One of the coronaviruses known as the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is the source of highly contagious disease identified as COVID-19. In late December 2019, this virus initially broke out in Wuhan, China, and rapidly spread to other countries worldwide [1]. From infection without symptoms to mild, moderate, and severe pneumonia with failure in many organs, which can be deadly to the afflicted persons, it presents a broad spectrum of clinical manifestations [2]. Concerns about the safety of newly developed and marketed vaccines, as well as possible long-term side effects, have significantly increased among the public. It is essential to evaluate the acceptability rate, perspectives, and worries regarding receiving COVID-19 vaccinations to look into the frequency side effects of vaccines among the Iraqi population in Baghdad province [3]. COVID-19 infection and vaccinations both cause immunological responses. By carefully managing the damage caused by SARS-CoV-2, a coordinated immune response may be generated using inactivated viruses or mRNA-based adenoviral vectors [4]. Antigen-specific antibody responses are monitored following SARS-CoV-2 vaccination as markers of protective immunity. Immunoglobulin (IgM) antibodies are created shortly after the humoral immune system’s response to viral infections, offering prompt protective protection [5]. After that, Higher affinity immunoglobulin G antibodies are produced together with isotype class shift and maturation. The substantial IgG responses to SARS-CoV-2 elicited via the current mRNA vaccines have been extensively studied. However, there needs to be more consensus about the timing of vaccine-induced IgM responses and the role of previous immunity. The importance of IgM in protective immunity against COVID-19 was highlighted by the robust association seen between reducing levels of IgM against spike (S) protein and the receptor binding domain (RBD) and declining responses of neutralizing antibodies [6]. Following the infection, IgA plays a vital role in the early neutralization of the SARS-CoV-2 virus. In individuals with no measurable IgG and without symptoms recorded for a medical history, serosurveys have revealed positive results for IgA. A prior study found that individuals with mild or asymptomatic illnesses might have both transient or non-existent IgG positive and IgA positivity. Although there was no detectable IgA or IgG in their serum, a few individuals had mucosal IgA discharges [7]. This study aims to estimate the levels of SARS-CoV-2 Anti-Spike S1 RBD IgM, IgA, and IgG in serum among people in Baghdad after one month of receiving the Pfizer-BioNTech vaccine, COVID-19 infected and infected with the SARS-CoV-2 virus after vaccination patients respectively.

A. Sampling This study, a case-control analysis, was carried out between November 1, 2022 and January 13, 2023. Following thirty (30) days of Pfizer-BioNTech (BNT162b2) second dose vaccination, COVID-19 infection, and COVID-19 infection following Pfizer-BioNTech (BNT162b2) full vaccination before six months to one-year (6 months to one year) with a control group, one hundred twenty (120) volunteers were chosen for this study. Each group had thirty individuals, with a 50:50 male-to-female ratio, age ranging from 20 to 45 on average, and from various regions of Baghdad. In addition, research volunteers with a history of gastrointestinal illnesses, pharyngitis, urinary tract infections, or immune response issues during the previous year were not allowed to participate. The study groups’ individuals were divided into: The Control group (C) included persons not previously infected by COVID-19 or vaccinated with any vaccine related to the Coronavirus and did not suffer from any type of chronic diseases (DM, IHD, HT, or Asthma). The Pfizer-BioNTech (BNT162b2) vaccinated group (V) included persons who did not have previous infection with COVID-19 but were vaccinated with the Pfizer-BioNTech vaccine and were selected one (30) days after the second dose. The COVID-19 infected group (CoV) included individuals who were selected after one month of COVID-19 infection and were not vaccinated. The Pfizer-COVID-19 group (V-COV) included individuals who were vaccinated with the Pfizer-BioNTech (BNT162b2) vaccine six months to one year after the date of the second dose, and then after one month, they got COVID-19. B. Blood Sample Collection By the research’s protocol, each person in the study group had their vein punctured to get five milliliters of blood, which were then placed into gel tubes and left for around twenty minutes to allow the blood to clot at room temperature. The serum was extracted from the tubes and centrifuged for 10 minutes at 5000 RPM. The serum was then transferred into Eppendorf tubes to prevent contamination, freezing, and melting. The following Humoral immunological markers were measured using the ELISA technique: SARS-CoV-2 Anti-Spike S1 RBD IgM, IgA, and IgG. C. ELISA Protocol By the manufacturer’s protocol, serum levels of the COVID-19 S1 RBD IgG Kit for ELISA (catalog number IEQ-CoVS1RBD-IgG), SARS-CoV-2 S1 IgA Kit for ELISA (catalog number MBS398093), and SARS-CoV-2 S1 IgM Kit for ELISA (catalog number MBS7612291) were measured by using an indirect ELISA (enzyme-linked immunosorbent assay) test. The results were computed using a microplate reader (Paramedical/Italy ELISA system). Six standards were created for the sandwich, Anti-Spike S1 IgA, Anti-Spike S1 RBD IgG, and Anti-Spike S1 IgM, according to the manufacturing company’s instructions. These standards will be utilized for serum Anti-Spike S1 IgM titers (ng/ml), Anti-Spike S1 RBD IgG titers (Unit/ml), and Anti-Spike S1 IgA titers (ng/ml) to be quantified and analyzed. The concentration serum Anti-Spike S1 IgM titers, Anti-Spike S1 RBD IgG titers, and Anti-Spike S1 IgA titers were plotted for each calibrator regarding the serum Anti-Spike S1 IgM titers, Anti-Spike S1 RBD IgG titers, and Anti-Spike S1 IgA titers ELISA Kit to obtain the mean calculation. Data were counted by determining the mean absorbance for each duplicated measurement. D. Ethical Approval This study was given ethical approval by the Aliraqia Medical College Review Board / the Medical College / Al-Iraqia University. E. Statistical analysis The statistical program SPSS software for statistics v.26.0 (SPSS Inc., Chicago, IL, USA) was used to run the investigations. To determine if there are statistically significant differences, do a one-way ANOVA. Furthermore, a chi-square analysis was performed to determine the significance of the percentage differences. Statistics are deemed significant when P is less than 0.05.

