Two-years antibody responses following SARS-CoV-2 infection in humans: A study protocol.

The long-term antibody response to the novel SARS-CoV-2 in infected patients and their residential neighborhood remains unknown in Indonesia. This information will provide insights into the antibody kinetics over a relatively long period as well as transmission risk factors in the community. We aim to prospectively observe and determine the kinetics of the anti-SARS-CoV-2 antibody for 2 years after infection in relation to disease severity and to determine the risk and protective factors of SARS CoV-2 infections in the community. A cohort of RT-PCR confirmed SARS-CoV-2 patients (case) will be prospectively followed for 2 years and will be compared to a control population. The control group comprises SARS-CoV-2 non-infected people who live within a one-kilometer radius from the corresponding case (location matching). This study will recruit at least 165 patients and 495 controls. Demographics, community variables, behavioral characteristics, and relevant clinical data will be collected. Serum samples taken at various time points will be tested for IgM anti-Spike protein of SARS-CoV-2 and IgG anti-Spike RBD of SARS-CoV-2 by using Chemiluminescent Microparticle Immunoassay (CMIA) method. The Kaplan-Meier method will be used to calculate cumulative seroconversion rates, and their association with disease severity will be estimated by logistic regression. The risk and protective factors associated with the SARS-CoV-2 infection will be determined using conditional (matched) logistic regression and presented as an odds ratio and 95% confidence interval.

Introduction

It has been over two years since the currently ongoing Coronavirus Disease 2019 (COVID19) pandemic was announced as a public health emergency of international concern. COVID-19 has inflicted tremendous mortality and morbidity in a relatively short time [1-3]. The duration of a protective immune response after a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has not yet been fully studied. Still, the dynamics of the humoral immune response during an acute phase of SARS-CoV-2 infection are well-understood [4, 5].

The seroconversion takes place 6–14 days after the diagnosis of SARS-CoV-2 infection [6-9]. A significant increase in virus-specific antibody levels is observed on days 16–35 after the onset of symptoms [7, 10–13]. Some studies have reported that antibody levels peak within the first few months, then wane and remain stable for several months, indicating that the immunity may last longer [7, 11, 13–19]. Other studies suggest that the levels of SARS-CoV-2-specific IgG are durable and decline after 6–8 months [20, 21]. The latest study by Marcotte et al. reported that anti-SARS-CoV-2 RBD and S IgG increased at 15–28 days after symptoms onset in almost all of the patients, then gradually dropped by less than 4-fold from the antibody response until 6 months, but thereafter, persist in the majority of patients up to 15 months [22]. Tan et al. reported that IgM was detected on day 7 (peaked on day 28), and IgG was detected on day 10 (peaked on day 49) [23]. A previous study by Zhao et al. determined that seroconversion took place at later median times [24].

Severity levels of cases, e.g., asymptomatic, mild, or severe, were found to produce identifiable antibodies against MERS-CoV, SARS-CoV and SARS-CoV-2 [25]. Although differences in disease severity do not lead to significant kinetic changes of the antibodies, severe SARS-CoV cases with sequelae showed lower neutralizing antibodies [26]. Seroconversion rate, as well as peak antibody levels, increased according to disease severity in MERS-CoV infection, [27] and antibodies response seemed to be less likely to evoke in milder infections [27, 28]. Tan et al. showed that SARS-CoV-2 infection might indicate a probable opposing pattern to MERS-CoV, for which in both severe and non-severe patients, IgM emerged at the same time, while IgG emerged earlier in severe patients [23]. Marcotte et al. reported that the half-lives of anti-RBD and anti-S IgG antibody response were shorter in patients with mild/moderate than severe/critical disease [22].

Furthermore, in the general population, understanding risk factors for COVID-19 is needed to plan an appropriate disease prevention model. A surveillance system is needed to accommodate various challenges in data collection involving hundreds of respondents. Seroconversion and duration of immune response among patients with COVID-19 in Indonesia, particularly in a cohort setting, have never been reported.

This study protocol describes plans to observe and determine the kinetics of the antibody response up to two years following SARS-CoV-2 infection and identify the protective and risk factors of infection in the community. The ability to predict the likelihood of SARS-CoV-2 infection has the potential to inform decision-making at the individual, stakeholder, and government levels. Furthermore, evidence-based risk stratification tools can provide a platform for policymakers to prioritize population segments in national vaccination programs.

