key: cord-0932045-qayxa7hx
authors: Matsuba, Ikuro; Takuma, Tetsuo; Hatori, Nobuo; Takai, Masahiko; Watanabe, Yoshiyuki; Takada, Nobukazu; Kishi, Satoru; Matsuzawa, Yoko; Nishikawa, Tetsuo; Kunishima, Tomoyuki; Degawa, Hisakazu; Nishikawa, Masanori; Ono, Yoshiaki; Kanamori, Akira
title: Study on Continuation of Antibody Prevalence Six Months after Detection of Subclinical Severe Acute Respiratory Syndrome Coronavirus 2 Infections
date: 2021-11-06
journal: Intern Med
DOI: 10.2169/internalmedicine.8019-21
sha: 921df88fdbce47a2a89fdab3d334fa84a9f50a5c
doc_id: 932045
cord_uid: qayxa7hx

OBJECTIVE: To examine the continuation of antibody prevalence and background factors in antibody-positive subjects after asymptomatic infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: A study was carried out to investigate the SARS-CoV-2 antibody (IgG) prevalence. SARS-CoV-2 antibodies (IgG) were measured and analyzed with immunochromatographic tests. PATIENTS: Among 1,603 subjects, comprising patients, physicians, and nurses at 65 medical institutes in Kanagawa, Japan, 39 antibody-positive subjects received follow-up for 6 months. RESULTS: Of the 33 subjects who consented to the follow-up (23 patients and 10 medical professionals), continued positivity of IgG antibodies was confirmed in 11 of 32 cases (34.4%) after 2 months, 8 of 33 (24.2%) after 4 months, and 8 of 33 (24.2%) after 6 months. A significant difference was found in the sleeping time, drinking habits, hypertension, and use of angiotensin-receptor blockers on comparing subject background characteristics among three groups: patients with antibody production that continued for six months after the first detection of positivity, patients in whom antibody production stopped at four months, and patients in whom antibody production stopped at two months. CONCLUSION: The continuation rate of IgG antibody prevalence was 24.2% at 6 months after the first detection of antibody positivity in cases with asymptomatic coronavirus disease 2019 (COVID-19) infections. This percentage is low compared with the antibody continuation rate in patients who have recovered from symptomatic COVID-19 infection.

Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the coronavirus disease 2019 (COVID- 19) pandemic has affected 223 countries as of February 19, 2021 . There have been 109 million infections and 2.4 million deaths as of February 19, 2021 (1) . Worldwide vaccine developments aimed at stabilizing the situation have been building expectations, but the duration of antibody prevalence that can be obtained from such vaccines remains a point to be addressed.

There have been several reports thus far concerning the continuation of antibody prevalence in patients who have contracted SARS-CoV-2, and opinions are divided. Some have reported that antibody prevalence was maintained for four to six months (2) (3) (4) (5) (6) , while others say it diminished after a short period of time (7) (8) (9) . However, the antibody titers re-portedly increase with the severity of the infection (2, 4, 7, 10) , with more severe cases continuing to produce antibodies for a longer period of time than milder cases (4, 5) while being unlikely to cause aggravation after vaccination.

To what extent antibodies can be produced and how long they remain after the infection in asymptomatic cases have been attracting attention. While differing results have been reported concerning the continued production of antibodies generated by COVID-19, there are no reports involving the long-term follow-up evaluation of the antibody prevalence following subclinical infections. Of note, we previously reported that 2.4% of physicians, nurses, outpatients, and medical examinees generated antibodies from subclinical infections (11) .

We therefore investigated the presence of antibodies at two, four, and six months after the detection of antibody positivity in patients who tested positive for antibodies in the first test.

This study was registered with the Clinical Trials Registry (https://www.umin.ac.jp/;UMIN000040333; May 8, 2020) and performed in accordance with the study protocol, the Declaration of Helsinki, and the Ethical Guidelines for Clinical Studies of the Japanese Ministry of Health, Labor, and Welfare. This study was approved by the Ethics Review Board of the Kanagawa Physicians Association. All participants provided their written informed consent before participation.

This multi-center epidemiological study at 65 institutes in Kanagawa, Japan, enrolled patients from May 18 to June 24, 2020. Subjects received follow-up until January 31, 2021.

