key: cord-0304731-4pf66wmi authors: Tsukinoki, K.; Yamamoto, T.; Saito, J.; Sakaguchi, W.; Iguchi, K.; Inoue, Y.; Ishii, S.; Sato, C.; Yokoyama, M.; Shiraishi, Y.; Kato, N.; Shimada, H.; Makabe, A.; Saito, A.; Tanji, K.; Nagaoka, I.; Saruta, J.; Yamaguchi, T.; Kimoto, S.; Yamaguchi, H. title: Prevalence of Salivary IgA Reacting with SARS-CoV-2 among Japanese People Unexposed to the Virus date: 2022-01-10 journal: nan DOI: 10.1101/2022.01.09.22268986 sha: f2e2d030689676a7a0f0adba0afa14b50562f70c doc_id: 304731 cord_uid: 4pf66wmi While the COVID-19 pandemic caused by SARS-CoV-2 has posed a threat to public health as the number of cases and COVID-19-related deaths are increasing worldwide, the incidence of the virus infection are extremely low in Japan compared with many other countries. To explore the reason for this strange phenomenon, we hypothesized the high prevalence of natural secretory IgA in saliva as mucosal IgA reacting with SARS-CoV-2, and thus surveyed the positivity for, as well as levels of, such reactive salivary IgA in a cohort of Japanese people of a wide range of age. The major findings were that 95/180 (52.78 %) of overall individuals who had not been exposed to SARS-CoV-2 were positive for salivary IgA with the levels ranging from 0.002 to 3.272 ng/ml, and that there may be a negative trend in positivity for salivary IgA according to age. These results suggest a role of mucosal IgA in host defense against SARS-CoV-2 infection. SARS-CoV-2 primarily infects the mucosal surfaces of the nasopharynx and the upper respiratory tract (16, 17) , as well as the oral cavity (18) , at least until advanced stages of the disease (COVID- 19) when viral RNA may become detectable in the circulation. The virus also infects the glands and mucosae of the oral cavity which harbor epithelial cells expressing angiotensin converting enzyme 2 (ACE2) and several other receptors for the virus spike (S) proteins, particularly the receptor binding domain (RBD) (18) (19) (20) . These findings also underscore a crucial role of sIgA in protecting mucosal surfaces against SARS-CoV-2 by neutralizing the virus and/or impeding its attachment to epithelial cells at the initial stage of the virus infection. There is recent evidence showing that anti-SARS-CoV-2 immunity not only occurs after a natural infection, but may also precede such an active infection. Cross-reactivity to SARS-CoV-2 antigen peptides has been identified on T-cells and B-cells from pre-pandemic donors; S proteinreactive CD4+ T-cells are not only detected in a large fraction of patients with COVID-19 but also in a smaller but considerable fraction of the healthy individuals with no history of COVID-19 (21, 22) . Consistent with this, it was also demonstrated that antibodies, probably including polyreactive natural autoantibodies (23) , cross-reacting with SARS-CoV-2 have been detected in healthy individuals unexposed to the virus (9, 24, 25) , and that a cross-reactive human IgA monoclonal antibody, which binds to SARS-CoV-2 S proteins with resulting in competitive inhibition of ACE2 receptor binding, has been successfully developed (26) . All these findings led us to the hypothesis that lower population-based susceptibility to SARS-CoV-2 infection seen in Japan compared to G7 and many other countries is associated with higher prevalence of sIgA reactive with the virus among Japanese people. Therefore, as an initial step to approach this possibility, we implemented a cohort study with healthy Japanese participants unexposed to SARS-CoV-2, in which we measured concentrations of the virusreactive mucosal IgA (sIgA) using saliva samples because sIgA, as a secreted antibody, is noninvasively accessible in saliva (27, 28) . Measurements were also performed for SARS-CoV-2-reactive IgA and IgG concentrations in serum for comparison. Consent to the present study was provided and the questionnaire was completed by 139 adult participants (aged ≥ 20 years) themselves or by each legal representative of 41 juvenile participants (aged <20 years). Thus, both saliva and blood samples and only saliva samples were collected from all the enrolled adults and juveniles, respectively. All of the 180 participants had no clinical history nor current status consistent with COVID-19 and tested negative for SARS-CoV-2 by RT-PCR. All of the 180 individuals were tested whether they had detectable amounts of SARS-CoV-2reactive salivary IgA or not. The results showed that among them 95 