Methods

Aims of the study

The primary objective of this study is to prospectively observe and determine the kinetics of anti-SARS-CoV-2 antibodies for 2 years after infection in relation to disease severity. The secondary objective is to identify the risk and protective factors of SARS CoV-2 infections through a comparison of SARS-CoV-2 confirmed patients (case group) and their respective neighborhood individuals (control group). Further, we aim to develop a cohort system in managing responses to the pandemic, which will combine hospital-based and population-based longitudinal data in the Sleman District, Yogyakarta Province, Indonesia. Such a cohort system will contribute to the development of a fast, accurate, and secure data collection and management application.

Study design

This study comprises two sub-studies, i.e., a case-control study and a prospective cohort study. We will use the case-control design to determine the risk and protective factors of SARS-CoV-2 infections. Cases will be recruited from the Academic Hospital of the Universitas Gadjah Mada (UGM) in Yogyakarta. For each case, 3 control subjects, who are uninfected, will be recruited from an area within a 1-km radius from the case’s residence. We hypothesized that individual behavior, traveling, and societal structures might affect the risk of infection. Then, we will implement the prospective cohort design to observe the kinetics of antibody anti-SARS-CoV-2 in cases for two years following infection. We will use Chemiluminescent Microparticle Immunoassay (CMIA) method to quantify the peripheral blood IgM/IgG anti-Spike RBD titer of SARS-CoV-2. The case-cohort will provide parallel data, enabling us to correlate the seroconversion and dynamics of antibody responses with clinical data, demographics, and comorbidities. We hypothesized that the IgM/IgG titer to SARS-CoV2 will correlate with disease severity.

Study setting

The study is taking place in Sleman, a district within the Yogyakarta province, Indonesia. The district spans an area of 574.82 km2, which is about 18% of the province and is inhabited by 1,219,640 residents (December 2020). Geographically, Sleman is located between 110° 33′ 00″ and 110° 13′ 00″ East Longitude, 7° 34′ 51″ and 7° 47′ 30″ South Latitude (Fig 1) [29]. As of 31 August 2021, the Sleman District COVID-19 Task Force reported cumulative cases of 52,766, of which 6,131 were active, with 2,308 deaths [30].

Fig 1

Location of the research.

(a) the country of Indonesia (b) the island of Java (c) Yogyakarta province, and (d) Sleman regency © OpenStreetMap Contributor. This map is licensed under the Creative Commons Attribution-Share Alike 2.0 License (CC BY-SA). For further information, please visit http://www.openstreetmap.org/copyright.

Sample size

Fleiss’s formula was used to calculate the sample sizes of cases and controls with a ratio of 1:3 with 90% power and 95% confidence interval (CI) to detect an odds ratio of 2 [31]. The resulted sample size was 118 cases. The sample size was further adjusted to account for the expected rate of loss to follow-up and missing data of 40%. Thus, the final sample size for cases is 165, and 495 healthy individuals for control will be recruited from the Sleman population.

Study participants

Patients will be identified prospectively at the time of confirmation of the diagnosis in isolation wards at the UGM Academic Hospital. The inclusion criteria are patients older than 18 years old who are confirmed COVID-19, defined as positive SARS-CoV-2 reverse transcriptase-polymerase chain reaction (RT-PCR) results from any respiratory sample, and reside in Sleman District at the time of diagnosis. There are no exclusion criteria for the case group.

The controls comprise individuals who live within a 1-km radius of the case’s residence. The inclusion criteria are healthy adults (aged >18 years old) with no prior or current SARS-CoV-2 infection upon screening (using GeNose®) at the time of interview. The exclusion criteria are any eligible person who shows a flu-like syndrome on the screening day.

Control group sampling procedures will be done as follows: the case’s residence will be located using Google Earth. The longitude and latitude of the location point will be documented and then imported into Google Earth. Subsequently, a radius of 1 kilometer will be made from the point before generating aerial imagery referenced to 100-meter grids within the radius. Each grid cell will be assigned a number consecutively according to row, with the smallest number, one, assigned to the top left corner cell and the largest number assigned to the bottom right corner cell. Five grid cells will be randomly selected (among which 3 will stand as a backup if the land is vacant or the number of houses is too few). Houses in the selected grids will be numbered. Twenty households will be selected by random sampling. The field staff will visit the selected houses starting from the first selected house. The Kish grid method will be used to select an individual in each house as a potential control subject [32, 33].

