Assessment of S-Specific IgG and IgM Positive Rates in Healthy Hospital Staff Members Vaccinated with the Inactivated SARS-CoV-2 Vaccine

Abstract

Widespread vaccination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine makes the assessment of antibodies' positive rates essential. In this study, a total of 378 hospital staff members vaccinated with the vaccine were selected as research subjects. Serum-specific IgG and IgM against the SARS-CoV-2 spike protein (S) were detected, and S-specific IgG and IgM positive rates were analyzed in different age and sex groups, as was the serological pattern of IgG/IgM. The positive rates of IgG and IgM were 92.06% and 44.44%, respectively. The percentage of both IgG and IgM positive (IgG⁺IgM⁺) was 43.92%. A total of 182 vaccinees (46.90%) were IgG positive and IgM negative (IgG⁺IgM⁻), and 28 vaccinees (7.41%) were negative for both IgG and IgM (IgG⁻IgM⁻); 2 participants were positive for IgM alone (IgG⁻IgM⁺). In sex subgroups, the rate of IgM positivity was significantly higher in the male group than in the female group (p = 0.027). In different age subgroups, positive rates for IgG in the young group were significantly higher than those in the other group (p = 0.035). Furthermore, ratios of sample values to cutoff values (S/CO values) for IgG in vaccinees who were S-specific IgG positive were compared, and the S/CO values of IgG were significantly higher in the younger group than in the other group (p < 0.001). When comparing the influence of sex on two specific serological patterns (IgG⁺IgM⁻ and IgG⁺IgM⁺), a significant difference in positivity rates was detected (p = 0.011). Male vaccinees were more likely than females to have an IgG⁺IgM⁺ pattern.

Introduction

It has been over a year since the outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the epidemic has been largely contained in most parts of the world, the situation in some regions is still grim, especially after the sporadic emergence of new cases and potentially more aggressive variants (19). Vaccination is the safest, most cost-effective way to prevent COVID-19 disease and death and is also an important safeguard against anticipated aggressive variants. To prevent and reduce virus transmission, it is imperative to accelerate the development of vaccines and vaccination programs. In this regard, scientists have made unprecedented efforts to develop vaccines, with great progress.

According to the latest vaccine candidate research and development report of the WHO, as of September 17, 2021, there were 311 SARS-CoV-2 vaccine research and development projects worldwide, including 10 types of vaccines, such as subunit vaccines, viral vector vaccines, inactivated virus vaccines, and DNA vaccines. A total of 117 vaccine projects are in clinical trials, and 194 are in the preclinical research stage. The inactivated SARS-CoV-2 vaccine (Vero cell)-BBIBP-CorV manufactured by Beijing Institute of Biological Products (BIBP), China National Biotec Group (CNBG), and Sinopharm, which was used in this study, has completed a phase III clinical trial and is currently in phase IV (18,21).

In some countries, widespread COVID-19 vaccination has been initiated. Identifying individuals with protective immunity induced by vaccination will likely be of increasing importance (12). Compared with the detection of immune cell function and cytokines by flow cytometry, serological detection of antibodies is rapid and cost-effective. In addition, there is an increasing demand for rapid serological tests, including magnetic particle chemiluminescence and enzyme-linked immunosorbent assays, to assess the presence of protective antibodies (14). In this study, serum S-specific IgG and IgM were detected by the magnetic particle chemiluminescence method in 378 healthy hospital staff, who had completed COVID-19 vaccination, and the clinical significance was assessed.

Materials and Methods

Participants

A total of 378 hospital staff members who received the BBIBP-CorV vaccine were selected for this study (113 males and 265 females; between 18 and 59 years of age; mean age 38.18 years; median age 39 years; standard deviation 9.82). All participants were vaccinated according to the 0 + 28-day schedule. The vaccine was administered at a dose of 4 μg (0.50 mL). The route of administration was an intramuscular injection in the upper deltoid muscle. Due to the shortage of vaccines at the time, vaccines were administered in batches. The first dose of vaccine was administered from December 29, 2020, to January 10, 2021.

All participants were monitored through SARS-COV-2 nucleic acid testing once weekly for ∼9 months and screened for SARS-CoV-2 infection using epidemiological and vaccination history surveys before vaccination. None of the participants had been infected with SARS-CoV-2 and were vaccinated against COVID-19 for the first time.

The criteria to be a candidate for vaccination were screened strictly by national technical guidelines for COVID-19 vaccination in China. None of the participants had an adverse reaction. Individuals who were unvaccinated or given only the first dose of the vaccine were excluded. All participants provided written informed consent.

