Differences in the concentration of anti-SARS-CoV-2 IgG antibodies as a result of post-covid or/and post-vaccination immunization

At the end of 2020, population-based vaccination programs with new generation mRNA-based vaccines began almost all over the world. The aim of the study was to evaluate the titer of anti-SARS-CoV-2 IgG antibodies against the S1 subunit of the "spike protein" as a marker of the humoral response in 477 patients and the concentration of gamma interferon as an indicator of cellular response in 28 individuals. In our studies, we used the serological enzyme-linked immunosorbent assays. IgG were measured in weeks 2 and 3 after the rst dose and 1–5 weeks after the second dose of mRNA vaccine in seropositive and seronegative individuals, as well as in symptomatic and asymptomatic convalescents. High levels of antibodies are observed in 98% of our vaccinated cohort, and the presence of protective T cells was conrmed in all the studied groups. The humoral immune response is diversied and is visible as early as 2–3 weeks after the rst dose of mRNA vaccine. The level of protection increased signicantly after the second dose, with the increase being much greater in pre-vaccine healthy subjects and not much in convalescents. at the cellular level in all subjects. The response was variable, but we did not observe such wide differences as in the case of neutralizing antibodies.


Introduction
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a member of the subgenus Sarbecovirus, causes coronavirus disease 2019 . The rst cases occurred in late 2019 1

. On 11
March 2020, the World Health Organization (WHO) declared the SARS-CoV-2 outbreak a pandemic -the gravest global health crisis of our times. Now almost 2,800,000 deaths and 128,000,000 cases of SARS-CoV-2 infection have been reported worldwide as of March 31st 2 . An understanding of immune responses to SARS-CoV-2 and coronavirus vaccines is necessary, due to the rapid spread of the pandemic across the globe.
High-a nity antibodies are produced by B cells, which are activated by CD4 + T cells. The role of CD8 + T cells is to destroy infected cells 6 . SARS-CoV-2 speci c antibodies are directed against spike protein (S) and nucleocapsid (N). Special role play neutralizing antibodies against the S1 subunit with receptorbinding domain (RBD). RBD binds to angiotensin-converting enzyme 2 (ACE2), which make it easier for the virus to penetrate host cells. After SARS-CoV-2 infection, antibodies can be detected in patients after 3 days, when symptoms occur and seroconversion in most of them appear within 7-14 days. In the acute phase of the disease, IgM antibodies develop and peak at 14 to 35 days, then begin to decline over the next 21 to 35 days. IgG antibodies peak at around 21 to 49 days after infection occurs and, together with neutralizing antibodies, may persist for up to four months 7,8 . CD4 + T and CD8 + T cells speci c for SARS-CoV-2 infections recognize peptides associated with nucleocapsid, spike protein, membrane proteins (M) of the virus and are present in most COVID-19 patients 7 . Speci c CD4 + T cells differentiate into Th1 and Tfh cells. In addition to activating B cells, Tfh cells are crucial for the proper functioning of neutralizing antibodies, while Th1 cells produces IFNγ and cytokines. Furthermore CD4 + T cells help CD8 + T cells respond to infection and high concentration of speci c CD8 + T cells gives a better prognosis for COVID-titre and the duration of the immune system response, including the cellular response, are the basis for further research into better understanding the protective mechanisms, pathogenesis and prognostic factors of COVID-19 disease. This knowledge is also important for the development of effective treatments and vaccines 4, 5, 7, 9 . During a global pandemic, mRNA vaccines are the fastest available vaccines due to their short production time and low biological requirements 1  vaccines are based on the coronavirus mRNA, speci cally on the fragment encoding the S protein 10 . It is one of the structural proteins that initiate infection by attaching the virion to the host's cell membrane 10 .
Moreover, the S1 subunit of the S protein contains an immunologically relevant receptor binding domain (RBD) which is a key target of neutralizing antibodies 1 . According to clinical studies, after vaccination, subjects developed a strong dose-dependent antibody response to the S protein after the rst and second inoculations 11 . Neutralizing antibodies were found in all subjects after the second inoculation, and the antibody titers were equal to or greater than the neutralizing antibody titers of COVID-19 patients 11 .
The immune response to COVID-19 is poorly understood. There is insu cient knowledge about safe and immunologically effective vaccination strategies against SARS-CoV-2. There is no de nite answer to which vaccination strategies will be most effective 14 . Moreover, there is still insu cient information on the short-and long-term effects of vaccination. The aim of the research was to determine the humoral and cellular responses in vaccinated persons and in convalescents.

