key: cord-0899629-xdvuae7v
authors: Goto, A.; Miyakawa, K.; Nakayama, I.; Yagome, S.; Xu, J.; Kaneko, M.; Ohtake, N.; Kato, H.; Ryo, A.
title: Analysis of humoral immunity against emerging SARS-CoV-2 variants: a population-based prevalence study in Yokohama, Japan
date: 2022-03-27
journal: nan
DOI: 10.1101/2022.03.26.22272766
sha: 01f7d17e824b61621cb102257357172165b54274
doc_id: 899629
cord_uid: xdvuae7v

Background Little is known about the population prevalence of antibodies against emerging immune escape variants of SARS-CoV-2. Methods A population-based prevalence study was conducted in Yokohama City, the most populous municipality of Japan. Quantitative measurements of immunoglobulin G against SARS-CoV-2 spike protein (SP-IgG) and qualitative measurements of neutralization antibodies against the Omicron BA.1 and BA.2 variants were performed. Results Of 6,000 randomly selected residents aged 20-74, 1,277 participated in the study during a period from January 30 to February 28, 2022. Of them, 3% had prior diagnosis of COVID-19, 96% received at least two-doses of SARS-CoV-2 vaccines, and 94% were positive for SP-IgG. The positive rates of neutralizing antibodies were 28% to Omicron BA.1 and BA.2 variants in a random sample of 10% of participants (n=123) and 100% to BA.1 and BA.2 among participants who received the third vaccination at least 7 days before (n=66). Conclusions In this population-based prevalence study in Japan, most had SP-IgG antibodies but the overall neutralizing antibody positive rate was 28% against the Omicron BA.1 and BA.2 variants. The population-level insufficient humoral immunity against the Omicron variants may explain the outbreak of COVID-19 during this period in Japan.

Little is known about the population prevalence of antibodies against emerging immune escape variants of SARS-CoV-2.

A population-based prevalence study was conducted in Yokohama City, the most populous municipality of Japan. Quantitative measurements of immunoglobulin G against SARS-CoV-2 spike protein (SP-IgG) and qualitative measurements of neutralization antibodies against the Omicron BA.1 and BA.2 variants were performed.

Of 6,000 randomly selected residents aged 20-74, 1,277 participated in the study during a period from January 30 to February 28, 2022. Of them, 3% had prior diagnosis of COVID-19, 96% received at least two-doses of SARS-CoV-2 vaccines, and 94% were positive for SP-IgG. The positive rates of neutralizing antibodies were 28% to Omicron BA.1 and BA.2 variants in a random sample of 10% of participants (n=123) and 100% to BA.1 and BA.2 among participants who received the third vaccination at least 7 days before (n=66).

. CC-BY-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. In this population-based prevalence study in Japan, most had SP-IgG antibodies but the overall neutralizing antibody positive rate was 28% against the Omicron BA.1 and BA.2 variants. The population-level insufficient humoral immunity against the Omicron variants may explain the outbreak of COVID-19 during this period in Japan.

SARS-CoV-2, seroprevalence, neutralizing antibodies, Omicron, Omicron sub-lineage . CC-BY-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. Global Access (COVAX) [1] is leading a global collaboration to accelerate the development and manufacture of COVID-19 vaccines and guarantee fair and equitable access to every country in the world.

Vaccination against COVID-19 was almost completed by the end of 2021 for those who wished to receive two doses of the vaccine in Japan. However, the antibody titers decrease 6 months to 1

year after vaccination, and even individuals who have received two doses of the vaccine can be infected (i.e., breakthrough infection) [2, 3] . Under these circumstances, the third vaccination (booster shot) was administered in December 2021 after an interval of 6-8 months from the completion of the second vaccination. In Japan, approximately 80% of the total population have received at least two doses of the vaccine against SARS-CoV-2, and approximately 33% have received the third shot of the vaccination as . CC-BY-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) [4] , which is similar to vaccination rates in other high-income countries [5] .

However, there is an emerging concern that the variants of concern (VOC), particularly the Omicron variant (B.1.1.529 or BA.1), would escape the antibodies elicited by vaccination against SARS-CoV-2 [6, 7] . In fact, Japan has been experiencing a resurgence of COVID-19 cases [8] with the Omicron variant becoming dominant since January 2022 [9] , despite most of the adult population have completed two doses of vaccines [10] .

From public health perspectives, evaluation of a population-level immunity to SARS-CoV-2, including the Omicron BA.1 and the newly emerged Omicron sub-lineage BA.2 (B.1.1.529.2) variants, is essential to facilitate evidence-informed decision-making regarding COVID-19. However, only a few studies have reported the population-level prevalence of antibodies against SARS-CoV-2 [11] and VOC, such as Delta (B.1.617.2) and Omicron BA.1 [12] . Moreover, to the best of our knowledge, there is a lack of data on population immunity against the newly emerged variant, the sub-lineage BA.2 of the Omicron variant, at present. Since Omicron BA.2 has begun to dominate globally[13], an investigation of the immunity against BA.2 at a population level is urgently warranted.

