Effectiveness of COVID-19 vaccines in preventing asymptomatic infection: a cross-sectional study based on nucleocapsid antibody assessment

Abstract

Objective: To investigate the status of asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in medical facilities.

Methods: A cross-sectional study survey was conducted among healthcare workers toward the end of the eighth wave of the coronavirus disease 2019 (COVID-19) epidemic in Japan. Participants completed a self-administered questionnaire providing details their COVID-19 and COVID-19 vaccination history and provided a blood sample for SARS-CoV-2 nucleocapsid (N) antibody testing as evidence of past SARS-CoV-2 infection. Participants were categorized into four groups, according to whether they had received 0–2, 3, 4, or 5 doses of vaccine. The prevalence of N antibodies was compared between groups.

Results: Of 494 participants, 288 reported no history of SARS-CoV-2 infection, of whom 185 accepted serological testing for past infection. Of the 185, 14 (7.6%) showed serological evidence of past infection, indicating asymptomatic infection. The risk of asymptomatic COVID-19 was inversely associated with the number of doses of COVID-19 vaccine received.

Conclusions: The cumulative effect of vaccination on preventing asymptomatic COVID-19, suggests that repeated COVID-19 vaccination of healthcare workers may serve as an infection control measure.

 Introduction

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), originated in China in December 2019 and rapidly spread worldwide. Subsequently, SARS-CoV-2 has repeatedly mutated, particularly since the introduction of vaccination, and various variants of concern, including the Alpha, Delta, and Omicron variants, have emerged. The incidence of SARS-CoV-2 infection has repetitively increased and decreased in waves according to the changes in infectivity, individual susceptibility, and behavioral restrictions^1,2).

COVID-19 includes flu-like symptoms, such as a fever, cough, sputum production, fatigue, anosmia, taste disturbances, gastrointestinal symptoms, dizziness, and tinnitus. Immunological protection against COVID-19 can be induced by either previous infection or vaccination^3,4). Originally, patients were diagnosed with COVID-19 based on characteristic symptoms; therefore, those with asymptomatic infections were overlooked.

However, as with many infectious diseases, some cases of SARS-CoV-2 infection are asymptomatic. Despite having extremely mild or no symptoms of SARS-CoV-2 infection, individuals with asymptomatic infection play an important role in SARS-CoV-2 transmission^5,6). Their SARS-CoV-2 viral loads are similar to those of symptomatic individuals, as determined by the PCR testing of nasopharyngeal swabs⁷⁾. Therefore, understanding the prevalence and characteristics of individuals with asymptomatic infection is important in healthcare settings.

In Japan, the surge in infection caused by the highly contagious Omicron variant from October 2022 to March 2023, known as the eighth wave, had a major impact on the general population, including children and frontline medical workers. Unlike other regions (such as Europe and the USA) where COVID-19 vaccination of healthcare workers was initiated later, in Japan vaccination of healthcare workers was prioritized and most healthcare workers had received several doses of vaccine before the first major wave (the eighth wave) of SARS-CoV-2 infection in Japan⁸⁾. Therefore, prior vaccination may have masked COVID-19 symptoms and resulted in cases of asymptomatic infection among healthcare workers.

However, the impact of the eighth wave on the incidence of asymptomatic infection is unclear. Furthermore, studies regarding the effectiveness of vaccines in preventing infections are based on patient awareness of their COVID-19-associated symptoms⁹⁾. The effect of vaccinations on asymptomatic infections is also unclear.

Therefore, we used the SARS-CoV-2 nucleocapsid (N) test, which is positive for several months post infection^10–13), to investigate the incidence of asymptomatic infection among healthcare workers and the effect of the number of doses of vaccine received on the incidence of asymptomatic infection during the eighth wave of COVID-19.

 Material and Methods

We conducted a cross-sectional study on the prevalence of COVID-19 in healthcare workers at our medical facility. Participants completed a questionnaire, which included questions on age, sex, height, weight, body mass index (BMI), smoking status, alcohol consumption, number of COVID-19 vaccine doses received, and COVID-19 history as of March 2023. The evaluation of vaccination safety was managed by the infection control team in our medical facility.