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Human subjects. The research group consisted of 477 adult volunteers, 362 women and 115 men. All human subjects were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the study. The patients represented research subgroups (Table 1): 24 healthy and unvaccinated individuals; 15 persons with  con rmed SARS-CoV-2 infection but no symptoms (unvaccinated); 82 persons who had COVID-19 and  had multiple symptoms of infection (unvaccinated); 19 persons who were 2 or 3 weeks after the rst dose   of the vaccine (did not have COVID-19); 203 persons who were vaccinated with the second dose within 1-5 weeks before blood sampling for analysis (did not have COVID-19); 124 patients who were con rmed to be infected with SARS-CoV-2 and who were vaccinated at corresponding time points. Quantitative determination of IgG antibodies against SARS-CoV-2. The starting material for analyses was serum obtained after centrifuging whole blood in clot tubes for 5 min at 4 000 rpm. The serum was then carefully removed from the cell pellet and used. Depending on the subgroup of patients, sera were diluted 5-fold, 10-fold, 20-fold, 50-fold or 100-fold. An enzyme-linked immunosorbent assay (ELISA) was used to quantify the in vitro quanti cation of human IgG antibodies to SARS-CoV-2. The tests were performed using the commercial automated analyzers EUROIMMUN Analyzer I-2P and the Anti-SARS-CoV-2 QuantiVac ELISA kit (IgG) EUROIMMUN (Lübeck, Germany), according to the manufacturer's instructions. The test reaction wells were coated with the S1 domain of the SARS-CoV-2 spike protein recombinantly Cellular response analysis -cell stimulation. The starting material for analyses was fresh whole blood collected in lithium heparin tubes. Plasma was obtained using the SARS-CoV-2 IGRA stimulation tube EUROIMMUN (Lübeck, Germany), according to the manufacturer's instructions. The kit contains three test tubes for one sample. The rst (BLANK) does not contain any activating ingredients for immune cells.
The plasma obtained from it is used to determine the individual interferon-gamma background. The second (TUBE) is coated with the components of the S1 domain of the SARS-CoV-2 virus protein. If activation-capable cells are present in plasma, they are stimulated to secrete interferon gamma during incubation in this tube. The third (STIM) is coated with a mitogen that induces non-speci c interferongamma secretion. The plasma obtained from this tube is used to verify whether the sample contains a su cient number of cells and whether they have a su cient ability to activate. 500 µl of whole blood was pipetted into three set tubes and incubated at 37°C for 24 h. After this time, the tubes were centrifuged for 10 min at 12 000 rpm. The plasma interferon-gamma concentration from the BLANK tube represents the individual interferon gamma background and therefore must be subtracted from the plasma concentration obtained in the TUBE and STIM tubes. This BLANK subtraction must be performed individually for the TUBE and BLANK samples of each whole blood sample. After subtracting BLANK, the interferon-gamma concentration in the STIM tube must be signi cantly higher than the BLANK value alone in order to consider that the immune cell count and stimulation in the whole blood sample are su cient.
Cellular response analysis -interferon-gamma ELISA. The level of interferon gamma was determined by an ELISA test performed on a commercial automatic EUROIMMUN Analyzer I-2P with the use of the Interferon-gamma ELISA kit, according to the manufacturer's instructions. The test kit contains a microplate with reaction wells coated with anti-interferon-gamma monoclonal antibodies. In the rst reaction step, calibrators, controls and plasma samples diluted in sample buffer (1:5) were added to the coated reaction wells to bind interferon-gamma and incubated for 120 min at RT. After this time, the wells were washed with washing buffer (5 x 450 µl). In the next step of the reaction, 100 µl of biotin-labeled anti-interferon-gamma antibodies were added and incubated again for 30 min at RT. The washing was repeated (5 x 450 µl), then 100 µl of streptavidin-HRP was added and incubated 20 min at RT. Photometric determination of the colour intensity was carried out at a wavelength of 450 nm. The colour intensity was proportional to the interferon-gamma concentration. The results were expressed in mlU/ml. Statistical analysis. One measurement was performed for each patient, and based on the medical questionnaire, the patient was assigned to the group data according to vaccination and COVID-19 status.
The mean, median, minimum and maximum value were determined for each group in the statistical description. In order to determine the signi cance of differences between experimental subgroups, a oneway analysis of variance (ANOVA, p < 0.05) was performed. Spearman's test (two-tailed) was used to determine the correlation. people who became ill with COVID-19 within 2 weeks of taking the rst dose, 1 who became ill 4 weeks after taking the rst dose, and 2 people who tested positive at 4 and 6 weeks after taking the second dose of mRNA vaccines. These people fell ill a few days after the serological test, which showed that they had high antibody titers 262.2 BAU/ml and 1106 BAU/ml, respectively).
