Seroepidemiological assessment of SARS-CoV-2 vaccine responsiveness and associated factors in the vaccinated community of the Casablanca-Settat Region, Morocco

Assessing the prevalence of SARS-CoV-2 IgG positivity through population-based serological surveys is crucial for monitoring COVID-19 vaccination efforts. In this study, we evaluated SARS-CoV-2 IgG positivity within a provincial cohort to understand the magnitude of the humoral response against the SARS-CoV-2 vaccine and to inform evidence-based public health decisions. A community-based cross-sectional seroprevalence study was conducted, involving 10,669 participants who received various vaccines (two doses for BBIBP-CorV/Sinopharm, Covishield vaccine, and Pfizer/BioNTech, and one dose for Johnson & Johnson's Janssen COVID-19 vaccine). The study spanned 16 provinces in the Casablanca-Settat region from February to June 2022, during which comprehensive demographic and comorbidity data were collected. We screened samples for the presence of IgG antibodies using the SARS-CoV-2 IgG II Quant assay, which quantifies antibodies against the receptor-binding domain (RBD) of the spike (S) protein, measured on the Abbott Architect i2000SR. The overall crude seroprevalence was 96% (95% CI: 95.6–96.3%), and after adjustment for assay performance, it was estimated as 96.2% (95% CI: 95.7–96.6). The adjusted overall seroprevalences according to vaccine brands showed no significant difference (96% for BBIBP-CorV/Sinopharm, 97% for ChAdOx1 nCoV-19/Oxford/AstraZeneca, 98.5% for BNT162b2/Pfizer-BioNTech, and 98% for Janssen) (p = 0.099). Participants of older age, female sex, those with a history of previous COVID-19 infection, and those with certain chronic diseases were more likely to be seropositive among ChAdOx1 nCoV-19/Oxford/AstraZeneca and BBIBP-CorV/Sinopharm vaccinee groups. Median RBD antibody concentrations were 2355 AU/mL, 3714 AU/mL, 5838 AU/mL, and 2495 AU/mL, respectively, after two doses of BBIBP-CorV/Sinopharm, ChAdOx1 nCoV-19/Oxford/AstraZeneca, BNT162b2/Pfizer-BioNTech, and after one dose of Janssen (p < 0.0001). Furthermore, we observed that participants vaccinated with ChAdOx1 nCoV-19/Oxford/AstraZeneca and BBIBP-CorV/Sinopharm with comorbid chronic diseases exhibited a more pronounced response to vaccination compared to those without comorbidities. In contrast, no significant differences were observed among Pfizer-vaccinated participants (p > 0.05). In conclusion, our serosurvey findings indicate that all four investigated vaccines provide a robust humoral immune response in the majority of participants (more than 96% of participants had antibodies against SARS-CoV-2). The BNT162b2 vaccine was found to be effective in eliciting a strong humoral response compared to the other three vaccines. However, challenges still remain in examining the dynamics and durability of immunoprotection in the Moroccan context.


Study design, participants and settings
This cross-sectional seroepidemiological study encompassed 10,669 individuals aged 14 years and above.Conducted in 16  Within each province, a purposive selection was made of public primary health care centers.In each health center, a local leader (a physician) and an investigator (nurse) were assigned to conduct the survey.Participants included consenting individuals visiting the public primary health care centers or blood donors at the transfusion center during the survey period.
Each subject was informed about the study before being sampled and comprehensive demographic and comorbidities data were completed.In accordance with general data protection rules, subject details were kept confidential and after sample collection, full names were removed and were replaced with participant codes.The study protocol was in accordance with the Helsinki declaration and received an approval from the ethics committee of Ibn Rochd hospital in Casablanca (N°01/22) and written informed consent was obtained from each participant.
A staff member conducted face-to-face interviews with consenting individuals using a well-designed questionnaire, which was then completed on a computer.Each participant assigned a unique eleven-digit identification number.The questionnaire aimed to gather socio-demographic information and potential risk factors for SARS-CoV-2 infection.Variables encompassed self-reported sociodemographic data, along with conditions such as hypertension, diabetes, respiratory disease, smoking, alcohol intake, drug addiction, cancer, cardiovascular disease, chronic renal failure, neurological disease, mental health conditions, systemic disease, infectious syndrome, metabolic syndrome, history of prior COVID- 19, and details about COVID-19 vaccination types.
The data collected through the questionnaire were subsequently integrated with the results of laboratory antibody tests using Microsoft Excel 2021 software.
Following the questionnaire completion, a nurse utilized an aseptic procedure to collect 4 ml of EDTA blood samples from consenting participants.The collected samples were promptly transferred to the COVID-19 serology laboratory at the Pasteur Institute of Morocco.Upon arrival at the laboratory, the samples underwent centrifugation at 2500 rpm for 15 min.
The exclusion criterion encompassed any participant experiencing a decline in willingness post-consent or reporting contraindications to venepuncture.