Therefore, in this population-based prevalence study in Yokohama City, the most populous . CC-BY-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 March 27, 2022. ; https://doi.org/10.1101/2022.03.26.22272766 doi: medRxiv preprint municipality of Japan, we evaluated population-level humoral immunity against emerging immune escape variants such as Omicron BA.1 and BA.2. The survey was undertaken from January 30 to February 28, 2022, during the middle of the sixth wave of COVID-19 in Japan, which was driven by the emergence of the Omicron BA.1.

In total, 6,000 residents were randomly selected from a Japanese population aged 20-74 year, from Yokohama City, Japan. With a population of approximately 3.7 million, Yokohama City is the most populated basic municipality in Japan and is located in Kanagawa Prefecture next to Tokyo. The study invitations were mailed to these residents in January 2022. Among those, 1,277 individuals (546 men and 731 women; response rate: 21.3%), who did not have a confirmed diagnosis of COVID-19 within 2 weeks of the study entry, participated in this study during a period from January 30 to February 28, 2022. The participation rate was relatively high among middle-aged (45-59) women with response rate being 30% or more (Supplemental Figure 1 ). All participants provided written informed consent. The study was approved by the Institutional Review Board of the Yokohama City University.

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Each participant provided approximately 7 ml of blood samples and completed a questionnaire with questions regarding their prior COVID-19 diagnosis, vaccination against SARS-CoV-2, and lifestyle and social factor at study entry. Participants who provided incomplete answers, such as incorrect vaccination date, were supplemented with an additional questionnaire or underwent telephone interview. Therefore, there were not missing data on variables listed in Table 1 .

Following prior publications [14] [15] [16] [17] [18] , IgG antibodies against spike protein (SP-IgG) or total Ig antibodies against nucleocapsid protein (NP-total Ig) were measured using the commercial chemiluminescent enzyme immunoassay (AIA-CL SARS-CoV-2 SP-IgG and NP-total Ig antibody detection reagents, Tosoh, Japan) to assess prior infection (NP-total Ig) or vaccination against SARS-CoV-2 (SP-IgG or NP-total Ig). An index of ≥ 1.0 for these antibodies were considered positive according to the manufacturer's instructions. The limit of detection (LOD) for SP-IgG and NP-total Ig was 0.1, and those with values below LOD were assigned a value of 0.05 for statistical analyses. For the assessment of neutralizing antibodies, a rapid qualitative neutralizing assay was performed as previously reported [15] using the spikes of D614G as a reference and three variants (Delta, Omicron BA.1, and BA.2) among a random sample of 10% (n=123) of the total study population and 79 participants who received the booster shot of the SARS-CoV-2 vaccine. Sera were assayed using the rapid qualitative . CC-BY-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 March 27, 2022. ; https://doi.org/10.1101/2022.03.26.22272766 doi: medRxiv preprint neutralization test (hiVNT) with the HiBiT-tagged virus-like particle carrying SARS-CoV-2 spike. At a fixed serum sample dilution of 1 in 20, if luminescence signal inhibition exceeds 40%, a sample is considered to possess neutralizing activity equivalent to the neutralizing titer of 51 or more in the HIVbased pseudovirus method [15] .

We computed an overall crude antibody prevalence rate and its 95% confidence interval (CI) for the positivity of SP-IgG antibody as defined as an index of ≥ 1.0 and a weighted prevalence rate and 95% CI to adjust for differences in participation rate across age and sex and represent the overall population aged 20-74 of Yokohama City. For the weighted prevalence rate, we calculated the participation rate by sex and age group in 5-year increments. Subsequently, we computed the weighted prevalence rate and its 95% CI with weights with the reciprocal of the participation rates, using the survey package [19] in R. For the crude prevalence, the Clopper-Pearson method was used to estimate CI using the binom.test function in R.

Characteristics such as age, number of vaccinations (none, one, two, or three does), and number of days since vaccination in relation to SP-IgG index were investigated by stratification and linear regression analyses, after excluding those with prior COVID-19 diagnosis or positive NP-total Ig, . CC-BY-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 March 27, 2022.

which indicates prior infection with SARS-CoV-2. In regression analyses, after excluding those with prior COVID-19 diagnosis or positive NP-total Ig, log-10 transformed SP-IgG index was regressed on age or days since the last vaccination among those who completed two doses of vaccines.

Among randomly selected 10% subpopulations (n=123) of the total participants, we examined the positive rates (95% CIs using the Clopper-Pearson method) for neutralization antibodies against the Delta, Omicron BA.1, and BA.2 variants with D614G as a reference strain. In addition, we examined the positive rates neutralization antibodies against these variants among those who received the three doses of vaccines against SARS-CoV-2 at least 7 days before, after excluding those with prior COVID-19 diagnosis or positive NP-total Ig. All statistical analyses were performed with R version 4.1.2 (The R Foundation for Statistical Computing).