Data collection

Demographic data (basic demographic data, mobility, travel history, number and composition of households, and house size), community variables (environmental density, local transmission in the neighborhood, contact history), behavior (adherence to health protocols, tobacco use, transportation, and sleep patterns), comorbidity (diabetes, cancer, stroke, hypertension, obesity, heart disease, chronic obstructive pulmonary disease, chronic liver disease, chronic kidney disease, and autoimmune disease), pregnancy and COVID-19 vaccination status, and anthropometric parameters will be collected from medical records (case group) and direct interviews (control group). The data collection process in the community will be a collaborative effort involving local stakeholders and volunteer cadre groups.

The Hospital’s laboratory information of the case group (SARS-CoV2 RT-PCR of nasopharyngeal/oropharyngeal swab, complete blood count, electrolyte, blood glucose, ferritin, CRP, procalcitonin, liver transaminases, serum, BUN, serum creatinine, chest X-ray and/or computerized tomography (CT)-scan), clinical management (drugs, intravenous fluid, supplements, oxygenation, ventilator support, and other intensive care support), and comorbidity (diabetes, cancer, stroke, hypertension, obesity, heart disease, chronic obstructive pulmonary diseases, pregnancy) will be retrieved from hospital records. The severe group will be categorized as patients who are dyspnea, hypoxia (SpO2 <93% in room air), require ICU level care and other organ support (such as inotropes and renal replacement therapy), or die after enrolment during hospitalization. Non-severe groups will be categorized as patients who do not meet the criteria for severe disease. Data of any resolution/persistence of symptoms, travel history, compliance with health protocols, any possible COVID-19 re-infection, and COVID-19 vaccination status will be recorded in each subsequent monitoring time point.

Blood samples collection and storage

A 3 mL venous blood sample will be collected from all consenting participants. Serial peripheral blood samples will be collected on the day of diagnosis when discharged from the hospital and at weeks 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 72, and 96 after discharge. For monitoring samples, the blood will be collected at the outpatient clinic or a nearby community health center by making an appointment beforehand to meet with the research nurse/research officer. Once collected in an EDTA-containing vacutainer, blood samples will be transferred to the project laboratory (Biobank Unit at the Faculty of Medicine, Public Health and Nursing UGM, Yogyakarta, Indonesia). Plasma will be collected by centrifugation at 4,000 rpm for 10 minutes [34]. All specimens will be processed and stored on the same day as the sample receipt. Plasma will be stored at -80 °C until testing.

CMIA for IgM and IgG anti-SARS-CoV2

Examination of IgM anti Spike protein of SARS-CoV2 and IgG against Spike RBD of SARS-CoV-2 will be performed using Chemiluminescent Microparticle Immunoassay (CMIA) from Abbot (Cat. 6R87 for IgM and 6S60 for IgG). Two types of immunoassays are used in this study. Both are automated, two-step immunoassays. The SARS-CoV-2 IgM assay is used for the qualitative detection of IgM antibodies SARS-CoV2, and the SARS-CoV-2 IgG II Quant assay is used for the qualitative and quantitative determination of IgG antibodies against SARS-CoV-2. Serum or plasma (separated by dipotassium EDTA or tripotassium EDTA or lithium heparin or sodium heparin or sodium citrate) can be used in this assay. Both of the assays have an identical principle in the procedure. The sample, SARS-CoV2 antigen coated paramagnetic particles, and assay diluent are mixed and incubated. The IgM and IgG antibodies against SARS-CoV2 present in the sample will bind to the SARS-CoV2 antigen-coated microparticle. The detection of IgM and IgG are processed separately. After the washing step, anti-human IgM or IgG acridinium-labeled conjugate is added to the mixture and incubated. Following a wash cycle, Pre-Trigger and Trigger Solutions are added. The resulting chemiluminescent reaction is measured as a relative light unit (RLU) [35, 36].

Data management plan

All respondents will have unique identification. Research nurse/field officers will enter data into eSynthesis (version 1.9.8—A survey tool. Sleman, Yogyakarta, Indonesia), an application installed on their mobile tablet. Supervisors and data managers will check the suitability of the data to ensure data quality. Maintaining data quality will include calibrating instruments regularly, and having a validation feature in the application. A red warning sign will appear if the input format does not match or if there are data that have not been filled in. The data will be cleaned several times by the data manager until ready for analysis. Data types consist of text, number, audio, and image data. The data consist of primary and secondary data. Primary data will be obtained by measuring directly and interviewing respondents in the field. Secondary data will be taken from the respondents’ medical records. Data will be stored on institutional servers restricted to a particular research team. The study’s data processing (collection, storage) is based on the UGM Academic Hospital and the Health and Demographic Surveillance System Sleman (HDSS Sleman) data protection concepts.