Comparisons of antibody levels between subgroups. Neutralizing antibody concentrations ranging from 0 to 38 400 BAU/ml were analysed in the study. Anti-SARS-CoV-2 antibody levels were variable (Fig. 1, Table 2). Concentrations below 25.6 BAU/ml (negative result) were found in people who were not vaccinated and did not suffer from SARS-CoV-2 infection, 12 SARS-recovered (infection con rmed in October and November 2020), as well as in 3 people, who had only taken the rst dose of mRNA vaccine. Relatively low primary humoral immunity was found in 3 patients 2 weeks after taking the second dose (78 BAU/ml for an 86-year-old woman, 89 BAU/ml for an 80-year-old woman, and 106.02 BAU/ml for a 46-year-old man, respectively).
In seronegative subjects, in the third week after immunization with the preparation, the mean level of antibodies was higher than in seropositive subjects without vaccination, which con rms the effectiveness of the vaccines in inducing a humoral response. 10 to 14 days after the second dose, a 10-fold increase in neutralizing antibodies is obtained. The highest levels of neutralizing antibodies were found in vaccinated probands who underwent SARS-CoV-2 infection, both after the rst and second doses, regardless of the week of vaccination (Table 2, Fig. 3). Two weeks after the rst dose of vaccination, the median level of antibodies in seronegative subjects was lower than in seropositive subjects without vaccination. In the third week after taking the rst dose, vaccinated convalescents had a titer of IgG antibodies more than 11-fold higher than in those who had not received the rst dose in the same period (348.00 BAU/ml vs. 3989.00 BAU/ml; p = 0.008).
The median (as well as the mean, data in the supplement) individual seropositive anti-SARS-CoV-2 IgG concentrations after the rst dose was even higher than the median (and mean concentrations) of seronegative subjects each week after the second dose of mRNA vaccine (Fig. 3).
Correlation of the antibody titer with age and gender. The correlation between the concentration of IgG antibodies and the age of the study participants and their gender was analysed. There was no signi cant correlation (p > 0.05) of the titre of antibodies against the S protein, although a lower concentration of antibodies of this class was noticeable in men compared to women (Fig. 2B,C; Table in the supplement) in each of the analysed subgroups. Considerable disproportion in the size of the groups should be taken into account. Similarly, for each of the compared categories, no signi cant correlation was found between age and the concentration of anti-SARSs-CoV-2 IgG (p > 0.05). Nevertheless, a noticeable trend was the highest concentration values for people aged 36-45 yrs. and 46-55 yrs. (Fig. 2A). In people over the age of 65, slightly lower antibody titers were found, but the difference was noticeable after the age of 80 and in those with chronic diseases (especially: diabetes, thyroid disease, ulcerative enteritis; data not shown).
Monitoring of the humoral response in the rst weeks after vaccination. For the 8 vaccinated persons (COVID laboratory employees), who had not been infected with SARS-CoV-2, regular weekly determinations of IgG antibody levels were performed to understand the dynamics of immunization. Last assay was performed at an interval of 1 month. All of them showed a several-fold increase in the level of neutralizing antibodies compared to the previous measurement until the second week after receiving the second dose of the mRNA vaccine. The greatest changes in IgG concentration were noted in the rst and second weeks after taking the second dose (5-10 fold increase). Between the second and third weeks after the second dose of the vaccine (6 weeks after the rst dose of vaccine), all probants had a signi cant decrease in anti-SARS-CoV-2 antibody titers (Fig. 3). In the third week after the rst dose, the mean concentration was 998.89 BAU/ml (range 83.83-2845 BAU/ml). In the second week after the second dose, it was highest with a mean of 6,056.18 BAU/ml (range 1889-12650.50 BAU/ml). After 10 weeks of vaccination, antibody levels had dropped to a mean level of 1758.66 BAU/mL (range 320.00-3840.00 BAU/ml).
Humoral immunity and cellular immunity. Cellular immunity analysis was performed for selected patients. Th cell activity was analysed indirectly by measuring the concentration of interferon gamma secreted by activated lymphocytes after 24 h in vitro stimulation. The mean concentration of INF-in the non-antigen stimulated samples was 18.17 mlU/ml (range 0.50-89.08 mlU/ml). After stimulation with S1 antigen, the concentration of interferon gamma in the samples increased signi cantly (p < 0.001). The mean concentration was 1625.00 mlU/ml (range 11.75-2499.25 mlU/ml). In convalescents, the mean Despite the high titer of antibodies and the concentration of interferon gamma (Fig. 4), 3 people in this group were infected with the SARS-CoV-2 virus. One of these patients was reinfected with the virus within 6 months of COVID-19 disease and 4 weeks after receiving the vaccine dose, which con rmed the lack of su cient immune protection.