Smokers were defined as those who smoked ≥5 cigarettes per day. Drinkers were defined as those who consumed ≥20 g of alcohol per day on ≥5 days per week. Participants were defined as having previous SARS-CoV-2 infection, based on one or more of the following criteria: 1) a positive polymerization chain reaction (PCR) or antigen test result confirming SARS-CoV-2 infection; or 2) a history of a sore throat, cough, or a fever of ≥38°C for >1 day, and close contact with an individual with SARS-CoV-2 infection between February 2020 and March 2023. Close contacts were defined as those who satisfied the same criteria for SARS-CoV-2 infection/COVID-19 used to define previous infection in study participants.

Serological tests for SARS-CoV-2 N antibodies are not affected by vaccination and are positive in those who have been infected with SARS-CoV-2. The participants who reported no history of infection in the questionnaire survey underwent serological testing for the N antibody. N antibody testing was performed using an Architect SARS-CoV-2 Immunoglobulin G (IgG) Detection Kit (Abbott, Inc., Abbott Park, IL, USA). Antibody levels <0.5 units were considered negative according to Abbott Diagnostics Product Information Letter PI1060-2020¹⁴⁾.

The prevalence of N antibodies was assessed according to the number of doses of COVID-19 vaccine received. Participants were categorized into four groups based on whether they had received 0–2, 3, 4, or 5 doses of vaccine.

The distribution of continuous variables was visually assessed using a histogram. The demographic and clinical characteristics of the participants were reported as means and standard deviations (SDs) for normally distributed continuous variables, as medians and interquartile ranges (IQRs) for non-normally distributed continuous variables, and as frequencies and percentages for categorical variables.

The Mann–Whitney U-test or Kruskal–Wallis test was used to test for differences in the non-normally distributed continuous variables between the groups. One-way analysis of variance was used to test for differences in the normally distributed continuous variables between the groups.

The prevalence of N antibody positivity according to the number of vaccine doses received was tested for trend using the Cochran–Armitage trend test. p-values <0.05 were regarded as statistically significant. The Bonferroni–Dunn test was used as a post-hoc test if the Cochran–Armitage test was statistically significant.

Multivariable logistic regression was used to assess the association between the number of doses of COVID-19 vaccine received and SARS-CoV-2 N antibody test positivity in participants without a history of COVID-19 or SARS-CoV-2 infection. The multivariable logistic regression model included age <40 years, male sex, smoking, alcohol consumption, BMI ≥25 kg/m², and number of doses of COVID-19 vaccine as predictors. Two-tailed p-values <0.05 were regarded as statistically significant. All statistical analyses were performed using the SPSS, version 29.0.1 (IBM Corp., Armonk, NY, USA).

Ethical approval was obtained from the Institutional Review Board of Hakujikai Memorial Hospital (approval number: #Hakujikai45), and the study was carried out in compliance with the relevant laws and guidelines and the ethical standards of the Declaration of Helsinki. Written informed consent was obtained from all participants for publication of their de-identified data.

 Results

By March 2023, a total of 494 staff working in our medical facilities had completed the questionnaires. The demographic and clinical characteristics of all participants (N = 494) and participants without a history of SARS-CoV-2 infection (N = 288) are shown in Table 1. Among 288 participants who reported no history of SARS-CoV-2 infection in the questionnaire survey, 185 accepted serological testing for past infection, of whom 14 (7.6%) showed serological evidence of infection, indicating past asymptomatic infection.