Serological testing for S-specific IgM and IgG antibodies

Blood samples were collected in vacuum-drying tubes without additives and then centrifuged at 3,000 rpm for 10 min to separate serum. Two batches of serum samples were collected and stored at −80°C in a freezer. The first batch of 245 samples was taken on Day 0 before vaccination and tested on April 8, 2021. The second batch of 378 samples was collected 14 days after the completion of two doses of vaccine (Day 42) and tested on the same day.

Serum S-specific IgM and IgG antibodies were assessed using 2019-nCoV IgG and IgM antibody detection kits (magnetic particle chemiluminescence method; Zhengzhou Antu Biological Engineering Co., Ltd, Zhengzhou, China) and chemiluminescence automatic analyzer (Autolumo A2000 Plus, Zhengzhou, China). The kits were approved for detecting 2019-nCoV IgM and IgG antibodies by the National Medical Products Administration of China. The test procedures were conducted according to the instructions of the instruments and kits.

Calculation method of antibody S/CO values by Autolumo A2000 Plus

The cutoff value was calculated as follows: Cutoff value = the average luminous value of the positive control × the cutoff coefficient. The cutoff coefficient of the kit for IgG is 0.20 and that of IgM is 0.10.

The S/CO value was calculated as follows: S/CO value = luminescence value of sample/Cutoff value.

The result was considered positive at S/CO ≥1.00 and negative at S/CO <1.00.

Statistical analysis

SPSS 23.0 software was used for statistical analysis. All participants were grouped by sex and age. The young group consisted of 208 vaccinees between 18 and 39 years of age, and the other group consisted of 170 vaccinees 40 to 59 years of age. The chi-square test was used to compare rates of positivity between each age and sex subgroup after weighting. For seropositive subjects, an analysis of normal distribution was evaluated using one-sample K-S test, and S/CO values did not conform to a normal distribution. Therefore, the Mann–Whitney U test was used to compare S/CO values between each group. p < 0.05 was considered statistically significant.

Results

Cohort description

Among 378 vaccinees, the rate of S-specific IgG positivity was 92.06% (348/378), and S/CO values ranged from 1.03 to 138.19 (mean value 23.61; median value 16.74). The rate of S-specific IgM positivity was 44.44% (168/378); the S/CO value ranged from 1.01 to 81.04 (mean value 5.08; median value 3.08). A total of 166 vaccinees (43.92%) showed both S-specific IgG and IgM positivity; 182 were positive for S-specific IgG alone, and the S/CO value of IgG ranged from 1.15 to 79.57, with a mean value of 16.85 and a median value of 13.02.

One male and one female vaccinee were S-specific IgM positive and IgG negative. Twenty-eight patients (7.41%) were negative for both S-specific IgG and IgM (double negative), ranging in age from 25 to 57 years (Table 1). Fifteen of the double-negative vaccinees underwent blood withdrawal again on day 62; only three became IgG positive, and the others remained negative.

Table 1.

Distribution of Sex and Serological Antibody Patterns

	IgG⁺IgM⁻ ^a	IgG⁺IgM⁺	IgG⁻IgM⁻	IgG⁻IgM⁺
Female (N = 165)	141	107	16	1
Male (N = 113)	41	59	12	1
Total (N = 378)	182 (48.15%)	166 (43.92%)	28 (7.41%)	2 (0.53%)

+positive; −negative.

The baseline of antibody levels

To exclude false-positive results, S-specific antibodies in 245 blood samples (82 males and 163 females; mean age 37.62 years) collected before vaccination were evaluated. Two (0.82%) female participants (32 and 45 years) were found to be false positive for S-specific IgG, with S/CO values of 1.18 and 14.03. Further examination of the serum sample with higher false-positive S/CO value showed high levels of total protein, IgG, IgA, C3, and uric acid. After the elimination of false-positive samples, the S/CO values of S-specific IgG ranged from 0.01 to 0.22 (mean value 0.02). Only one 54-year-old male vaccinee presented a false-positive (0.41%) result for S-specific IgM (S/CO value, 3.04). The S/CO value for S-specific IgM seronegative participants ranged from 0.01 to 0.17 (mean value 0.04).

Sex bias for S-specific antibodies

To analyze the influence of sex on the rate of S-specific IgG/IgM antibody positivity, rates were compared between male and female subgroups. For S-specific IgG, the rate in the male group was 88.50% and that in the female group was 93.58%, with no significant difference between the groups (χ ² = 2.81, p = 0.094). However, the rate of S-specific IgM positivity differed significantly (χ ² = 4.89, p = 0.027), with the male group being significantly higher than the female group (57.15% vs. 38.51%). The S/CO value of S-specific IgM was also significantly higher in the male group (p < 0.001) (Fig. 1A).