individuals (52.78 %) tested positive as shown in Table1. The fraction that tested positive did not differ with gender, but varied by age; higher and lower positivities were observed for the youngest age group (<20 years) and the oldest age group (≥51 years), respectively, compared to the two intermediate age groups (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) years and 36-50 years groups). Thus, it was suggested that there is a negative trend of positivity for saliva IgA according to age. No association was found for salivary IgA between test results and comorbidities, history of any vaccinations or daily oral care and/or dietary habits (data not shown). We observed a broad range of reactive salivary IgA concentrations with an over 1,600−fold difference between the extremes (0.002 and 3.272 ng/ml) among the 95 test-positive individual . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; samples. However, the results of 90 of the 95 samples (94.7 %) clustered around a median of 0.102 ng/ml (Fig.1) . Similar distribution patterns and medians of levels for test-positive samples were also observed in each age group; medians for <20 years-, 20-35 years-, 36-50 years-and >50 years-groups were 0.111, 0.134, 0.067 and 0.121 ng/ml, respectively. Three test-positive samples with exceptionally high reactive salivary IgA levels between 1.03 and 3.27 ng/ml were derived from individuals in the 20-35 years-group (1 sample) or the 26-50 years-group (two samples). One hundred and thirty-nine adult participants provided matched saliva and serum samples collected during the same visit. All samples were tested multiplex assay for measuring levels of SARS-CoV-2-reactive IgA in saliva, as well as those of equivalent IgA and IgG in serum. Sixtyeight (48.92 %), 24 (17.27 %) and 17 individuals (12.23 %) were positive for SARS-CoV-2reactive salivary IgA, serum IgA and serum IgG, respectively ( Table 2 ). The fraction that tested positive for each Ig equivalent varied by age and BMI. A negative trend of test positivity according to age and BMI were observed for serum IgG and salivary IgA, respectively, while the proportion of test positivity for serum IgA and serum IgG in the fraction of BMI at a reference interval (20) (21) (22) (23) (24) (25) was higher than the other two BMI fractions (Table 2) . We also conducted correlation analysis to learn possible correlation of the test positivity for saliva IgA with that for serum IgA and/or serum IgG. The results showed no significant correlation with the value for either serum Ig equivalents (Table 3) . Our study aimed to survey the presence of sIgA in saliva, together with IgA and IgG in serum, capable of reacting with SARS-CoV-2 among Japanese people of a wide range of age from infants to the elderly who had been unexposed to the virus. The results evidenced the presence of salivary IgA reactive with a SARS-CoV-2 S1 antigen at detectable levels in a substantial fraction of enrolled individuals of all ages. Indeed, 95/180 (52.78 %) of overall individuals were positive for anti-SARS-CoV-2 salivary IgA. In addition, we found that there may be a negative trend in positivity for the virus-reactive saliva IgA according to age; the highest and the lowest prevalence of positivity were observed for the youngest age group (<20 years, 65.85 %) and the oldest age group (≥51 years, 40.38 %), respectively, compared to two intermediate aged adult groups. Recently, a huge body of evidence has accumulated that susceptibility to SARS-CoV-2 infection generally increases with age. Compared to younger / middle-aged adults, children are less susceptible to the virus infection, while estimated susceptibility in older adults is considerably higher (29) (30) (31) (32) (33) (34) (35) (36) (37) . Particularly, low fragility of children and adolescents against SARS-CoV-2 has become a matter of epidemiological and virological concern and intensive studies have been performed to identify the immune mechanisms implicated. As a result, several protective mechanisms including differences in the timing and nature of the induced cytokine responses and high amounts of ACE2 expressed, as well as trained immunity acquired from frequent viral infections and/or routine vaccinations, have been hypothesized (38) . High positivity for SARS-CoV-2-reactive salivary IgA in children and adolescents, compared to adults, as observed in the present study may contribute to lower their susceptibility to the virus. So far as concerned with adult individuals, our results are in accordance with the data precedently presented by Tsukinoki and colleagues, who described a rate of positive SARS-CoV-2 cross-reactive saliva IgA as high as 46.7 % among the virus-uninfected professional workers of Kanagawa Dental University Hospital with being significantly lower in prevalence . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; of positivity for individuals aged ≥ 50 years compared with those aged ≤ 49 years (39) . The results of these two studies indicating that there is a decrease in SARS-CoV-2-reactive saliva IgA with increasing age are in line with preceding researches (40) (41) (42) . There are numerous reported human studies which indicated the association of low levels of or decreases in salivary IgA with incidence of upper respiratory infections, mostly common cold, in various study cohorts, such as infants (43), healthy children (44) (45) (46) (47) , children with adenoid hyperplasia (48) , children with Down's syndrome (49) , elite athletes (50) (51) (52) (53) , individuals during intense physical training (54, 55) and those who had undergone tonsillectomy or adenoidectomy in childhood (56) . These results suggest that individuals who are deficient in salivary IgA, particularly those who are negative for salivary IgA reactive with SARS-CoV-2, may be at higher risk for the virus infection. In this context, unexpectedly high rates of SARS-CoV-2reactive salivary IgA positivity seen in unexposed Japanese participants could be a part at least of the reason for extremely low incidence of COVID-19 in this country. We also found that SARS-CoV-2-reactive antibodies are contained not only in salivary IgA but also in serum IgA and serum IgG. However, there was no correlation in the positivity or level of the virus-reactive antibodies between saliva IgA and both of the two Ig isotypes existing in serum. This may be probably due to different mechanisms involved in regulation of generation and/or dynamics of each type of Igs. It is well accepted that sIgA, a major component of salivary IgA, is generated and released at mucosal inductive site tissues, while the serum counterpart is, like IgG, derived from a distinct source, the bone marrow (57, 58) . Our postulate is that all of salivary IgA, serum IgA, and serum IgG antibodies reacting with SARS-CoV-2 might work cooperatively through binding to a common site RBD on S1 proteins of the virus, even though their binding affinities are different. The present study has both strengths and limitations. By implementing this immunological survey in a community-based cohort study with a wide age range of healthy Japanese people who had been unexposed to SARS-CoV-2, we were able to collect preliminary but unprecedented epidemiological data regarding the prevalence and levels of assumable polyreactive natural salivary IgA autoantibodies that exhibit reactivity with the virus. To the best of our knowledge, our survey, along with the preceding one (38) , is the only available study which has provided data useful for considering a putative role mucosal natural IgA antibodies in protecting the human host from SARS-CoV-2 infection. Supportably, the presence of natural polyreactive sIgA autoantibodies acting as the frontline of mucosal defense against various infections has been demonstrated (59) . Our study has several limitations. Firstly, our sample size was not large with limiting the robustness of our findings in saliva, as well as in serum. If such samples could be increasingly available, statical power for the analyses presented here will be increased. Secondly, we do not know whether and what levels of the salivary IgA detected in the presented study are protective through neutralization against SARS-CoV-2 infection. Thirdly, because of the lack of follow-up data, it was also unable to directly correlate negativity for or low levels of SARS-CoV-2-reactive salivary IgA with the feasibility to have the virus infection. Nevertheless, the results obtained from the present study still lead us to favor the hypothesis that "natural" salivary IgA could contribute to lower the susceptibility to SARS-CoV-2 infection. According to the latestly publicized data, there is no significant difference in the completion rate of vaccination among the G7 Countries ranging from 60.1 % for USA to 77.7 % for Japan (as of December 6, 2021) (60). Despite that, the mean number of new COVID-19 cases per 1,000,000 people during the first week of December, 2021 for Japan was extremely low of 0.81 compared . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; with the equivalents for other six G7 Countries as follows: 358.56 (USA); 686.26 (UK); 656.85 (France); 622.66 (Germany); 242.71 (Italy); and 83.79 (Canada) (4). Considering these global epidemiological data and our findings that a larger fraction of Japanese people are conferred with SARS-CoV-2-reactive salivary IgA, it is probable that mucosal immunity due to natural sIgA autoantibodies play a positive role in achieving herd immunity. In conclusion, our preliminary results, obtained with SARS-CoV-2 unexposed people reflecting the general population in Japan, showed the presence of natural sIgA autoantibodies reactive with the virus at a considerably high rate. This may be related with low incidence of COVID-19 in Japan. This epidemiological humoral immunity survey was a cross-sectional cohort study undergoing in the Kantoh District of Japan that is located in the middle area of the Main Island of the country constituted of Metropolitan Tokyo and three neighboring Prefectures (Kanagawa, Chiba and Saitama). This survey was conducted in two groups of individuals invited to participate in the present study. Group I volunteers aged between 20 and 75 years were recruited from the general public in the Kantoh District. Group II consisted of children and adolescents under treatment for their dental conditions, university students and dentists aged between 3 and 71 years in Kanagawa Dental University Hospital, Yokosuka, Kanagawa, all of whom were inhabitants of the Kantoh District. The inclusion criteria were: to have Japanese racial background; to have had no history or experience of diagnosed COVID-19, SARS-CoV-2 PCRpositive, nor COVID-19-related symptoms; and to have had not experienced common cold-like symptoms during the preceding two weeks. Individuals were excluded if: they have had received anti-SARS-CoV-2 vaccination; they were under treatment for systemic diseases or injuries; they had oral mucosal diseases with local bleeding; or they had participated in another clinical study within one month prior to the current study period. Approval to undertake the study was obtained from the Kanagawa Dental University The study was registered on April 1, 2021 in the database of the University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR) that meets the JCMJE standards. The study ID was UMIN000043717. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; All eligible participants were requested to visit one of the following four study centers: Medical Station Clinic (Tokyo); Kanagawa Dental University Hospital; Kanagawa Dental University Yokohama Clinic (Kanagawa); and Sato Dental Clinic (Kanagawa), on April 26 -August 1, 2021 for collection of both saliva and blood samples (in adult participants aged ≥ 20 years) or for collection of saliva samples only (in children or adolescent participants aged <20 years). All the participants were also instructed to visit the indicated study center early in the morning on the appointed day and after arrival, to refrain from taking any foods and drinks, as well as from tooth-brushing, for at least one hour before the collection of saliva samples, and then to take a cup of water for cleaning the oral cavity. One hour later, saliva samples were collected from all participants at the defined time period in the morning (9:30-10:30) to avoid diurnal variation of saliva flow. Sample collections were performed using Salivettes or Salikids (Sarstedt AG & Co., KG, Numbrecht, Germany). The kit comprises a plastic coat tube and a sponge roll attached with a string for preventing aspiration. The sponge roll was placed under the tongue for 2 minutes to impregnate with a sufficient amount of saliva. Immediately after removing from the mouth, the sponge roll was inserted in a coat tube to cool on ice. The tube was centrifuged at 3,000 x g for 5 minutes at 5 °C to yield clear supernatant fluid (testing saliva sample). The testing saliva samples thus obtained were dispensed into several portions. One hundred μ l of the sample was immediately mixed with 500 μ l of inactivation solution for a SARS-CoV-2 PCR test to confirm test-negative. The residual samples were stored at -80 °C. Blood samples were collected from an antecubital vein and allowed to stand for 15 minutes at room temperature. The blood tubes were centrifuged at 2,000 x g for 15 