Serological assay
Serological tests aimed at assessing immune status and monitoring antibody response in individuals who received the COVID-19 vaccine were conducted at the Viral Hepatitis Laboratory of the Pasteur Institute of Morocco.Given that the receptor-binding domain (RBD) serves as the principal immunodominant determinant of the trimeric surface spike glycoprotein, immune responses against RBD are considered the most reliable indicators of both past infection and vaccine-induced immunity 10,11 .We employed the SARS-CoV-2 IgG II Quant assay, a chemiluminescent microparticle immunoassay (CMIA), for both qualitative and quantitative determination of IgG antibodies specific to the receptor-binding domain (RBD) of the S1 subunit of the spike protein of SARS-CoV-2 in human plasma.This assay was conducted using the Abbott Architect i2000SR instrument manufactured by Abbott Laboratories, based in Abbott Park, Illinois.
Results were interpreted according to the manufacturer's criteria: negative if the cutoff value was < 50 AU/ml and positive if the cutoff value was ≥ 50 AU/ml.The assay is characterized by a documented sensitivity of 91.6% and specificity of 99.4%, which is deemed acceptable for conducting SARS-CoV-2 seroprevalence surveys, as previously reported 12 .The analytical measurement interval for the SARS-CoV-2 IgG II Quant assay is specified as ranging from 9.9 to 40,000 AU/ml.

Statistical analysis
Descriptive statistics were presented in terms of frequency and percentage for categorical variables, while continuous variables were expressed as median and interquartile range (IQR).Both crude and adjusted seroprevalences were reported as proportions with corresponding 95% confidence intervals (CI).The sensitivity and specificity values, namely 91.6% and 99.4% respectively, were calculated based on a prior assessment described in the serological assay 12 .Bayes rule applied to estimate the adjusted seroprevalence (π) as a function of crude seroprevalence (p), sensitivity (r), and specificity (s) according to the formula: π = [p − (1 − s)/r − (1 − s)] as reported previously 13,14 .
The chi-square test was employed to examine associations among categorical variables.Univariate and multivariate logistic regression models were employed to estimate odds ratios (ORs), considering IgG antibody status as the outcome variable.Independent variables included sex (male/female), age, hypertension, diabetes, respiratory disease, cancer, cardiovascular diseases, chronic kidney disease, physical distancing, mask-wearing behavior, and past history of confirmed COVID-19 disease (present/absent).Variables with a p-value < 0.05 in the univariate analysis were subsequently included in the multivariate analysis.
Mann-Whitney U Test and Kruskal-Wallis test were employed for comparing nonparametric continuous variables across different groups.Statistical analyses were carried out using R software for Windows and Graph-Pad PRISM version 6e (GraphPad Software, San Diego, CA, USA).A p-value less than 0.05 was considered statistically significant, and two-tailed comparisons were performed.

Ethics approval
This study was conducted in accordance with the Declaration of Helsinki.The study was approved by the ethics committee of Ibn Rochd hospital in Casablanca (N°01/22).

Basic characteristics of the study participants and overall seroprevalence among the vaccinated community
The distribution of participants based on socio-demographic data and potential risk factors for SARS-CoV-2 infection is presented in Table 1.Out of the total, 10,669 blood samples were assessed (12% of participants refused to participate in the serological survey), comprising 7,380 women and 3,475 men, with a median age of 44 years (ranging from 14 to 102 years) (Table 1).

Seroprevalence in the BBIBP-CorV (Sinopharm)-vaccinated community
We determined an adjusted seroprevalence rate of 96% (95% CI: 95.5% to 96.4%) among participants who received the BBIBP-CorV vaccine (Table 2).Logistic regression analysis revealed several variables associated with a higher risk of detecting anti-SRAS-CoV-2 antibodies (Table 3).Specifically, a higher prevalence of antibody positivity was linked to age.Additionally, the odds of being antibody-positive are 0.59 times lower for male participants than for females.A higher prevalence of antibody positivity was associated with hypertension, mask-wearing, and PCR-confirmed COVID-19 disease (Table 3).Furthermore, the analysis by chronic kidney disease revealed that participants with kidney disease had a significantly lower probability of being seropositive than participants without kidney disease (Table 3).www.nature.com/scientificreports/

Seroprevalence in the Covishield/AstraZeneca vaccinated community
For participants vaccinated with ChAdOx1-nCov-19, the adjusted seroprevalence was 97% (95% CI: 96-97.9)(Table 2).Logistic regression analysis assessing the association between SARS-CoV-2 seropositivity and demographic characteristics and chronic diseases between February and June 2022 is presented in Table 4.In univariable analysis, participants in the age groups of more than 14-19 years and those with a PCR-confirmed diagnosis (OR = 2.65; 95% CI: 1.05-6.68;p = 0.038) tended to be significantly more likely to be seropositive for anti-SARS-CoV-2 antibodies (Table 4).In contrast, in the multivariable analysis, only participants with a PCR-confirmed diagnosis were more likely to be seropositive for anti-RBD (OR = 2.69; 95% CI: 1.17-7.78;p = 0.036) (Table 4).

