F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.126577.2 Study Protocol Articles The dynamics and determinants of specific systemic and mucosal antibody responses to SARS-CoV-2 in three adult cohorts in the Ecuadorian Andes: a study protocol

[version 2; peer review: 1 approved, 2 approved with reservations]

 Leon-Rojas Jose E. Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-8130-6460 a 1 2 Veloz Tatiana Investigation Writing – Review & Editing 3 Teran Jair Investigation Writing – Review & Editing 3 Perez Monica Investigation Writing – Review & Editing 3 Arias-Erazo Fernanda Formal Analysis Investigation Writing – Review & Editing 3 4 Villacis Lizet Investigation Writing – Review & Editing 5 Velez Jorge Investigation Resources Writing – Review & Editing 6 Recalde Ricardo Funding Acquisition Investigation Methodology Project Administration Resources Writing – Review & Editing https://orcid.org/0000-0001-5896-605X 2 5 Jiménez Patricia Funding Acquisition Investigation Methodology Project Administration Resources Writing – Review & Editing https://orcid.org/0000-0002-0666-177X 4 7 Martin Miguel Conceptualization Data Curation Formal Analysis Investigation Methodology Software Supervision Validation Visualization Writing – Review & Editing https://orcid.org/0000-0003-2053-1039 1 2 3 Chis Ster Irina Conceptualization Data Curation Formal Analysis Investigation Methodology Software Supervision Validation Writing – Review & Editing https://orcid.org/0000-0003-2637-1259 8 Cooper Philip Conceptualization Formal Analysis Funding Acquisition Investigation Methodology Resources Validation Writing – Review & Editing 3 8 Romero Natalia Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Methodology Project Administration Resources Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-2891-3496 2 3 Departamento de Pediatría, Obstetricia y Ginecología y Medicina Preventiva, Universitat Autonoma de Barcelona (UAB), Barcelona, Spain Red Grups de Recerca d’Amèrica i Àfrica Llatines (GRAAL),, Quito, Ecuador Medical School, Universidad Internacional del Ecuador, Quito, Ecuador Departamento de Ciencias de la Vida y de la Agricultura, Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador Medical School, Universidad Técnica de Ambato, Ambato, Ecuador Hospital de Especialidades Eugenio Espejo, Quito, Ecuador Grupo de Investigación en Sanidad Animal y Humana (GISAH), Quito, Ecuador St George's University of London, London, UK JoseEduardo.Leon@autonoma.cat

No competing interests were disclosed.

 23 2 2024 2022 11 1392 21 2 2024 Copyright: © 2024 Leon-Rojas JE et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction

There are limited longitudinal data on the systemic and mucosal antibody responses to SARS-CoV-2 from Latin America, a region severely affected by COVID-19, and where vaccine strategies have been implemented during the evolving pandemic.

 Objective

To evaluate determinants of seroprevalence and changes in levels of anti-SARS-CoV-2 antibodies longitudinally in adults with different levels of exposure to SARS-CoV-2 (defined a priori as low, medium, and high based on presumed occupational risk), in two Andean cities in Ecuador.

 Methods

Longitudinal cohort study of 1,000 adults aged 18 years and older with questionnaire data and sample collection done at 0, 3, 6, and 12 months during the period 2020-2023. Observations collected included WHO-ISARIC questionnaire and peripheral blood and saliva samples for measurement of IgG and IgA antibodies, respectively. Planned analyses are tailored to the longitudinal nature of the outcomes defined by participants’ antibody levels and aim at estimating their average trends with time since infection in each of the occupational groups, adjusted for demographics and calendar-time levels of SARS-CoV-2 infection in the general population. The latter reflect the impact of the national control measures such as vaccinations and movement restrictions.

 Importance

Understanding the duration and the dynamics of waning immunity to SARS-CoV-2, in the context of exposures to emerging virus variants and immunization, will inform the implementation of targeted public health strategies in the Latin American region.

 Ethics and Dissemination

This study will observe the bioethical principles of the Declaration of Helsinki. Informed written consent will be obtained. Samples from participants will be stored for up to three years after which they will be destroyed. The study protocol was approved by the Ecuadorian Ministry of Public Health Ethics Committee for COVID-19 Research. Antibody results will be provided to participants and participating institutions and to the national health authorities.

 COVID-19 SARS-CoV-2 Seroprevalence Antibody levels risk factors Ecuador Universidad Internacional del Ecuador UIDE-MAT-INP-MEM-21-0289 UIDE-DGIP-MAT-MEM-21-0288 UIDE-DGIP-MAT-MEM-20-0168 Corporación Ecuatoriana para el Desarrollo de la Investigación y la Academia CEDIA-CEPRAXVI-2022-17ANTICUERPOSIGGIGA CEDIA- CEPRA XVI-2022-17 ANTICUERPOS IGG IGA Grant given to NR, JLR, PJ and RR. UIDE-MAT-INP-MEM-21-0289 Grant given to NR, PC, MM, JLR. UIDE-DGIP-MAT-MEM-21-0288 Grant given to JLR, NR, PC, MM. UIDE-DGIP-MAT-MEM-20-0168 Grant given to NR, PC, MM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

The revised version contains clearer information regarding the laboratory tests performed for SARS-CoV-2 as well as more specific text on the objectives, introduction and limitations of the study. All of these changes were done in response to the reviewers suggestions.

 Introduction

The pandemic caused by the new coronavirus, SARS-CoV-2, the etiological agent of COVID-19, has caused 612 million confirmed cases and approximately 6.5 million deaths worldwide since December 2019 when the virus first emerged ( Johns Hopkins Coronavirus Resource Center). In Ecuador, the first confirmed case of COVID-19 was reported on 29 th February 2020 and as of 19 th September 2022, one million confirmed cases have been reported with 35,885 deaths. The highest burden of infection is among those aged 20 to 55 years age group, while death rates are greatest among those aged over 65 years ( Daily Report of the Ecuadorian Ministry of Public Health).