The inclusion criteria were as previously reported (11) . Subjects were those who tested positive in the first anti-SARS-CoV-2 antibodies (IgG) test and consented to followup.

Subjects were informed of the study in writing before giving their written consent and answered questionnaires. Their blood was then collected for antibody testing. They were informed of the need for follow-up if they tested positive in the first antibody test. After obtaining their written consent, their blood was collected for antibody testing at two, four, and six months after the detection of antibody positivity, with clinical symptoms recorded at each timepoint.

The study was terminated when subjects tested negative for antibodies or after six months.

The Cica Immuno-test SARS-CoV-2 IgG was used (12) . This is a reagent developed through collaborative research by Professor Akihide Ryo of the Department of Microbiology, Yokohama City University Graduate School of Medicine and Kanto Chemical, Tokyo Japan, which detects human anti-SARS-CoV-2 antibodies (IgG) contained in the serum of individuals infected with the novel coronavirus. The reagent is based on the principles of immunochromatography.

The test was performed immediately after blood collection. In brief, when a serum sample containing human anti-SARS-CoV-2 antibodies (IgG) is dropped on the sample pad of the test device, human anti-SARS-CoV-2 antibodies (IgG) move on the test strip in the test device and form complexes with goat anti-human IgG antibodies labelled with black particles (black particle-labelled antibodies) contained in the conjugate pad. These complexes then move to the membrane and bind to viral proteins fixed where a test line will appear. This accumulates black particle-labelled antibodies, which form the test line. If human anti-SARS-CoV-2 antibodies (IgG) are not present in the serum, black particle-labelled antibodies do not accumulate, and no test line appears. Whether human anti-SARS-CoV-2 antibodies (IgG) are present or not, black particle-labelled antibodies also bind to rabbit anti-goat IgG antibodies fixed where a control line appears. This accumulates black particle-labelled antibodies, and the control line appears. The control line is an indicator of the normal development of the serum.

This assay kit was verified by the National Institute of Health Sciences on a simultaneous performance evaluation test of an antibody assay kit against the new coronavirus, and the maximum dilution factor for which all cases were positive was 64-fold (13). The accuracy of this assay kit was equal to or better than that of many other assay kits.

Information, such as age and sex obtained in the first survey (11) , and the presence of clinical symptoms and IgG antibodies at two, four, and six months after the detection of antibody positivity, was collected.

The SARS-CoV-2 antibody (IgG) positivity rate was assessed two, four, and six months after the first detection of antibody positivity. Subjects were divided into three groups for a comparison: patients with antibody production that continued for six months after the first detection of positivity, patients in whom antibody production stopped at four months, and patients in whom antibody production stopped at two months.

The data were analyzed using the R software program, version 3.5.2 [R Foundation for Statistical Computing, Vi- enna, Austria (https://www.R-project.org/)]. When comparing the patients' background characteristic, Fisher's exact test was used for nominal variables, and an analysis of variance was used for continuous variables. If the answer to a questionnaire survey item was unknown, the datum corresponding to it was excluded from statistical tests. The two-sided significance level was 5%. Demographic characteristics are presented as the mean±standard deviation or the number of cases (%).

showing antibody positivity in the subclinical infection study, the 33 who consented to participate in the study received follow-up. The continued production of antibodies was observed in 11/32 cases (34.4%) at 2 months, 8/33 (24.2%) at 4 months, and 8/33 (24.2%) at 6 months. Of the subjects showing continued antibody production, two were found to have clinical symptoms, such as a cough, during the follow-up.

Table shows the participant characteristics of the three groups: patients with antibody production that continued for six months after the first detection of positivity, patients in whom antibody production stopped at four months, and patients in whom antibody production stopped at two months. There were significant differences among the three groups in sleeping time, drinking habit, hypertension, and use of angiotensin-receptor blockers (ARBs).

Seven of the 39 antibody-positive individuals in the initial test underwent polymerase chain reaction (PCR) testing. However, all of them were PCR-negative. Even in cases where the antibody test was positive, the possibility of current infection/onset could not be ruled out. Therefore, in some cases, PCR tests were performed, whereas in others, the tests were refused by public health centers/medical associations. Some patients did not receive a PCR test because they had no symptoms and did not want to get the check. Patients who were asymptomatic at the subsequent followup were required to pay for the antigen test and PCR test themselves. For this reason, it was up to the subject to decide whether or not to carry out the test. However, we continued to follow patients in whom antibody production had stopped, with no subsequent reports of infection.