Expected outcomes

We intend to measure the kinetics of antibody response to the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2 in a cohort of patients infected with SARS-CoV-2 (up to 2 years after detection of SARS-CoV-2 RNA). The association between antibody level and severity of illness will be measured across time. Risk and protective factors associated with SARS-CoV-2 infection will be measured at the time of case detection by comparing the proportion of factors between the case and control groups.

The type of data and statistical analyses planned

Continuous variables with normal distribution will be reported as mean with standard deviation (SD), and non-normal distributed variables as median with interquartile range (IQR). The Kaplan-Meier method will be used to calculate cumulative seroconversion rates. The association between antibody level and disease severity will be estimated by logistic regression. The characteristics of the case and control groups will be described as mean and SD or frequency and percentage. The factors (risk and protective) associated with the COVID-19 infection will be determined using conditional (matched) logistic regression and presented as OR and its 95% CI. This study will be reported in accordance with the Strengthening the Reporting Observational Studies in Epidemiology (STROBE) checklist [37]. Missing data and lost to follow-up patients will be reported. Missing data will be handled by using multiple imputation models [38].

Ethical considerations and declarations

This study was reviewed and approved by the Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine, Public Health, and Nursing UGM (KE/FK/0882/EC/2020). Written informed consent will be documented from all respondents prior to enrolment into the study. The authors are responsible for the content and writing of the paper.

The status and timeline of the study

The study started enrolment in January 2021 and is recruiting a cohort of study participants. This multi-year research project will be conducted for three years with respective targets and indicators. The first and second years of the study focus on participant enrolment, monitoring, and laboratory analysis. Study results are planned to be disseminated in the third year of study.

Discussion

Antibody-based detection can help determine prior SARS-CoV-2 infections in populations and may allow for vaccination monitoring in the future [39]. Examination of antibodies against SARS-CoV2 is important for several reasons: confirmation of present and past infection, evaluation of patients with characteristic COVID-19 symptoms but negative nucleic acid amplification tests (NAATs), seroepidemiological studies on COVID-19, assessment of the development of antibody-mediated protective immunity and investigation of the immune response and immunopathology of COVID-19.

This study is designed to observe the dynamics of antibody anti-SARS-CoV-2 for two years following SARS-CoV-2 infections. Potential key points relevant to the current pandemic include unknown seroconversion and seroreversion in hospitalized patients. Time to seroconversion may be related to the severity of the disease. A study from North America showed that in symptomatic COVID-19 patients, the median time to seroconversion of SARS-CoV-2 RBD (IgG and IgM) among hospitalized patients was 4 days earlier compared to non-hospitalized patients, proposing an association between antibody kinetics and disease severity [8]. SARS-CoV-2 RBD and S IgG can be detected at approximately the same time as IgM (usually within 2 weeks after symptom onset) [22]. SARS-CoV-2 RBD and S IgG titers peaked 14–30 days after infection. Even though they decreased over time, the IgG antibody response stabilized after 6 months and was still detected in the majority of patients at 6–15 months [22, 40–42].

IgG responses to the SARS-CoV-2 spike RBD showed minimal seroreversion, which was sustained for more than 3 months. Meanwhile, the IgM responses were short-term, and most patients sero-reverted within 2.5 months following symptom onset [8]. Patients with pneumonia, supplemental oxygen requirement, and intensive care unit admission had minimal neutralizing antibody decay at 180 days post symptoms onset (persistent group) compared to patients with the milder disease who showed seroreversion in less than 180 days. However, 27% of patients with pneumonia also sero-reverted in less than 180 days (rapid waning group) [43].

As the SARS-CoV-2 delta variant surge occurred in mid-2021, several hamlets and sub-districts in the study area implemented lockdown [44]. During the high transmission months, obstacles were encountered, which included patients’ reluctance to enroll because of fear that their infection status could be exposed. Patients also found no compelling reason to make a follow-up visit to the nearest healthcare facility after hospital discharge. Control group recruitment was also hindered by a reluctance to visit healthcare facilities, compounded by fears of getting infected. In addition, many potential control subjects refused testing because of fears that the test result would be positive. Recruitment of the control group is counted on the social and health system of the local community. As in many places in Indonesia, community leaders and health cadres have significant influence at the hamlet level. They would be the contact point because of their understanding and familiarity with their local community. By utilizing cadres or community leaders as the key persons to reach the potential respondents, the response is expected to be higher [45].