The presence of specific IgG antibodies to SARS-COV-2 indicates past exposure to SARS-COV-2 and vaccination, given that most serological assays using the spike protein cannot distinguish between these two forms of exposure. Additionally, since the virus invades the body through the mucosal surfaces, the role of mucosal immune responses measured by specific IgA, has been neglected compared to the measurement of systemic IgG responses. 1 Timely seroprevalence studies in the population are useful in monitoring rates of exposure to natural infections (prior to vaccination programs), and vaccine coverage and immunogenicity following the implementation of vaccination programs. 2 Measuring the waning immunity across different groups in the population will add to our understanding of the epidemiology of the infection and will help identify population groups with increased infection susceptibility. 1 – 3

Numerous seroprevalence studies have been conducted in the Latin American region, both population-based studies and studies focusing on specific populations (healthcare (HCW) or first-line workers, prison workers, indigenous populations, elderly populations, etc). 4 – 15 The reported seroprevalence is highly heterogenous between studies (10,4% – 68,1 %), varying by study population, characteristics of the COVID-19 epidemic at the time of sampling, and differences in sociodemographic factors. 4 – 15 Factors associated with increasing risk of seropositivity include ethnicity (indigenous or Afro-descent), presence of COVID-19 symptoms, past history of COVID-19 or close contacts, lower educational level, and working night shifts (for HCW) or in a prison. 4 – 15

Among these studies, a cross-sectional study in 2020 of 3,124 children aged 5 to 17 years in 10 Colombian cities reported highly variable seroprevalence ranging from 25.0% in Medellin to 63.9% in Guapi, the latter a coastal city with a high proportion of Afro-Colombians with the poorest living conditions. 10 Further, a cross-sectional study in 2021 from Brazil of six indigenous groups in the Amazon region showed a high average seroprevalence for anti-SARS-CoV-2 IgG (68.1%). 11 Finally, a systematic review analysing 15 seroprevalence studies from Latin America and the Caribbean reported a highly variable seroprevalence between indigenous populations ranging 4.2% to 81.7%. 13

There are nine published studies estimating seroprevalence rates for IgM/IgG antibodies to SARS-CoV-2 from different regions of Ecuador ( Table 1) covering the period from April 2020 to April 2021 with seroprevalence estimates ranging 7.7% to 64%. 16 – 24 Six of these studies are derived from samples nested within the same observational adult cohort from Atahualpa, a coastal community in Santa Elena Province and it is uncertain the degree of overlap between samples. 16 – 21 Two of four longitudinal studies using the Atahualpa cohort with follow-up of 1 16 and 12 18 months, estimated seroincidence (i.e., incidence rate ratio for seroconversion) of 7.4 16 to 9.9 18 per 100 person years, respectively.

Summary of seroprevalence estimates for IgM/IgG antibodies to SARS-CoV-2 from Ecuador, ordered by sampling date.
Author (Year) Location Design Date of Sampling Sample size Age (mean yrs) Test Seroprevalence
Del Brutto (2021) 21 Atahualpa, Santa Elena Province, coastal rural Longitudinal April 2020 - May 2021 277 70.6 IgM/IgG Rapid Test 63.2% (95% CI 57.4-68.6)
Del Brutto (2020) 17 Atahualpa, Santa Elena Province, coastal rural Cross-Sectional May 2020 673 59.2 IgM/IgG Rapid Test 45%
Del Brutto (2020) 20 Atahualpa, Santa Elena Province, coastal rural Cross-Sectional May 2020 319 70.5 IgM/IgG Rapid Test 44%
Del Brutto (2020) 16 Atahualpa, Santa Elena Province, coastal rural Longitudinal May-June 2020 362 59.9 IgM/IgG Rapid Test 7.7% IRR 7.4/100 person months (95% CI 4.7-10.2)
Del Brutto (2021) 19 Atahualpa, Santa Elena Province, coastal rural Longitudinal May 2020 – April2021 519 61.8 IgM/IgG Rapid Test 63%
Del Brutto (2021) 18 Atahualpa, Santa Elena Province, coastal rural Longitudinal May 2020 - April 2021 673 59.2 IgM/IgG Rapid Test 64% IRR 9.9/100 person months (95% CI 8.9-10.8)
Acurio-Páez (2021) 22 Cuenca, Azuay Province, Andes, rural/urban Cross-Sectional Aug -Nov 2020 2,457 39.0 IgM/IgG Rapid Test 13.2% (95% CI 12.0-14.6%)
Zambrano (2021) 23 Babahoyo, Guayas Province, coastal urban Cross-Sectional Sept 2020 - Jan 2021 100 pregnant women 24.8 IgM/IgG ELISA 32%
Vallejo-Janeta (2022) 24 5 coastal districts in Esmeraldas, Province Cross-Sectional Oct 2020 1250 45.3 IgM/IgG Rapid Test 11.7% (95% CI: 10.0-13.6);

IRR - incidence rate ratio for seroconversion; yrs - years; 95% CI - 95% confidence intervals.

In most regions, vaccination has been a major component of national mitigation strategies for COVID-19 resulting in marked reductions in hospitalizations and deaths worldwide. 25 In Ecuador, vaccination was initiated on 21 st January 2021 as part of the national vaccination strategy “Plan Vacunarse”, consisting of four phases: Phase 0, a pilot phase, started in January 2021 and targeted first line workers and very high-risk individuals; Phase 1 started in March 2021 and included individuals with risk of severe disease such as the elderly; Phase 2 started in June 2021 and included the adult populations living in provinces with a high incidence of COVID-19; and Phase 3 started in September 2021 including adult populations in the other provinces. 26 Booster doses (third and fourth doses) started in October 2021 and April 2022, respectively ( Vaccination Report Ministry of Public Health). As of May 2022, 83% of the population had received a complete 2-dose vaccination schedule (with one of CoronaVac, Pfizer-BioNTech, AstraZeneca, and Cansino), and 35.6 % has received at least one booster shot ( Vaccination Report Ministry of Public Health). Figure 1 shows confirmed cumulative SARS-COV-2 infections and monthly incidence per 1000 population over the period February 2020 through May 2022.

Confirmed cumulative SARS-COV-2 infections and monthly incidence per 1000 population over the period February 2020 to May 2022.

Legend: Arrows indicate the initiation of different phases in the national vaccination programme. Data from: https://www.salud.gob.ec/coronavirus-covid19-ecuador/.

Here, we present the protocol for a longitudinal study of seroprevalence and changes in systemic (plasma IgG) and mucosal (salivary IgA) antibodies being done in three adult cohorts of different infection risk justified by presumed different infection patterns, in two Andean cities in Ecuador, a low-middle income country. In addition, we will explore the effects of natural infections and vaccinations, as well as other relevant determinants (e.g. age, sex, and occupation), on seroprevalence and time trends since infection on antibody levels.

