Heterogeneity assessment of vaccine‐induced effects using point‐of‐care surrogate neutralization test for severe acute respiratory syndrome coronavirus 2

Abstract Introduction Coronavirus disease (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has become a global pandemic even after vaccination. We aimed to identify immunological heterogeneity over time in vaccinated healthcare workers using neutralization antibodies and neutralizing activity tests. Methods Serum samples were collected from 214 healthcare workers before vaccination (pre) and on days 22, 90, and 180 after receiving the first dose of BNT162b2 vaccine (day 0). Neutralization antibody (NAb, SARS‐CoV‐2 S‐RBD IgM/IgG) titers and two kinds of surrogate virus neutralization tests (sVNTs) were analyzed (UMIN000043851). Results The NAb (SARS‐CoV‐2 S‐RBD IgG) titer peaked on day 90 after vaccination (30,808.0 μg/ml ± 35,211; p < 0.0001) and declined on day 180 (11,678.0 μg/ml ± 33,770.0; p < 0.0001). The neutralizing activity also peaked on day 90 and declined with larger individual differences than those of IgG titer on day 180 (88.9% ± 15.0%, 64.8% ± 23.7%, p < 0.0001). We also found that the results of POCT‐sVNT (immunochromatography) were highly correlated with those of conventional sVNT (ELISA). Conclusions Neutralizing activity is the gold standard for vaccine efficacy evaluation. Our results using conventional sVNT showed large individual differences in neutralizing activity reduction on day 180 (64.8% ± 23.7%), suggesting an association with the difference in vaccine efficacy. POCT‐sVNT is rapid and user‐friendly; it might be used for triage in homes, isolation facilities, and event venues without restrictions on the medical testing environment.


| INTRODUC TI ON
COVID-19 is widespread throughout the world, with a global cumulative caseload of over 100 million in early 2021; just 6 months later, the caseload increased to 200 million. Eight months later, on April 17, 2022, the number of COVID-19 cases worldwide exceeded 500 million. 1,2 The persistent spread of infection worldwide is accelerating due to mutant strains such as the delta and omicron variants. 3,4 In the future, it is hoped that SARS-CoV-2 infection prevention measures such as coronavirus vaccination, the development of therapeutic agents for COVID-19, and advances in testing methods will limit and control the spread of infection; however not yet been de- Thus, we specifically analyzed the correlation between conventional surrogate virus neutralization test (sVNT) and point-of-care surrogate neutralization test (POCT-sVNT) to explore the potential of POCT-sVNTs.

| Healthcare workers enrolled in clinical trials
This clinical study was performed on healthcare workers at clinics and hospitals affiliated with the Kawasaki Physicians Association for 6 months (April 10, 2021, to November 10, 2021). The study was a prospective observational study, carried out using the optin method, and was approved by the ethics committee of the Kanagawa Prefecture Medical Association (approval code: #R3-0311). Subgroups, such as age and gender groups, were not defined.
We decided on two factors as exclusion criteria (recent COVID-19 vaccination and disagreement). Informed consent for the questionnaire and the blood test was obtained from all participants. Details of our study design and results have been uploaded to the University  Injection side reaction   symptoms (tears, cough, chest pain, tachycardia, chest tightness,   pharyngeal strangulation, oral itching, arrhythmia, abdominal pain,   tunnel vision, shortness of breath, anxiety, nasal congestion, nasal itching, vomiting, nasal discharge, sore throat, rash, itching, and dizzyness, diarrhea, fever, headache, fatigue, muscle pain); (3) smoking status; (4) habitual alcohol intake; and (5) blood type.

