Gender, Immunological Response, and COVID-19: An Assessment of Vaccine Strategies in a Pandemic Region of Oaxaca, M é xico

: COVID-19 is no longer a public health emergency of international concern, but long COVID’s effects are yet to be fully understood. Hence, globally, SARS-CoV-2 is still a profound threat to public health and of perilous nature as a zoonotic disease. Timely vaccination provided to individuals worldwide during the pandemic phase was under a certain degree of control; however, few studies have reported the effectiveness of vaccines administered in Mexico, and its surveillance is paramount. Furthermore, an unknown proportion of Mexican individuals have not yet received any vaccine, and the circulation of the Omicron, Pirola, and FLiRT variants is ongoing. A cross-sectional serology survey study design was employed, involving 150 individuals from Southern Mexico (Oaxaca) whose humoral immune responses after vaccination were tested by an ELISA; the receptor-binding domain of the SARS-CoV-2 spike protein served as a recombinant antigen in the ELISA. One hundred thirty-nine out of 150 individuals (92.6%; 95%-CI = 87–95%) examined were positive for the ELISA, but in 11 individuals, the vaccines did not induce any immune response. Interestingly, the immune responses (antibody prevalence and levels) of females (58%) were higher (T= − 2.21; p -value = 0.02) than those of males (41%). However, in this sample population of Southern Mexico, age, vaccine type, comorbidity, and body mass index did not have any effect ( p > 0.05) after COVID-19 vaccination. Taking all results together, here, we present factors that affected immune responses of individuals during the first vaccination campaign in Oaxaca, Mexico; however, vaccine surveillance during the post-pandemic phase needs further investigation.


Introduction
Globally, COVID-19 vaccination was paramount in providing acquired immunity against SARS-CoV-2 and was under certain control during the pandemic.The SARS-CoV-2 virus has generated multiple variants with mutations in the spike protein due to amino acid changes, which can affect key viral virus transmissibility and antigenicity, likely in response to the changing immune profile of the human population.Further understanding of these spike mutations is critical to assessing their potential to evade immune responses after infection or vaccination [1].These mutations may confer an infectious advantage leading to increased transmission rates and potential impact on disease severity [2].Thereby, epidemiologic studies of COVID-19 vaccines focused on how vaccines were distributed [3], their outcomes [4], the factors that influenced their administration [5], and their effects within various populations [6,7].The variables in such studies were the vaccine type [8], dosing schedules [9], adverse events [10], vaccine effectiveness against different variants [11], and the overall vaccination population coverage.Insights gained from these studies were instrumental in shaping vaccination campaigns, prioritizing specific populations for vaccination, and adjusting vaccination strategies in response to emerging variants [12].Moreover, these studies contributed to further understanding of how vaccination reduces the severity of illness, hospitalization, and mortality rates [13,14].
The spread of COVID-19 in Mexico has proven challenging, primarily because of the high prevalence of cardiometabolic diseases and the longstanding sociodemographic and healthcare disparities of the Mexican epidemiologic scenario [15].In particular, the older male population faced a significantly elevated mortality rate throughout the pandemic, contributing to Mexico's national case fatality rate of 9.95%, the highest ever recorded worldwide [16].Therefore, the Ministry of Health of Mexico adopted, at the onset of the pandemic, a comprehensive vaccination strategy [17], offering a range of twelve different types of vaccines, such as BNT162b2 Pfizer BioNTech, AZD1222 Vaxzervria, Ad5-nCoV Convidecia, AZD1222 Covishield, Gam-COVID-Vac, BBV152 Covaxin, AZD1222 ChAdOx-1-S, CX-024414 Spikevax, Ad26.COV2-S Janssen, Abdala, Soberana Pl, and Soberana 02 BIOCEN to citizens to ensure widespread coverage of vaccine doses among the Mexican population.
Most individuals from Oaxaca, Mexico were vaccinated against COVID-19 during 2021 and 2022.Therefore, a cross-sectional serology survey study design was performed, involving 150 individuals from Oaxaca who had received the first dose of the COVID-19 vaccine at that time; the effectiveness of the humoral immune responses of individuals after vaccination and variables such as age, gender, comorbidity, body mass index, and type of vaccine that could have affected acquired immunity were assessed using statistic parametric methods.Here, we report findings that will guide further COVID-19 vaccination strategies during the post-pandemic phase of COVID-19 in Southern Mexico and elsewhere.