Seroprevalence in the BNT162b2/Pfizer-BioNTech vaccination community
For participants vaccinated with BNT162b2, the overall adjusted prevalence of anti-SRAS-CoV-2 antibodies was 98.5% (95% CI: 95.0-100.0)(Table 2).Logistic regression analysis showed that participants with hypertension who received BNT162b2 were more likely to be seronegative compared to female participants and those without hypertension (Table 5).www.nature.com/scientificreports/

Seroprevalence in the JCovden-vaccinated community
The adjusted seroprevalence among participants vaccinated with the Janssen/Johnson & Johnson's COVID-19 vaccine was 98% (95% CI: 85.2-100.0).Owing to the small sample size (n = 42), logistic regression analysis of the association between SARS-CoV-2 seropositivity and demographic characteristics and comorbid conditions was not conducted.
In fully vaccinated participants with BNT162b2/Pfizer, stratification by demographics, comorbidities, and history of COVID-19 showed no significant differences in anti-RBD antibody concentrations (Fig. 4A-D,F).In contrast, an elevated antibody level was observed in participants who reported wearing a mask (median = 6753 AU/mL) compared with those who did not report wearing a mask (median = 4909 AU/mL) (p = 0.046) (Fig. 4E).
Stratification of participants vaccinated with JCovden/Johnson & Johnson's COVID-19 vaccine showed no association by gender (p = 0.456), and no correlation between age and anti-RBD antibody levels was noted (p = 0.362).However, stratification of participants by comorbidities was not performed due to the limited sample size (n = 42).