Table 1.Demographic and clinical characteristics of all participants and participants without a history of SARS-CoV-2 infection

Characteristic	All participants (N = 494)	Participants without a history of SARS-CoV-2 infection (N = 288)
Age (years), median (IQR)	38 (27–49)	39 (28–52)
Sex, n (%)
Female	363 (73.8)	201 (69.8)
Male	131 (26.2)	87 (30.2)
Smoking status, n (%)	79 (16.1)	52 (18.1)
Alcohol consumption, n (%)	80 (16.2)	49 (17.0)
Body mass index (kg/m2), mean (SD)	23.2 (4.3)	22.9 (5.9)
Number of vaccinations, median (IQR)	4 (3–4)	4 (3–4)
History of COVID-19/SARS-CoV-2 infection, n (%)
No	288 (58.5)
Yes	204 (41.5)

IQR, interquartile range; SD, standard deviation

The prevalence of N antibody positivity was significantly lower in participants who had received 3 (10.8%; p < 0.05), 4 (4.8%; p < 0.01), and 5 doses (3.0%; p < 0.01) of COVID-19 vaccine than that in those who had received 0–2 doses of vaccine (36.4%; Table 2 and Fig. 1). The decreased prevalence of N antibody positivity according to the number of doses of vaccine received showed a significant trend (Cochran–Armitage trend test p < 0.05).

Table 2.Prevalence of N antibody-positive test results, according to the number of COVID-19 vaccine doses received

Number of doses of COVID-19 vaccine	Number of antibody (+) tests	Number of antibody (-) tests	Total number of tests	Antibody prevalence (%)
0–2	4	7	11	36
3	4	33	37	11
4	5	99	104	4.8
5	1	32	33	3
Total	14	171	185	7.6

According to the Cochran–Armitage test for trend, the N antibody prevalence decreased significantly as the number of COVID-19 vaccine doses increased (p < 0.05).

Fig. 1.  Graph depicting the prevalence of asymptomatic severe acute respiratory syndrome coronavirus-2 infection, as determined by nucleocapsid (N) antibodies, according to the number of COVID-19 vaccine doses received.

The results of the unadjusted and multivariable (adjusted) logistic regression analysis of factors associated with SARS-CoV-2 N antibody positivity in participants without a history of COVID-19/SARS-CoV-2 infection are shown in Table 3. Multivariable logistic regression analysis of factors associated with a positive N antibody test in participants without a history of COVID-19/SARS-CoV-2 infection (Table 3) revealed that participants who had received 3 doses of COVID-19 vaccine had a significantly lower probability of N antibody positivity than those who had received 0–2 doses of vaccine (odds ratio [OR]: 0.16, 95% CI: 0.03–0.95, p = 0.044). Moreover, participants who had received 4 or 5 doses of vaccine also had a significantly lower probability of N antibody positivity than those who had received 0–2 doses of vaccine (4 doses: OR: 0.07, 95% CI: 0.01–0.37, p = 0.002; 5 doses: OR: 0.04, 95% CI: 0.003–0.54, p = 0.015).

Table 3.Unadjusted and adjusted logistic regression of factors associated with a positive SARS-CoV-2 nucleocapsid (N) antibody test result in participants without a history of COVID-19/SARS-CoV-2 infection

Covariates	N antibody positive N (%)	Unadjusted analysis		Adjusted analysis
		OR (95% CI)	p	OR (95% CI)	p
Age (years)
<40	7 (8.0)	1.00		1.00
≥40	7 (7.2)	0.90 (0.30–2.68)	0.850	1.41 (0.41–4.86)	0.590
Sex
Male	4 (8.5)	1.00		1.00
Female	10 (7.2)	0.84 (0.25–2.82)	0.777	1.42 (0.36–5.69)	0.617
Smoking status
No	10 (6.5)	1.00		1.00
Yes	4 (13.3)	2.23 (0.65–7.65)	0.220	2.39 (0.61–9.32)	0.209
Alcohol consumption
No	10 (6.6)	1.00		1.00
Yes	4 (11.8)	1.88 (0.55–6.40)	0.312	1.13 (0.28–4.54)	0.862
Body mass index (kg/m2)
<25	12 (8.8)	1.00		1.00
≥25	3 (6.4)	0.79 (0.21–2.95)	0.723	0.80 (0.19–3.33)	0.757
Number of doses of vaccine
<3	4 (36.4)	1.00		1.00
3	4 (10.8)	0.21 (0.04–1.06)	0.059	0.16 (0.03–0.95)	0.044
4	5 (4.8)	0.09 (0.02–0.41)	0.002	0.07 (0.01–0.37)	0.002
5	1 (3.0)	0.06 (0.01–0.57)	0.015	0.04 (0.003–0.54)	0.015