Progression into Acute Respiratory Distress Syndrome (ARDS) and multi-organ dysfunction are not the only problems leading to morbidity and mortality of COVID19 [46-48]. Clinical outcomes also depend on the adequacy of the clinical management. The lack of available resources, equipment, and properly trained healthcare workers, in addition to the shortage of medicines and limited healthcare infrastructure, have contributed to adverse outcomes [49-51]. Treatment variability described in the current study is due to multiple changes in the national clinical guidelines or other factors affecting the availability of resources (e.g., drugs and other supporting treatments). The variability may be reflected in the patients’ characteristics and correlated with clinical outcomes.

This study has some limitations. As a long-term longitudinal study, retaining participants for the entire length of the 2-year study duration can be burdensome. The willingness of respondents to continue participating may decrease over time [52]. For those who refuse to visit a healthcare facility for monitoring, household visits will be done. This is expected to reduce attrition among participants [52, 53]. Another attempt to maintain participation is by offering incentives that include free basic medical check-ups, such as a registered nurse’s examination of body temperature and blood pressure, for every follow-up visit [53]. Even though household visits is effective, this would lead to increased logistic burdens [52].

This study anticipated an absence in neutralizing antibodies (Nabs) measurement, arguably a better surrogate of immune protection [22, 54]. However, previous studies showed that Nabs levels are directly proportional to the antibody levels of RBD-IgG [8].

A previous study analyzed pseudo-neutralizing antibodies targeting the SARS-CoV-2 S protein in infected persons collected between 0- and 2.5-months post-symptoms. Over the course of infection, all individuals tested developed detectable Nabs levels. Nab titers were correlated with the concentration of anti-RBD IgG (r = 0.87). Furthermore, similar to anti-RBD IgG responses, Nab titers plateaued and remained detectable at later time points [8].

We also admit problems with comparing the screening methods for the patients and the controls. Limitations in study expenditure forced us to employ a more affordable screening method for the controls. We use the patients’ breath-print analyses (GeNose C19®) instead of the nucleic acid amplification test (NAAT). GeNose C19® utilizes breath-borne volatile organic compound (VOC) biomarkers coupled with machine learning to discriminate between COVID-19 and non-COVID-19 respiratory infections accurately [55, 56]. Recent publications on GeNose showed a sensitivity of 86%-94% and specificity of 88%-95% [55]. A systematic review and meta-analysis study showed that VOCs-based breath analysis had a cumulative sensitivity of 98.2% (97.5% CI: 93.1%−99.6%) and specificity of 74.3% (97.5% CI: 66.4%−80.9%) [57].

Previous studies revealed public health challenges surrounding the stigma of being infected with COVID-19 in Malawi and Nigeria [58, 59]. Empirical observation of local communities in Sleman found that swabbing for PCR and antigen testing might have enhanced the stigma, where understandably, breath analysis would be a more acceptable option.

The study presented here will provide current and local evidence, even though it may not be generalizable on a global scale. The study outcomes will provide data for a better understanding of the serological measurement of SARS-CoV-2 infection, improving insights into the at-risk population and post-infection long-term follow-up for the kinetics of anti-SARS-CoV-2 antibody.

8 Mar 2022

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Reviewer #1: Thank you for inviting me to review this manuscript. Although this manuscript seems interesting, there are several lacking points, especially within the Materials and Methods which need further clarification.

1) The number of references cited within the text paragraph should be ordered from their appearance in the text not from the alphabetical order. Please re-arrange all references within the text paragraph to match the journal's guidelines.

2) In Materials and Methods, I think between the exposed group and the control group is not comparable. If the participants in exposed group are those who have positive results from SARS-CoV-2 RT-PCR, then the control group should consist of those who have negative results from SARS-CoV-2 RT-PCR test. If the control group only use GeNose as diagnostic methods and not RT-PCR, it will cause bias in the results. As we all know, GeNose accuracy in diagnosing Covid-19 is still questionable, because it has low specificity and also may results in false negative results. Many of those who have negative GeNose results can still showed a positive results when tested with RT-PCR test as gold standard diagnostic methods. Therefore, the authors should revise this.

3) Moreover in the Materials and Methods, the inclusion criteria for the exposed groups is all patients with confirmed Covid-19 through RT-PCR test which can include all variety of age (pediatrics, teenagers, adults, older people). Meanwhile, in the control groups, the authors only include patients who are >18 years old. This can also cause bias in the results because of age gap between exposed and control groups. Please revise this.

4) In Materials and Methods, the authors should state about the guidelines they used in this study (e.g., STROBE guidelines).

