The COVID-19 pandemic, declared by the World Health Organization (WHO) in March 2020, continues to be a major problem with an enormous impact. The fight against SARS-CoV-2 has focused the efforts of healthcare professionals and scientific researchers, with the aim of tackling the problem at all levels, from prevention, protecting the population by means of vaccines, to caring for cases [1] [2] .

In December 2020, the first vaccines were authorized and began to be administered in various countries. According to the WHO, by 21 February 2022, a total of 10,407,359,583 doses had been administered [3] . Vaccination coverage in Spain is high, with 93.0% of the population aged over 12 fully vaccinated and more 54% of the general population with booster doses [4] .

Since its emergence, the SARS-CoV-2 virus has been constantly evolving. To date, the WHO has designated five variants as of concern, namely, Alpha, Beta, Gamma, Delta and Omicron, considering their impact on transmission, the severity of illness or their ability to evade immune protection [5] [6] [7] .

Vaccine efficacy ranges from 50% to 95% [8] . The long-term protection of antibodies against subsequent reinfection after COVID-19 and/or vaccination has not yet been fully established. Understanding of antibody kinetics against SARS-CoV-2 and its vaccines is evolving rapidly [9] . Monitoring the immune response against SARS-CoV-2 is essential to evaluate long-term vaccine efficacy. Immunoglobulin G (IgG) antibodies constitute an appropriate tool to reach this goal, especially regarding the antibody trend induced by the new class of mRNA vaccines against SARS-CoV-2, which is still insufficiently defined [10] .

The WHO suggests many pivotal questions remain about the effectiveness of vaccines in real-world settings, which can only be answered in studies on post-introduction vaccine effectiveness [11] . Independent experts agree that knowledge about the protection provided by these vaccines will emerge in the coming months [12] . It is worth noting that the length of the protection provided by the vaccines may vary, as shown by immunization against other diseases such as flu [13] .

The TAG-CO-VAC (WHO Technical Advisory Group on COVID-19 Vaccine Composition) believes that vaccines against COVID-19 are necessary and should be developed and studies are needed to monitor the immune and clinical response of the vaccines [13] . Questions remain unanswered as regards the duration of immunity and whether the new variants appearing will be neutralized by the antibodies generated by current vaccines [14] . Vaccines against SARS-CoV-2 are highly efficient against severe forms of the disease, hospitalization and death. However, insufficient protection against several circulating viral variants could suggest a decrease in immunity and the need for additional vaccine doses [15] . Many countries are administering a third dose of COVID-19 vaccines, but the evaluation of vaccine-induced immunity is insufficient [16] .

Healthcare and socio-health professionals have been significantly affected by the COVID-19 pandemic. The report published by the Spanish Ministry of Health (10 February 2022) revealed 204,094 cases of infection since the onset of the pandemic [17] . Spain has one of the largest percentages of infected healthcare employees, which justifies the prioritization of immunization in this population, with their being one of the first groups to be vaccinated [18] . Healthcare workers (HCWs) were among the first group of people vaccinated [19] . Characterization of the kinetics of antibody response to vaccination is important to devise future vaccination strategies and studies on workers in the healthcare sector have pioneered both the assessment of the occupational risk of COVID-19 and the surveillance of the immune and clinical responses to the vaccines administered to date.

To identify the sociodemographic, clinical, and epidemiological characteristics associated with occupational exposure to SARS-CoV-2 in a population of healthcare workers.

To determine seroprevalence and to measure levels of antibodies (IgG) against SARS-Cov-2 at 6 and 9 months after vaccination.

Below, we present the results of the study, which respond to our aims. The sociodemographic characteristics of the study population are mean age 46.45 years; (SD = 9.95); Range = 41.74; Minimum value = 23.9 years; Maximum value = 65.8 years; Median (Mn) = 45.4 (9 subjects did not report their age). Table 1 shows distribution by age group, sex, education level, occupation, and area of work (hospital, primary care and socio-health care). There is a notably high proportion of women, 40 to 49 years is the largest age group (42.6% of total), a majority have university studies and a large percentage are nursing professionals. Thus, the predominant profile is that of a female nurse working in a hospital and aged below 50.