 Protocol General objective

To estimate changes in and determinants of seroprevalence and levels of anti-SARS-CoV-2 antibodies over 12 months of follow-up in adults aged 18 years and older, with varying levels of exposure (defined a priori as low, medium, and high based on presumed occupational risk) and associated factors, in two Ecuadorian Andean cities.

 Specific objectives

Identify risk factors (including cohort membership) associated with baseline seroprevalence and IgG antibody levels in the three cohorts (pre-vaccination)

Evaluate changes in seroprevalence and IgG antibody levels over at least 12 months of follow-up.

Identify effects of determinants on longitudinal changes in specific IgG antibody levels over at least 12 months of follow-up. Potential determinants will include cohort membership, sociodemographic factors, vaccination (including type of vaccine administered), number of vaccine doses, natural (past exposures) infections, and their timing.

Estimate the pattern of decline in anti-SARS-CoV-2 IgG antibodies among seropositives and impact of vaccinations and other relevant determinants on this decline.

To estimate the development and longevity of the mucosal immune response through the measurement of levels of anti-SARS-CoV-2 IgA in saliva and the impact of relevant determinants on changes in antibody levels.

Methods

Study design

Prospective longitudinal cohort ( Figure 2).

Recruitment and follow-up of 3 cohorts by calendar time.

Legend: Syringes show introduction of COVID-19 vaccine doses, red drops show sample collections, and dotted red drops show timings of future sample collections.

 Definition of the study population and eligibility criteria

Three cohorts from two Ecuadorian Andean cities, based on assumed risk of occupational exposure to SARS-CoV-2, are being recruited in two Andean cities, Quito, and Ambato. These three cohorts are presumed to differ in the probability of exposure to SARS-CoV-2. Eligibility criteria for entry into the 3 cohorts are provided in Table 2.

High Risk (HR): Defined as being of high exposure risk through an occupational setting of health care workers and support personnel working in the emergency service, ICUs and hospitalization areas at a major public hospital (Hospital Eugenio Espejo) in Quito, at altitude of 2,800 m in northern Andean region, with 2.9 million inhabitants.

Medium Risk (MR): Population whose occupation puts them at a considerable risk of contracting SARS-CoV-2 (merchants, farmers or those that do not self-isolate due to the nature of their work). The population selected was Huachi Grande parish in the city of Ambato, at altitude of 2,600 m, in central Andean region, with 500,000 inhabitants.

Low Risk (LR): Population with a presumed low risk of SARS-CoV-2 infection due to the characteristics of their occupation (staff and students at a private university, with the means to self-isolate) and living within the metropolitan district of Quito. This cohort is composed of teachers, administrative staff, and the student community of Universidad Internacional del Ecuador (UIDE), Quito.

Eligibility criteria for three adult cohorts.
Characteristic Cohort 1 Cohort 2 Cohort 3
Level of risk at recruitment/Setting/study population High Risk: Health care workers in public hospital Medium risk: merchants, farmers, and highly mobile workers Low risk: staff and students at private university
Inclusion criteria

Adults aged 18 years or older

Healthcare workers

Hospital workers

Adults aged 18 years or older

Inhabitant of Huachi Grande

Adults aged 18 years or older
Exclusion criteria

Age <18 years

Pregnancy or breastfeeding

Religious beliefs that prevent participation

Unable to provide informed consent

Data and sample collection

Questionnaires

Data are being collected digitally by trained interviewers using a standardised tool developed specifically for the COVID-19 pandemic ( ISARIC-WHO Global COVID-19 Clinical Platform “Rapid core case report form (CRF)” questionnaire, subsections 1b, 1f, and 3a) at baseline (month 0) and every three months either via telephone or face-to-face interviews. This tool collects data on socio-demographics, history of signs and symptoms related to COVID-19, and previous diagnoses and treatments. The main determinants that will be considered during analysis will be age, sex, ethnicity, history of COVID-19, history of PCR testing, symptoms, time since diagnosis, time since last vaccination, type of vaccine, and number of doses.

 Clinical samples

Saliva and blood are being collected at baseline and 3-monthly intervals (0, 3, 6, and 12 months). Blood samples are being collected into 5 ml Vacutainer tubes with EDTA as anticoagulant (Vacutainer, BD Biosciences), and plasma separated by centrifugation and stored in 0.5 mL aliquots at -20 °C. Saliva sample (0.5-1 mL) are being collected directly into 2 ml microtubes after asking the participant to think of food, centrifuged to remove residual food debris, and stored in 0.5 mL aliquots at -20 °C.

 Measurement of anti-SARS-CoV-2 IgG and IgA antibodies

Presence and levels of IgG antibodies directed against the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein will be measured in stored plasma using an ELISA essay adapted from Guevara et al., 2021. 27 Specific IgA antibodies against SARS-CoV-2 RBD will be measured in saliva samples using an ELISA-based assay modified from Costantini et al., 2022. 28 Specific IgG and IgA quantitation will use standard curves of serum pools of positive samples calibrated against an WHO international standard (National Institute for Biological Standards and Control, Potters Bar, United Kingdom). Flat bottom 96-well microtiter plates (Nunc Maxisorp™, Thermo Fisher Scientific, USA) were coated overnight at room temperature with recombinant RBD (50 ng/well) in phosphate-buffered saline solution (PBS pH 7.2). Wells content was discarded and washed with PBS containing 0.1% Tween-20 (PBST) and wells blocked with 100 μL of blocking buffer (PBS-0.1% Tween 20 and 4% bovine serum albumin) to each well and incubated for 30 minutes at 37 °C. After blocking, the plates were washed and 100 μL of serum samples diluted with 5% non-fat milk in PBST was added to each well for reaction at 37 °C for 1 hour. Dilutions from 1:200 to 1:1000 were used to capture a broad range of antibody levels and achieve optimal measurement of antibody levels. After washing, 100 μL of horseradish peroxidase (HRP)-labelled goat anti-human IgG (Invitrogen, USA) antibody diluted 8000-fold with 5% non-fat milk in PBST was added to each well and incubated at 37 °C for 30 minutes. Post-wash, 100 μL of SIGMAFAST o-phenylenediamine dihydrochloride substrate solution (Sigma-Aldrich, USA) was added to each well for reaction at room temperature (RT) for 10 minutes in the dark. The reaction was stopped by the addition of 100 μL of 3M HCl. Optical density (OD) at 490 nm was measured.

