Antispike Immunoglobulin-G (IgG) Titer Response of SARS-CoV-2 mRNA-Vaccine (BNT162b2): A Monitoring Study on Healthcare Workers

The secretion of IgG SARS-CoV-2 antispike antibodies after vaccination with BNT162b2 and the protection represent the response of the human organism to the viral vector symptomatic infections. The aim of the present investigation was to evaluate the immune reaction in health workers of the Polyclinic of Bari to identify the relationship of antispike titers with blood type, sex, age, and comorbidities. This prospective observational study (RENAISSANCE) had as its primary endpoint the assessment of serologic response to BNT162b2 at three blood titers: the first at 60 days after the second dose (3 February 2021); the second titer at 75 days after the first titer; and the third titer at 130 days after the second titer. Out of 230 enrolled staff members, all responded excellently to the mRna Pfizer (BNT162b) vaccine. Only one patient, 40 days after the second dose (3 February 2021), was positive on the swab control performed on 15 March 2021, although completely asymptomatic, and was negative on the subsequent molecular swab performed on 30 March 2021. All the patients responded to the mRNA Pfizer (BNT162b) vaccine with an antispike IgG level above 500 BAU/mL at the first antispike protein essay (60 days after the second dose on 3 April 2021); at the second titer (75 days after the first titer on 20 June 2021), 4 (1.7% of 230 enrolled) patients showed an antispike IgG level under 500 BAU/mL; at the third titer (130 days after the second titer on 30 June 2021, which means 9 months after the second dose), 37 (16.1% of 230 enrolled) patients showed an antispike IgG level under 500 BAU/mL. The data analysis demonstrated that patients belonging to blood group 0, regardless of their rhesus factor, showed the strongest level of antibodies compared to the other groups. No dependency was found between low antibodies level and sex or age. Molecular swab controls were performed every 15th of the month continuously. However, the enrolled patients’ activity was at high risk because they carried out medical activities such as dental and surgical as well with droplets of water vaporized by the effect of turbines, piezosurgery. The vaccination campaign among health workers of the Policlinico of the University of Bari “Aldo Moro” led to an excellent serological response and the complete absence of COVID-19 incident cases, so the antibody response was excellent. The COVID-19 vaccine booster shot should be administered after 9 months and not without prompt antispike titer detection to assess if any sign of waning immunity is present in that specific patient.


Introduction
SARS-CoV-2 represents a viral vector with a very critical airborne transmission capability [1,2]. In fact, the air droplets' release seems to be one of the most effective diffusion ways for COVID-19 infection [3]. Therefore, many authors indicated healthcare workers and dentists as very critical subjects for viral vector exposure due to the medical environment and the prolonged contact with potentially infected patients [4,5]. In fact, this condition seems to be associated with a confined working environment with lots of aerosol generation and risk of being infected from salivary droplets which contain the SARS-CoV-2 virus [5][6][7].
Many studies have demonstrated that after infection, a decline in serum anti-SARS-CoV-2 antibodies occurs, decreasing rapidly in the first 120 days after infection and then more slowly in the following 210 days while maintaining significant antibody levels for at least 11 months after infection [8][9][10][11][12]. In an analysis of 3689 adults aged ≥18 years who were admitted to 21 US hospitals in 18 states from 11 March to 15 August 2021, the efficacy values of Moderna and Pfizer-BioNTech mRNA vaccines (VE) and Jannsen vaccine were assessed. Values were estimated at 15-40 days after receiving the second dose of Moderna and Pfizer-BioNTech vaccine or the single dose of Janssen vaccine [13]. The EV levels were 93% for Moderna and 88% for Pfizer, respectively, while the single dose Janssen vaccine had a slightly lower EV of 71%. These results suggest that the double-dose protection of the mRNA vaccines (Pfizer-BioNTech and Moderna) is greater than the 1-dose Janssen. Moderna vaccine showed an efficacy of 93% at 2-17 weeks (median = 66 days) after receiving the second dose of vaccine and 92% at >17 weeks (median = 141 days) (p = 1.000). In contrast, the Pfizer-BioNTech vaccine showed a significantly reduced VE of 91% (median = 69 days) and 77% (median = 143 days), respectively. Moderna also produced higher levels of post-vaccination anti-RBD antibodies than the Pfizer-BioNTech vaccine. The VE of the Moderna vaccine is better than Pfizer-BioNTech vaccines, because the mRNA content in the Moderna vaccine is greater, as are the time intervals between doses (28 days for Moderna and 21 days for