FIG. 1.

(A) In the IgM positive cohort, the S/CO value of S-specific IgM was significantly higher in the male group. (B) In the IgG positive cohort, the S/CO values of S-specific IgG in the young group were significantly higher. (C) The S-specific IgG S/CO value in the both IgG and IgM positive (IgG⁺IgM⁺) cohort was significantly higher than that in the IgG positive and IgM negative (IgG⁺IgM⁻) cohort. Data were shown as mean with SD. SD, standard deviation.

Age bias for S-specific antibodies

Significant differences in the rates of S-specific IgG positivity between different age groups were observed (χ ² = 4.44, p = 0.035), although IgM rates did not differ significantly (χ ² = 0.39, p = 0.533). The rate of S-specific IgG positivity was higher in the young group, and S/CO values of S-specific IgG in the young group were significantly higher (p < 0.001) (Fig. 1B). There was no significant difference in S-specific IgM S/CO values between groups of S-specific IgM-positive vaccinees (p = 0.623).

The impact of sex and age on dominant patterns of serum S-specific IgG/IgM

Serological patterns for gG⁺IgM⁻ (48.15%) and IgG⁺IgM⁺ (43.92%) after the COVID-19 vaccine were maintained. Chi-square test results showed a significant difference in rates of IgG⁺IgM⁻ and IgG⁺IgM⁺ positivity between the male and female subgroups (χ ² = 6.41, p = 0.011). Males were more likely to have IgG⁺IgM⁺ patterns, and the S-specific IgG S/CO value in the IgG⁺IgM⁺ cohort was significantly higher than that in the IgG⁺IgM⁻ cohort (p < 0.001) (Fig. 1C). Nevertheless, no significant difference was found between the different age groups (χ ² = 0.07, p = 0.788).

Discussion

SARS-CoV-2 is a β coronavirus and shows homology with SARS-CoV and MERS-CoV(2). SARS-CoV-2 contains four structural proteins: the spike protein (S), nucleocapsid protein (N), an envelope protein (E), and membrane protein/matrix protein (M).

The S protein consists of two subunits: S1 and S2. The receptor-binding domain (RBD) of the S1 subunit is responsible for recognizing and binding to host cells through the specific receptor angiotensin-converting enzyme 2 (ACE-2) (20). The S2 subunit mediates fusion between the host cell membrane and the virus (16). The M and E proteins are mainly involved in the viral assembly process. The N protein, a highly immunogenic protein, is involved in RNA synthesis and the viral budding process. The S protein is the main target of most SARS-CoV-2 vaccines, and the neutralizing antibodies stimulated by it block the recognition and binding of the S protein or RBD to the host cell receptor, thus protecting against SARS-CoV-2 infection (15,17).

The target antigen of the kits used in this study is a recombinant S protein, and specific antibodies against S were detected. The study of Louise showed that the frequency of inhibition by a rapid surrogate virus neutralization test strongly correlates with spike-specific IgG titers detected by both commercial and in-house enzyme-linked immunosorbent assay and microneutralization test titers (a gold standard correlate of protection) (14). However, the disadvantage of our study is that the neutralization effect of the detected antibodies was not verified. The vaccine used in this study is an inactivated vaccine with robust immunogenicity that effectively induces a humoral immune response.

Indeed, only 7.41% of the vaccinees were negative for both S-specific IgG/IgM antibodies, and 92.59% were positive for at least one antibody (S-specific IgG or IgM). The phase 1/2 trial of the BBIBP-CorV inactivated vaccine showed a robust humoral immune response in 100% of vaccine recipients (21). The rates of positivity for specific antibodies in our study were lower than the clinical trial, and the main reasons may be the different detection reagents used and the single postvaccination time point evaluated in our study. Neutralizing antibodies were tested during the clinical trial, but diagnostic reagents were used to detect S-specific IgG/IgM in our study; different viral epitopes may also have been used for detection.

IgM and IgG antibodies against SARS-CoV-2 are exclusion indicators for suspected COVID-19 cases according to the eighth edition of the “Guideline of diagnosis and treatment for COVID-19” by the Chinese National Health Commission, and complement the false negatives of the nucleic acid test. Therefore, the high rates of IgG and IgM positivity in this study suggest that it is of great significance to inquire about vaccination history in the clinical diagnosis of COVID-19. False positivity may occur due to the positive judgment value of the reagent itself, the presence of interfering substances (rheumatoid factor, anisotrophil antibody, complement, lysozyme, etc.), or specimen factors (such as hemolysis, contamination by bacteria, long storage time, incomplete coagulation, etc.).