Study Population, Inclusion Criteria, and Vaccines
From June 2021 through June 2022, a cross-sectional serology survey study was conducted in Oaxaca, Mexico (N-17.06542• , W-96.72365 • ).One hundred and fifty individuals, aged 18 and older, were enrolled; inclusion criteria were that individuals had received at least one type of vaccine distributed by the Ministry of Health during the former COVID-19 vaccination campaign at that time.Participants completed a standardized questionnaire approved by the Ethics Committee of the Instituto Politécnico Nacional-México city (protocol code CBE/006/2020; date of approval 21 December 2020); containing clinical and demographic information, including any history of SARS-CoV-2 infection, proof and/or evidence of the type of vaccine administered, dosage vaccine details, vaccine-related side effects, gender, age, weight, height, and any comorbidity or absence.Various types of the vaccine were available during the study at that time, such as BNT162b2 (Pfizer BioNTech, Mainz, Germany) mRNA-1273 (Moderna, Boston, MA, EE.UU.), ChAdOx1nCoV-19 (As-traZeneca, Oxford, UK), Ad5-nCoV (CanSino, Tianjin, China), CoronaVac (Sinovac, Beijing, China), and Gam-COVID-vac/Sputnik (Moscow, Russia).However, most individuals in this study had received CanSino, AstraZeneca, or Sputnik.

Sample Collection
Venous blood samples were collected from participating individuals at least three months after receiving the first dose of vaccine.The blood sample was allowed to clot and then centrifuged to remove the clot and blood cells.The supernatant (serum) was stored at −80 • C for further analysis.

The Enzyme-Linked Immunosorbent Assay (ELISA)
The determination of IgG antibodies against SARS-CoV-2 was performed following the instructions of the V2G ® kit provided by the manufacturer: Unidad de Desarrollo e Investigación en Bioterapeúticos (UDIBI) of Instituto Politécnico Nacional, México city.The ELISA detects human serum antibodies of the IgG type against SARS-CoV-2 with 99.33% sensitivity and 97.82% specificity [18].Briefly, blood samples (serum) were diluted at a 1:100 ratio and placed into a 96-well plate previously coated and incubated with the RBD antigen of the spike protein of the SARS-CoV-2 virus.Six subsequent washes using a solution of phosphate buffered saline-Tween 20 (PBS-Tween 20) were performed to remove nonbinding proteins from serum sample.The antigen-antibody-RBD complex was revealed using a secondary anti-human IgG-HRP antibody and the excess was eliminated through washing.Colorimetric development was carried out using 3,3 ′ ,5,5 ′ -tetramethylbenzidine (TMB) as substrate.The reaction was stopped with methanesulfonic acid, as indicated in the kit, and the optical density (OD) was measured at 450 nm in an ELISA microplate reader using the SkanIt Software 6.0, ver.6.0.0.44 (Varioskan LUX 1.00.37;Thermo Fisher Scientific, Waltham, MA, USA).
External controls, provided by the UDIBI manufacturer, consisted of one positive and one negative control, which were run within the same ELISA plate.Serum from an individual with COVID-19 (positive on the test) and serum from an individual with no history of COVID-19 disease served as internal controls.The interpretation of ELISA followed UDIBI manufacturer's technical specifications.Briefly, a negative outcome was determined when the optical density (OD) value was ≤0.5, indicating the absence of IgG antibodies associated with SARS-CoV-2 in the sample.Conversely, a positive outcome was determined when the OD value was ≥0.6, indicating the presence of IgG antibodies linked to SARS-CoV-2 in the sample.