Pfizer) [14,15]. However, in this study, variant-specific VE, including Delta variants (B.1.617.2 and AY underlines), were not assessed. In another study of 3975 healthcare personnel, 204 (5%) were positive for SARS-CoV-2, of which 5 were vaccinated ≥2 weeks after the second dose, 11 were partially vaccinated ≥2 weeks after the first dose and <2 weeks after the second dose, and 156 were not vaccinated. Persons (5%) became infected. Vaccine efficacy was 91% with full vaccination and 81% with partial vaccination. In addition, in partially or fully vaccinated infected subjects, the mean viral RNA load was 40% lower than in unvaccinated subjects, as well as 58% fewer febrile symptoms and a shorter illness of 2.3 days [14]. The VE of the mRNA vaccines towards the Delta variant were reduced, and this was higher than in the Pfizer vaccine (Moderna 76%, Pfizer 42%) [16,17]. In conclusion, however, the data ensure that all COVID-19 vaccines approved or self-released by the FDA ensured substantial defense against hospitalization for COVID-19 at 99% [18]. The association of the improved antibody response linked to the longer time interval between the first and second administration (see Moderna/Pfizer) can be correlated to the concept of the binding energy or receptor affinity that a B-cell has for a given antigen. This is the same affinity as the antibodies secreted by the same B-cell after antigenic stimulation. Thus, with the attendance of small doses of antigen, cells with high-affinity receptors will be stimulated more, resulting in the secretion of high-affinity antibodies. Conversely, larger doses of antigen will induce lower affinity antibodies. This relationship between immunogen dose and antibody affinity explains why, as time passes after immunization and the concentration of antigen in the body decreases, antibody affinity increases. Since it is mainly B cells with high affinity receptors that are stimulated, the average affinity of antibodies in the serum increases. The attendance of antibodies aids this selection by competing with cellular receptors for the antigen. During the secondary response, the increased affinity of the antibodies may be because of the stimulation of B cells with high affinity receptors, which occurred during the primary response when antigen concentrations were progressively decreasing over time. It is hypothesized that the secondary response would result from the stimulation of cells qualitatively different from those of the primary response [17,[19][20][21][22][23][24][25]. The role of the booster dose of the primary cycle is to achieve a greater immune response and to ensure a good level of defense against infection [26]. The main goal of immunization during the COVID-19 pandemic is to improve the clinical course by avoiding hospitalization and reducing mortality. Therefore, the third dose should only be done if it is clear that there is no protection against these disease outcomes of the disease over time. The FDA and CDC suggest a booster dose at least 4 weeks after the second dose of Moderna or Pfizer, or 60 days after the first dose of Janssen/Johnson & Johnson for people who have comorbidities associated with immunosuppression [28][29][30][31][32]. On 30 July 2021, Israel became the first to give a booster dose of Pfizer against COVID-19 to all persons aged over 60 years who had been immunized at least 150 days previously. At 2 weeks after the booster dose, there was an 11.4-fold chance of infection and a >10-fold lower chance of severe disease. Against the Delta variant, the efficacy of Pfizer's third dose was about 95%, a similar value to the efficacy of the original vaccine, which had been reduced from 85% to 75% against severe forms [33]. The EMA is evaluating the use of the third dose for Moderna. Data from the pharmaceutical company's studies have shown significant anti-pal responses 15 days after the third dose of the Moderna vaccine (a 50 microgram booster dose of mRNA-1273): more than 40-fold against the Delta variant (B.1.617.2), 32-fold against Beta (B.1.351), and 43.6-fold against Gamma (P.1). In addition, the reactogenicity of a third BNT162b2 mRNA COVID-19 vaccine was analyzed. A study conducted on seniors and immunocompromised individuals reported that local and systemic side effects were analogue to those who received prior doses. [34] Bensouna et al. analyzed the humoral immunity after a booster dose of Pfizer in 69 persons cured with either hemodialysis or peritoneal dialysis. In this analysis, the third dose was performed at least four weeks after the second dose. Results showed a substantial rise in the antibody level in the sample. However, there was not a significant rise in the antibody level after a booster dose in persons who were undergoing chemotherapy or in those with initial high anti-S1 antibodies [35]. The target of the present investigation was to assess the short and long term immune profiling vaccine response in healthcare workers.