minutes. The resultant serum samples were stored at -20 °C until they were transported to the assay laboratory in Kanagawa Dental University where all samples were collected, stored at -80 °C and immunologically analyzed. Enzyme-linked immunosorbent assays (ELISAs) to measure the binding of IgA in saliva, as well as IgA and IgG in serum, to SARS-CoV-2 spike-1 were performed using an assay system modifying the human IgA ELISA quantitation set (#88-102; Bethyl Laboratories, Montgomery, Texas, USA) that had been reported by Yamamoto et al (61) . In this kit, anti-IgA or anti-IgG antibodies are adsorbed on the plate as capture antibodies beforehand. For the measurement of IgA in saliva, spike-1-mFc recombinant protein (#40591-V05H1; Sino Biological, Beijin, China) was used as the SARS-CoV-2 antigen based on previous reports (39) . However, since that antigen reacts with IgG, spike-1-His recombinant protein (#40591-V08B1; Sino Biological, Beijin, China) was used to measure the cross-antibodies in serum. Both antigens are SARS-CoV-2 spike 1 subunit proteins containing RBD. Biotin-labeling of the antigens was performed using a labeling kit (#BK01; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions. Half-well plates added with saliva samples at 1:500 dilution or serum samples at 1:100 dilution in carbonate-bicarbonate buffer were incubated for 1 hour at 25 °C, and then washed with washing buffer. To the wells thus coated with diluted saliva or serum samples for measurement of SARS-CoV-2-reactive IgA or IgG antibodies, 100 μ l per well of biotin-labeled antigen at a concentration of 1 μ g/ml was added, and incubated for 1 hour at 25 °C. Wells were washed five times with washing buffer, and then plates were incubated with horseradish peroxidase (HRP) solution A (Bethyl laboratories) for 1hour at 25 °C. Plates were developed by addition of the HRP substrate, TMB (Bethyl Laboratories, Inc., Waltham, Massachusetts, USA), for 15 minutes at 25 °C. Then the developing reaction was quenched by adding stop solution. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. ; ODs were measured at 450 nm in a microplate absorbance reader (Bio-Rad Laboratories, Hercules, CA, USA). As positive control, commercially available two antibody products, i.e. spike-neutralizing IgG antibody (cat#40592-R001, Sino Biological; from 0 to 20 μ g/ml) and spike-neutralizing IgA antibody (cat#E-AB-V1027, Elabscience, Houston, Texas, USA; from 0 to 2 μ g/ml) for detecting SARS-CoV-2-reacting IgG and IgA, respectively. A positive control and a negative control PBS were added to every assay plate for validation. Background absorbance value for negative control was subtracted from absorbance value for each saliva or serum sample to account for non-specific binding of biotin-labeled antigen to wells without antibody. The detection limits for saliva IgA, serum IgA and serum IgG were 0.002, 0.163 and 1.0 ng/ml, respectively; thus, the tests for saliva IgA, serum IgA and serum IgG were considered positive when the values observed were equal to or above the detection limits. At saliva collection with or without blood drawing, participants were asked to complete a questionnaire containing: current demographic characteristics (e.g. age, gender, body-mass index [BMI], nativity, physical health, comorbidities such as pollinosis and other allergic disorders), oral care (particularly, daily frequency of tooth-brushing), history of vaccination for any viral diseases (e.g. influenza, type-B viral hepatitis, rubella and mumps), current medication and dietary habits including consumption of fermented foods and/or supplementary diets, as well as COVID-19-related symptoms. Comparison of positivity for SARS-CoV-2-reactive Igs among different participant groups was analyzed using the chi-squared test. The correlation between saliva IgA, serum IgA, and serum IgG concentrations reactive to SARS-CoV-2 was evaluated by Pearson's correlation coefficient. The results with P-values less than 0.05 were considered statistically significant. Analyses were performed using IBM SPSS Statistics version 27 (IBM, USA). References and Notes . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 10, 2022. ; Table 1 . Proportion positive for salivary IgA reactive with SARS-CoV-2 in all participants of the study cohort stratified by gender and age groups . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted January 10, 2022. 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