| Neutralization antibodies (NAbs) measurement
Each protein (Anti-SARS-CoV-2 S-RBD protein Human IgM/IgG) was coated onto microplates (Anti-SARS-CoV-2 S-RBD protein Human IgM/IgG ELISA kit, Proteintech Group Inc.). A sufficient number of microwell strips were placed in a holder to run the controls and samples. A 100 μl each of standard and 1:100 diluted samples were added to the microwells and incubated for 30 min. Then, 100 μl of the 1x HRP-conjugated anti-human IgG/IgM secondary antibody was added into the microwells and incubated for 30 min. Each well was washed 4 times by dispensing 350 μl of diluted wash solution into each well, and 100 μl of the substrate was added into the microwells and incubated for 10 min. A 100 μl of the stop solution was then added to each of the microwells. The plate was read at 450 and 620 nm immediately after adding the stop solution. The best fit standard curve was determined by regression analysis using the four-parameter logistic curve fit (4-PL). 5

| POCT-sVNT assay
COVID-19 S1 RBD IgG/neutralizing Ab Test is a rapid chromatographic immunoassay for the qualitative detection of S1 RBD IgG/ neutralizing antibodies to SARS-CoV-2 in whole human blood, serum, or plasma to evaluate individual immunity after infection or vaccination (The RapiSure COVID-19 S1 RBD IgG/neutralizing Ab Test, BioFront, Seoul, Korea). We added 25 μl of serum to each specimen well of the test device and 60 μl of the buffer separately and waited for the colored lines to appear. The test result was read at 10-15 min. When two colored bands appear on the membrane, the COVID-19 S1 RBD IgG test results are said to be positive. One band appears in the control region (C), and another in the test region

| Statistical analysis
Questionnaire results were calculated using the unpaired t-test. The Correlations between NAbs and sVNT were analyzed using Pearson correlation coefficients. The Spearman's rank correlation coefficient was used to analyze correlations between NAbs, sVNT, and POCT-sVNT. All data presented were derived from two independent experiments.

| Clinical information and samples collection
There were significant differences in gender (p < 0.0001) and age (Male 50.2 + 15.7, Female, 44.6 + 13.5, p = 0.02) owing to the inclusion of clinical nurses (Table 1).

| POCT surrogate virus neutralization test (POCT-sVNT) assay
Two independent researchers visually confirmed and quantified the density of the test band and came to a consensus. Examples of the positive and negative results for the control group are shown in Figure 3C. The inhibition rates of conventional sVNT and POCT-sVNT were highly correlated (r = 0.63, p < 0.00001) ( Figure 3B).
Results showed no significant differences in age and adverse reactions on day 180, but there were significant differences in smoking/ nonsmoking and habitual alcohol/nonalcohol intake (p = 0.0350, p = 0.0011) ( Figure 4B).

| Questionnaire
There were no significant differences in age for appearance of symptoms after receiving the first dose of the vaccine (presence, n = 144, 43.9 + 13.6: absence, n = 70, 49.9 + 14.6), and after receiving the second dose of the vaccine (presence, n = 181, 45.3 + 13.8: absence, n = 33, 48.9 + 16.3); however, the percentage of people with symptoms was higher after the second dose (1st 67%, 2nd 85%) ( Figure 4A, Table 1). Most participants complained of muscle pain, fatigue, headache, and fever after the first and second doses ( Figure 4A).

| DISCUSS ION
We inferred that changes in various symptoms after coronavirus mRNA vaccination and various individual factors, such as smoking history, alcohol consumption, age, and sex, might be associated with a vaccine effect that decreases over time (i.e., reduced neutralizing activity). NAbs were measured by sVNT and POCT-sVNT over time. Results showed no significant differences in age and adverse reactions after vaccination, but there were significant differences in smoking/nonsmoking and habitual alcohol/nonalcohol intake (p = 0.0350, p = 0.0011) ( Figure 4B). Furthermore, for the neutralization capacity analysis, the SARS-CoV-2 spike receptorbinding domain (RBD) was an important site for establishing SARS-CoV-2 infection through the human angiotensin-converting enzyme 2 (ACE2) receptor. The inoculated coronavirus mRNA vaccine sets this spike protein as a target; hence, the SARS-CoV-2 S1 RBD IgM and IgG antibody titers were analyzed as the neutralizing antibodies (NAbs) against the virus S1 RBD. 6 Figure 1A). [9][10][11] Furthermore, when compared to SARS-CoV-2 S1 RBD IgG levels  Figure 1A).