To assay saliva samples, the same procedure as mentioned above was followed, with the exception that the saliva samples were centrifuged at 14,000 rpm for 8 minutes at 4 °C and supernatants were diluted 1:1 to 1:4. As secondary antibody, 50 μl of Goat Anti-Human IgA alpha chain (HRP) (Abcam, United Kingdom) antibodies were added at dilution of 1:2000 with 5% non-fat milk in PBST and incubated for 30 minutes at 37 °C. The development procedure was the same as for IgG.

 Follow up

The initial sample was collected before vaccination in all cohorts; after baseline evaluation subsequent sampling was done as follows:

at 3 months after baseline if the participant had not been vaccinated.

at 30-45 days after receiving the two-dose vaccine schedule or boosters’ doses.

Follow-up was done on site (university, hospital, or parish administrative building) or through home visits at the sampling follow-up times (0, 3, 6, and 12 months depending on vaccine dose timings). Subjects were contacted also by phone at monthly intervals to collect data on new infections and vaccinations and to provide antibody results, as well as address any doubts or concerns about the study. All evaluations were done by a dedicated and trained study team.

 Statistical considerations

At the time of study planning, there were no reliable longitudinal data to estimate minimum sample size needed to understand the dynamics of waning immunity in different risk populations. Given the rapid dynamics of unfolding events, convenience sampling will be done in each of the three occupational risk cohorts with the aim of recruiting a total of 1,000 adults. Our data are exploratory in nature and the estimates will serve as input for planning future prospective comparative studies.

Cross sectional variables will be summarized according to their nature, i.e. means, standard deviations, medians, inter-quartiles, ranges and frequencies and proportions for categorical variables. Data summaries will be done for each risk group to understand the characteristics of each risk group.

Presence and levels of IgG/IgA antibodies will define binary and continuous longitudinal outcomes, respectively. Generalized estimation equations (GEEs) will be used to fit population averaged models under the most general assumptions of unstructured variance covariance matrix. It is likely that the distributions IgG/IgA antibody levels will be highly skewed and logarithmic transformation will be required to satisfy such models’ assumptions. The continuous longitudinal outcome will quantify patterns in trends in the monthly average waning immunity across groups and their characteristics – trends are expected to be nonlinear. The binary outcomes will evaluate the associations between presence/absence of antibodies and epidemiologically plausible independent variables through odds ratios. Mixed models will also be done and the estimates will be compared with those obtained by the GEEs fit. All analyses will be adjusted for levels of the infection in the general populations by calendar time, and by associated periods of interventions. Time-varying covariates are expected to be defined based on these later interventions. P-values less than 0.05 will indicate statistically significant (potentially adjusted) associations and 95% confidence intervals will assess the uncertainty of the estimates.

Multivariable models will be built for each risk group accounting for all potential explanatory variables and their interactions - particularly with time at follow-up. The most parsimonious model will be constructed based on the largest complete observations dataset using likelihood criteria such as quasi-likelihood value under the independence model criterion (QIC) for GEEs or likelihood-ratio type criteria for mixed models. GEE estimation is based on completely at random missing observations assumption whilst mixed models are based on missing at random assumption. The attrition patterns will be investigated, and sensitivity analyses to the estimates obtained on complete datasets using the two estimation techniques will be performed and discussed.

 Ethics and dissemination

The study protocol was approved by the Ecuadorian Ministry of Public Health Ethics Committee for COVID-19 Research (approval ID 020-2020- MSP-CGDES-2020-0172-O). The study results will be disseminated through presentations at conferences and to key stakeholder groups including policy makers, and through postgraduate theses and peer-review publications. All participants were required to accept participation by reading and signing an informed consent at the time of the first sampling.

 Discussion

We are following up three adult longitudinal cohorts with presumed distinct exposure risks to SARS-CoV-2 based on occupation, in two Andean cities of Ecuador, to study determinants of seroprevalence and changes in systemic and mucosal antibody levels against SARS-CoV-2.

Seroprevalence testing can be done using the detection of specific IgG and or IgM antibodies. IgM antibodies are detectable first from day 5-7 days post-infection with a peak at day 28 while IgG become detectable after 5-10 days and peak at 49 days. 1 , 29 , 30 In contrast, specific IgG antibodies following vaccination against SARS-CoV-2 are detectable from about a week following the first dose and peak at approximately 30 days after the second dose, remaining detectable for 3-6 months. 31 , 32 For example, a cross-sectional study in 56,261 Chilean individuals reported an IgG seroprevalence of 28.1% three weeks after the first dose and 77.4% four weeks after the second dose of the CoronaVac vaccine; and a prevalence of 79.4% three weeks after the first dose and 96.5% four weeks after the second dose of the BNT162b2 Pfizer vaccine. 7 In healthcare workers (HCW) receiving the BNT162b2 Pfizer vaccine, a study showed that median serum levels of antibodies reached a peak 1 month after the second dose and were 37% and 57% lower at 3 and 6 months, respectively. 33 Finally, a study, also in HCW, showed that antibodies remained high at three months after the third vaccination dose and that antibodies were quantitatively higher in those HCWs that had a history of COVID-19 infection and a third vaccine dose. 34

Our study aims to measure changes in antibody levels in the context of continuous (documented) exposures to natural infection and widespread vaccination with blood collections done as follows: 1) before the first vaccine dose; 2) at 30-60 days after the second dose; 3) 30-60 days after the third dose (when possible); and 4) 30-60 days after the fourth dose (where possible). These sampling times will allow us to measure effects of COVID-19 vaccination and booster doses with the three vaccines licensed for use in Ecuador (CoronaVac, Pfizer and AstraZeneca) on seroprevalence and antibody levels.