General Characteristics
Since March 2020, the staff involved in patient care has undergone periodic screening with molecular tests for SARS-CoV-2 infection at the university hospital consortium polyclinic of Bari. Subsequently, the vaccination campaign with the vaccine BNT162b2 mRNA COVID-19 (Pfizer, New York, NY, USA) began at the same institution. For the purposes of this analysis, hospital staff were asked to assess antibody dynamics after vaccination. In this analysis, 230 healthcare workers from different departments were included (90 dentistry, 72 radiology, 34 forensic medicine, 34 internal medicine) of which 23  The antibody levels of the recruited healthcare workers were evaluated. Overall, the antibody response was assessed circa ten months after vaccination. Anti-SARS-CoV-2 Spike IgG antibodies were assessed with the LIAISON ® SARS-CoV-2 TrimericS IgG assay (DiaSorin, Saluggia, Italy), which can show both binding and neutralizing antibodies to the trimeric Spike glycoprotein. Subjects were engaged from 11 January 2021 (first dose) and 3 February 2021 (second dose). The occurrence of vaccine-associated viral infections was assessed by RT-PCR on symptomatic/contact cases through 30 September 2021. All health care workers enrolled in the research were given a nasopharyngeal swab every 15th of each month to assess the onset of COVID-19 after the second vaccine. All enrolled in the research always performed swabs every 15th of the month starting in May 2020. Therefore, the enrolled healthcare workers were screened and did not contract SARS-CoV-2 already from 15 May 2020. The last sampling for assessing the antibody levels was carried out 270 days after the first dose of the Pfizer vaccine. The subjects enrolled in the analysis were monitored with molecular swab on the 15th of each month and none were positive. Thus, the registered healthcare professionals had all been screened and certainly did not contract SARS-CoV-2.

Statistical Analysis
The sample size calculation was performed considering an effect size f 2 : 0.05; α error: 0.05 and power (1-β): 0.80, and 3 predictors. The determined population output was 222 subjects which was increased by 4% as an eventual drop-out compensation.
Descriptive statistical analysis was conducted by the program Microsoft Excel (Microsoft, Redmond, WA, USA) by calculating the average, max, and min level of the antibody titers for different groups of patients by blood type, number of vaccination shots, titers test titers, gender and age, as well as the correlation coefficients by test titers. The normal distribution of the study data was assessed through the Kolmogorov-Smirnov test. The Kruskal-Wallis followed by the Dunn's post-hoc test was conducted to evaluate the mean differences of the study groups.
The level of significance was p < 0.05.

Results
All the patients responded to the mRNA Pfizer (BNT162b) vaccine with an antispike IgG level above 500 BAU/mL at the first antispike protein Essay (2 months after the second dose on 3 April 2021): 100% of personnel had anti-S IgG titers ≥2000 BAU/mL, 19.2% between 1500-2000 BAU/mL, 9.8% between 1000-1500 BAU/mL, and 3.4% ≤1000 BAU/mL ( Figure 1A). body titers for different groups of patients by blood type, number of vaccination shots, titers test titers, gender and age, as well as the correlation coefficients by test titers. The normal distribution of the study data was assessed through the Kolmogorov-Smirnov test. The Kruskal-Wallis followed by the Dunn's post-hoc test was conducted to evaluate the mean differences of the study groups.
The level of significance was p < 0.05.