Numerous studies have measured the immune response in healthcare workers to both SARS-CoV-2 infection and vaccines. Drawing on this bibliography, we compared our data with those from studies focused on similar variables (those assessing humoral immune response following vaccination with Pfizer and with follow-ups at several months). The healthcare professionals included in other studies have similar profiles to those of our sample, with an average age of 54.4 years and a mean of 46.3. The mean ages typically range between 40 and 48 [23] [24] [25] , although some studies use a younger population with a mean age of between 33 and 37 [26] [27] and [28] . In all cases, there is a greater proportion of women, ranging from just over 50% [26] , around 60% - 69% [24] [25] [27] and more than 70% [28] , as in our study, with 76% of women. Some of the works include populations of which more than 80% are women [23] [29] and [30] . The findings on age-related variations in the immune response are conditioned by this limited diversity in the ages of the health service employees. As regards the sex-related variations, given the largely female populations and the scant number of men in some groups, the differences reported are, arguably, not always significant or clear.

The incidence of previous exposure to SARS-CoV-2 in healthcare workers is typically higher than among the general population in their respective countries, which is in line with the results of our study. The incidence data vary greatly and tend to be reported as a percentage of persons infected, ranging between 7% and 25%. The range of values is as follows: studies with the lowest incidence rates of 7% and 7.8% [27] [30] [31] ; around 10% [32] ; others with figures similar to our 19.3%, such as 19.22% [23] ; and others with higher incidence rates of 23% [14] 25% [26] and 32.1% [33] .

Based on a cutoff point of 50 AU/mL for IgG Spike positivity (RBD), 100% of the participants in our study had antibodies following vaccination, in line with other studies [14] [30] . These results are described in some studies as seroconversion, as they present data compared with baseline levels, finding that all vaccinated employees without prior COVID-19 infection have positive IgG levels [9] , or percentages close to 100% [30] [31] [32] [36] . Discrepant data were also reported with 22.9%, being seronegative [19] .

Terpos et al. followed up a group of healthcare workers for several months after vaccination, finding persistent but attenuated anti-SARS-CoV-2 humoral immunity at 3 months after the second vaccination with BNT162b2 in healthy individuals [32] [34] . Several studies have established peak level circulating antibodies at 3 - 4 weeks after the second vaccination, and there is a considerable consensus on the decrease in levels over time, especially after the third month [26] . Additionally, most of the studies present dispersion in the values in coherence with our findings.

The study by Rode et al. reported that most of the participants present positive values, from 50 to 2000 AU/mL was the most representative range of antibody titers in almost 80% of subjects [35] . The interquartile range for our data at 6 months was slightly higher although the conclusions of immune response efficacy are mostly similar.

Among the factors related to differences in IgG levels on which there is more evidence, the most notable are age and previous infection. Regarding age, evidence suggests that younger individuals tend to present higher levels of immunoglobulin G anti-SARS-CoV-2 (19, 29 and 30). In our work, no association was found between IgG level and age.

We found no differences in IgG levels by sex, which is consistent with the findings of other studies (35 and 39). However, some studies have detected higher IgG levels in women in the initial immune responses after vaccination (9, 19, 21, 30, 32 and 34).

Comparisons of antibody levels in persons with previous infection and subsequent vaccination confirm hybrid immunity is more robust in IgG measurements a few weeks after vaccination. Individuals with prior infection present higher IgG levels at all time points [31] and these differences are maintained various months after vaccination [9] [25] [35] , although they are more pronounced in the early weeks post-vaccination. Seropositivity was significantly higher in healthcare workers with prior COVID-19 infection, according to the cross-sectional study by El-Ghitany et al. [33] . In our study, the differences in IgG levels remained significantly higher in persons with prior infection both 6 and 9 months after vaccination.

Hansen et al. found that a single dose of the BNT1622b vaccine induces a robust antibody response in individuals with previous infection, which, in immunogenicity, is equivalent to a double dose of the vaccine [30] . Consequently, it is considered that a single dose might be sufficient in individuals with previous infections, regardless of the time elapsed since the diagnosis [24] . Nonetheless, a generalized decline in antibody levels over time has been found in vaccinated individuals both with and without prior infection.

