were still noted in a minority of patients across all three groups (data not shown). As shown in Table 3 Moreover, salivary IgG to Spike-f and NC-C were highly correlated in this cohort (r=0.88, p<0.0001, Spearman correlation test), with concordant serostatus in the majority of samples (Figure 1b) . Significant, albeit moderate, correlations were also seen between IgA to Spike-f

and NC-C (r=0.62, p<0.001), and between Spike-f specific IgA and IgG (r=0.45, p<0.001) (Figure 1b) .

Next, we applied this assay platform to evaluate a second independent cohort: Cohort 2.

Participants here were self-reporting symptom-free individuals visiting the University Dental

Clinic's premises of Karolinska Institutet and the Eastman Institute in Stockholm. A total of 147 individuals from May to November 2020 participated and donated saliva samples.

Samples were collected and tested using the same standard operating protocol as for Cohort 1. Shown in Figure 2a and based on antigen-specific cutoffs calculated on 12 negative controls, antibody reactivities to Spike-f and NC-C in this cohort were as follows:

IgG was detected in 14% to Spike-f and 15% to NC-C, while 11% had detectable IgG to both antigens; for IgA, 14% and 6% of the samples showed reactivity to Spike-f and NC-C respectively, while only 6% showed reactivity to both. Salivary positivity was particularly enriched among participants with a self-reported recent history of COVID-19-like symptoms (14 days to three months prior to sampling time). Significant reactivities of IgG (p=0.004, and p=0.01) and IgA (p<0.0001, and p=0.044) to either Spike-f or NC-C was found to associate with a recent history of symptoms compared to pre-pandemic controls (Figure 2a) . Adding risk factors with symptoms further enriched the salivary IgG positivity. This includes recent A c c e p t e d M a n u s c r i p t 12 Covid-19 contact, travelling abroad, or clinical duties which increased IgG positivity to 23%, 15% or 13% respectively to Spike-f; and 23%, 19% or 17% to NC-C (Figure S1b) .

A correlation analysis (Figure 2b) 

Next, the effects of virus inactivation by heat treatment (HT) at 56°C for 1 h, 1% Triton X-100

(Triton) as well as room temperature (RT) (identical aliquots left out for indicated time) on the antibody results were determined (Figure 3) . Both HT and Triton showed little change in the cut-off (based on the ten included negative controls). A good correlation between treated and non-treated samples was noted (Figure 3 and S2 Severe COVID-19 symptoms have been shown to induce strong antibody responses in 99% of convalescent individuals, but published data also shows that these antibody responses tend to decline slower than in mild symptomatic cases (6, 9, 16, 19) . This may be attributed to the fact that tests developed earlier in the pandemic were based on detection of samples from severe COVID-19 cases resulting in sub-optimal sensitivity to mild infections (24) .

Furthermore, many of the initial test kits used the nucleocapsid as a target antigen and antibodies against it have been shown to decline more rapidly (25) , as also demonstrated here. In this study, we deliberately recruited convalescent samples from mild COVID-19

patients, in order to evaluate the multiplex antibody platform was capable to detect SARS-CoV-2-specific antibodies in saliva in such patients. In the present study, saliva reactivities were compared against blood serology using certified diagnostics (including anti-N pan-Ig M a n u s c r i p t 14 ECLIA), which show high performance in detecting late convalescent blood samples. In fact, our result is in line with a recent South Korean group reporting this diagnostic antibody assay is, among several others, effective in detecting SARS-CoV-2 antibodies in blood (90%) up to eight months after either asymptomatic infection or mild symptomatic cases (26) . Here, the persistence of salivary IgG to structural viral proteins in the saliva samples nine months postrecovery from mild COVID-19 is intriguing, and possibly explained by a secondary exposure or spill-over from blood circulating responses. More studies are therefore warranted to clarify the mechanism underlying the magnitude of salivary responses with better matched study participants. It has been shown that the mucosal antibody response is triggered slightly earlier than the systemic response upon infection (10) . Information is still limited about the duration and kinetics of mucosal antibodies secreted into the mouth and nose, particularly in this patient group. A sensitive salivary antibody detection assay with the capability to identify infections with various severities would contribute to improving the current understanding of mucosal antibodies to SARS-CoV-2. For instance, such studies may compare low versus high avidity antibodies and their relation to neutralization or disease enhancement (10, 27, 28) . The advantage of multiplexed assays for antibody detection is that they allow to minimize the sample consumption and increase the throughput by maintaining high sensitivity and specificity. One examples is the recent large-scale screenings of crossreactivities to multiple pandemic or endemic coronaviruses (29) , and capacity to contribute at-home testing for COVID-19 telemedicine diagnosis and monitoring as proposed recently by Torrente-Rodríguez et al. (30) The hypothesis that antibodies towards previously known coronaviruses may block SARS-CoV-2 has raised questions about their functionality. However, such antibodies are known to be protective for only around six months after infection, and would therefore have disappeared by the time of emergence of SARS-CoV-2 (31,32). Clearly, further assessment of neutralizing capacity against Sars-Cov-2 virus and related coronavirus in human saliva is A c c e p t e d M a n u s c r i p t 15 necessary. Other important applications for saliva immunoassays include evaluation of vaccine-induced mucosal immunity, which is in fact ongoing in our laboratories for monitoring of local antibody recognition of virus mutations or vaccine-escape mutants. Since mouth and nose are the first ports of entry for SARS-CoV-2, sensitive and accurate methods for quantitative measurements of local immunity will lead to better means to combat COVID-

One limitation of our study was the relatively small sample size and the predominantly male population. Another weakness was that blood samples were not analyzed in the same way as saliva and as several diagnostic assays were used, only binary data was provided. Also, because of the cross-sectional design, we could not obtain baseline or longitudinal saliva samples. Moreover, we could not assess individual possibilities of re-exposure or reinfection.

However, it is unlikely that humoral immunity was boosted because in Stockholm, where the study took place, the period June-Nov 2020 (second-wave) showed an increase in the daily incidence rate of COVID-19 from 30 to 400 cases/100,000 population (33). In conclusion, despite waning immunity concerns, the present study shows that our multiplex bead-based immunoassays can detect antibodies against SARS-CoV-2 in late convalescence saliva up to nine months after mild COVID-19. 

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All study participants who took interest in this study.

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