Other than vaccination or natural infection, additional factors have been reported to have significant effects on anti-SARS-Cov-2 antibodies. Front-line professionals such as doctors, nurses, hospital workers, police officers, among others, are more likely to acquire COVID-19 and therefore, would be expected to have higher seroprevalence rates than the general population. 35 – 37 A study involving 569 Canadian HCWs reported an IgG seropositivity duration of 415 days and 213 days for symptomatic and asymptomatic individuals, respectively; with obesity, age (over 55 years), and non-Caucasian ethnicity associated with longer seropositivity. 38 Finally, ethnicity is also a factor affecting seroprevalence with indigenous populations having the highest reported seroprevalence (ranging from 4.2 – 81.65%) and populations of African descent having 68% prevalence. 35 – 37 These differences might be explained by greater levels of migration (particularly in seeking work) and more menial work opportunities that don’t allow self-isolation, and a consequently greater risk of exposure to SARS-CoV-2. This is exemplified by a study done in Colombia where the global prevalence in the populations studied was 32.25%, while in Pacific coastal city of Guapi (where a high proportion of the population is of African descent with high rates of temporary migration), the IgG seroprevalence was 68%. 10

There are relatively few longitudinal data on mucosal antibody (IgA) response to SARS-CoV-2 from settings in low and middle-income countries. A systematic review of 38 original studies measuring specific IgA reported a seroprevalence of 86.5% among PCR positive SARS-CoV-2 patients and that the production of specific IgA was detectable during the first 10 days following infection, and remained detectable for prolonged periods (e.g. 75 days after symptom onset). 39 Finally, a study that analysed 100 communities in Wuhan, China showed that the presence of specific IgA antibodies decreased rapidly over time, as for IgM, especially those infected but who were asymptomatic. 40

Our study will evaluate differences in seroprevalence between cohorts with different occupational exposures (HCWs, highly mobile workers, and university staff and students) believed to have differing a priori risks of exposure to SARS-CoV-2. To our knowledge, there are no published longitudinal studies analysing cohorts with differing occupational risks for changes in systemic and mucosal antibodies to SARS-CoV-2. We hope to identify relevant factors related to changes in antibody levels including the effect of vaccination (including vaccine type and dose), occupation, and natural infections (and re-infections) with SARs-CoV-2.

Study limitations

The study is subject to several potential limitations. Significant losses to follow-up can lead to selection bias and these are being minimized by close coordination of project activities with community leaders and institutional authorities, and regular contacts with study participants (at 3-monthly intervals). Recall bias is being minimized through these regular follow-ups to collect information on potential infections with SARS-CoV-2 and vaccination dates. Misclassification of data collection is being minimized by using a widely used standardized instrument (ISARIC WHO), and of antibody measurements by the use of validated detection assays blind to potential exposures. Because of high expected seroprevalence through vaccination, we are measuring antibody levels, thus maximizing the potential power of the study. Misclassification of exposure to SARS-CoV-2 is unavoidable and may be systematic because of limited access to PCR-based assays – underreporting of confirmed infections will likely be least in the hospital-based cohort. Similarly, the use of different vaccines of differing immunogenicity likely will influence maximal antibody levels: vaccine type and dose will be considered as a potential confounder or determinant in different longitudinal analyses. The receipt of multiple vaccine doses at regular intervals (up to 3 doses during 12 months or so of follow-up) may limit the ability to measure rates of decline in antibody levels. Further, the measurement of IgG specific for spike protein and not for other SARS-CoV-2 antigens will limit our ability to distinguish vaccine-induced antibodies from those induced by natural infection.

 Conclusions

We are undertaking a prospective longitudinal cohort study to evaluate the effects of SARS-CoV-2 and vaccine exposures on longitudinal changes in systemic (plasma IgG) and mucosal (salivary IgA) antibodies to SARS-CoV-2. To our knowledge, this is one of very few such longitudinal studies being done in a low-middle income country setting and will contribute to our understanding of the impact of natural infectious exposures versus vaccination on population immunity, and the need for further booster vaccinations in different population groups.

 Data availability

No data are associated with this article.

 Acknowledgements

The authors would like to thank Corporación Ecuatoriana para el Desarrollo de la Investigación y Academia - CEDIA for the financial support given to the present research, development, and innovation work through its CEPRA program, especially for the CEPRA XVI-2022-17 ANTICUERPOS IGG IGA fund.

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Viruses. 2022;16(3):386–394. 34962079 10.1111/irv.12952 Del Brutto OH Costa AF Mera RM : Late incidence of SARS-CoV-2 infection in a highly-endemic remote rural village. A prospective population-based cohort study. Pathog. Glob. Health. 2020;114(8):457–462. 32988333 10.1080/20477724.2020.1826152 Del Brutto OH Costa AF Mera RM : SARS-CoV-2 in Rural Latin America. A Population-based Study in Coastal Ecuador. Clin. Infect. Dis. 2021;73(2):314–317. 10.1093/cid/ciaa1055 Del Brutto OH Costa AF Recalde BY : Factors Associated With a Persistent Seronegative Status 1 Year After a SARS-CoV-2 Massive Infection Outbreak in Community Dwellers Living in Rural Ecuador: A Prospective Population-based Study. J. Prim. Care Community Health. 2021;12. 34715744 10.1177/21501327211054989 Del Brutto OH Mera RM Del Brutto VJ : Risk for Subsequent SARS-CoV-2 Infection and Severe COVID-19 Among Community-Dwellers With Pre-Existing Cervicocephalic Atherosclerosis: A Population-Based Study. J. Prim. 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Public Health. 2021;18(9):4657. 33925680 10.3390/ijerph18094657 Zambrano H Anchundia K Aviles D : Seroprevalence of SARS-CoV-2 immunoglobulins in pregnant women and neonatal cord blood from a highly impacted region. Placenta. 2021;115:146–150. 34626893 10.1016/j.placenta.2021.10.001 Vallejo-Janeta AP Morales-Jadan D Paredes-Espinosa MB : Sustained COVID-19 community transmission and potential super spreading events at neglected afro-ecuadorian communities assessed by massive RT-qPCR and serological testing of community dwelling population. Front. Med. 2022;9. 36059834 10.3389/fmed.2022.933260 Baradaran HR Dehghanbanadaki H Moradpour F : The effect of COVID-19 mRNA vaccines against postvaccination laboratory-confirmed SARS-CoV-2 infection, symptomatic COVID-19 infection, hospitalization, and mortality rate: a systematic review and meta-analysis. Expert Rev. Vaccines. 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Med. 2020;5(5):908–920. 32428207 10.1093/jalm/jfaa079 Notarte KI Guerrero-Arguero I Velasco JV : Characterization of the significant decline in humoral immune response six months post-SARS-CoV-2 mRNA vaccination: A systematic review. J. Med. Virol. 2022;94(7):2939–2961. 35229324 10.1002/jmv.27688 Hua Q Zhang H Yao P : Immunogenicity and immune-persistence of the CoronaVac or Covilo inactivated COVID-19 Vaccine: a 6-month population-based cohort study. Front. Immunol. 2022;13. 36032136 10.3389/fimmu.2022.939311 Salvagno G Henry B Pighi L : Total anti-SARS-CoV-2 antibodies measured 6 months after Pfizer-BioNTech COVID-19 vaccination in healthcare workers. J. Med. Biochem. 2022;41(2):199–203. 35510202 10.5937/jomb0-33999 Padoan A Cosma C della Rocca F : A cohort analysis of SARS-CoV-2 anti-spike protein receptor binding domain (RBD) IgG levels and neutralizing antibodies in fully vaccinated healthcare workers. Clin. Chem. Lab. 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Viruses. 2022;16(5):916–925. 35510653 10.1111/irv.12997 Rangel-Ramírez VV Macías-Piña KA Servin-Garrido RR : A systematic review and meta-analysis of the IgA seroprevalence in COVID-19 patients: Is there a role for IgA in COVID-19 diagnosis or severity? Microbiol. Res. 2022;263:127105. 35816990 10.1016/j.micres.2022.127105 Yang J Ma L Guo L : Seroprevalence and dynamics of anti-SARS-CoV-2 antibodies: a longitudinal study based on patients with underlying diseases in Wuhan. Respir. Res. 2022;23(1):188. 35841095 10.1186/s12931-022-02096-5 10.5256/f1000research.162983.r250296 Reviewer response for version 2 Nicolai Eleonora 1 Referee Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy

Competing interests: No competing interests were disclosed.

 26 2 2024 Copyright: © 2024 Nicolai E 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

I appreciated your work highlighting the importance of the change in T lymphocytes and antibodies for different immune conditions. I found your work well written. I have only a few minor concerns:

In the Contribution to the field paragraph, there are few typo errors:

"In this work, we evaluated that the impact of preexisting immunity on the cellular immunity and humoral immunity in COVID-19 patients." Delete "that".

"Further comparison found that the duration of hospitalization and the coronavirus negative conversion time in the mixed immunity subgroup were also shorter than those in the to the natural immunity subgroup and the acquired immunity subgroup." Delete "to the".

In figure 3 are reported values within 4 weeks. It would be interested to extend the monitoring time.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Yes

Are the datasets clearly presented in a useable and accessible format?

Yes

Reviewer Expertise:

NA

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

 10.5256/f1000research.139005.r216384 Reviewer response for version 1 Lassaunière Ria 1 Referee https://orcid.org/0000-0002-3910-7185 Department of Virus & Microbiological Special Diagnostics, Statens Serum Institute, Copenhagen, Denmark

Competing interests: No competing interests were disclosed.

 15 11 2023 Copyright: © 2023 Lassaunière R 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The well-written protocol addresses relevant questions regarding antibody dynamics, factors associated with seroconversion, longitudinal changes in IgG and IgA levels, and the effect of vaccination on waning antibody levels. A strength of the study is the longitudinal design, inclusion of participants residing in different regions of Ecuador and of various occupations, and assessment of mucosal antibody responses. The authors address the limitations in the study including recall bias using interviews to collect data on a 3-monthly basis; however, it may be difficult to overcome this bias for exposures before enrolment. Furthermore, studying anti-spike antibodies only will not be sufficient to distinguish between infection and vaccination.

Specific comments:

Specific objective 3 – in addition to the factors listed, vaccine modality is also an important factor to consider. The authors list different vaccines administered to the Ecuadorian population including a whole inactivated vaccine (CoronaVac), mRNA vaccine (Pfizer-BioNTech), and adenoviral-vectored vaccines (AstraZeneca and Cansino), that could be considered.

As the authors mention, it will not be possible to differentiate between infection-induced and vaccination-induced antibodies using anti-spike antibodies. With the exception of whole inactivated vaccines, approved COVID-19 vaccines do not include the nucleocapsid protein, a viral protein that induces antibody responses in those who become infected. Did the authors consider using an anti-nucleocapsid ELISA in addition to distinguish vaccine-induced responses from infection-induced responses?

The risk of SARS-CoV-2 infection for farmers/merchants and university students/staff may be quite difficult to segregate considering the multitude of other factors contributing to transmission/acquisition of SARS-CoV-2, beyond the possibility to self-isolate. For example, farmers and merchants likely spend more time outdoors, with short interactions with other people in well-ventilated areas, unlike students who are much more social and aggregate in environments with a large number of people for longer periods of time, likely poorly ventilated environments. Since the authors will consider cohort membership for identifying risk factors for baseline seroprevalence, is it necessary to categorize the cohorts according to exposure risk?

Do the authors anticipate a confounding effect of the antigen used in the ELISA after individuals may have been infected with Omicron that contains >12 amino acid changes in the RBD?

It is unclear if this study protocol is written for a prospective study with planned recruitment and sampling, a retrospective study with planned antibody analyses on samples already collected, or an ongoing study. At the time of writing, were the cohorts being recruited (page 6), or were recruited and sample collection done and subjects contacted (page 8)?

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Yes

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Infectious diseases, Virology, Immunology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Leon-Rojas Jose Universitat Autonoma of Barcelona, Spain

Competing interests: No competing interests to declare

 21 2 2024

Comment 1: The well-written protocol addresses relevant questions regarding antibody dynamics, factors associated with seroconversion, longitudinal changes in IgG and IgA levels, and the effect of vaccination on waning antibody levels. A strength of the study is the longitudinal design, inclusion of participants residing in different regions of Ecuador and of various occupations, and assessment of mucosal antibody responses. The authors address the limitations in the study including recall bias using interviews to collect data on a 3-monthly basis; however, it may be difficult to overcome this bias for exposures before enrolment. Furthermore, studying anti-spike antibodies only will not be sufficient to distinguish between infection and vaccination.

Response 1: Thank you for your comments, we agree that some recall bias will be present in exposure before the enrolment, although given that COVID-19 created significant distress in the country, we believe that participants were likely to have good recall of pre-study COVID-19-related illnesses. We agree that measuring RBD IgG can result in confounding of the antibody levels between infection or vaccination.

Comment 2: Specific objective 3 – in addition to the factors listed, vaccine modality is also an important factor to consider. The authors list different vaccines administered to the Ecuadorian population including a whole inactivated vaccine (CoronaVac), mRNA vaccine (Pfizer-BioNTech), and adenoviral-vectored vaccines (AstraZeneca and Cansino), that could be considered.

Response 2: The vaccine received will be considered in the analysis and we have made this clearer in the text.

Comment 3: As the authors mention, it will not be possible to differentiate between infection-induced and vaccination-induced antibodies using anti-spike antibodies. With the exception of whole inactivated vaccines, approved COVID-19 vaccines do not include the nucleocapsid protein, a viral protein that induces antibody responses in those who become infected. Did the authors consider using an anti-nucleocapsid ELISA in addition to distinguish vaccine-induced responses from infection-induced responses?