To verify factors responsible for false-positive results, we tested 245 serum samples before vaccination, and the false-positive rates of IgG and IgM were 0.82% and 0.41%, respectively. It is worth noting that participants who had a false-positive result showed a more than fourfold increase in antibodies after vaccination; thus, they were considered to have a positive response to vaccine in the subsequent analysis. The study of Jason Rosado showed that when true seroprevalence is less than 2%, the relative error is minimized when specificity is 100% (13), indicating that the specificity of such kits is high, with a baseline of S/CO values less than 0.05.

IgM is a short-term acute antibody produced by antigen stimulation, the levels of which rise and fall over a relatively short period. Positivity for the SARS-CoV-2-specific IgM antibody usually begins at 3–5 days after COVID-19 onset (4). Besides, IgM tends to appear at ∼10 days after infection with other SARS-associated coronavirus (5). Regardless, the regulation of IgM antibody production after COVID-19 vaccination has not been reported.

Nevertheless, it is not uncommon for inactivated vaccines to induce IgM antibodies. For example, in addition to producing high titer virus-specific IgG antibody after trivalent inactivated influenza virus vaccination, virus-specific IgM antibodies were found in 90–92% of vaccinees, and the IgM antibody persisted as long as 6 months in 55–62% of subjects after vaccination (8). Moreover, anti-HBS IgG antibodies can be detected from day 4 to 21 after hepatitis B virus vaccination, but specific anti-HBS IgM is present before the detection of anti-HBS IgG, the titer of anti-HBS IgM is always low, and the half-life of this antibody is short (less than 21 days) (10). In our study, the rate of S-spike IgM positivity reached 44.57% on day 42, which may be caused by the half-life of S-spike IgM being longer than 15 days in some populations.

Studies have also shown that antigen-specific IgM can enhance the immune response to inactivated vaccines (1), and some scholars have even proposed the hypothesis that IgM can be used as an immune adjuvant (7), which may explain the phenomenon that the level of S-spike IgG in the IgG⁺IgM⁺ pattern group was significantly higher than that in IgG⁺IgM⁻ in our study. To a certain extent, the rate of IgM positivity may reflect differences in humoral immunity of a population to the vaccine. The rate of S-spike IgM positivity in male vaccinees is higher than in females, which may be due to the different activation statuses of B cells between the sexes (9).

IgG antibodies are an important immune marker that can provide long-term protection when stimulated by antigens. The reason why blood was drawn on the 14th day after the second dose of vaccine was that many studies have reported IgG antibodies could be detected around the 14th day after the onset of COVID-19 symptoms (4,6,11,22). The rate of IgG antibody positivity was as high as 92.06%, indicating that this cohort had a good response to the vaccine. Compared with sex, age had a stronger effect on the rate of positivity and even S-spike IgG S/CO values.

The relatively low IgG levels in older adults may be related to their lower cellular immune response to inactivated vaccines (e.g., HBV, trivalent inactivated influenza vaccine) (23). In particular, the quality of the B cell response changes with age, for example, older people have been shown to have fewer “IgM memory” cells, regulatory B cells, and plasma cells (3). Despite no significant difference in the rate of S-specific IgG positivity between the sexes in this study, the average titer of S-specific IgG in females was higher than that in males, indicating that females had a higher level of IgG antibody response (23).

Finally, it should be noted that this study is a descriptive analysis, which inevitably has certain limitations. SARS-CoV-2 is also a novel virus, and many unknown factors may not have been considered in this study.

Conclusion

Vaccine immunoreactivity was very high, with a 92.59% overall positive rate for S-specific IgG and IgM. In addition to S-specific IgG, males tended to produce S-specific IgM. In addition, IgM may promote the secretion of IgG, as based on the significantly increased IgG S/CO values among vaccinees with the IgG⁺IgM⁺ pattern. Young vaccinees have a greater rate of S-specific IgG positivity and higher S-specific IgG levels.

Footnotes

Acknowledgments

The study was approved by the Medical Ethics Committee of Guangzhou Twelfth People's Hospital. Sincere thanks to all the staff of Guangzhou Twelfth People's Hospital. Thank you for your support.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the Natural Science Foundation of Guangdong (grant numbers: 2018A030313533), the Science and Technology Program of Guangzhou (grant number: 201707010156, 202102080398, 202102010049), and the Medical Science and Technology Research Fund Project of Guangdong Province (grant numbers: A2020140).

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