Results
All the patients responded to the mRNA Pfizer (BNT162b) vaccine with an antispike IgG level above 500 BAU/mL at the first antispike protein Essay (2 months after the second dose on 3 April 2021): 100% of personnel had anti-S IgG titers ≥2000 BAU/mL, 19.2% between 1500-2000 BAU/mL, 9.8% between 1000-1500 BAU/mL, and 3.4% ≤1000 BAU/mL ( Figure 1A).  Figure 1B). At the third titer (130 days after the second titer on 30 June 2021, which means 9 months after the second dose) 37 (16.1% of 230 enrolled) patients showed antispike IgG level under 500 BAU/mL; this percentage adds to the 1.7% of the second titer for a total 17.8% that fell below the above threshold. (Figure 1C). Seven months after the conclusion of the vaccination program, only one subject (0.43% of 230 enrolled) had SARS-CoV-2 infection, but without any symptoms and negativization after 15 days. Our descriptive analysis (Figures 1 and 2 points to the fact that patients belonging to blood group 0, regardless of their rhesus factor, showed the strongest titer of antibodies compared to group A, B, and AB in each of the three titers ( Figure 1 and Table 1). Age range of the analysis was the following (Figure 2): 1. Between the age of 20 and 30 years old there were 45 subjects (19.57% of 230 enrolled).   Figure 1B). At the third titer (130 days after the second titer on 30 June 2021, which means 9 months after the second dose) 37 (16.1% of 230 enrolled) patients showed antispike IgG level under 500 BAU/mL; this percentage adds to the 1.7% of the second titer for a total 17.8% that fell below the above threshold. (Figure 1C). Seven months after the conclusion of the vaccination program, only one subject (0.43% of 230 enrolled) had SARS-CoV-2 infection, but without any symptoms and negativization after 15 days. Our descriptive analysis (Figures 1 and 2 points to the fact that patients belonging to blood group 0, regardless of their rhesus factor, showed the strongest titer of antibodies compared to group A, B, and AB in each of the three titers ( Figure 1 and Table 1). Age range of the analysis was the following (Figure 2): Between the age of 60 and 70 years of age there were 46 subjects (20% of 230 enrolled).  In the age group between 50 and 60, we detected an increase of antibody levels in all three titers compared to the other groups, which showed instead approximately close average antibody levels between the different titers (Figures 3-6 and Table 1). No dependency with the antibodies level was found on gender ( Figure 7 and Tables 2 and 3   In the age group between 50 and 60, we detected an increase of antibody levels in all three titers compared to the other groups, which showed instead approximately close average antibody levels between the different titers (Figures 3-6). No dependency with the antibodies level was found on gender (Figure 7, Tables 2 and 3).

Discussion
The population sampling of the present investigation was conducted in order to include according to a more equal distribution healthcare workers from a medical/surgical interventional area and doctors from a non-interventional medical area. The differences of healthcare work exposure could produce a sensible critical point in the population enrollment and a potential limit of the study. On the contrary, this approach is able to produce a more consistent sample size and consequently a higher statistical power.
Immunological memory is a property of both T and B lymphocytes [4,[36][37][38][39]. In an antiviral response, cytotoxic T lymphocytes selectively eliminate the infected cells; neutralizing antibodies secreted by plasma cells preventing the virus from infecting them [37,[40][41][42][43][44][45][46][47][48][49][50][51][52]. Virus-specific T helper cells are required to generate immunological memory, particularly for long-lasting bone marrow plasma cells (BMPC), which secrete antiviral antibodies when the virus has disappeared for long-lasting immunity [28,36,[53][54][55][56][57][58][59][60][61]. The bone marrow (BM) is one of the main immunological organs in the human body. BMPCs are detected in the BM and in gut-associated lymphoid tissues (GALT), which produce antibodies for a lot of time [40,62]. Studying the serum values of patients convalescing from COVID-19 at 1, 4, 7, and 11 months, it was detected that infection with SARS-CoV-2 provokes a transient and early response with a high production of extrafollicular (spleen and lymph nodes) antibodies, which decrease relatively quickly [63][64][65][66]. Subsequently, more stable serum antibodies secreted by long-lasting BMPC are detected. In fact, analysis of bone marrow aspirates obtained approximately 7 and 11 months post-infection revealed S-specific anti-SARS-CoV-2 BMPC [67][68][69][70]. Consequently, circulating anti-S IgG titers at 210-240 days after symptom begin in convalescent individuals is related with the concentration of anti-S IgG BMPC present in the bone marrow aspirate [40,62]. All convalescent subjects who received a dose of mRNA vaccine increased all components of the humoral reaction. The data confirm that BMPC expressing specific antibodies are long-lasting, have serum neutralizing activity against new variants of concern, and are cleared and produced extensively after vaccination. These data suggest that immunity in convalescent persons will be very long-lasting. Individuals who contracted COVID-19 and received mRNA vaccines will produce antibodies and memory B cells that will also be protective against circulating SARS-CoV-2 variants [71][72][73][74][75]. Research at the Washington University School of Medicine in St Louis, Missouri, on the value of the memory B-cell response, analyzed fragments from the lymph nodes of vaccinated patients and found 'germinal centres', i.e., tiny areas of B-cell refinement, which, over time, synthesized increasingly powerful immune cells, thus being able, through this evolutionary process, to fight the Delta variant and other worrying SARS-CoV-2 variants. The persistence of these germinal centers was detected at 15 weeks post immunization [13].