Response 3: We agree that recent studies indicate that assays to measure levels of IgG nucleocapsid antibodies may not be as robust and replicable as those used to measure spike protein and might wane faster which is a limitation in a longitudinal analysis 1-4. Our study protocol, which was written early in the epidemic and approved by the emergency bioethics committee of the Ecuadorian Ministry of Health, only allowed us to measure spike protein antibodies. In retrospect, we agree that the ability to measure antibodies against other SARS-CoV-2 antigens would have allowed us to distinguish natural immunity from that acquired after vaccination. This limitation has been added to our manuscript.

Additionally, the ethics committee that approved our protocol was disbanded soon after the emergency had been resolved and we were therefore unable to modify the protocol to allow us to measure these antibodies.

Further, the measurement of IgG specific for spike protein and not for other SARS-CoV-2 antigens will limit our ability to distinguish vaccine-induced antibodies from those induced by natural infection.

References:

Santosh Dhakal, Tong Yu, Anna Yin, Nora Pisanic, Zoe O Demko, Annukka A R Antar, Andrea L Cox, Christopher D Heaney, Yukari C Manabe, Sabra L Klein, Reconsideration of Antinucleocapsid IgG Antibody as a Marker of SARS-CoV-2 Infection Postvaccination for Mild COVID-19 Patients,  Open Forum Infectious Diseases, Volume 10, Issue 1, January 2023, ofac677,  https://doi.org/10.1093/ofid/ofac677

Van Elslande J, Oyaert M, Ailliet S, et al.  Longitudinal follow-up of IgG anti-nucleocapsid antibodies in SARS-CoV-2 infected patients up to eight months after infection. J Clin Virol 2021; 136:104765.

Karger AB, Brien JD, Christen JM, et al.  The serological sciences network (SeroNet) for COVID-19: depth and breadth of serology assays and plans for assay harmonization. mSphere 2022; 7:e0019322.

Owusu-Boaitey N, Russell TW, Meyerowitz-Katz G, Levin AT, Herrera-Esposito D. Dynamics of SARS-CoV-2 seroassay sensitivity: a systematic review and modelling study. medRxiv 2022.09.08.22279731 [Preprint]. September 9, 2022. Available at: https://doi.org/10.1101/2022.09.08.22279731.

Comment 4: The risk of SARS-CoV-2 infection for farmers/merchants and university students/staff may be quite difficult to segregate considering the multitude of other factors contributing to transmission/acquisition of SARS-CoV-2, beyond the possibility to self-isolate. For example, farmers and merchants likely spend more time outdoors, with short interactions with other people in well-ventilated areas, unlike students who are much more social and aggregate in environments with a large number of people for longer periods of time, likely poorly ventilated environments. Since the authors will consider cohort membership for identifying risk factors for baseline seroprevalence, is it necessary to categorize the cohorts according to exposure risk?

Response 4: Thank you for your insightful comments. We have categorized the cohorts according to perceived exposure risk.  For example, university students during sampling time were self-isolating in their homes with strict curfews mandated by the Ecuadorian government while the merchant/farmer group, rather than working on the fields, depended on face-to-face selling in markets and cattle fairs, places characterized by the agglomeration of people.

Comment 5: Do the authors anticipate a confounding effect of the antigen used in the ELISA after individuals may have been infected with Omicron that contains >12 amino acid changes in the RBD?

Response 5: We do expect that the Omicron variant will result in cofounding; however, its important to note that in Ecuador, the Omicron variant was starting to become dominant on the 7 th of January of 2022, reaching its peak at the end of the month. Until then, all groups had at least two follow-up samples collected.

Comment 6: It is unclear if this study protocol is written for a prospective study with planned recruitment and sampling, a retrospective study with planned antibody analyses on samples already collected, or an ongoing study. At the time of writing, were the cohorts being recruited (page 6), or were recruited and sample collection done and subjects contacted (page 8)?

Response 6: At the time of writing, all participants had been recruited and were being followed-up. As you can see in figure 2, in the HR cohort sample collection was finalized, whereas in the MR and LR cohorts, 3 of 4 samples were finalized and the final one was ongoing.

 10.5256/f1000research.139005.r191772 Reviewer response for version 1 Lin Chun-Yang 1 Referee https://orcid.org/0000-0002-3064-0178 The University of Tennessee Health Science Center, Memphis, TN, USA

Competing interests: No competing interests were disclosed.

 1 8 2023 Copyright: © 2023 Lin CY 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

In the study protocol, Leon-Rojas et al. proposed a longitudinal cohort study to follow 1000 adults with varying SARS-CoV-2 exposure and to monitor the change in antibody levels against SARS-CoV-2 spike. Participant demographic information and baseline samples are collected at month 0. The clinical samples and relative information will be collected over the following 3, 6 and 12 months or when the participant receive the booster dose after 30-45 days. The antibody level in the samples is measured by ELISA and calibrated to NIBSC positive samples. With the study they want to understand 1) whether there are risk factors related to the level of SARS-CoV-2 exposure. 2) the change in SARS-CoV-2 spike antibody levels over a period of at least 12 months. 3) whether there are determinants that can affect antibody levels; 4) whether vaccination or other determinants affect antibody regression. 5) whether the relevant determinant also influences the SARS-CoV-2 IgA level in saliva. The results are analyzed by generalized estimating equations to construct a multivariable model for determinant identification.

Major points:

The introduction provides a comprehensive overview of the seroprevalence studies that have been conducted in Latin America. However, the rationale for the study is not explicitly stated until the discussion section. It would be helpful to emphasize the unique aspects of this study, such as the fact that it is a longitudinal study being conducted in a low-middle income country, in the introduction. This would help to make the study more relevant to the target audience.

The authors propose to use SARS-CoV-2 RBD ELISA to determine the determinants of antibody levels. However, it is important to note that the antibody levels to SARS-CoV-2 RBD can be influenced by both natural infection and vaccination. If the authors do not closely monitor the participants, such as by conducting routine nasal swabs for PCR detection, the effect of vaccination or asymptomatic infection on the change in antibody levels could be confounded. It is therefore recommended that the authors include the detection of antibody to viral internal proteins, such as N, ORF8, or M proteins in the ELISA (1-2), in order to distinguish between the effects of infection and vaccination.

