Evaluation of Natural and Vaccine-Induced Anti-SARS-CoV-2 Immunity: A Comparative Study between Different Groups of Volunteers

(1) Background: The production of anti-SARS-CoV-2 antibodies should help minimize the severity of COVID-19 disease. Our focus was to investigate and compare different vaccination schedules, monitoring circulating S-RBD Ab (antibodies anti—Spike protein—Receptor Binding Domain) levels after administering two doses in naïve patients. Likewise, vaccine-stimulated immunity in naïve and previously infected patients was compared. (2) Methods: We included 392 patients. Sera were evaluated by Elecsys anti-SARS-CoV-2 S. Statistical analyses were conducted by MedCalc and JASP. (3) Results: In COVID-19 patients, the median value of Ab levels was 154 BAU/mL, stable up to 9 months after the infection. From the data observed in vaccinated patients, higher median values were recorded in COVID-19/Pfizer BioNTech (18913 BAU/mL) than in other groups (Pfizer BioNTech: 1841; ChadOx1 961; heterologous vaccination: 2687) BAU/mL. (4) Conclusions: In conclusion, a single booster dose given to previously infected patients raised an antibody response much higher than two doses given to naïve individuals and heterologous vaccination generated a robust persistent antibody response at high levels, steady up to three months after administration.


Introduction
Understanding the immune response to the SARS-CoV-2 is critical to improve diagnostic pathways and vaccine platforms and providing perspective on the future course of the pandemic. Antibodies are the only immune memory component able to provide a "sterilizing" immunity through neutralizing antibodies (nAbs) that can block the virus even before the infection of our cells [1]. Nowadays, different kinds of commercial kits are available to analyze various circulating immunoglobulins produced versus the virus proteins, such as Anti-Nucleocapsid (N) or Anti-Spike-SARS-CoV-2 antibodies (S-RBD Ab). In this work, the focus is on the circulating S-RBD antibodies.
The production of anti-SARS-CoV-2 antibodies should prevent the virus enter to the upper respiratory and oral cavity cells and would minimize the severity of COVID-19 disease to a regular cold or asymptomatic disease [2]. This finding is the main focus of current clinical trials of COVID-19 vaccines. Although the mechanism of protection has still to be clarified [3], a recent study showed a strong correlation between circulating levels of anti-SARS-CoV-2 Spike IgG antibodies and the symptom onset after a doubled-dose vaccination [4].
Since 1 January 2021 in Italy, the first doses of BNT162b2 vaccine (Pfizer/BioNTech, Comirnaty) were delivered [5]. After a short period, the ChAdOx1 nCoV-19 vaccine was distributed to the population. Both have a double-dose administration, the prime and boost approach, but with different modalities and principles. Strong evidence has been reported to achieve a high immunity, which was demonstrated, for example, to be effective in preventing 95% of COVID-19 cases [6].
However, after the European Medicines Agency (EMA) linked the ChAdOx1 nCoV-19 vaccine with rare, yet severe and sometimes fatal, adverse thromboembolic events mainly in younger people [7], Italy and other countries in Europe halted their distribution of this vaccine to either parts or all of their population, recommending a first-dose ChAdOx1 nCoV-19 vaccine followed by the BNT162b2 as second dose, called heterologous vaccine [8]. Current studies suggest that heterologous vaccine built a robust immune response [7,9], which appears to be similar to or even greater than homologous (BNT162b2 or ChAdOx1 nCoV-19) vaccine response. Moreover, others studied the S-RBD Ab level after vaccination following infection, which may provide stronger protection than either natural or vaccineinduced immunity alone [10]. This last one is called hybrid immunity.
Our aim was to investigate different vaccination schedules and compare antibody levels, between BNT162b2, ChAdOx1, and heterologous schedule, monitoring S-RBD Ab levels after administering two doses in naive patients. Likewise, vaccine-stimulated immunity in naive and previously infected patients was compared to natural immunity developed in mild or moderate symptoms patients.