Our study discovered that after 270 days after the second dose, most of the enrolled patients still showed a significant antispike titer. This is in contrast with what Yinon M.
Bar-On et al. showed in their analysis [76]. We think that antispike titers greater than 500 BAU/mL can still deliver protection as it should be noted that a decline in serum antibodies does not mean that there is a lowering of immunity but rather a rising of it with the development and persistence of SARS-CoV-2 memory CD8 + T cells, SARS-CoV-2 memory B Cells and SARS-CoV-2 memory CD4+ T cells in the bone marrow [77]. According to the age variable, no significant differences were detected between the the study groups at titers 1, 2, and 3 (p > 0.05). In fact, the groups seemed to produce similar fluctuations and a consistent decrease in the antispike IgG levels. Similar evidence was detected according to the he blood type groups that only the O negative blood groups seemed to produce a more consistent level of antispike IgG (p < 0.05) at titer 1 and titer 2. No differences in titer 3 were detected between the blood types in the present investigation (p > 0.05). This correlation with COVID-19 protection activity has been suggested by several authors but the association is not completely cleared and is controversial according to the current literature. In addition, very few data are reported in relation to the vaccination effectiveness. Rana et al. reported on a single center study that the A, B, and Rh+ blood groups were susceptible to COVID-19 infection in comparison to blood groups O and Rh− [78]. Very similar findings were reported on different populations groups such as household and children [79].
Certain subsets of individuals might also carry some form of protection having a much higher antibody's titer compared to other subsets: in our study, we demonstrated that patients belonging to group zero may have this "enhanced" protection due to a higher antispike titer that lasts longer over time. The subset of patients aged 50-60 might have this increase in antibodies' level because of some undetected comorbidity that in our hypothesis could lead to this immunological picture. Any other study at the moment does not support this finding. No dependency with the antibodies' level was found with gender. This is in contrast with what Shachor-Meyouhas et al. [80] showed in their analysis where the male sex was identified as a risk factor for lower antibody level in an observational timeline of 3 months after the second shot. The strength of our analysis is that it extended over 9 months after the second shot. The COVID-19 vaccine booster shot should therefore be administered after 9 months and not without prompt antispike titer detection to assess if any sign of waning immunity is present in that specific patient. It must be hence noted that the majority of the subjects enrolled in our study were protected against COVID-19 even after 9 months after the first dose of the vaccine despite their activity being a high risk because they carried out medical, dental, and surgical activities, and with droplets of water vaporized by the effect of turbines, piezosurgery. More studies are required to assess waning immunity kinetics in specific subsets of persons with specific traits such as comorbidities and other anamnestic data.
In healthy adults, two 30 µg doses of BNT162b2 elicited high neutralizing titers and robust, antigen-specific CD4 + and CD8 + T cell responses against SARS-CoV-2 [57,58]. Therefore, it revealed 95% efficacy among phase 2-3 study subjects aged 16 years or older [57]. Although BNT162b2 is a two-dose regimen, early protection after a single dose has been reported in clinical trials and based on real data [59,60]. A significant titer decay has been detected by Israel et al. [81] in a preliminary report on a wide population screening on BNT162b2 vaccinated subjects, reporting different antibody kinetics between vaccinated patients and convalescents. At 6 months after vaccination the 16.1% patients reported an antibody titer below the seropositivity threshold of <50 AU/mL, while 10.8% convalescent subjects were below <50 AU/mL 9 months after COVID-19 infection. A high titer of autoimmune antibodies in COVID-19 patients has been registered, although it is not clear how these antibodies help in the progression of the disease and its clinical picture. These antibodies were studied in a retrospective study of 115 hospitalized COVID-19 patients who had different clinical manifestations; the reaction of autoimmune antibodies to common antigens such as erythrocyte lysate, lipid phosphatidylserine (PS), and DNA was tested. In up to 36% of patients, a large quantity of IgG autoantibodies against erythrocyte lysate was detected.