Some works have found an impact on immune response levels of other factors, such as chronic diseases, smoking and high BMI. Nonetheless, the findings are inconclusive [9] [26] . El-Ghitany et al. found a relationship with smoking, with antibody positivity being significantly lower in smokers (61.9%) compared to non-smokers (87.7%) (p = 0.003) [33] . Our study also confirms that smoking inhibits immune response

As regards BMI, the studies by Hansen et al. [30] and de El-Ghitany et al. provide no conclusive data to support a relationship between high BMI and impaired immune response [33] . Papadopoulos et al., however, found an association between older participants, higher BMI and the presence of autoimmune diseases with negative effects on the development of anti-SARS-CoV-2 antibodies 9 months after full-vaccination [36] .

Our findings point to durable immunity despite the decline in antibody levels 9 months post-vaccination. Other works coincide with our findings. Studies indicate a decline from peak levels in neutralizing antibody titers, although these remained detectable in most participants 6 months post-vaccination [29] [37] [38] . The data reported by Rode et al. at 6 months after full vaccination (mean IgG 966.0 AU/mL) are similar to those in our study [35] .

The recent systematic review by Notarte et al., characterizing the kinetics of anti-SARS-CoV-2 antibodies following the second dose of a primary cycle of mRNA vaccination, revealed that the peak humoral response was reached at 21 - 28 days after the second dose. Subsequently, serum levels progressively decreased at 4 - 6 months post-vaccination. and the results showed that, regardless of age, sex, serostatus and presence of comorbidities, there is an antibody decay [39] .

The studies that offer findings more than 6 months after vaccination (in general they do not exceed 8 or 9 months) show antibodies largely remain active, despite a notable decline in levels [37] [40] , with important variations according to groups of subjects [41] .

There is concern about whether the vaccines will be less effective against the new variants of the virus (Delta, Omicron, Omicron B.A.2). Although studies have already been published on Omicron and the Delta and Beta variants and RNA vaccines are reported to provide protection against severe and lethal forms of COVID-19 but infection persists against these SARS-CoV-2 variants [37] [41] .

There is considerable agreement on implementing booster doses in high-risk populations, such as daily alcohol drinkers, frail elderly, smokers and other groups, in whom both a greatly diminished humoral and cellular immune response has been evidenced, and in which booster doses may be warranted [16] [41] [42] . Some studies point towards the need to customize additional booster doses to achieve an adequate neutralizing response against the new circulating variants, which justifies the decisions on the third dose implemented in European countries and those that may be recommended by health authorities in the coming months [34] .

Immunosurveillance studies estimate the duration of immunity and are especially necessary for designing public health responses to the general population, healthcare workers and, particularly, specific population groups with a compromised immune response [43] [44] . It is even noted that the heterogeneity of responses to vaccines suggests that personalized recommendations based on COVID-19 history and lifestyle are necessary [45] .

The incidence of COVID-19 disease in healthcare workers is higher than in the general population.

A high proportion, close to 100% of immunized individuals was detected, with positive IgG levels at both 6 and 9 months after vaccination.

The immune response was found to be more robust in certain groups of individuals, with evidence of a clear, positive association with prior COVID-19 infection in vaccinated persons.

Non-smokers develop a more powerful immune response and present higher IgG levels compared to smokers.

Antibody levels remain positive 9 months after full vaccination despite the evidence of a decline in IgG levels.

The external validity of the study might be limited by the mean age of the study population, given that it did not include individuals at age extremes. Additionally, as a healthy adult population, comorbidity was low.

Prior COVID-19 infection was self-reported by the participants. In the initial questionnaire and at the second sample taking, the participants reported any prior infection, providing data on the date of the diagnostic test (PCR o antigen test).

Data and sample collection were affected by circumstances beyond the control of the researchers, primarily changes in the employment status of the workers or a lack of response despite having agreed to participate in the study.

Between the first data and sample collection and the second, 18 subjects (12%) were lost, which may increase the error in the estimates.

The diagnostic method used to detect anti-SARS-Cov2 serum levels did not allow the neutralizing capacity of these antibodies to be determined. Additionally, we were unable to differentiate the antibodies generated by the vaccine (anti-S, RBD), from those generated naturally by participants following infection (anti-N assays).