5) In Materials and Methods, the technique used for calculation of the sample size should be explained more. The authors may look into these studies for references (1. http://dx.doi.org/10.1136/bmjopen-2021-050824, 2. https://doi.org/10.1186/s12889-018-5364-2)

6) In Materials and Methods (sample size), the authors have mentioned "To achieve the case-control ratios of 1 : 3, a correction for non-response, serologically positive control, and control who had a flu-like syndrome was made." What do the authors mean by this statement? Please explain more about this statement.

7) It would be better if the protocol of this study has been registered in international registries, such as ClinicalTrials.gov.

Reviewer #2: Manuscript Number: PONE-D-21-34739

Title: Two years antibody responses following SARS-CoV-2 infection in humans: a study protocol

Journal: Plos One

The author appraised this paper by observing the kinetics of anti-SARS134 CoV-2 antibody for 2 years after infection in relation to disease severity.

However, your article is inadequately presented. Furthermore, there are many grammatical mistakes and spelling mistakes as well. Although the article has scientific rigor, several major flows need to be corrected before publication.

Major comments:

General Comments:

1. Many non-scientific and incorrect/wrong information/sentences are there, which may mislead the readers.

2. Every section of the manuscript must be written scientifically according to the published literature with appropriate references.

3. English is weak. The authors need to improve their writing style. The whole manuscript needs to be checked by native English speakers (certificate mandatory).

3. Spacing, punctuation marks, grammar, and spelling errors should be reviewed wholly.

4. The research questions could be defined more precisely. This is probably due to language concerns.

Title:

1. The present title of the article looks okay.

Abstract:

1. The purpose and significance of this research must be explained in the abstract more clearly.

2. The abstract section is unsuitable—no focus point in the abstract section.

Introduction:

1. The introduction section is inapplicable. Need to change the introduction considerably. Try to include the existing research limitations also, how the present research unravels those limits.

2. The study's gaps should be clearly defined in the introduction section with the applicable references.

3. The flow of the introduction is not perfect and unspecific. My advice is to make the sentences more lucid and legible for more productive comprehension.

4. In the introduction section, there are many redundant sentences repeating in the whole paper, making me feel the paper like a "cut and paste" from several other resources.

5. The paragraphs are not logically arranged; there are unnecessary repeats.

6. Arrange the sentences: “There is an urgent need for assays that…”.

7. Many research programs still use paper-based…Not clear.

8. Delete I, we, our throughout the manuscript.

9. The first four paragraphs of the introduction section is not good. Need to merge and concise the whole introduction into four paragraphs.

10. The introduction section seems missing important information. This section needs profound modification with more Up-to-date references.

11. Aim of the study not clear.

12. The information which authors provided is old.

Materials and Methods:

1. Exclusion criteria…what was the purposes?

2. Blood samples collection and storage. Need details.

3. Data management plan: The writing is not good.

4. Safety considerations. Is this necessary?

5. Very few references are in the material and methods.

6. The type of data and statistical analyses planned: Not clear.

7. Patient and public involvement and ethics approval. Number is missing.

8. Need to add references for different sections.

Discussion:

1. The current discussion is poor.

2. unknown of sero-conversion and sero-reversion of antibodies: Need to elaborate.

3. Many text repetitions are found in the discussion section. It is highly recommended to emphasize findings and assumptions that support or disagree with other work(s).

4. Addendum, repetition of the results should be avoided in the discussion section.

5. It is still unclear how long the immunity lasts after patients recover from SARS-CoV2 infection…Is this okay?

6. Understanding about long-term antibody responses is currently limited…What’s the basis?

7. There had also been frequent adaptations…need more insights with references.

8. Challenges include a nationwide restriction: poor writings.

9. Our study targets a large sampling…. not justified.

Figures:

1. The resolution of figures can be improved.

References:

1. For better understanding, I feel this manuscript needs more detailed background information and a precise explanation of their study in the Introduction and Discussion section.

2. Several published articles related to antibody responses following SARS-CoV-2 infection in humans must be included within the relevant text part of the manuscript.

**********

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Reviewer #1: No

Reviewer #2: Yes: Talha Bin Emran

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19 Apr 2022

Thank you for giving us the opportunity to submit a revised version of our manuscript entitled, “Two years antibody responses following SARS-CoV-2 infection in humans: a study protocol” to PLOS ONE.

We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. I am grateful to the reviewers for their insightful comments on our paper. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have highlighted the changes within the manuscript and you can identify the changes from track changes. Here is a point-by-point response to the editor and to the reviewers’ comments and concerns.