Patients
All the sera were obtained from private clinical laboratory analysis LIFEBRAIN COSENZA Srl, Cosenza (CS), Italy, collected in the period May-September 2021. At the time of laboratory acceptance, short interviews were conducted to enroll patients in the study and to divide them into different groups, as described below. Patients previously infected with COVID-19 joined group 1. Group 2, 3, and 4 patients were selected after they completed a questionnaire, declaring that they had never been infected by SARS-CoV-2 before vaccination. Moreover, all the samples were collected after the second dose administration. In group 5, patients were included who had previously been infected with COVID-19 and with one-single booster dose of Pfizer BioNTech. Table 1 summarizes the principal characteristics of each group, as anamnestic data, division between infection or vaccine, the number of subjects enrolled, the period spend after infection or the vaccination, and the monitored period.
The sera collected were centrifugated for 10 min at 2000× g and then processed as fresh samples.
We included 392 patients, 185 males and 207 females, aged 10 to 87 years. All patients with Anti-SARS-CoV-2-S antibody values < 0.82 BAU/mL were excluded from the study. Table 2 details the inclusion and exclusion criteria adopted to enroll in the study volunteer subjects.
The study was approved by the Ethical Committee of University of Rome "Tor Vergata", (approval number: R.S.44.20). Informed consent was obtained from all the subjects enrolled in the study. The study has been conducted following the Declaration of Helsinki (2013).

Antibody Dosage Anti Spike-SARS-CoV-2
The Elecsys anti-SARS-CoV-2 S (Roche S-RBD tAb) is an electro-chemiluminescence immunoassay (ECLIA) for the in vitro quantitative determination of total antibodies (IgG/IgA/IgM) to the SARS-CoV-2 S-RBD protein in human serum and plasma, performed by fully automated Roche Cobas E602 analyzer (Roche Diagnostics GmbH, Mannheim, Germany).
The assay is a three-step test process that uses a recombinant protein representing the RBD of the S antigen in a double-antigen assay format, which favors the detection of high-affinity antibodies against SARS-CoV-2. Patient samples are incubated with a mix of biotinylated and ruthenylated RBD antigens to form double antigen immune complexes. After the addition of streptavidin-coated microparticles, DAGS complexes bind to the solid phase. The reagent mixture is then transferred to the measuring cell, where the microparticles are magnetically captured. Electrochemiluminescence is induced by applying a voltage and measured with a photomultiplier. The signal yield increases with the antibody titer. The cut-off value in arbitrary units (AU/mL), the conversion factor to obtain BAU/mL, the cut-off value in BAU/mL and the linearity range in AU/mL are respectively: 0.8, 1.0288, 0.823, and 0.40-250, as declared by the manufacturer.

Statistical Analysis
For normal data distribution, parametric tests were used, such as analysis of variance with Bonferroni post hoc test for more than two variables. For the not-normal data distribution non-parametric test, the Kruskal-Wallis test (variables with more than two categories) was used to verify differences between groups. All statistical analyses will be performed using MedCalc Version 18.2.18 (MedCalc Software Ltd., Ostend, Belgium) and JASP 0.15.0.0 (JASP program, University of Amsterdam, Amsterdam, The Netherlands).

Results
The patients were divided into five groups, as illustrated in Table 1. The criteria for inclusion and exclusion are summarized in Table 2. Box whisker plots (Figures 1 and 2) were used to graphically represent the distribution of the data. The data were not normally distributed (verified by applying the Shapiro-Wilk test) and were characterized by median and percentiles values. months after the infection. All the data were not significant between each other (p: ns); from the fourth month, an increase is observed which reaches a maximum value after six months (3773 BAU/mL), and a slight decrease in the following months.
As can be seen from Figure 2A, the antibody levels in group 1 declined rapidly in the first month after the vaccination plan and then stabilize over time. The median value of the first month highlighted an antibody peak at 3744 BAU/mL (IQR: 2064-7948 BAU/mL), which significantly (p < 0.01) declined by 39% in the second month, 2271 BAU/mL (IQR: 1366-3479 BAU/mL) and dropped by 66% at the third month (p < 0.01) 1280 BAU/mL (IQR: 682-1904 BAU/mL).
In Figure 2C, antibody levels of heterologous vaccine patients have been shown; after the first month after booster administration, the median value, 5617 BAU/mL (IQR: 2625-8992 BAU/mL), was 1.5 times higher than group 2 (p = ns) and 10 times higher than group 3 (p < 0.01). After 2 months, the median value achieved, 2572 BAU/mL (IQR: 1820-4273 BAU/mL), was 2 times higher than group 3 (p < 0.01). No significant differences were noted in the second month between group 4 and group 2 median value (p = ns). In the third month, a median value of 3715 BAU/mL (IQR: 2202-4941 BAU/mL) was observed; 2.6 times higher than group 2 (p < 0.01) and 10 times higher than group 3 (p < 0.01).
Regarding the last group, in Figure 2D, the results obtained from patients with previous SARS-CoV-2 infection were analyzed; these patients were given a single dose of Pfizer-BioNTech vaccine after a period ranging from 4 to 10 months after infection. These   Figure 3 shows overall S-RBD antibody levels from vaccinated patients for each group (3A) and divided for the 3 months (3B). In violin bow Figure 3A, it was highlighted that group 5 S-RBD Ab distribution was significantly greater than other groups (p < 0.01).