Statistical Analysis
Point estimate of SARS-CoV-2 IgG antibody rate in the sample population and adjusted Wald 95% confidence intervals (CIs) surrounding point estimate were determined using the Wald method [19].Antibody rates were expressed as percentages.A Kolmogorov-Smirnov test was performed to corroborate the dispersion of the antibody levels data; however, data were transformed to exponential values (antibody levels) to satisfy the condition of normality.We refer to antibody levels as the result obtained from the post-immunoassay optical density reading, both below and above the established cut-off value.The dependent variable was the response to COVID-19 vaccines measured as antibody levels, whereas the independent variables were gender, age, type of vaccine received, and comorbidity.To evaluate the effect of gender and comorbidities on antibody levels after vaccination, two Student's t tests were performed using proc TTEST, and three ANOVA tests using proc ANOVA were performed to assess the effects of the type of vaccines, age groups, and body mass index (BMI).All statistical analyses were performed with SAS OnDemand for Academics (2021) and graphs constructed with RStudio software (version 4.3.1).

Results
Overall, 150 serum samples were collected in Oaxaca, and a biobank containing serum samples of vaccinated individuals was developed.One hundred thirty-nine out of one hundred fifty (92.6%; 95%-CI = 87-95%) individuals examined were positive for the ELISA.Of the 150 participants, 88 (58.7%) were female and 62 (41.3%) were male.Of the 88 females, 83 (94.3%; 95%-CIs = 51-67%) were ELISA positive whereas of the 62 males, 56 (90.3%; 95%-CIs = 32-48%) were positive (Table 1).Variation in the antibody rate between males and females was noted (p < 0.05).It is acceptable that the rates are different at α 0 ± 0.05 because that of males (90.3%) did not lie between 51% and 67%, the confidence interval estimated for females (Table 2).Moreover, the antibody levels of the two sexes also varied (t = −2.21;p-value = 0.02; Table 3; Figure 1A).In Mexico, at the outset of the vaccination campaign, the Ministry of Health administered the CanSino vaccine; namely, 118 individuals in this study of Oaxaca received this vaccine, of which 107 (90.6%) individuals tested positive and the remaining negative.Additionally, 16 individuals received AstraZeneca and 16 others received SinoVac, Pfizer, Sputnik V, and Moderna.All but the CanSino vaccines induced immune response, as 100% of such individuals generated vaccine antibodies (Table 1).As noted in Figure 1B, individuals vaccinated with CanSino showed a widespread range of OD values (=0.26 to 2.24).In contrast, individuals administered AstraZeneca (range = 0.71 to 1.79) and other vaccines (Sinovac, Pfizer, Sputnik V, and Moderna) had a more reduced one (range = 1.12 to 1.99).However, no variation (F = 0.73; p-value = 0.48; Table 3) was found in the immune responses of individuals to each type of vaccine.
To analyze whether the age (years old) groups of individuals affected the immune responses, five age classifications were constructed.Individuals aged 18-30 years old had the highest percentage of antibody response (100%), followed by the group of 70 years and older (94%).The age group showing the lowest percentage of antibody response was the 31-45 year old group (86%; Table 1).However, no variation was seen in the antibody In Mexico, at the outset of the vaccination campaign, the Ministry of Health administered the CanSino vaccine; namely, 118 individuals in this study of Oaxaca received this vaccine, of which 107 (90.6%) individuals tested positive and the remaining negative.Additionally, 16 individuals received AstraZeneca and 16 others received SinoVac, Pfizer, Sputnik V, and Moderna.All but the CanSino vaccines induced immune response, as 100% of such individuals generated vaccine antibodies (Table 1).As noted in Figure 1B, individuals vaccinated with CanSino showed a widespread range of OD values (=0.26 to 2.24).In contrast, individuals administered AstraZeneca (range = 0.71 to 1.79) and other vaccines (Sinovac, Pfizer, Sputnik V, and Moderna) had a more reduced one (range = 1.12 to 1.99).However, no variation (F = 0.73; p-value = 0.48; Table 3) was found in the immune responses of individuals to each type of vaccine.
To analyze whether the age (years old) groups of individuals affected the immune responses, five age classifications were constructed.Individuals aged 18-30 years old had the highest percentage of antibody response (100%), followed by the group of 70 years and older (94%).The age group showing the lowest percentage of antibody response was the 31-45 year old group (86%; Table 1).However, no variation was seen in the antibody rates among age groups because the rates and confidence intervals overlapped (Table 2); again, no difference (F = 0.98; p-value = 0.44; Table 3; Figure 1C) was noted in the antibody level immune responses of individuals among age groups.