Antibody positivity was maintained in 11/32 cases (34.4%) at 2 months after the detection, in 8/33 (24.2%) at 4 months after detection, and in 8/33 (24.2%) at 6 months after detection in this study. We continued to follow patients in whom antibody production had stopped and considered them to be free from infection, as they had no suggestive symptoms.

There have been several reports on the short-term continued antibody production after subclinical infection. In a 2month follow-up study in India, only 57 of 201 (28.36%) subjects in whom antibody positivity had been previously detected showed IgG antibody positivity when they were tested again 2 months later (9). Long et al. conducted an 8week follow-up of subclinical patients, noting that the rate of continued positivity was 60% (18/30) (8) . In our study, we found the rate of continued positivity was 34.4% at 2 months after the first detection of antibody positivity. This result was in the middle of previously reported values. In the present study, we followed cases for up to six months after the infection, an unprecedented follow-up duration for subclinical infection case; we consequently noted a low rate of continued antibody production. Follow-up studies have produced diverse results in symptomatic patients who were followed from the recovery phase onward. In a study in which quantitative tests were performed with antibody titers, the titer values remained stable in most cases for three months and were still sufficient for protecting from viruses even after five months, despite some decrease (5) . In a large-scale study in Iceland, the antiviral antibody titer values with two types of pan-Ig antibodies increased for two months after the diagnosis of infections with quantitative PCR, with such values maintained for up to four months after the diagnosis (2) . In a study in Chicago, IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD) remained detectable for 4 months in 75% of 24 subjects who had shown IgG antibody positivity at the outset (3).

Although not conducted to verify antibody titers, a UK study found that 89 of 11,052 medical professionals who had not had antibodies at baseline developed symptoms of COVID-19 during a 30-week study, while none of the 1,246 who had had antibodies at baseline developed COVID-19 symptoms (6) . Their report further suggests that antibody production could continue for at least six months.

It is impossible to simply compare these reports with our study because ours dealt with subclinical infections, and follow-up was started after the detection of antibody positivity. However, of note, antibodies became undetectable after a short period of time, a point that was presumably influenced by the severity of symptoms. The acquisition of high antibody titers that drop slowly facilitates continued antibody production.

Regarding the acquisition of the high antibody titers, Chen et al. performed a study involving 92 subjects with symptomatic COVID-19 in the US, noting a significant correlation between the volume of anti-SARS-CoV-2 IgG and the age and self-claimed severity (4) . Other studies reported similar results (2, 7) . According to Long et al., IgG production continued for a longer time in symptomatic patients than in subclinical patients (8) . Researchers in Japan also reported that the virus-neutralizing antibody titers were found to be higher in more severe cases than in milder ones, with more cytokines and chemokines produced in more serious patients (10) . These findings are supported by other study reports (14, 15) . Goto et al. also reported that a regression analysis indicated that a high body mass index, a fever, and the need for mechanical ventilation or extracorporeal membrane oxygenation were significantly associated with elevated neutralizing antibody titers (16) . There is likely a correlation between the degree of virus-neutralizing antibody titers and the seriousness of symptomatic cases when antibodies are produced. The antibody titers in our study were probably low because the cases were asymptomatic.

Antibody production reportedly continued for a longer duration in cases that recovered quickly despite the seriousness at the time of infection than other cases (4). Furthermore, in a study from New York, when antibody titers were assessed in patients who had recovered from COVID-19, the titers dropped more slowly in patients with higher titers during the recovery phase, and the group with low titers included some who even showed antibody negativity by three months after the onset of symptoms (5) . In a study on subclinical infections in India, 8 of 243 patients complained of symptoms suggesting COVID-19 infections within 1 month after the first confirmation of antibody positivity. These patients took PCR tests and were all diagnosed with COVID-19. At that time, antibody production was not detected in seven of these eight cases (9) . Other studies have also suggested that antibodies tend to decrease if the initial antibody production is low (17) (18) (19) (20) (21) .

Considering the factors contributing to higher titer values and continued high titers, it seems reasonable to say that the antibody production continuation ratio is low in cases with subclinical infections compared with symptomatic cases.