The anti-spike S1 RBD IgM serum levels mean were significantly increased (P value <0.05) in the control group compared to all groups Figure 1 and also the same results were observed between each group with each other’s Table 1. Figure 1: Mean of Anti-Spike S1 RBD IgM serum level for all groups in this study Table 1: Association of Anti- spike S1 RBD IgM serum level in all groups of study Group N Mean Std. Deviation   Antispike IgM Vaccinated non infected 30 1.3940 1.90448 <0.0001 Infected non-vaccinated 30 21.4640 10.07719 Antispike IgM Vaccinated noninfected 30 1.3940 1.90448 <0.0001 Infected vaccinated 30 14.9520 6.28225 Antispike IgM Vaccinated noninfected 30 1.3940 1.90448 0.04 Control 30 .6440 .12489 Antispike IgM Infected non-vaccinated 30 21.4640 10.07719 0.004 Infected vaccinated 30 14.9520 6.28225 Antispike IgM Infected non-vaccinated 30 21.4640 10.07719 <0.0001 Control 30 .6440 .12489 Antispike IgM Infected vaccinated 30 14.9520 6.28225 <0.0001 Control 30 .6440 .12489 The immunoglobulin G and immunoglobulin A serum titers were significantly increased (P value <0.05) in all groups which compared to the control group Figures 2 and 3. There was a significant difference (P value <0.05) among the (V) group and both the (CoV) and (V-CoV) groups while there were non-significant difference (P value <0.05) between the (CoV) group and the (V-CoV) group Table 2 and 3. Figure 2: Mean of Anti-Spike S1 RBD IgG level in the serum for all groups in this study Figure 3: Mean of Anti-Spike S1 RBD IgA serum level for all groups in this study Table 2: Association of Anti- spike S1 RBD IgG serum level in all groups of study Group N Mean Std. Deviation P value Antispike IgG Vaccinated non infected 30 618.6713 82.63897 <0.0001 Infected non-vaccinated 30 821.7997 263.02618 Antispike IgG Vaccinated non infected 30 618.6713 82.63897 <0.0001 Infected vaccinated 30 913.9780 172.09034 Antispike IgG Vaccinated non infected 30 618.6713 82.63897 <0.0001 Control 30 51.3340 24.16198 Antispike IgG Infected non-vaccinated 30 821.7997 263.02618 0.11 Infected vaccinated 30 913.9780 172.09034 Antispike IgG Infected non-vaccinated 30 821.7997 263.02618 <0.0001 Control 30 51.3340 24.16198 Antispike IgG Infected vaccinated 30 913.9780 172.09034 <0.0001 Control 30 51.3340 24.16198   Table 3: Association of Anti-Sike S1 RBD IgA serum level in all groups of study Group N Mean Std. Deviation P value Antispike IgA Vaccinated noninfected 30 357.28373 44.068095 <0.0001 Infected non-vaccinated 30 531.72100 149.560504 Antispike IgA Vaccinated noninfected 30 357.28373 44.068095 <0.0001 Infected vaccinated 30 574.31000 104.749120 Antispike IgA Vaccinated noninfected 30 357.28373 44.068095 <0.0001 Control 30 25.80333 9.386283 Antispike IgA Infected non-vaccinated 30 531.72100 149.560504 0.2 Infected vaccinated 30 574.31000 104.749120 Antispike IgA Infected non-vaccinated 30 531.72100 149.560504 <0.0001 Control 30 25.80333 9.386283 Antispike IgA Infected vaccinated 30 574.31000 104.749120 <0.0001 Control 30 25.80333 9.386283