This research was funded by Albacete Provincial Council, Call resolution code 1780 (03/02/2022) and application 30591.

“Conceptualization, V.D-U. and E.N-R. and T.P-D. and JD.P-S and C.O-M.; methodology, V.D-U. and E.N-R. and T.P-D. and JD.P-S and C.O-M. and IM.S-O and J.L-S and L.R-F and A.A-E; software, IM.S-O and V.D-U and E.N-R and C.O-M.; formal analysis, IM.S-O and V.D-U and E.N-R; investigation, V.D-U. and E.N-R. and T.P-D. and JD.P-S and C.O-M. and IM.S-O and A.A-E ; resources, E.N-R. and T.P-D. and A.A-E and F.G-A ; data curation. V.D-U. and E.N-R. and T.P-D. and JD.P-S and C.O-M. and IM.S-O and A.A-E and F.G-A and J.L-S and L.R-F; writing—original draft preparation, V.D-U. and E.N-R. and T.P-D. and C.O-M. and IM.S-O; writing—review and editing, V.D-U. and E.N-R. and T.P-D. and JD.P-S and C.O-M. and IM.S-O and A.A-E and F.G-A and J.L-S and L.R-F; supervision, V.D-U; project administration, V.D-U. and E.N-R. and T.P-D and F.G-A.; funding acquisition, V.D-U. and E.N-R. and T.P-D and F.G-A. and C.O-M. All authors have read and agreed to the published version of the manuscript.”

The project (Code 2021-27 prospective OsD) was approved by the Clinical Research Ethics Committee of the University of Castilla-La Mancha (UCLM) and the Albacete Health Service Area (4/27/2021), as well as by the Spanish Medicines Agency (05/21/2021). The Castilla-La Mancha Health Service (SESCAM) gave its approval to the study on June 11, 2021. It was published in the Spanish Registry of Clinical Trials, which is mandatory for this type of design: observational study of drugs (OsD). All the participants gave their signed informed consent to participate in the study.

Informed consent was obtained from all subjects involved in the study.

Data is available on request due to restrictions ethical.

All the research team would like to thank the management of the ICM Almansa; without their help, it would not have been possible to conduct this study. This work has received funding from the Provincial Council of Albacete, the Faculty of Nursing of Albacete (UCLM) and the Integrated Care Management of Almansa. We would also like to thank the following: Simón Gómez-Biedma, head of the laboratory service at Almansa General Hospital. The laboratory service at the Hospital of Almansa and the microbiology service at Albacete University Hospital. Nurse Verónica Ortiz Díaz, for her unconditional help in collecting samples. Pedro Delicado-Useros for his attention and advice on sampling and statistical inference. All the participants for their selfless collaboration.

• High adherence to the SARS-CoV-2 vaccination regimen was observed among health personnel (almost 100%).

• The acquired immune response is positive (IgG > 50 AU/ml), from the first dose of the vaccine (seroconversion close to 100% of cases).

• IgG levels remain high at 6 and 9 months after administration of the complete regimen, although there is a significant decline at 9 months (around 28.9%) in individuals with repeated measures.

• The cumulative incidence of COVID-19 in healthcare workers was 19.33% in the year prior to vaccination and 2.3% in a similar period after the start of vaccination.

The study of factors associated with immunity has identified that smoking favors a weaker immune response, as evidenced by a significantly decreased IgG level in the smoking population.

The authors declare they have no conflicts of interest.

Delicado-Useros, V., Navarro-Rodenas, E., Sánchez-Onrubia, I.M., Ortega-Martínez, C., Alfaro-Espín, A., Pérez-Serra, J.D., García-Alcaraz, F., Lozano-Serra, J., Robles-Fonseca, L. and Pérez-Domenech, T. (2023) Evolution of Acquired Humoral Immunity after Full Vaccination against SARS-CoV-2. IgG Levels in Healthcare Workers at 6 and 9 Months. World Journal of Vaccines, 13, 13-32. https://doi.org/10.4236/wjv.2023.132002

Lost values not included. Percentages are calculated over valid data. Own preparation. Data on the study population from ICMAlmansa (Albacete) 2022.