Discussion
Covid vaccines produced by P zer/BioNTech use mRNA technology. According to the characteristics of vaccine, the minimum time needed to obtain full immunity after the second dose for the Comirnaty vaccine is 7 days. Onset of protection observed approx. 14 days after vaccination. Clinical trials have shown almost 95% of effective in preventing COVID-19 in people without prior infection. After the rst dose, the effectiveness of the preparation was estimated at approx. 52% 15 . How long immunity induced by SARS-CoV-2 infection remains unclear at this stage, but antibodies are expected to last for least six months (as in the case of a COVID-19) to potentially several years. There is also insu cient information on protection against the emerging new variants of coronaviruses.
In this study, we analysed the elements of the immune response primarily in vaccinated (seropositive and seronegative) individuals and compared them with determinants of immunity in convalescents (vaccinated and unvaccinated). The basic determinant of immunization as a result of disease or vaccination in our study was the analysis of the humoral response expressed by the concentration of IgG antibodies against the S protein of the SARS-CoV-2 virus. The purpose of this analysis was to screen the primary immune response and thus cross-sectional analysis of the dynamics of B cell responses in each of the 16 compared patients groups. In the second stage, the activation of T lymphocytes was analysed in selected people (in 3 representative groups). Based on the obtained results, a high degree of heterogeneity of immune responses was found. After vaccination, the parameters of the humoral response were measurable in all probands, which con rmed the effectiveness of the mRNA vaccine in activating B lymphocytes to produce antibodies and T lymphocytes to secrete gamma interferon.
Additional assessment of the cellular immune response (detection of interferon gamma, including the determination of pathogen-responsive T-cell activity), con rmed post-vaccination and post-covid immunization at the cellular level in all subjects. The response was variable, but we did not observe such wide differences as in the case of neutralizing antibodies.
Primary humoral immunity, one of the indicators of which is the presence of IgG antibodies, appeared 2 weeks after receiving the mRNA vaccine in 66.8% of people vaccinated with the rst dose of Comirnaty, and after 3 weeks in all 11 people in our study group. Researchers working on the clinical trials for the Comirnaty vaccine observed a vaccine effectiveness of 52% between the time of the rst and second doses, which is a 21-day period. Based on independent UK studies, it is estimated that the P zer/BioNTech vaccine may be more effective after the rst dose than previously thought. In this study, it was observed that the effectiveness of the rst dose of the vaccine 15 days after receiving it was actually closer to 89 to 91 percent 16 . Researchers University of She eld and University of Oxford, in cooperation with the UK Coronavirus Immunology Consortium (UK-CIC), tentatively conclude, based on observational studies conducted in the UK, in which healthcare workers were vaccinated against COVID-19, that the rst dose of the vaccine may provide immune protection against a severe course of COVID-19. The study was conducted on a group of 237 people, some of whom had previously been infected with SARS-CoV-2, and some had never suffered from COVID-19. Above mentioned researchers, similar to our observations, obtained the strongest immune response in those who had have been infected SARS-CoV-2 before vaccination. After one dose of the P zer/BioNTech vaccine, the levels of T-cells in the plasma clearly increased compared to the levels seen in people who had been vaccinated but previously uninfected to coronavirus 17 .
Based on the concentration of anti-SARS-CoV-2 antibodies, it was found that patients who experienced symptomatic SARS-CoV-2 infection, both after receiving the rst and second doses, regardless of the week of vaccination, have signi cantly higher antibody titer compared to seronegative people. Our observations are consistent with studies Angyal et al. 17 , who published data showing that in people who got vaccinated after contracting COVID-19, antibody responses after the rst dose of P zer/BioNTech vaccine were 6.8 times higher, and T cell responses 5.9 times higher than in people who had never had the disease before. In contrast, among those who did not get sick but received a single dose of mRNA vaccine, the level of protection was similar or higher than that observed after natural infection. These researchers also did not nd any correlation between age and the intensity of the humoral or cellular The group of SARS-recovered patients included both those with high serum levels of antibodies and those whose IgG titre may suggest a loss of immunity acquired after COVID-19, and thus indicate the need for vaccination. Antibody levels below < 35.2 BAU/ml (negative or uncertain result) were detected in symptomatic convalescents (10 patients) who had been ill 5-6 months prior to serological examination.