CI: confidence interval; OR: odds ratio

 Discussion

Of the healthcare workers who reported no history of SARS-CoV-2 infection, 7.6% had serologic evidence of asymptomatic infection within the past several months during the eighth wave. This indicates that asymptomatic infection is not uncommon and even if authorities lower the COVID-19 infectious disease crisis alert level, maintaining infection control measures in medical institutions is advisable. In addition, this cross-sectional study suggests that the prevalence of asymptomatic SARS-CoV-2 infection, which poses a risk of spreading infection^5–7), decreased according to the number of COVID-19 vaccine doses received. After COVID-19 was first detected in late 2019 in China¹⁵⁾, SARS-CoV-2 caused major outbreaks in most of countries other than Japan during 2020 before the introduction of mass vaccination. Therefore, most studies regarding the effectiveness of vaccines have evaluated hybrid immunity, based on a combination of SARS-CoV-2 infection and vaccination, and have focused primarily on symptomatic cases¹⁶⁾. To our knowledge, this study is the first to focus on vaccine efficacy against asymptomatic SARS-CoV-2 infection, rather than symptomatic infection, which has been demonstrated in numerous studies.

Furthermore, because 10% or more of individuals with COVID-19 experience long-term sequelae for ≥2 months after infection^17,18), providing repeated booster doses of vaccine at regular intervals should be considered to prevent such sequelae. However, the value of administering additional doses of vaccine is a subject of debate owing to concerns regarding the adverse effects of vaccination. Some people are reluctant to receive multiple doses of COVID-19 vaccine²⁾. Our results reveal that administering additional booster doses of the vaccine has a cumulative protective effect against SARS-CoV-2 infection. These findings can inform vaccination policy and might help to overcome vaccine hesitancy. Regular COVID-19 vaccination is expected to continue to play an important role in preventing the spread of infection and maintaining social life, especially in medical institutions.

Estimating the magnitude and durability of protection induced by vaccination in the population is challenging, because of the following: 1) varying incidence rates and timing of past infection and vaccination, 2) multiple types of vaccine used, 3) differing numbers of vaccine doses received, and 4) SARS-CoV-2 variants of concern evading pre-existing immunity^1,2). Evaluating the effectiveness of multiple doses of vaccine in preventing asymptomatic SARS-CoV-2 infection is challenging. Nevertheless, using N antibodies, we were able to investigate the prevalence of asymptomatic infection among individuals without a history of SARS-CoV-2 infection. Serological antibody tests for SARS-CoV-2 infection have different characteristics depending on the manufacturer^10–12). The Abbott SARS-CoV-2 N antibody test used in this study detects antibodies immediately after infection, and these N antibodies remain positive for several months¹³⁾. Therefore, this period is likely to have been long enough for IgG to wane, particularly in those with asymptomatic infection during the earlier stage of the pandemic. Thus, the cases of asymptomatic infection detected in this study are likely to reflect SARS-CoV-2 infections that occurred between December 2022 and March 2023, during the eighth wave in Japan, during which the Omicron variant was prevalent in Japan. The characteristics of the N antibody used in this study matched the timing of the investigation into the eighth wave caused by the Omicron strain, thus fortuitously providing valuable insights into the incidence of asymptomatic infection.