Discussion
The evolution of the COVID-19 vaccination strategy in Mexico during the pandemic phase was somehow effective and under certain control.Here, a cross-sectional serology survey study on the evolution of the vaccination strategy in the population of Oaxaca against COVID-19 was conducted.Noteworthy in this study is that females had higher antibody rates (Table 2) and levels (t = −2.2;p-value = 0.02, Table 3; Figure 1A) of immune response compared to males.Although many variables can affect the development of antibodies, one key variable is gender, as sexual differences predispose males and females to divergent antibody responses to infectious diseases, particularly those of a viral nature [20].Moreover, females generally display higher levels of antibody responses to bacterial and viral vaccines compared to males [21].This can be attributed to the innate and adaptive immune systems of females who typically exhibit faster responses to vaccines.Several factors, including hormonal, behavioral, and genetic ones contribute to this difference [22].The increased immune reactivity in females presents several advantages, such as the potential to administer a lower antigen dose for vaccination against a specific viral strain and the possibility of cross-protection against related viral strains or variants [21].However, only three out of the multiple studies that documented vaccine efficacy have provided disaggregated results based on gender.Notably, none of the three studies reported a significant difference in the antibody response rates of males and females [23].However, in a study conducted with Mexican subjects, gender and differences in the percentage of neutralizing antibodies against the SARS-CoV-2 were found: Females had a higher percentage of antibody levels than males [24], which is in agreement with the present findings.
Most individuals (90.6%) tested in Oaxaca developed antibodies against the first dose of the CanSino vaccine in 2021.The CanSino vaccine is produced using genetic engineering techniques with recombinant virus technology [25].It was documented that in Mexico, 19% of the population examined did not respond to the CanSino vaccine [26].This finding coincides with the present report where the CanSino vaccine elicited a lower percentage of antibody development (10% of individuals did not respond) against the RBD region of the SARS-CoV-2 virus antigen in comparison to that of other vaccines assessed here, where an immune response was induced in all individuals (100%) after vaccination.Furthermore, in clinical trials, the CanSino vaccine was less effective at reducing symptomatic cases of COVID-19 than that of other vaccines [27].Individuals vaccinated with CanSino showed a widespread range of OD values (Figure 1B), either due to a more heterogeneous variable immune response or the higher sample size (n = 118; Table 1) examined in comparison to those from AstraZeneca (n = 16) and the other (Sinovac, Pfizer, Sputnik V, and Moderna) vaccines.Therefore, other variables such as age were also assessed.
The COVID-19 vaccines might exhibit reduced efficacy in older individuals in comparison to younger ones [28].Nevertheless, vaccines are effective and provide substantial protection in older individuals [29], but there is a need for booster doses in older individuals.Older adults and individuals with weakened immune systems often stand to gain from supplementary doses to uphold optimal protection levels [30].We did not find a significant effect of any of the five age groups on antibody rates and levels from the immune response (Tables 2 and 3).Although it has been observed that older adults have poorer immune responses to COVID-19 vaccines than younger ones [31], our results did not seem to support this phenomenon.The logical explanation is as follows: Firstly, the study had a small sample size consisting of 150 individuals examined.Secondly, many other factors such as gender, comorbidity, racial differences, type of vaccine, and duration since vaccination could contribute to the variation in immune responses to vaccination [32].The type of vaccine is what most influences the production of an immune response [33]; however, in our population studied, the majority of the individuals in the sample (n = 118) received the CanSino vaccine.Thus, the effect of comorbidity on antibody rates and levels from the immune response was also assessed.
Some studies reveal lower immunogenicity of COVID-19 vaccines in populations with comorbidities, who are overweight, or are obese compared to healthy subjects.Therefore, it seems that there is an association between comorbidities and lower antibody levels [34].Individuals with comorbidities face an elevated risk of severe COVID-19 disease and associated mortality [35].Reduced antibody levels against SARS-CoV-2 following vaccination of individuals with type 2 diabetes, cancer, end-stage kidney disease, and other immune-compromised conditions in comparison to those of healthy individuals were documented [36].Overweight or obese individuals seem to elicit less of an immune response after COVID-19 vaccination, likely because metabolic dysfunction [35].Here, no variation in the antibody rates and levels from individuals with no or ≥1 comorbidity, who are overweight, or are obese was observed (Table 3), indicating that comorbidity or metabolic dysregulation could not affect the immune response after immunization using different biologics of the Oaxaca population.However, this might also be because of study limitations, as only serum samples from 150 individuals were examined and collected during a specific timeframe (June 2021 through June 2022).Hence, additional surveillance is needed in Mexico to document whether comorbidity or metabolic disorders are paramount in determining vaccine responsiveness [37].In general, vaccine surveillance is needed during the post-pandemic phase and, in particular, to better understand the main factors modulating responsiveness after vaccination.Such studies are currently underway.