This observation in accordance with the reports contained in ECDC Technical Report 21 . The currently available results of cohort studies con rm that the protective effect of natural SARS-CoV-2 infection ranges from 81-100%, begins on day 14 after infection, and lasts for a period of ve to eight months 21,22 . Unfortunately, relatively low titers of IgG antibodies were also determined in 5 asymptomatic survivors after a period of several weeks after the positive test results, which may indicate a high risk of viral reinfection. People who have had COVID-19 should be vaccinated to ensure long-term and strong immunity. Chia et al. 23 , noted that in convalescents group it is possible to distinguish ve different patterns of the dynamics of neutralizing antibodies, and their modelling may in uence the prediction of individual immunity longevity in convalescents, and thus the decision to vaccinate within this group of patients. Persistence of neutralizing antibodies in SARS-recovered related to the severity of the disease (we also observed this relationship in our study) and the sustained levels of proin ammatory cytokines, chemokines and growth factors. Chia et al. 23  Hence, as the authors emphasize, there is a need to induce a vaccine immune response.
In the serological test, 3 people patients had signi cantly lower IgG anti-SARS-CoV-2 antibodies compared to the other vaccinated persons, the level of IgG antibodies (78-106.02 BAU/ml). Based on these results, it can be concluded that these people have not acquired signi cant humoral immunity and may still be at risk of coronavirus infection despite vaccination. Hence the need to conduct special serological surveillance in people aged 65 + and/or people with coexisting chronic diseases, and perhaps to consider the need to take further doses of the preparation ensuring protective properties. Worrying is also the fact of con rmed cases in 3 despite vaccination, high rates of cellular and humoral responses.
However, no variant of the virus has been identi ed that overcame the immune response mechanisms in these individuals. SARS-CoV-2 infections and vaccine infections have been reported sporadically 25,26 , but raise important issues regarding the duration of immunity after natural infection and the extent of protection after vaccination, as well as the transmission of the virus by these individuals.
Testing the concentration of antibodies to S protein in both convalescents and vaccinated patients enables the analysis of the course of the humoral immune response in COVID-19. Quantitative testing of anti-SARS-CoV-2 IgG antibodies allows to determine whether the patient has responded to vaccination, and if so how intensely. It also enables the assessment of the humoral immunity acquired after undergoing SARS-CoV-2 infection. By testing anti-SARS-CoV-2 antibodies, it is possible to determine the concentration of antibodies that provide protection against infection, as well as to make rational decisions about booster doses of COVID-19 vaccines. The greatest bene t of the research is the ability to quantify the acquisition of humoral immunity to SARS-CoV-2 as a result of coronavirus infection and/or vaccination. Antibody testing is not required in the context of vaccination, but knowledge of the immune status before and several weeks after the last dose of vaccination may nevertheless allow inferring the immune response to immunization and provide an indication of the degree of immunity obtained against COVID-19. Due to the lack of data on the persistence of immunity acquired as a result of a vaccine reaction, it is also important to monitor the level of antibodies over time (especially among healthcare professionals, people over 65 years and chronically ill), as the future may also indicate the need (or lack thereof) taking a booster dose. The results of the conducted research could be useful in the future for the development of new recommendations of the vaccination program, hence the need to continue them on a larger scale. The diversity of immune responses shows the need for research, the inherent element of which will be immunological monitoring of the durability of disease resistance or protection against its severe course in vaccinated people and/or susceptibility to reinfection in COVID-19 convalescents. By analysing the level of antibodies, it is possible to identify people who are already immunized as well as people who have not acquired immunity as a result of vaccination, and those who may have lost the acquired immunity after contacting SARS-CoV-2. It seems that such a test should be an integral part of the assessment of immunological parameters, especially before making an informed decision about vaccination or its delay in convalescents, as well as the assessment of the durability of immune protection, important from the perspective of making a decision to take booster doses a few months after the initial administration of the vaccine against COVID-19. Figure 1 Comparisons of antibody levels between the analysed patients subgroups. Blue colour indicates the concentration of anti-SARS-CoV-2 IgG antibodies in seronegative individuals before vaccination. Individuals with con rmed SARS-CoV-2 infection prior to receiving the mRNA vaccine dose, as well as seropositive unvaccinated individuals, are marked in green.

Figure 3
IgG-anti-SARS-Cov-2 concentrations in the rst 10 weeks after receiving the mRNA vaccine for 8 seronegative individuals.

Figure 4
Comparison of anti-SARS-CoV-2 IgG antibody titers against S1 protein and gamma interferon concentrations released after stimulation of Th lymphocytes in 28 individuals (patients who recovered