Comparison of Antibody Levels over Time between All Groups
Higher median values were recorded in group 5, 18,913 BAU/mL (IQR: 11,447-25,720 BAU/mL) than in other groups. The median value of Pfizer BioNTech was 1841 BAU/mL  Figure 1 shows the persistence of antibody anti-SARS-CoV-2 levels in patients affected by COVID-19 (group 1), who were tested from 1 to 9 months after infection with SARS-CoV-2. The patients analyzed were mostly in an age range of 40-50 years old (age mean 43.36, SD 17.74), with no significant prevalence of morbidity in males, 43% (57 out of 133), compared to females, 57% (76 out of 133). The antibody levels were very heterogeneous (median 154 BAU/mL) with a minimum value at 6 months post-infection of 3.5 BAU/mL and a maximum value of 3773 BAU/mL. The antibodies increase began to be observed since the first month after the disease, with maximum values between the fourth and fifth months. A moderate antibodies decrease was observed in the following months. The data showed that antibody levels persist over time remaining relatively stable up to 9 months after infection (median 107.4 BAU/mL; Interquartile Range (IQR): 81.6-291.1 BAU/mL). All data showed no significance (p: ns).
As can be seen from Figure 2A, the antibody levels in group 1 declined rapidly in the first month after the vaccination plan and then stabilize over time. The median value of the first month highlighted an antibody peak at 3744 BAU/mL (IQR: 2064-7948 BAU/mL), which significantly (p < 0.01) declined by 39% in the second month, 2271 BAU/mL (IQR: 1366-3479 BAU/mL) and dropped by 66% at the third month (p < 0.01) 1280 BAU/mL (IQR: 682-1904 BAU/mL).
In Figure 2C, antibody levels of heterologous vaccine patients have been shown; after the first month after booster administration, the median value, 5617 BAU/mL (IQR: 2625-8992 BAU/mL), was 1.5 times higher than group 2 (p = ns) and 10 times higher than group 3 (p < 0.01). After 2 months, the median value achieved, 2572 BAU/mL (IQR: 1820-4273 BAU/mL), was 2 times higher than group 3 (p < 0.01). No significant differences were noted in the second month between group 4 and group 2 median value (p = ns). In the third month, a median value of 3715 BAU/mL (IQR: 2202-4941 BAU/mL) was observed; 2.6 times higher than group 2 (p < 0.01) and 10 times higher than group 3 (p < 0.01).

Discussion
The immunity duration in people who have tested positive for COVID-19 nowadays is one of the most discussed topics in the scientific world and, to date, there is still no definitive and unambiguous answer in this regard. What we know is that following primary infection, over 90% of patients develop a positivity for antibodies against SARS-CoV-2, which, although decreasing over time, remain detectable for several months [2,11].
In the present work, the circulating S-RBD Ab persistence was analyzed in 122 patients who contracted the SARS-CoV-2 virus, up to 9 months after infection. A retrospec-

