Systemic COVID-19 vaccination also enhances the humoral immune response after SARS CoV-2 infection. An approach to criteria for COVID-19 re-immunization is needed. Do we need a third dose?

Systemic vaccination of the BNT162b2 mRNA stimulates humoral response. The aim of our study was to compare the intensity of humoral immune response, measured by SARS CoV-2 IgG, SARS CoV-2 IgM, and neutralization S-RBD IgG antibodies level, post COVID-19 vaccination versus post SARS COV-2 infection. We analysed 1060 people in the following groups: convalescents, healthy vaccinated, vaccinated with COMIRNATY, AstraZeneka, Moderna, Johnson&Johnson and vaccinated SARS CoV-2 convalescents. A concentration of SARS CoV-2 IgG, SARS CoV-2 IgM, and neutralizing S-RBD IgG was estimated in Bialystok Oncology Center laboratory by chemiluminescent immunoassay- CLIA, MAGLUMI. Results: 1. We observed a raise of antibodies response in both, convalescent SARS CoV-2 and COVID-19 vaccinated groups 2. The level of all antibodies’ concentrations in vaccinated COVID-19 convalescents was signicantly higher. 3. We differentiated an asymptomatic SARS CoV-2 convalescents from control group. Based on our analysis we suggest that it is important to monitor SARS CoV-2 antibodies concentrations as an indicator of asymptomatic COVID-19 infection, and as an equivalent of effectiveness of humoral response in convalescents and vaccinated people. Taking into consideration the time-limited nature of the effects of post infection SARS CoV-2 recovery or vaccination, among others physiological half-life, we suggested monitoring IgG antibodies level as a criterium for next vaccination.


Introduction
Vaccination is the best form of prevention of infectious diseases. The P zer-BioNTech vaccine BNT162b2 (COMIRNATY) 1 has been recognized so far as the most effective vaccine in preventing SARS Co-V-2 infection and severe COVID-19 disease 2,3,4 . A rising level of speci c antibodies -IgM and IgGand neutralizing S-RBD IgG as an immune humoral response is an effect of vaccination. A similar effect is observed post SARS CoV-2 exposure (COVID- 19) 5 . It has been proven that in symptomatic SARS CoV-2 infections, the level of neutralizing S-RBD antibodies correlates with the severity of COVID-19 disease and with hospitalisation 5, 6 . Such a correlation was observed neither in asymptomatic convalescent  patients nor in healthy vaccinated individuals after the rst dose of systemic double vaccination 7,8 . There was evidence that after a second dose of systemic vaccinations with mRNA P zer-BioNTech, the antibody neutralizing level was lower than that in vaccinated convalescent COVID-19 patients at regular times8. Higher levels of naturalizing antibodies after SARS CoV-2 exposure and COVID-19 vaccinations are seen in autoimmune disease 9,10 . Seroconversion to neutralizing antibodies is mostly gradual and dependent on a patient's clinical state resulting from immune defence mechanisms, such as neutralization, complement activation, and cell cytotoxicity (ADCC) 7,11,12 . In extremely rare situations, SARS CoV-2 infection gives rise to systemic in ammatory response syndrome (SIRS) with systemic in ammatory multiorgan dysfunction (MODS) 13 . Depending on the phase and intensity of the in ammatory response, giving convalescents serum plasma antibodies for COVID-19 treatment may reduce or strengthen the in ammation 14 . There is much controversy involving convalescent-derived serum plasma for COVID-19 therapy 26 . Clearly, serum plasma antibodies are very effective at an early stage of COVID-19 disease, when the in ammation process has not yet involved elements of the humoral response 12,15,26 .
The evaluation of systemic vaccinations against SARS CoV-2 invariably raises questions about seroprotective antibody concentration levels and their half-life 11 . Aging of the immune system is, on the one hand, a risk factor for contracting COVID-19 and, on the other hand, determines poor postvaccination response 16 . The question then arises whether elderly individuals require an additional dose of the vaccine 16,17 . The same question can be asked about immunosuppression, people with immunode ciency syndromes, post transplantation patients, or oncologic or dialysis patients. Many published papers prove the safety of BNT162b2 vaccination in dialysis patients 18 , patients after lymphoma therapy 19 , and the safety of a third dose in kidney transplant recipients 20 . In healthy individuals, partial seroprotection, a mean of approximately 53% (32-68%, con dence interval 95%), is reached 14 days after the rst dose of BNT162b2 vaccine. Seven days after the second dose, seroprotection reached 95% 16

Materials And Methods
In our study, we observed a group of 1063 people, 783 females (74%) and 277 males (26%), in the age bracket between 18 and 89 years old. People over 50 years old constituted 45% (N=479) of the group.
Because of different postvaccination reactions, three subjects were considered early hype responders IgM and IgG and were excluded from further evaluation. Final group of 1060 subjects (Table 1 After a retrospective analysis of concentrations of SARS CoV-2 IgG antibodies in group G0, we identi ed subgroup G01 of 43 persons who had higher than cut-off value (0,2 AU/ml) concentrations of speci c IgG antibodies. The existence of speci c SARS CoV-2 antibodies was recognized as evidence of viral contact, and the patients were quali ed as SARS CoV-2 convalescents with no symptomatic COVID-19 history. G0=111. Among 43 analysed cases (G01), 13 persons were in the seroconversion phase (both positive IgM>1,0 AU/ml and IgG>0,2 AU/ml), 30 persons were in a late phase producing secondary antibodies (positive IgG, negative IgM), but 8 persons were in early phase of humoral viral response (positive IgM, negative IgG). Table 1.

