The Healthcare Study Examines the Humoral Anti-S1 Antibody Response Following mRNA Vaccination, Comparing Individuals with and without Prior SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), a member of the RNA coronaviridae family, emerged as the causative agent of the coronavirus disease in 2019 (COVID-19). This virus exhibits a notable affinity for the angiotensin-converting enzyme 2 (ACE2) receptor found on epithelial cells, particularly in organs like the lungs, facilitating infection and subsequent tissue damage. Upon infection, the innate and adaptive immune systems mount responses to combat the virus (Catanzaro et al. 2020; Esmaelizadeh and Elahi 2020; Mortaz et al. 2020; Vabret et al. 2020; Sette and Crotty 2021).

The adaptive immune response initiates with antigen-presenting cells recognizing and presenting viral antigens, thereby stimulating and orchestrating cellular immune responses against SARS-CoV-2. Activation of T helper (Th) cells prompts the secretion of Th1 cytokines such as interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin 2 (IL-2). Notably, cytotoxic T lymphocytes (CD8⁺) play a pivotal role in viral clearance through targeted elimination of infected cells. Among the most immunogenic antigens fostering the induction of memory T and B cells are the spike (S) protein and the matrix (M) protein from the viral capsule. Longitudinal studies have demonstrated a decline of approximately half in the memory CD8⁺ T-cell population within 6 months post-SARS-CoV-2 infection, contrasting with a similar decline in memory CD4⁺ T cells observed 3 months after patient recovery (Dan et al. 2021).

T follicular helper cells specific to SARS-CoV-2 within lymph nodes play a crucial role in stimulating B lymphocytes to generate antigen-specific antibodies and establish a memory B-cell subset. Notably, seroconversion from immunoglobulin IgM class (IgM) to IgG antibodies occurs in approximately 90% of patients infected with SARS-CoV-2, often within 10 days post-onset of clinical symptoms. Many of these patients demonstrate neutralizing activity against the spike (S) protein and its receptor binding domain (RBD), crucial for blocking viral attachment to the ACE2 receptor. However, the production kinetics and titers of antibodies vary considerably among symptomatic SARS-CoV-2 cases.

In a cohort study involving 6062 healthcare workers exposed to SARS-CoV-2, specific antibodies were detected in 212 individuals, particularly among those of younger age who exhibited typical symptoms such as anosmia, dry cough, and myalgia with fatigue (Ebinger et al. 2020; Garcia-Beltran et al. 2021).

The significance of neutralizing antibodies in combating SARS-CoV-2 is evident, as they aid in reducing viral load through cell-mediated mechanisms. Nevertheless, observations from patients with X-linked agammaglobulinemia, who recovered from SARS-CoV-2 infection without antibody production, underscore the essential role of antibodies in virus clearance. Additionally, concerns have been raised regarding the potential adverse effects of antibodies, including inflammation enhancement (antibody-dependent enhancement) and organ damage, particularly in the lungs (Vabret et al. 2020; Bettini and Locci 2021; Fraley et al. 2021; Garcia-Beltran et al. 2021; Sette and Crotty 2021; Vashishtha and Kumar 2021).

Antibodies targeting the S1 protein serve as vital indicators for evaluating the efficacy of immunization strategies (Tregoning et al. 2020; Bettini and Locci 2021; Vaquero et al. 2021; Vashishtha and Kumar 2021; Melgoza-González et al. 2022; Salleh et al. 2022). Following administration of mRNA vaccines, individuals not previously infected experience a robust germinal center response, accompanied by increases in plasma cell numbers and expansion of the memory B-cell population. Conversely, individuals with prior SARS-CoV-2 infection exhibit heightened responses following the initial vaccine dose, with a majority of antibodies demonstrating neutralizing capabilities. Notably, the magnitude of antibody production correlates with the proliferation of specific B memory cells (Laidlaw and Ellebedy 2022; Padoan et al. 2022; Roltgen et al. 2022; Van Elslande et al. 2022).

Studies have scrutinized antibody synthesis during SARSCoV-2 infection, post-vaccination in non-infected individuals and post-infection followed by vaccination. Sequential vaccine doses consistently elevate antibody production, with a notable prevalence of the IgG1 subclass. Vaccination induces a response primarily in the IgA1 subclass, with no significant increase observed in IgM levels post-vaccination (Vaquero et al. 2021; Salleh et al. 2022). Furthermore, individuals recovering from SARS-CoV-2 infection exhibit enhanced vaccine responses compared to those without prior infection, particularly when following similar vaccination schedules. Incorporating a third dose into the vaccination regimen significantly boosts specific antibody production, regardless of prior infection status (Ebinger et al. 2021; Fraley et al. 2021; Vaquero et al. 2021; Padoan et al. 2022; Salleh et al. 2022). Enhanced vaccination schedules, including third and fourth doses, prompt robust responses, stimulating the production of memory T and B cells alongside a substantial proportion of neutralizing antibodies (Amanat et al. 2021; Padoan et al. 2022; Roltgen et al. 2022).