Comments from editor

Comment 1: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Response: We have revised the whole manuscript based on the guidelines given. Thank you for pointing this out. The changes can be found throughout the manuscript, including affiliations, font, paragraphing, spacing, figures, and references. We corrected the reference format and completed them by adding DOI.

Comment 2: Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information

Response: Thank you for your important concern. We have, accordingly, specified the consent (line 268), with highlights. This study did not include minors.

Comment 3: We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"The study has received financial grant support from the Ministry of Finance of the Republic of Indonesia LPDP (RISPRO/KI/B1/TKL/5/15867/2/2020).

EA received the award.

The funders had and will not have a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Thank you for your suggestion. We deleted funding statement from the Acknowledgments section.

Comments from reviewer(s):

Reviewer #1

Comment 1: The number of references cited within the text paragraph should be ordered from their appearance in the text not from the alphabetical order. Please re-arrange all references within the text paragraph to match the journal's guidelines.

Response: Thank you for your valuable feedback. We edited the reference citation system according to the journal’s guideline.

Comment 2: In Materials and Methods, I think between the exposed group and the control group is not comparable. If the participants in exposed group are those who have positive results from SARS-CoV-2 RT-PCR, then the control group should consist of those who have negative results from SARS-CoV-2 RT-PCR test. If the control group only use GeNose as diagnostic methods and not RT-PCR, it will cause bias in the results. As we all know, GeNose accuracy in diagnosing Covid-19 is still questionable, because it has low specificity and also may result in false negative results. Many of those who have negative GeNose results can still showed a positive results when tested with RT-PCR test as gold standard diagnostic methods. Therefore, the authors should revise this.

Response: Thank you so much for your review. We put deep thought into this issue.

We realize that the study design was not the highest standard. Ideally, both groups, case and control used the same diagnostic tool to screen SARS-CoV-2 infection. However, there were several aspects that we considered which are as follows:

1. When we developed the proposal, GeNose was set by our government as a screening tool for domestic travel requirements (such as trains, ships, planes) according to Formal Information Letter of Indonesia COVID-19 National Taskforce no 12 in April 2021, so it has been considered a commonly used screening tool in public and has higher acceptability than PCR or antigen test (by using respiratory swabs specimen).

2. Respiratory swab sampling has a negative stigma in our society (we also conveyed this in the discussion line 355-357). We have conducted a preliminary study, inviting healthy residents to be screened using a respiratory swab. However, out of the 15 residents who were invited, only 1 accepted the invitation. We repeated this method for groups of residents in different sub-districts, and the results were similar. We confirmed back to the candidates, that actually they did not refuse to participate in the research, but they refused to come because they did not want to be swab tested.

3. In seeking control of healthy people, we invited candidate respondents who met the inclusion criteria to the public health center (Puskesmas) or a public hall close to their residence. Not all of these public places can be easily set-up to conduct a respiratory swab (it requires additional PPE, and the set-up process is inflexible). The stakeholders in charge of the public health center and public halls also suggested that we use other less invasive diagnostic tool to increase the recruitment rate, namely GeNose.

4. PCR or antibodies testing are much more costly than GeNose. For patients (case group), the testing cost is included in the regional outbreak health scheme. Nevertheless, by utilizing healthy people as controls, this cost must be borne by us, which is one of our limitations. As an illustration, at that time, the PCR test cost was around USD 95, the antigen swab was around USD 45, and GeNose was only USD 5.5. GeNose also showed a sensitivity of 86%-94% and specificity of 88%-95%. (Nurputra et al., 2021). We also conveyed this in the discussion section line 344-353.

5. Furthermore, a systematic review and meta-analysis study showed that VOCs-based breath analysis had a cumulative sensitivity of 98.2% (97.5% CI: 93.1%−99.6%) and specificity of 74.3% (97.5% CI: 66.4%−80.9%) (Subali et al., 2022).

6. We discussed the use of this GeNose as one of the weaknesses of the study.

Comment 3: Moreover in the Materials and Methods, the inclusion criteria for the exposed groups is all patients with confirmed Covid-19 through RT-PCR test which can include all variety of ages (pediatrics, teenagers, adults, older people). Meanwhile, in the control groups, the authors only include patients who are >18 years old. This can also cause bias in the results because of age gap between exposed and control groups. Please revise this.