Conclusions
The present findings confirmed the utility of our enzyme-linked immunoassay for COVID-19 vaccine surveillance.Further studies are needed to fully understand the vaccination platform's efficacy on immune response in the diverse Mexican populations.The present study extends beyond the conventional evaluations conducted by vaccine companies.Routine surveillance of vaccination campaigns is paramount to fight against the Omicron, Pirola, FLiRT, and other emerging and re-emerging SARS-CoV-2 variants effectively and efficiently.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.Before individual blood sampling, research procedures were explained, and a capsule summary of the research was provided.Written informed consent has been obtained from the individual(s) to publish this paper.
Data Availability Statement: Dataset available on request.

Figure 1 .
Figure 1.Data distribution of IgG RBD antibody levels (transformed to exponential values) to SARS-CoV-2 in 150 individuals of Oaxaca, Mexico: (A) Gender; (B) Type of vaccine; (C) Age group; and (D) Comorbidity.Horizontal lines indicate median values; boxes indicate quartiles 1 and 3; whiskers indicate the 1.5 interquartile range; and black dot circles indicate outliers.

Figure 1 .
Figure 1.Data distribution of IgG RBD antibody levels (transformed to exponential values) to SARS-CoV-2 in 150 individuals of Oaxaca, Mexico: (A) Gender; (B) Type of vaccine; (C) Age group; and (D) Comorbidity.Horizontal lines indicate median values; boxes indicate quartiles 1 and 3; whiskers indicate the 1.5 interquartile range; and black dot circles indicate outliers.

Table 1 .
The number of total samples and number of positive samples for gender, type of vaccine, age group, comorbidity, and body mass index (BMI).
1SinoVac, Pfizer, Sputnik V, and Moderna.2Individuals were classified as those who reported having no comorbidity at all (absence) and those individuals that reported to have at least one comorbidity or more.The comorbidities that individuals reported were chronic illness such as diabetes, hypertension, asthma, chronic heart problems, kidney problems, and/or hypothyroidism.

Table 2 .
The point estimate (antibody rate) of individuals for gender, age group, and body mass index (BMI) and the adjusted Wald 95% confidence intervals (ICs) surrounding point estimate.Only the number of positives (n = 139 for gender and age group and n = 82 for BMI) of the total sample was considered.

Table 3 .
The statistical parameters resulting from two Student's t tests for gender and comorbidity effect on antibody levels and two ANOVA tests for the type of vaccine and age group effect on antibody levels after vaccination.Condition of normality was satisfied after data (n = 150) were transformed to exponential values and Kolmogorov-Smirnov (KS) test was not significant (KS-value = 0.068; p-value = 0.0896).

Table 3 .
The statistical parameters resulting from two Student's t tests for gender and comorbidity effect on antibody levels and two ANOVA tests for the type of vaccine and age group effect on antibody levels after vaccination.Condition of normality was satisfied after data (n = 150) were transformed to exponential values and Kolmogorov-Smirnov (KS) test was not significant (KS-value = 0.068; p-value = 0.0896).