Augusto et al.¹⁹⁾ investigated the association between five human leukocyte antigen (HLA) loci and the clinical course of COVID-19. They have found that individuals with asymptomatic infection are more likely to possess specific HLA alleles, such as HLA-B*15:01. This suggests that genetic factors play a role in asymptomatic infection. Moreover, Augusto et al.¹⁹⁾ reported that in the final model adjusted for age and sex, the prevalence of HLA-B*15:01 in individuals with asymptomatic and symptomatic infection was 19.9% and 9.4%, respectively. The odds ratio for asymptomatic infection in individuals with HLA-B*15:01 compared with individuals without HLA-B*15:01 was 2.40 (95% CI 1.54–3.64). Therefore, HLA-B*15:01 is thought to provide some protection against symptomatic infection; however, asymptomatic infection cannot solely be attributed to genetic factors. Nakajima et al.²⁰⁾ analyzed HLA haplotypes in the Japanese population using high resolution allele typing. The prevalence of HLA-B*15:01 in the Japanese population was reported as 9.37%. The impact of genetic factors on the prevalence of asymptomatic infection and the efficacy of vaccines in preventing asymptomatic individuals in Japan requires further investigation.

 Limitations

This study has some limitations. First, it was a single-center study, and the sample size was limited. Second, because the study was limited to medical workers, the study results may not be generalizable to the general population. Third, among the 288 participants diagnosed with asymptomatic infection, only 185 (64%) consented to be tested for antibody levels. Thus, there is the possibility of selection bias. Participation in the study was voluntary, and the reason for non-participation is unknown. Nevertheless, because the testing costs were borne by our organization, non-participation cannot be attributed to economic factors.

 Conclusion

Of the individuals who reported no history of SARS-CoV-2 infection, 7.6% had serological evidence of recent asymptomatic infection which posed a risk of spreading infection for several months during the eighth wave. Therefore, because asymptomatic infection is not uncommon, maintaining infection control measures in medical institutions is advisable even after the COVID-19 infectious disease crisis alert level has been lowered. Furthermore, the risk of asymptomatic COVID-19 was inversely associated with the number of doses of COVID-19 vaccine. Therefore, the cumulative effect of vaccination on preventing asymptomatic COVID-19, suggests that repeatedly administering COVID-19 vaccine to healthcare workers may have value as an infection control measure.

Further studies are needed to determine the durability of the immune response to vaccination, not only the transition of S antibodies following vaccination but also through the development of improved tests that can predict the duration of a clinically protective N antibody-positive response after infection, to inform guidelines regarding the optimal timing and number of booster vaccines required, while monitoring the medium- to long-term side effects of vaccination.

 Acknowledgments

This research has been internally funded by the Hakujikai General Incorporated Foundation and performed by the BML Biomedical Laboratories. The views expressed are those of the authors and not necessarily those of Hakujikai General Incorporated Foundation or the BML Biomedical Laboratories.

 Author Contributions

KM, SS, YY, KT, TT and KO designed the study. SS, YY, CN and MS collected the data. KM, SS and YY performed the statistical analysis, and drafted the manuscript. All authors read and gave final approval of the submitted manuscript.

 Conflict of Interest

None to declare.