While there have been a range of results reported, it is difficult to simply compare these varied results because the testing methods are not standardized. In a previous study (8) in which virus antibody responses stopped 8 weeks later in 40% of subjects, the antibodies were measured using different methods from those in another study in which antibody production continued for a long time (5) . This is due in part to the former study targeting NPs and a single linear epitope in spike proteins. Stable virus-neutralizing antibody titers were reported in a study in which the antibody production was found to stop early. Therefore, the mechanism is considered to depend on the target antigens. The results from a study in Chicago suggested a greater sensitivity of RBD IgG assays than other assays, as the test results differed between nucleocapsid and RBD IgG in the presentation of serum antibody positivity (3). Chia et al. investigated the performance of N, S1, and RBD proteins from SARS-CoV-2 and SARS-CoV using four different test platforms and reported that although RBD protein showed the greatest specificity, the N proteins of both viruses had very high cross-reactivity and were unsuitable for the detection of virus-specific antibodies (22) . An N-terminal region that is highly homologous with other viruses was deleted from the N protein used as the antigen in the immunochromatographic kit used in the present study. Therefore, our kit will naturally produce fewer false-positive results than other kits (23) . It is necessary to closely check the testing methods when interpreting study results.

Furthermore, the present findings suggest that antibody production is difficult to sustain if a patient drinks alcohol routinely. Simou et al. reported that the risk of communityacquired pneumonia rises to 8% with an increase in daily alcohol consumption for every 10-20 g (24) . Another study reported that alcohol might impair immunity (25) (26) (27) . Kageyama et al. reported that frequent alcohol consumption was associated with a reduction in the serum anti-SARS-CoV-2S antibody titer after vaccination (28) . Since the antibody titer of the vaccine is evaluated based on the S protein, there may be a difference in assays based on the N protein. However, our study showed the same tendency. Alcohol may affect the continued production of antibodies to SARS-CoV-2.

In addition, more patients had hypertension and used ARB in the group showing continued antibody production than in the group not showing the continuation of antibody production. SARS-CoV-2 binds to angiotensin-converting enzyme (ACE) 2 receptors present on the surface of cells for intrusion (29) . Some studies have suggested that ACE inhibitors and ARBs may prevent aggravation of COVID-19 (30) (31) (32) , but another paper reported that they didn't affect (33) . If a patient uses ARB internally and more ACE2 receptors are produced, it may aid in maintaining antibody prevalence. However, other studies have reported that, in most cases, antibody prevalence continued for a longer period as the severity increased. Further verification is required with large-scale and long-term studies.

Results concerning the relationship between antibody titers and sex vary by study, although we found no marked differences by sex in the duration of antibody production. Some studies have suggested that men tend to produce IgG antibodies with high antibody titers (2, 7, 34) , while another study reported opposite results (35) . Further results are expected in the future.

If vaccines are developed, the challenge would be the continuation of the antibody production effect. The lack of continuous immunity to seasonal coronaviruses has also been shown epidemiologically (36, 37) . It would be important to sustain the immune response of both T and B cells (38) .

Several limitations associated with the present study warrant mention. The exact time of infection is unknown because the subjects were patients with asymptomatic infections. Accordingly, the actual period of continued antibody production is also unknown. In addition, we did not determine the titer values. Therefore, a quantitative assessment was not possible, and our study results cannot be simply compared with other studies. Although it is meaningful to observe changes in IgG antibody titers in chronological order, there was no general-purpose kit for evaluating antibody titers available at the beginning of this study. However, the IgG qualitative kit was considered sufficient for evaluating the presence or absence of subclinical infection. Finally, while we investigated the alcohol habits, we were unable to investigate the amount of alcohol intake.

The IgG antibody production rate was 24.2% (8 of 33 cases) among patients with asymptomatic infections of SARS-CoV-2 at 6 months after the first detection of antibody positivity. This was lower than the rate among patients who recovered from symptomatic infections. In addition, there were significant differences in drinking habit, hypertension, and the use of ARBs among three groups: patients with antibody production that continued for six months after the first detection of positivity, patients in whom antibody production stopped at four months, and patients in whom antibody production stopped at two months. The duration of the continuation of antibody production is a point that remains to be explored in vaccine development.