Anti-DNA and anti-PS antibodies recorded when the patients were admitted to hospital showed an interconnection with the severity of the disease: the positive predictive values were 85.7 and 92.8, respectively. Persons with good values for at least one of the two autoantibodies were rated at 24% of the total severe cases. Recent studies reveal that coagulation, neutrophil levels, markers of cell damage, and erythrocyte size are strongly correlated with anti-DNA antibodies. Anti-DNA and anti-PS autoantibodies can potentially be considered predictive biomarkers in the typology of a clinical course of COVID-19. Long COVIDs are those who present with the persistence of symptoms or the development of new symptoms related to SARS-CoV-2 infection, at least 28 days after diagnosis. Symptoms may be constant or intermittent and may be multi-organ [82]. Dyspnea, tachycardia, and extreme tiredness are more frequent despite the normalization of the inflammatory parameters. Negative RT-PCR diagnostic test values, undoubtedly related to fibrosis induced by cytokine storms in the acute phase, led to chronic pulmonary and cardiac damage, with reduced flow in spirometry tests, high titers of troponin T (TnT), and brain natriuretic peptide (BNP). There are values thought to be because of fibrosis remodeling with transforming growth factor (TGF)-beta secretion in the chronic phase, with overlapping results in other diagnostic tests (ultrasound or chest CT) [83][84][85]. Symptoms include night sweats, temperature changes, gastrointestinal tract disorders [86], constipation/soft stools and peripheral vasoconstriction due to autonomic nervous system dysfunction [87]. In a study at the Policlinico Universitario di Bari, the characteristics and risk factors of 35-day long COVID (35-LC) were investigated over one year from 8 March 2020 to 15 March 2021. The analysis assessed the age, gender, and symptom characteristics of the first week. A distinction was made between persons with a short course of infection (less than 10 days) (<10 days COVID) and those who had been symptomatic for at least 28 days (28 days COVID or 28-LC). Adverse outcomes were not shown to be localized. Instead, they were present in several systems, including the immune system (e.g., Guillain-Barré syndrome, rheumatoid arthritis, pediatric multisystem inflammatory syndromes, such as Kawasaki disease), and the hematological system (vascular hemostasis), depression and anxiety and a condition called 'brain fog', which causes difficulties in attention and concentration. Molecular mechanisms associated with these disease outcomes/symptoms have been correlated [88]. Under well-being conditions, the host's microbiome/virome [89] ecosystem is held in check by an effective host immune defense and persists in a state of equilibrium or homeostasis. In fact, dysbiosis leads to dozens of chronic metabolic changes [90]. Microbiome/virome dysbiosis may favor the growth of opportunistic pathogens. Immune dysregulation induced by SARS-CoV-2 may lead to an imbalance in the body's existing microbial and viral ecosystems that may cause long-term multi-thyroid functional alterations with multiple symptoms [91]. In fact, almost all organisms in human microbiome/virome communities are 'pathobionts', namely they are able to change their gene pool to become pathogenic organisms under conditions of unbalance and immunosuppression [92]. It is also possible that, after becoming infected, SARS-CoV-2 persists in certain parts of the body or tissues in some persons, causing chronic symptoms [93][94][95].

Conclusions
The present study findings seems to suggest no differences of the different variables evaluated among the selected population groups. Blood groups A, B, and Rh+ seem to produce a similar response to the vaccination treatment with similar trends in a mediumshort follow up. Blood groups O− seem to indicate an higher antispike IgG titer mediumshort terms that could potentially support the higher protection against the SARS-CoV-2 infection. Therefore, long term studies with a larger sample size are needed to assess the relationship of between blood groups and the response to the SARS-CoV-2 vaccines.  Informed Consent Statement: Informed consent was obtained from the subjects involved in the study. Written informed consent was obtained from the patients to publish this paper.

Data Availability Statement:
All experimental data to support the findings of this study are available upon request by contacting the corresponding author. The authors have annotated the entire databuilding process and empirical techniques presented in the paper.