References

• 1.   Bergeri  I,  Whelan  MG,  Ware  H, et al. Global SARS-CoV-2 seroprevalence from January 2020 to April 2022: a systematic review and meta-analysis of standardized population-based studies. PLoS Med 19; e1004107: 2022. DOI: 10.1371/journal.pmed.1004107.
• 2.   Sallam  M. COVID-19 vaccine hesitancy worldwide: a concise systematic review of vaccine acceptance rates. Vaccines (Basel) 9; 160: 2021. DOI: 10.3390/vaccines9020160.
• 3.   Polack  FP,  Thomas  SJ,  Kitchin  N, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med 383; 2603–2615: 2020. DOI: 10.1056/NEJMoa2034577.
• 4.   Jeffery-Smith  A,  Rowland  TAJ,  Patel  M, et al. Reinfection with new variants of SARS-CoV-2 after natural infection: a prospective observational cohort in 13 care homes in England. Lancet Healthy Longev 2; e811–e819: 2021. DOI: 10.1016/S2666-7568(21)00253-1.
• 5.   Alene  M,  Yismaw  L,  Assemie  MA, et al. Magnitude of asymptomatic COVID-19 cases throughout the course of infection: a systematic review and meta-analysis. PLoS One 16: e0249090: 2021. DOI: 10.1371/journal.pone.0249090.
• 6.   Kronbichler  A,  Kresse  D,  Yoon  S, et al. Asymptomatic patients as a source of COVID-19 infections: a systematic review and meta-analysis. Int J Infect Dis 98; 180–186: 2021. DOI: 10.1016/j.ijid.2020.06.052.
• 7.   Lavezzo  E,  Franchin  E,  Ciavarella  C, et al. Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo’. Nature 584; 425–429: 2020. DOI: 10.1038/s41586-020-2488-1.
• 8.   Arashiro  T,  Arai  S,  Kinoshita  R, et al. National seroepidemiological study of COVID-19 after the initial rollout of vaccines: before and at the peak of the omicron-dominant period in Japan. Influenza Other Respir Viruses 17; e13094: 2023. DOI: 10.1111/irv.13094.
• 9.   Bobrovitz  N,  Ware  H,  Ma  X, et al. Protective effectiveness of previous SARS-CoV-2 infection and hybrid immunity against the omicron variant and severe disease: a systematic review and meta-regression. Lancet Infect Dis 23; 556–567: 2023. DOI: 10.1016/S1473-3099(22)00801-5.
• 10.  National SARS-CoV-2 serology assay evaluation group. Performance characteristics of five immunoassays for SAR-CoV-2: a head-to-head benchmark comparison. Lancet Infect Dis 20; 1390–1400: 2020. DOI: 10.1016/S1473-3099(20)30634-4.
• 11.   Fox  T,  Geppert  J,  Dinnes  J, et al. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database Syst Rev 11; CD013652: 2022. DOI: 10.1002/14651858.CD013652.pub2.
• 12.   Mizoue  T,  Yamamoto  S,  Tanaka  A, et al. Sensitivity of three antibody assays to SARS-CoV-2 nucleocapsid protein in relation to timing since diagnosis. GHM Open 2; 51–53: 2022. DOI: 10.35772/ghmo.2021.01030.
• 13.   Harris  RJ,  Whitaker  HJ,  Andrews  NJ, et al. Serological surveillance of SARS-CoV-2: six-month trends and antibody response in a cohort of public health workers. J Infect 82; 162–169: 2021. DOI: 10.1016/j.jinf.2021.03.015.
• 14.   Lumley  SF,  Wei  J,  O’Donnell  D, et al. The duration, dynamics, and determinants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses in individual healthcare workers. Clin Infect Dis 73; e699–e709: 2021. DOI: 10.1093/cid/ciab004.
• 15.   Zhu  N,  Zhang  D,  Wang  W, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382; 727–733: 2020.
• 16.   Miyoshi  Y,  Chimed-Ochir  O,  Yumiya  Y, et al. Effect of vaccine booster dose on coronavirus disease 2019 prevention by age group: Analysis of data collected at polymerase chain reaction centers in Hiroshima Prefecture, Japan. Hiroshima J Med Sci 72; 1–4: 2023.
• 17.   Nalbandian  A,  Sehgal  K,  Gupta  A, et al. Post-acute COVID-19 syndrome. Nat Med 27; 601–615: 2021. DOI: 10.1038/s41591-021-01283-z.
• 18.   Subramanian  A,  Nirantharakumar  K,  Hughes  S, et al. Symptoms and risk factors for long COVID in non-hospitalized adults. Nat Med 28; 1706–1714: 2022. DOI: 10.1038/s41591-022-01909-w.
• 19.   Augusto  DG,  Murdolo  LD,  Chatzileontiadou  DSM, et al. A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection. Nature 620; 128–136: 2023. DOI: 10.1038/s41586-023-06331-x.
• 20.   Nakajima  F,  Nakamura  J,  Yokota  T. Analysis of HLA haplotypes in Japanese, using high resolution allele typing. MHC 8; 1–32: 2001. (in Japanese). DOI: 10.12667/mhc.8.1.

Abbreviations

BMI

body mass index

CI

confidence interval

COVID-19

coronavirus disease 2019

HLS

human leukocyte antigen

IG

immunoglobulin

IQR

interquartile range

N

nucleocapsid

OR

odds ratio

PCR

polymerization chain reaction

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

SD

standard deviation