Discussion
The immunity duration in people who have tested positive for COVID-19 nowadays is one of the most discussed topics in the scientific world and, to date, there is still no definitive and unambiguous answer in this regard. What we know is that following primary infection, over 90% of patients develop a positivity for antibodies against SARS-CoV-2, which, although decreasing over time, remain detectable for several months [2,11].
In the present work, the circulating S-RBD Ab persistence was analyzed in 122 patients who contracted the SARS-CoV-2 virus, up to 9 months after infection. A retrospective analysis of the data revealed a quite heterogeneous antibody response, which could depend on the severity of the disease (asymptomatic, mild, moderate, or severe) and inter-individual variability [2,9]. However, the antibody levels persist over time, relatively stable up to 9 months after infection, at a medium-low level (median 107.4 (81.6-291.1) BAU/mL) ( Figure 1). Furthermore, in some patients, the circulating S-RBD Ab levels are even increased over time, which contrasts with other recent publications that have found a slight decrease in the antibody levels [2,10]. This could have several explanations. Firstly, the test used evaluates total Ig rather than IgG, which could mean that test evaluates all the circulating immunoglobulin classes (IgA, IgG, and IgM). Second, the S-RBD Ab test inherently favors the detection of high-affinity antibodies [11]. Therefore, the increase in signal in the RBD test may reflect the maturation of antibody affinity over time, while the total amount of S-RBD Ab effectively remains stable or even slightly decreases [12]. The results obtained in this study are consistent with the data obtained in recent papers [2,6,9,13], where the persistence of circulating antibodies against the Spike protein and/or the domain RBD was observed in convalescent patients for 5, 6, 8, and 11 months, respectively, after the onset of symptoms.
For vaccinated patients, the data obtained in this work showed antibody levels decline after the immunological peak (Pfizer-BioNTech after 21 days, ChAdOx1 nCoV19 after two months), while from the fourth month onwards, the titer stabilizes over time. However, despite the first vaccine, the ChAdOx1 nCoV19 antibody response seemed more contained. After one month, the level of circulating antibodies S-RBD Ab is significantly lower than Pfizer-BioNTech. Otherwise, heterologous vaccination elicited a robust antibody response. Comparing the results obtained with homologous vaccination patients, a statistically significant difference was observed in circulating S-RBD Ab levels, even after 30 days. Still, after three months from the administration of the booster, the heterologous antibody levels continued to be significantly higher than Pfizer-BioNTech and/or ChAdOx1 nCoV19. The data obtained in the present work, therefore, provide evidence of strong immunogenicity of the heterologous vaccination capable of generating a robust persistent antibody response at high levels steady up to three months after administration. Several hypotheses explain these disparities between the groups, including differences in the age-populations tested, the different lifestyles, and biological variability of immune response after the SARS-CoV-2 vaccine. Moreover, the specific and technical properties of vaccine composition could lead to the results obtained [14].
From the data observed in group 5, the administration of one-single dose of booster developed a stronger antibody response, much more if compared to other groups. Several studies were in agreement with these findings [13,15], highlighting that a single booster dose given to previously infected patients raised an antibody response much higher than two doses given to naïve individuals. Furthermore, monitoring the antibodies levels after vaccine administration or after the booster dose is the key to better understanding the overtime antibodies stability and to determine if their protective action is level dependent. It should be also considered whether periodic booster doses could or could not overstimulate the immune systems [16] and these studies should investigate the cell immune memory response, too [17].
The assertion on the most effective vaccination strategy as the heterologous one is referred to naïve patients [2,9]. When the infection occurs the effect on immune system stimulation is higher than vaccine alone. Several works have already demonstrated how administration of a single dose on previously infected patients results in higher Abs production respect to double-dose vaccinated patients [14,15].
The limitation of this study is the imbalance in the different groups between the number of enrolled volunteers, based on the Italian vaccine campaign. Nevertheless, despite the limitations, the data obtained are consistent with the literature [18].
Another limit was the short period evaluated for vaccinated people, regardless some subjects, as they were volunteers, did not want to continue antibody monitoring.

Conclusions
The data obtained highlighted the importance of establishing the right schedule of vaccination. It has been seen that ChadOx1 nCoV19 patients did not develop a proper anti-SARS-CoV-2 immunity, as well as Pfizer BioNTech patients, whose antibody levels have risen as fast as they have decayed. For this, a third dose seems to be increasingly needed to stimulate immunity and to achieve strong (and protective) antibody levels.
On the other hand, data obtained in previously infected patients suggest just a single dose of vaccine may be sufficient. However, further investigations on reactogenicity and antibody kinetics are needed.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.