This research was financially supported by the Kanagawa Physicians Association, Kanagawa Prefecture Medical Association, and Kanagawa Prefecture.

World Health Organization, coronavirus disease (COVID-19) pandemic

Humoral immune response to SARS-CoV-2 in Iceland

Patterns and persistence of SARS-CoV-2 IgG antibodies in a US metropolitan site

Quick COVID-19 healers sustain anti-SARS-CoV-2 antibody production

Robust neutralizing antibodies to SARS-CoV-2 infection persist for months

Antibodies to SARS-CoV-2 are associated with protection against reinfection

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

A prospective study on rapidly declining SARS-CoV-2 IgG antibodies within one to three months of testing IgG positive: can it lead to potential reinfections?

The trend of neutralizing antibody response against SARS-CoV-2 and the cytokine/ chemokine release in patients with differing severities of COVID-19: all individuals infected with SARS-CoV-2 obtained neutralizing antibody

Survey of the current status of subclinical coronavirus disease 2019 (COVID-19)

Research team led by YCU professor successfully develops a rapid diagnostic test for COVID-19 that can detect antiviral antibodies in patient serum

led-by-ycu-professor-s uccessfully-develops-a-rapid-diagnostic-test-for-covid-19-that-can-d etect-antiviral-antibodies-in-patient-serum/ 13. National Institute of Health Sciences. The report of a simultaneous performance evaluation test of an antibody assay kit against the new coronavirus

Serum IgA, IgM, and IgG responses in COVID-19

SARS-CoV-2 neutralizing antibody responses are more robust in patients with severe disease

Sustained neutralizing antibodies 6 months following infection in 376 Japanese COVID-19 survivors

Decline of humoral responses against SARS-CoV-2 spike in convalescent individuals

Humoral response dynamics following infection with SARS-CoV-2. medRxiv

Persistence of serum and saliva antibody responses to SARS-CoV-2 spike antigens in COVID-19 patients

Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients

Longitudinal observation and decline of neutralizing antibody responses in the three months following SARS-CoV-2 infection in humans

Serological differentiation between COVID-19 and SARS infections

Whole nucleocapsid protein of SARS-CoV-2 may cause false positive results in serological assays

Alcohol and the risk of pneumonia: a systematic review and meta-analysis

Alcohol, injury, and cellular immunity

Chronic consumption of alcohol increases alveolar bone loss

Chronic ethanol exposure enhances the aggressiveness of breast cancer: the role of p38gamma

Antibody responses to BNT162b2 mRNA COVID-19 vaccine in 2,015 healthcare workers in a single tertiary referral hospital in Japan

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury

Renin-angiotensin system inhibitors and severity of coronavirus disease-2019 in Kanagawa, Japan: a retrospective cohort study

Antihypertensive drugs and COVID-19 risk: a cohort study of 2 million hypertensive patients

Renin-angiotensin system blockers and susceptibility to COVID-19: an international, open science, cohort analysis

Influential factor and trend of specific IgG antibody titer in coronavirus disease 2019 convalescents

A comparison study of SARS-CoV-2 IgG antibody between male and female COVID-19 patients: a possible reason underlying different outcome between sex

Seasonal coronavirus protective immunity is short-lasting

Human coronavirus NL63 molecular epidemiology and evolutionary patterns in rural coastal kenya

A systematic review of SARS-CoV-2 vaccine candidates

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We would like to thank the physicians and staff of the Kanagawa Physicians Association involved in the research and the patients participating in this study. We thank Dr. Masaaki Miyakawa from Director of Kanagawa Prefecture Medical Association, Dr. Masakazu Kikuoka from Chairman of Kanagawa Prefecture Medical Association, Dr. Yutaka Hatori from Director of Japan medical Association for his great cooperation. We also thank Professor Akihide Ryo from Department of Microbiology, Yokohama City University School of Medicine for his advice with the testing device. We also thank Professor Ung-il Chung, Professor Hiroto Narimatsu, Junior Associate Professor Sho Nakamura and Junior Associate Professor Xuemin Fang all from School of Health Innovation, Kanagawa University of Human Services for their advice with protocol. We thank Yoshiyuki Makino from Healthcare New Frontier Promotion Headquarters Office, Policy Bureau, Kanagawa Prefectural Government for his support.