This study aimed to analyze the titer and kinetics of anti-S1 protein antibodies (including IgG, IgA, and IgM) across various vaccination regimens (from first to third dose), particularly in relation to prior SARS-CoV-2 infection. Conducted among healthcare workers at a pediatric hospital, the research seeks to provide insights into antibody responses among this specific population.

Under “The Staff Vaccination Program,” employees from the University Children Hospital in Krakow participated in a study evaluating antibody production targeting the SARS-CoV-2 spike S1 protein following vaccination with the BioNTech-Pfizer BNT162b2 vaccine in two doses (later adjusted to include a third dose by the government). The cohort comprised 43 men and 606 women, totaling 649 participants. Data on participants’ SARS-CoV-2 exposure history, PCR results, COVID-19 symptoms, comorbidities, medical treatment, vaccination tolerance, and complications were self-reported. Ethical approval was obtained from the Jagiellonian University Medical College Ethics Committee.

Serum samples were collected from the participants according to the following schedule:

• Before the administration of the first vaccine dose in select participants.

• Within 3–4 weeks after the first vaccine dose.

• About 2–4 weeks after the administration of the second or third vaccine dose. The second dose was administered approximately 3 weeks after the first, with the final dose given 3–5 months later. The vaccination program commenced in late December 2020, initially utilizing the Pfizer-BioNTech BNT162b2 vaccine. In March 2021, some participants received the AstraZeneca ChAdOx1 nCoV-19 vaccine and were subsequently excluded from the study (Figure 1). Samples characteristics are shown in Table 1.

Fig 1.

Study design schema. COVID-19, coronavirus disease in 2019; SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2.

Serological analyses were conducted at AESKU.Diagnostics GmbH & Co. KG’s facilities in Wendelsheim, Germany, using AESKULISA® SARS-CoV-2 nucleocapsid protein (NP) IgM, IgA and IgG, as well as AESKULISA® SARS-CoV-2 S1 IgM, IgA and IgG immunoassays. Tests were performed following the manufacturer’s instructions. Test results >12 U/mL were considered positive, while results <8 U/mL were deemed negative. Antibody activities were quantified using recommended dilutions, with results referenced to the international standard preparation for easier comparison with other studies.

Participants were divided into two groups:

• Group 1 comprised individuals with evidence of prior infection, confirmed by positive SARS-CoV-2 polymerase chain reaction (PCR) or serological test results prior to vaccination, and positive serological test results after the second dose.

• Group 2 included staff without evidence of prior infection, including those with negative PCR or serological test results.

The average concentration of IgG anti-NP antibody in participants with prior SARS-CoV-2 infection was greater after the first and third doses of vaccination than before vaccination (mean values 27.4 U/mL and 24.8 U/mL, respectively, compared to 20.6 U/mL) but lower after the second dose (mean value 16.6 U/mL) (Table 2 and Figure 2). GraphPad Prism version 6.01 was used for statistical analyses. Kruskal-Wallis test was conducted to identify any significant changes in categorical variables over time and between groups. Non-parametric Mann-Whitney (Wilcoxon) test was used to compare quantitative data over time or between groups, respectively. Participants without SARS-CoV-2 contact had low antibody levels (below the limit of positive results). There was a statistically significant difference between participants after contact with SARS-CoV-2 and those without infection at all the assayed points. Positive or borderline anti-NP IgG antibody activity was considered a marker for previous infection with SARS-CoV-2 (Salleh et al. 2022). Individuals from the non-infected group with positive or borderline anti-NP IgG antibody levels at the time of vaccination were included in the “infection” group and were tested for reinfection with SARS-CoV-2. In patients without SARS-CoV-2 infection, the increase in the anti-NP antibody levels after vaccination may be the result of the period between infection and vaccination, persistent immune memory of the virus proteins, and nonspecific stimulatory activity of the vaccine. The production of this type of antibody by memory B cells against the NP protein is stimulated by vaccination as an effect of the general immune system being stimulated by the vaccines. Observation of antibody production in the majority of patients recovered from severe infection 1 year after infection and in patients after a mild course supported the induction of a persistent memory B-cell population (Van Elslande et al. 2022). The mean IgA antibody level against the NP was negative in both groups during the study. In some individuals from the group after prior infection, IgA antibodies were detected, with the highest value of 39.7 U/mL after the second dose of vaccination. The presence of anti-NP IgM antibodies was observed in individual participants, although the mean value remained below the cutoff point throughout the study. Despite this, the group of individuals with prior evidence of SARS-CoV-2 infection exhibited a slightly elevated average level of anti-nucleoprotein IgM. The maximum IgM antibody activity recorded was 36.2 U/mL in one participant after the second dose of vaccination.