Materials
Our study materials were blood specimens taken through venepuncture sampling. The concentration of antibodies was evaluated 4 hours after blood collection. If immediate assessment was not possible, the serum was collected and stored at -80 °C.

Methods
All 1063 participants had elevated levels of IgM and IgG antibodies oriented speci cally towards SARS CoV-2; in 546 subjects, IgG anti-S (S-RBD) antibodies were detected by chemiluminescent immunoassay-CLIA (MAGLUMI, Snibe Diagnostic, Shenzhen China).
The results greater or equal to 1.00 AU/ml SARS CoV-2 IgG, IgM and S-RBD were considered to be reactive and recognized as positive according to the manufacturer's protocol.
The Bioethics Commission of Medical University of Bialystok gave consent for our research.

Statistical Analysis
For the case-control analyses, nonparametric statistical methods were applied because of a lack of considered variables with a normal distribution. Comparing two groups to each other, the Mann-Whitney test was adopted, whereas to assess more than two groups, the Kruskal-Wallis test was applied. The results that were statistically essential were assumed to be p<0,05. PQStat Software 2021 and Tibco statistic 13.3 was used.

Results
After evaluating the concentrations of speci c antibodies in the control group of healthy unvaccinated subjects, we identi ed a subgroup of SARS CoV-2 convalescents (G01) after an asymptomatic COVID-19 infection. The concentration of antibodies from IgG class in that subgroup was comparable to the concentration observed in COVID-19 convalescents with fully symptomatic disease con rmed by a positive RT PCR test.
Our research showed the presence of SARS CoV-2 antibodies in both the convalescent and COVID-19 vaccinated groups, wherein the concentration of antibodies decreased with time in both groups.

Discussion
The evaluation of concentrations of IgG and IgM antibodies speci c to SARS CoV-2 that we conducted for this study allowed us to identify a new subgroup among the healthy, unvaccinated controls: subjects who had a previous asymptomatic SARS CoV-2 infection. Every patient in this subgroup presented lower concentrations of speci c SARS CoV-2 IgG antibodies than the cut-off assumed for a positive result by laboratory tests, between 0,2 and 1,0 AU/ml.
Group G4 was exposed to the virus three times: the rst time when they were infected with SARS CoV-2 virus and the next two times when they were infected through immunization. The triple exposure of this group caused the largest rise in neutralizing antibodies among all groups observed. Every contact with antigen altered the immune system, causing a burst of antibody production lasting up to 14 days 22,23 .
Although group G5 (N=4), vaccinated people who developed fully symptomatic COVID-19 infection, was excluded from further statistical analysis due to the group's small size, the results we obtained for that group correlate with literature data, which shows the largest growth in antibody concentration in vaccinated and infected people 8,21 .
Who needs the third does of the vaccine? Synthesis of antibodies from the IgM class is the rst phase of the humoral response after initial contact with the antigen. After the second or repeated contact with the same antigen, IgG antibody synthesis occurs. Conducted analysis of concentrations of speci c SARS CoV-2 antibodies IgG and IgM and their mutual dependence let us discover early and late phases of humoral response to SARS CoV-2 infection as well as to COVID-19 vaccine 17,23 . Indirectly, it allowed us to identify individuals who had asymptomatic COVID-19 infections. Of course, such an evaluation should be based on clinical examinations, identi cation of infection symptoms of lack of thereof, serological investigation -presence or lack of virus antigens. One should remember that timing of blood sample collection is fundamental for correct evaluation of concentrations of antibodies IgM and IgG. This timing depends on the patient's clinical status and dynamic changes in the immunologic system, e.g., after SARS COV-2 infection, and can effectively optimize planning of the next vaccine dose against Delta variant 27 .
In Israel, an increase in SARS infection was observed among people vaccinated with the BNT1262b vaccine 146 days post vaccination 24 . This increase was the most pronounced in people over 60 24 . In our opinion, it is reasonable to start measuring speci c antibodies IgG (late effect) and IgM (early effect) for vaccination evaluation at serological window neutralizing antibodies. As we know so far, a humoral response to neutralizing antibodies post vaccination is rapid and decreasing. In addition, in some people without exposure to SARS CoV-2, humoral and cellular responses are detected late, which indicates widespread immunity to SARS in the population 25 . In summary, our results indicate the importance of evaluating and monitoring immunological response parameters in vaccinated healthy people and convalescents, which will help to establish the necessity for the third dose of can vaccine against SARS CoV-2.  Figure 1 presented concentrations (AU/ml) (a) antibodies SARS CoV-2 IgG (aquamarine) (b) neutralizing S-RBD IgG (green) (c) antibodies SARS CoV-2 IgM (red) in control-study group; G0, G01 controls, G1 convalescent, G2 vaccinated COMIRNATY, G3 vaccinated other vaccines, G4 vaccinated convalescent group, G5 twice passed COVID-19 vaccinated COMIRNATY (*p, ANOVA Kruskala-Wallisa) Figure 2 presented concentrations (AU/ml) (a,c) antibodies SARS CoV-2 IgG (aquamarine) and IgM (red) (b,d) neutralizing S-RBD IgG (green) in vaccinated groups and post infection SARS CoV-2 (COVID-19) with compare to unvaccinated and unexposed groups respectively (*p, Mann-Whitney).