IgG increases follow IgM increases in common viral infections.
However, this pattern does not always apply to patients with COVID-19, especially immunocompromised patients with longterm PCR positivity and those with negative antibodies approximately 2 months after infection and healing. 12 We also conducted neutralization tests (binding inhibitory activity) and measured the NAbs antibody titer to understand "virus recognizability and infection control ability." The conventional virus neutralization test (cVNT) is the gold standard for viral antibody testing as a neutralization test for evaluating the inhibitory binding activity of the SARS-CoV-2 spike protein RBD  (Figure 3A), but no correlation was found by age and gender ( Figure 2B). Furthermore, no significant differences were found in the presence or absence of adverse reactions or blood type. However, significant differences in smoking (smoking, 58.54% + 26.7: nonsmoking, 68.48% + 20.6, p = 0.04) and habitual alcohol intake (alcohol, 60.50% + 22.3: nonalcohol, 71.04% + 20.4, p = 0.001) were found; it was inferred that these two factors affected the fast binding inhibitory activity following vaccination ( Figure 3B). Furthermore, a comparative  investigation between sVNT and POCT-sVNT measurement showed a correlation on day 180 (r = 0.63, p < 0.00001) ( Figure 3B).
NAbs antibody titer measurement is thought to be effective in determining the "ability to recognize the virus" by antibodies produced from human-acquired immunity after regular vaccination; however, it has been reported that the number of antibody-producing cells produced by the novel COVID-19 vaccine (mRNA vaccine) gradually decreases and the antibody concentration drops to approximately one-fourth of the peak after half a year or more. 26  with regards to the neutralization of sVNT on day 180 ( Figure 1B); therefore, it may be preferable to conduct neutralization assays to measure the Nabs titer and determine "the ability not only to recognize the virus but also to control infection." There are many reports on whether to use neutralization assays to determine infection controllability following vaccination. 30 However, further verification is needed to determine whether the same could be said for infection controllability following vaccination. However, the number of people infected with the Omicron variant is currently increasing despite the second dose of mRNA vaccinations.
There is no indicator for "individual vaccine efficacy determination (virus recognition ability + infection control ability)." Given that we were able to investigate the correlation between sVNT and POCT-sVNT from our neutralization test results ( Figure 3B),

F I G U R E 4 Symptoms and reactions after 1st and 2nd vaccination, and habitual factors. (A)
The presence or absence of symptoms after the first and second coronavirus vaccination is shown in a Venn diagram and bar graph. Symptoms were more frequent after the second vaccination; myalgia, malaise, headache, and fever were the most common. (B) Significant differences were found only in smoking and drinking alcohol for cases with decreased neutralization on day 180. The p values presented in A were calculated using unpaired two-tailed Student's t-tests  POCT-sVNT may be a candidate for this, and large-scale prospective trials should be considered.
It is difficult to purchase expensive testing equipment and set up a mass sample analysis and testing systems in private medical institutions such as the facilities that participated in this study. In that sense, the existence of a point-of-care rapid neutralizing activity analysis kit that uses the immunochromatography method does not require a special environment. It can easily and rapidly conduct sVNT analysis. It might be thought that this would have a wide-ranging social implementation, such as triage for the third dose of vaccination in clinical practice, triage for the extent of isolation restrictions after returning to Japan, indicator for staff reassignment (e.g., staff with reduced vaccine effects move to backyard shifts), and indicator for addition to vaccine passports in places such as restaurants, movie theaters, and events.

| CON CLUS ION
We used human serum samples from before and after mRNA vaccination to conduct a NAbs analysis and sVNT analysis over time.
Specifically, our results showed large individual differences in reduction in neutralizing activity using conventional sVNT on day 180. In addition, we found that sVNT analysis might be helpful as an indicator for "possible virus recognizability and infection controllability" following vaccination. The more recently developed POCT-sVNT has a high correlation with the conventional sVNT, which suggests the possibility of personal use.

CO N FLI C T O F I NTE R E S T
The authors state that they have no conflict of interest (COI).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that provided the evidence for the study are available from the corresponding author upon reasonable request.

I R B A PPROVA L CO D E A N D N A M E O F TH E I N S TITUTI O N
The study was a prospective observational study, carried out by the opt-in method of each institution, and approved by the eth-