Fig 2.

Changes of IgM, IgA and IgG antibodies levels against NP produced post vaccination according to prior infection of SARS- Cov-2.

Interestingly, participants with detectable IgA and IgM antibodies, above the cutoff point, did not display any symptoms of disease and did not develop COVID-19 during the 6 months observation period following the last dose of vaccination. This suggests that the presence of these antibodies may confer some level of protection against SARS-CoV-2 infection or disease manifestation.

The most interesting observation was the antibody profile of the anti-S1 IgG immune response to the vaccination (Table 3 and Figure 3). The anti-S1 IgG antibody level in the group without prior SARS-CoV-2 infection was high after the first dose of vaccination (347.1 BAU/mL from 12.2 BAU/mL) and further increased after the second dose of vaccination, which was expected (mean value 2329.5 BAU/mL). The final dose of vaccination did not increase the mean IgG antibody level (1522.6 BAU/mL) in the noninfected group of participants. The highest individual anti-S1 IgG antibody levels after subsequent doses of the vaccine were 2672.8 BAU/mL after the first dose, 7937.0 BAU/mL after the second dose and 5304.3 BAU/mL after the final dose. The anti-S1 IgG antibody activity was already high before vaccination in the group with prior SARS-CoV-2 infection (symptomatic or asymptomatic), with a mean value of 88.3 BAU/mL (maximal value—456.2 BAU/mL) and an increase of up to 1696.2 BAU/mL after the first dose of vaccination. The individual maximal values were 7281.4 BAU/mL. However, the average anti-S1 IgG antibody level after the second dose was slightly greater than that after the first dose (mean value—1969.2 BAU/mL), with a decrease (mean value—1152.1 BAU/mL) after the final dose of vaccination. An explanation for this decrease might be the long duration (up to 6 months) between the second and third doses (final) and the still high level of IgG before the final dose (infection group 1, 164 participants; mean value, 330.6 BAU/mL; no-infection group 2, 377 participants; mean value, 163.7 BAU/mL). According to a separate analysis (data not shown), IgG antibody production after vaccination in participants with asymptomatic SARS-CoV-2 infection in comparison to that in the symptomatic group (although with mild symptoms) did not significantly differ.

Fig 3.

Changes of IgM, IgA and IgG antibodies levels against S1 protein produced post vaccination according to prior infection of SARSCov-2.

The systematic increase in IgG antibody concentration after two doses of vaccination in the group without prior contact with the virus aligns with expectations for a robust immune response following vaccination. However, the lack of a further increase in IgG antibody levels after the third dose, coupled with a decrease compared to the response after the second dose, suggests a potential plateau or diminishing returns in antibody production with additional doses. Despite variations in individual responses within both groups, all participants exhibited antibody production against the S1 spike protein, indicating a general immune response to the vaccination.

Significant differences in IgG antibody response were observed between the group after infection and the noninfected group, highlighting the impact of prior exposure to the virus on vaccination outcomes. Similarly, comparisons within the noninfected group revealed differences in IgG antibody responses to the first and second doses of vaccination. These findings underscore the importance of considering individuals’ prior infection status when assessing vaccine efficacy and designing vaccination strategies.

The production of IgA antibodies against the S1 protein was studied due to the specific role of IgA in mucous defense against pathogens, including viruses. In the group after prior infection, the response to vaccination was greater than that in the noninfected group. In contrast to the IgG profile, which shows a response in IgA antibody production, a systematic increase in antibody levels after each dose of vaccination was noted, with the highest mean value occurring after the final dose. Similar to IgG antibody production, statistically significant differences were noted between the group after prior infection and the noninfected group. The level of IgA antibodies was low, but the response to the vaccination was proportional and even greater than that of IgG (Staruszkiewicz et al. 2022).

The anti-S1 IgM antibody activity in all participants was assessed before vaccination and was generally below the cutoff value, with individual positive findings (up to 19.2 U/mL) within the group of participants after prior infection. After the first dose of vaccination, an increase in the average anti-S1 IgM antibody level was observed in both groups (6.3 BAU/mL in the group without prior infection and 8.8 BAU/mL in the group after prior infection), but a systematic increase was noted in the group without prior infection (0.7, 6.3 and 9.6 U/mL). Despite a slight increase in the IgM antibody level after the second dose of vaccination within the group without prior infection, the mean value of IgM antibody level was close to the cutoff level in both studied groups. The individual anti-S1 IgM antibody level after vaccination was high, up to 116.1 BAU/mL in one participant with prior infection and 105.6 BAU/mL in one participant without prior infection. The results showed that the vaccination induced a weak and short-lasting response in the IgM immunoglobulin class.