Among 1,277 participants (546 men, 43%; 731 women, 57%), most had received two doses (but not the third dose) of vaccination, accounting for 90.2%; 6.2% had completed the third (booster) dose, 0.2% had received the first dose, and 3.4% had not been vaccinated (Table 1) . Among them, 2.8% . CC-BY-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) 

In 1,092 individuals with no history of infection and who tested negative for NP-Ig total and received two doses of Pfizer-BioNTech BNT162b2 or Moderna mRNA-1273 vaccine, as more days elapsed since the second dose, the lower the SP-IgG index tended to be (Figure 2A) . At any number of days, the SP-IgG index values were higher among those who received the Moderna mRNA-1273 vaccine than among those who received the Pfizer-BioNTech BNT162b2 vaccine. In addition, the older the age, the more SP antibody titers tended to be low; this trend was prominent for the BNT162b2 vaccine ( Figure 2B ). In 1,092 individuals with no history of infection and who tested negative for NPtotal Ig and received two doses of the BNT162b2 or mRNA-1273 vaccine, those who received the BNT162b2 vaccine tended to have more adverse reactions, such as fever, than those who received the mRNA-1273 vaccine (Supplemental Figure 2) .

In 76 people with no history of infection and who tested negative for NP-total Ig and received the three doses of vaccines, high SP-IgG index values were observed in those who had received the booster shot at least 7 days before ( Figure 3 ). Approximately 46.1% of these individuals reported that they had the most severe adverse reactions after the third shot (Supplemental Figure 3) .

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Among 123 randomly selected participants, we evaluated the prevalence for neutralization antibodies against the D614G, Delta, Omicron BA.1, and Omicron BA.2 variants. Among these individuals, four were unvaccinated, 115 had received two doses, and four had received three doses of the vaccines. Approximately 87% had neutralizing antibodies to the reference D614G strain, 74% to the Delta strain, 28% to the Omicron BA.1, and 28% to the Omicron BA.2 ( Table 2 ). Among them, the SP-IgG index values were higher among those with positive D614G neutralization antibodies (Supplemental Table 2) .

. CC-BY-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. Our findings showing that only 28% of our population had neutralization antibodies to both . CC-BY-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. Our findings show that 94% of participants had SP-IgG antibodies, which is largely consistent with the fact that approximately 96% of our population had completed their second or third dose of vaccination, 2.8% had been diagnosed with COVID-19, and antibody titers are known to decline over time [21] . Because approximately 87% of the residents aged 20-79 in Yokohama City received the . CC-BY-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 strengths of this study include its population-based design with a relatively large sample size and detailed assessments of the demographic data, vaccination status, and previous diagnosis of COVID-19. Furthermore, we measured SP-IgG and NP-total Ig using highly accurate quantitative measurements. Additionally, we assessed the population seroprevalence of the neutralizing antibodies against the variants including Omicron BA.2. However, the present study has several limitations. First, the cross-sectional nature of the study limits the ability to investigate causality between measured variables. However, given the growing evidence regarding the effectiveness of vaccination on the immunity against and risk of contracting COVID-19, our findings provide an accurate description of the humoral immunity against variants, including assessments of the emerging variants such as Omicron BA.1 and BA.2. Second, the assay used to assess the positivity of the neutralization antibodies was qualitative. That is, we could only evaluate the presence or absence of the neutralization antibodies at a certain threshold, equivalent to the neutralizing titer of 51 or more in the HIV-based pseudovirus method [15] . However, our findings are consisitent with published studies using the quantitative assessments of . CC-BY-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 March 27, 2022. ; https://doi.org/10.1101/2022.03.26.22272766 doi: medRxiv preprint the neutralization antibodies [23, 24] . Third, the response rate of our study was approximately 21%, which is not sufficiently high. However, to adjust the differences in age and sex distribution between our study and the overall population of Yokohama City, weighted prevalence rates were estimated and the results were almost identical. Lastly, our study provides evidence regarding a certain period of time in Yokohama City, Japan, and the generalizability may be limited. However, because the vaccination has been similarly provided to the nationwide population in Japan and the vaccination rates in Japan are similar to other high-income countries [10] , the findings could be generalized to other populations.

To summarize, in this population-based cross-sectional study conducted in Japan, the majority of participants had SP-IgG antibodies, and the older they were and the more the number of days that had elapsed after the second vaccination, the lower their antibody titers tended to be. The overall prevalence of the neutralizing antibodies to Omicron BA.1 and BA.2 variants were only 28% from January to 

SY is an employee of Integrity Healthcare Co., Ltd. NO is an employee of Tosoh Corporation.

These companies did not have any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. HK received grants from Shionogi & Company, Limited, and Asahi Kasei Pharma & Co., Inc. Other authors stated no conflict of interest.

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We would like to acknowledge all the participants for their participation in this research. We thank Sho Katsuragawa, Toshiya Sakota, and the staff members at the Yokohama City University for their assistance. We also thank Mr. Mitsuru Kurata at the Prime Health Partners Co., Ltd. for his contribution to this study.

. CC-BY-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. . CC-BY-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)