Following vaccination or natural infection, antibody responses specific to an antigen are tracked and observed as measures of protective immunity [5] because antibody levels can indirectly reflect the effectiveness of immune responses [7]. Both T-cell mediated and humoral immune responses are induced by SARS-CoV-2 vaccines and natural infection, which act mainly through targeting the S protein of the virus, in particular the S1 domain, which in turn results in the production of immunoglobulins A, G, and M antibodies against the spike protein [8]. Although vaccination will reduce the severity of COVID-19 infection, antibodies induced during vaccination will eventually fade away [9]. IgM is crucial for defensive immunity against COVID-19 because there is a clear correlation between decreasing anti-S IgM titers and decreasing neutralizing antibody responses [6, 11 ]. In the current study, we observed that, in terms of titters, the IgM response in the (CoV) and (V-CoV) groups was higher than in the (V) group and control persons, which agrees with previous research investigations [13]. It is interesting to note that Ruggiero et al. [11] found that those who had not been exposed to COVID-19 and had received the Pfizer mRNA vaccination had unusual response patterns of anti-S IgM, which included either no immunoglobulins M, IgM developing after IgG, or IgM and IgG present at the same time. It is challenging to hypothesize the cause of vaccine-induced aberrant responses of IgM in possibly individuals who received naive COVID-19 vaccine. An earlier response of primary immune against asymptomatic virus infection, an earlier booster vaccination with increased IgM decay, or an IgM memory response from an earlier immunity to cross-reactive human corona-viruses may all be contributing factors to the absence of IgM entirely after two weeks of the entire vaccination. Due to the reported Th1-polarized responses, the accessory action of the vaccine’s lipid components in promoting early and widespread IgG class-switching is another likely explanation [14]. The endurance of non-class-switched immunoglobulin M+ memory B cells may be the cause of vaccinees’ virus-specific immunoglobulin M responses [13]. Avidity of Anit-spike S1 RBD IgG specific to SARS-CoV -2 levels was assessed after vaccination with Pfizer-BioN Tech approved mRNA vaccines or SARS-CoV-2 natural infection [15]. IgG is initially produced during seven days after vaccination or natural infection. According to previous studies, individuals with a high IgG level were less expected to develop symptoms, had a shorter improvement time, and had higher titers of IgM [16]. Severe COVID-19 instances were invariably linked to greater antibody production and neutralization titers, even in the sample population’s heterogeneity [17]. According to a complete cohort study conducted on recipients of the mRNA-based vaccine, the Anti S1 RBD IgG response elicited by COVID-19 vaccination peaked up to 15 days after the second dose. It gradually decreased until six months or even more after vaccination [9]. Viral neutralization is mediated by SARS-CoV-2 S IgA, which is generated by spontaneous infection and is a crucial part of natural immunity. It has been studied how the COVID-19 vaccine affects IgA responses, particularly in comparison to mRNA-based vaccinations. The COVID-19 mRNA vaccine elicits S1-specific immunoglobulin A with comparable kinetics to S1-specific immunoglobulin G, according to Chan et al. [18]. However, the serum of vaccines decreases more quickly after both the first and second doses. It has been found that the mRNA-based medication induces serum SARS-CoV-2 S1 RBD -specific immunoglobulin A. However, the medication may also generate S1-specific immunoglobulin A in the mucosa of the nasal cavity. Moreover, anti-S1 IgA was secreted in women’s milk [19] and vaccine recipients’ saliva [11] in response to mRNA-based vaccinations. Lipid nanoparticles, particularly ones carrying mRNA-based vaccines, have been discovered in distant organs, such as the lung [20]. In contrast, the intramuscular vaccination technique does not produce mucosal immunity [21]. This study revealed that Anti-spike S1-RBD IgA levels in (the CoV group, V-CoV group, and V group) are higher compared to the (C) group (non-vaccinated, non-infected individuals), indicating that the response of Anti spike S1 RBD IgA is more prominent due to natural infection [18, 22]. V-CoV individuals generated higher responses of IgG and IgA against S antigens than their V or CoV participants. It was observed that this variation in reactions persisted for five months following vaccination. Regardless of whether the disease develops before or after a vaccine, Bates et al. have shown an increase in humoral immune responses that include binding and neutralizing antibodies [23].

Specific SARS-CoV-2 anti-S1 RBD IgM, IgA, and IgG were significantly increased in CoV, V-CoV, and V groups individuals after one month of Pfizer-BioNTech complete vaccination, COVID-19 infection after Pfizer-BioNTech full vaccination, COVID-19 infection and.  

The authors declare no conflict of interests. All authors read and approved final version of the paper.

All authors contributed equally in this paper.