Discussion
To foster herd immunity within the community and mitigate the transmission of the virus, Morocco has initiated a mass vaccination program against SARS-CoV-2.The COVID-19 vaccines approved for use in Morocco have demonstrated positive safety and efficacy profiles 4,15 .The vaccines administered in our country have proven effective in preventing deaths and hospitalizations related to SARS-CoV-2 infection 4 .Certainly, understanding the humoral response after SARS-CoV-2 vaccination plays a crucial role in predicting protection against reinfection.This knowledge is instrumental in devising strategies to limit the pandemic and provides valuable insights for public health decision-makers, enabling them to plan more effective and targeted vaccination programs tailored to different populations 16,17 .In this study, the SARS-CoV-2 IgG II Quant assay was employed to quantify IgG antibodies to the receptor-binding domain (RBD).This assay demonstrated a strong correlation with SARS-CoV-2 protein S-specific neutralizing antibodies.These neutralizing antibodies are considered crucial for protection against the disease, reinforcing the reliability of the chosen assay in assessing the humoral response to SARS-CoV-2 [18][19][20] .Among the study participants, a notable proportion exhibited IgG positive antibodies, with a seroprevalence rate of 96%.This figure surpasses the reported seroprevalence in Moroccan healthcare workers (86.59%, using the Abbott Architect test) documented before the surge of the Omicron variant 21 .The observed increase in the antibody rate was primarily attributed to hybrid immunity, defined as the combination of two doses of a COVID-19 vaccine and at least one SARS-CoV-2 infection either before or after the initiation of vaccination [21][22][23] .Interestingly, the rate of anti-RBD antibody seroconversion (98.5%) was notably higher with the BNT162b2/Pfizer vaccine compared to other vaccine brands.Remarkably, these findings align with those reported in prior studies 24 .Median anti-RBD antibody concentrations were notably high in individuals vaccinated with mRNA (BNT162b2/Pfizer-BioNTech), followed by vector-type vaccines (ChAdOx1 nCoV-19/ Oxford/AstraZeneca and JCovden/Johnson & Johnson's COVID-19 vaccine), and an inactivated whole-virion vaccine (BBIBP-CorV/Sinopharm).These trends align with findings reported in previous studies [24][25][26][27] .Among participants in this study, a confirmed history of COVID-19 was found to be strongly associated with anti-RBD antibody titers across different vaccine brands.This association has also been documented by other research groups 22,26,28,29 .
We estimated an adjusted seroprevalence rate among participants vaccinated with BBIBP-CorV, the most widely used vaccine in Morocco, of 96%, which is lower than the seroconversion rate (> 99%) reported in the interim results of phase 3 vaccine trials 30 , yet, it is higher than the seroprevalence reported in the Kyrgyz population (91.6%) vaccinated with Sinopharm 31 .In our investigation, seropositivity rates and antibody concentrations www.nature.com/scientificreports/studies 32,33 , this discrepancy challenges the conventional notion that the post-vaccination humoral response is likely to be low or non-existent in older participants.Furthermore, we observed that participants vaccinated with BBIBP-CorV/Sinopharm with comorbid chronic diseases had a higher seroconversion rate, and comorbidities did not adversely affect their immune response to COVID-19 vaccines.The most significant finding of this study is the association between comorbidities and a higher antibody response to the Sinopharm vaccine.However, a previous study showed that hypertension, diabetes, and combined diseases did not negatively affect the immune response 34 .Due to a considerable number of participants having a history of exposure to SARS-CoV-2 infection, and considering that individuals with comorbid chronic diseases are more susceptible to COVID-19 and are more likely to be hospitalized for further treatment, the probability of developing severe disease following infection is heightened.This elevated risk is reflected in higher hospitalization and mortality rates 35 .Therefore, the heightened humoral response could be attributed to hybrid immunity, aligning with previous research indicating that advanced age is associated with a higher antibody response, potentially linked to multiple chemokine-induced hyperinflammation in COVID-19 patients 36 .
In those vaccinated with the Covishield/AstraZeneca vaccine, the adjusted seroprevalence was 97%, which is lower than that reported in the previous study and phase 2/3 trial of the ChAdOx1 nCoV-19 vaccine 37,38 , but similar to another report 39 .We observed a significant influence of age, but not gender, on seropositivity, and IgG levels increased with age, consistent with a previous study using the Covishield vaccine 40 .
A variety of comorbidities (hypertension, diabetes, heart, kidney, respiratory diseases, etc.) did not affect the seroconversion rate or have a negative effect on the humoral response after two doses of the Covishield vaccine.These data seem to be consistent with the results of previous serological surveys 15,40 .In contrast, a previous study showed no significant difference in the humoral response between participants with and without comorbidities 37 .On the other hand, IgG concentrations are lower in those with comorbidities than in those without 40 .
CoV-2.Previous data have shown that male gender and the presence of comorbidities predicted seronegativity for SARS-CoV-2 [46][47][48] .Whereas, in participants who received the BNT162b2/Pfizer vaccine, no correlation was observed between IgG antibody concentration and participant age and sex.These results appear to be in agreement with previous studies 29,46 .
Among the limitations of our study, we can highlight that our sample size was relatively small in the study groups vaccinated with BNT162b2/Pfizer and JCovden vaccines.Additionally, for COVID-19 convalescents, the lack of disease severity classification (from asymptomatic to critical) is noteworthy.Although the humoral response is weak, a robust post-infection cellular response is known to provide long-term protection against SARS-CoV-2 infection 49 .Moreover, it is generally accepted that higher antibody levels and neutralizing antibodies specific to the SARS-CoV-2 spike protein are likely to be protective against the disease.Therefore, it would be of interest to assess the cellular immune response and neutralizing antibodies.Additionally, data collection was conducted through a questionnaire, and there is no means to verify the accuracy of the reported chronic diseases, and their prevalence in the general population; this poses a potential limitation in our investigation.Lastly, the 12-13-year-old age group was not included in our study.Future research endeavors will address this age range in subsequent studies.

Conclusion
To our knowledge, this study is the largest investigation comparing the humoral response of four types of vaccines deployed in Morocco.The data generated by this cross-sectional serological survey could help inform and improve vaccination strategies.This is the first study in the region to assess not only post-vaccination seroprevalence but also the humoral response generated by the four main types of vaccines implemented by the Ministry of Health in Morocco to produce IgG antibodies against SARS-CoV-2.We suggest that further studies on IgG seroprevalence in individuals who received 3 and 4 doses, as well as monitoring the dynamics of the humoral response prospectively, be conducted in our country.

Figure 1 .
Figure 1.Anti-SARS-CoV-2 antibody levels among four vaccine brands.Data are presented as median and interquartile range for IgG antibody.Mann-Whitney and Kruskal-Wallis tests were used.

Table 2 .
Composition of the cohort with respect to vaccine type.*Adjusted seroprevalence based on the assay sensitivity and sepecifity.

Table 4 .
Univariate and multivariate analyses for the association between risk factors and SARS-CoV-2 seropositivity, based on a logistic regression model among 1,817 participants receiving two doses of ChAdOx1nCoV-19 vaccine from 16 provinces of the region of Casablanca-Settat, Morocco during February to June 2022.*Data are missing.

Table 5 .
Univariate and multivariate analyses for the association between risk factors and SARS-CoV-2 seropositivity, based on a logistic regression model among 208 participants receiving two doses of BNT162b2 vaccine from 16 provinces of the region of Casablanca-Settat, Morocco during February to June 2022.*Data are missing.