The authors describe the methods of ELISA measurement using two reference papers. However, it would be helpful to provide more detail about the procedure, such as whether the serum will be serially diluted for the measurement and what TMB substrate will be used. Additionally, the authors should consider the huge different antibody levels that could be observed between vaccination and natural infection, which might out of ELISA detection range. If all samples are diluted to the same extent, the differences between the antibody levels in the vaccinated individuals may be limited because of saturating antibody binding.

The authors mention that demographic and related information will be collected using WHO CRF Questionnaires. However, they do not specify which determinants they plan to include in the analysis. It would be helpful to provide more detail about the specific determinants that the authors plan to investigate, as this information would be relevant to the target audience.

​​

 Overall, the study protocol is well-designed and has the potential to provide valuable insights into the determinants of antibody levels against SARS-CoV-2 in a low-middle income country. The suggestions that I have made are intended to improve the clarity and relevance of the study.

Is the study design appropriate for the research question?

Yes

Is the rationale for, and objectives of, the study clearly described?

Partly

Are sufficient details of the methods provided to allow replication by others?

Partly

Are the datasets clearly presented in a useable and accessible format?

Not applicable

Reviewer Expertise:

Mucosal immunology, virology, host-pathogen interactions, B cell, transcriptome analysis, influenza virus, SARS-CoV-2

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

References : Antibody Response to SARS-CoV-2 Membrane Protein in Patients of the Acute and Convalescent Phase of COVID-19. Front Immunol .2021;12: 10.3389/fimmu.2021.679841 679841 34421894 10.3389/fimmu.2021.679841 : Accurate Diagnosis of COVID-19 by a Novel Immunogenic Secreted SARS-CoV-2 orf8 Protein. mBio .2020;11(5) : 10.1128/mBio.02431-20 33082264 10.1128/mBio.02431-20 Leon-Rojas Jose Universitat Autonoma of Barcelona, Spain

Competing interests: No competing interests to declare

 21 2 2024

Comment 1: The introduction provides a comprehensive overview of the seroprevalence studies that have been conducted in Latin America. However, the rationale for the study is not explicitly stated until the discussion section. It would be helpful to emphasize the unique aspects of this study, such as the fact that it is a longitudinal study being conducted in a low-middle income country, in the introduction. This would help to make the study more relevant to the target audience.

Response 1: Thank you for your recommendation, we have added a highlighted sentence in the introduction section.

Comment 2: The authors propose to use SARS-CoV-2 RBD ELISA to determine the determinants of antibody levels. However, it is important to note that the antibody levels to SARS-CoV-2 RBD can be influenced by both natural infection and vaccination. If the authors do not closely monitor the participants, such as by conducting routine nasal swabs for PCR detection, the effect of vaccination or asymptomatic infection on the change in antibody levels could be confounded. It is therefore recommended that the authors include the detection of antibody to viral internal proteins, such as N, ORF8, or M proteins in the ELISA (1-2), in order to distinguish between the effects of infection and vaccination.

Reponse 2: We appreciate the recommendation of including antibody detection against internal viral proteins; we agree that measuring RBD IgG can result in confounding in antibody levels between naturally-acquir ed and vaccine-induced immunity. However, our study was performed at the early stages of the pandemic in Ecuador with limited access to COVID-19 testing. In order to try to mitigate this, we have conducted a strict follow-up of participants and recorded the reported PCR results or clinical diagnosis of COVID-19 at each new sample that will be considered in the analysis.

Comment 3: The authors describe the methods of ELISA measurement using two reference papers. However, it would be helpful to provide more detail about the procedure, such as whether the serum will be serially diluted for the measurement and what TMB substrate will be used. Additionally, the authors should consider the huge different antibody levels that could be observed between vaccination and natural infection, which might out of ELISA detection range. If all samples are diluted to the same extent, the differences between the antibody levels in the vaccinated individuals may be limited because of saturating antibody binding.

Response 3: Additional information on the procedure is now provided in the text: 

Flat bottom 96-well microtiter plates (Nunc Maxisorp™, Thermo Fisher Scientific, USA) were coated overnight at room temperature with recombinant RBD (50 ng/well) in phosphate-buffered saline solution (PBS pH 7.2). Wells content was discarded and washed with PBS containing 0.1% Tween-20 (PBST) and wells blocked with 100 µL of blocking buffer (PBS-0.1% Tween 20 and 4% bovine serum albumin) to each well and incubated for 30 minutes at 37°C. After blocking, the plates were washed and 100 µL of serum samples diluted with 5% non-fat milk in PBST was added to each well for reaction at 37°C for 1 hour. Dilutions from 1:200 to 1:1000 were used to capture a broad range of antibody levels and achieve optimal measurement of antibody levels . After washing, 100 μL of horseradish peroxidase (HRP)-labelled goat anti-human IgG (Invitrogen, USA) antibody diluted 8000-fold with 5% non-fat milk in PBST was added to each well and incubated at 37°C for 30 minutes. Post-wash, 100 μL of SIGMAFAST o-phenylenediamine dihydrochloride substrate solution (Sigma-Aldrich, USA) was added to each well for reaction at room temperature (RT) for 10 minutes in the dark. The reaction was stopped by the addition of 100 μL of 3M HCl. Optical density (OD) at 490 nm was measured.

To assay saliva samples, the same procedure as mentioned above was followed, with the exception that the saliva samples were centrifuged at 14,000 rpm for 8 minutes at 4°C and supernatants were diluted 1:1 to 1:4. As secondary antibody, 50 μl of Goat Anti-Human IgA alpha chain (HRP) (A bcam, United Kingdom) antibodies were added at dilution of 1:2000 with 5% non-fat milk in PBST and incubated for 30 minutes at 37°C. The development procedure was the same as for IgG.

Comment 4: The authors mention that demographic and related information will be collected using WHO CRF Questionnaires. However, they do not specify which determinants they plan to include in the analysis. It would be helpful to provide more detail about the specific determinants that the authors plan to investigate, as this information would be relevant to the target audience.

​​

We have included a sentence in the Questionnaires section explaining the main determinants to be considered. We would like to point out that, as specified in the text, we’re using subsections 1b, 1f, and 3a of the WHO CRF.

Overall, the study protocol is well-designed and has the potential to provide valuable insights into the determinants of antibody levels against SARS-CoV-2 in a low-middle income country. The suggestions that I have made are intended to improve the clarity and relevance of the study.

Thank you for your kind remarks, we have included your suggestions.