Response: Thank you so much for your review. We revised inclusion criteria for case group (lines 148-151, highlighted):

“The inclusion criteria are patient age more than 18 years old who is confirmed COVID-19, defined as positive SARS-CoV-2 reverse transcriptase- polymerase chain reaction (RT-PCR) results from any respiratory sample, and reside in Sleman District at the time of diagnosis”

Comment 4: In Materials and Methods, the authors should state about the guidelines they used in this study (e.g., STROBE guidelines)

Response: Thank you for your suggestion. This study will be reported in accordance with the Strengthening the Reporting Observational Studies in Epidemiology (STROBE) checklist (lines 260-262, highlighted).

Comment 5: In Materials and Methods, the technique used for calculation of the sample size should be explained more.

Response: Thank you for your review. We explained the calculation of the sample size (lines 139-144, highlighted).

“Fleiss’s formula was used to calculate the sample sizes of cases and controls with a ratio of 1:3 with 90% power and 95% confidence interval (CI) to detect an odds ratio of 2.30 The resulted sample size was 118 for cases. The sample size was further adjusted to account for the expected rate of lost to follow-up and missing data of 40%. Thus, the final sample size for cases is 165, and 495 healthy individuals for controls will be recruited from the Sleman population.”

Comment 6: In Materials and Methods (sample size), the authors have mentioned "To achieve the case-control ratios of 1 : 3, a correction for non-response, serologically positive control, and control who had a flu-like syndrome was made." What do the authors mean by this statement? Please explain more about this statement.

Response: Thank you so much for your review. We revised the whole paragraph (line 139-144, highlighted). In brief, we recruited 3 controls for each 1 case, with inclusion criteria they are healthy adults (aged >18 years old) with no prior or current SARS-CoV-2 infection upon screening (at the time of interview).

Comment 7: It would be better if the protocol of this study has been registered in international registries, such as ClinicalTrials.gov

Response: Thank you for your suggestion. We are in the process register to ClinicalTrials.gov through the administrator at our university.

Reviewer #2

General Comments:

Comment 1: Many non-scientific and incorrect/wrong information/sentences are there, which may mislead the readers.

Response: Thank you. We revised the whole manuscript, and consulted this manuscript to native English speaker registered in our university.

Comment 2: Every section of the manuscript must be written scientifically according to the published literature with appropriate references.

Response: Thank you. We added recent references to support introduction/background and discussion sections

Comment 3: English is weak. The authors need to improve their writing style. The whole manuscript needs to be checked by native English speakers (certificate mandatory). Spacing, punctuation marks, grammar, and spelling errors should be reviewed wholly.

Response: Thank you. We consulted this manuscript to native English speaker registered in our university. The certificate is attached to this letter.

Comment 4: The research questions could be defined more precisely. This is probably due to language concerns.

Response: Thank you for your suggestion. We incorporate the research question together with the aim of the study which can be found in the abstract (line 39-42, highlighted).

“We aim to prospectively observe and determine the kinetics of the anti SARS-CoV-2 antibody for 2 years after infection in relation to disease severity and to determine the risk and protective factors of SARS CoV-2 infections in the community.”

and in the ‘Aims of the study’ section (line 98-107).

“The primary objective of this study is to prospectively observe and determine the kinetics of anti-SARS-CoV-2 antibody for 2 years after infection in relation to disease severity. The secondary objective is to identify the risk and protective factors of SARS CoV-2 infections through comparison of SARS-CoV-2 confirmed patients (case group) and their respective neighborhood individuals (control group).”

Title:

Comment 1: The present title of the article looks okay.

Response: Thank you for your positive review

Abstract:

Comment 1: The purpose and significance of this research must be explained in the abstract more clearly.

Response: Thank you. We revised the abstract. The purposes are stated in line 39-42 (highlighted) and the significance of current study was stated in line 36-39 (highlighted).

“The long-term antibody response to the novel SARS-CoV-2 in infected patients and their residential neighborhood remains unknown In Indonesia. This information will provide insights into the antibody kinetics over a relatively long period as well as transmission risk factors in the community. We aim to prospectively observe and determine the kinetics of the anti SARS-CoV-2 antibody for 2 years after infection in relation to disease severity and to determine the risk and protective factors of SARS CoV-2 infections in the community.”

Comment 2: The abstract section is unsuitable—no focus point in the abstract section.

Response: Thank you for your correction. We edited the abstract structure according to journal’s guidelines.

Introduction

Comment 1: The introduction section is inapplicable. Need to change the introduction considerably. Try to include the existing research limitations also, how the present research unravels those limits.

Response: Thank you for your suggestions. As you see in our revision, we re-structured the introduction majorly. Other studies observed not more than 13 months, while the current study will monitor immune response until two years following infection.

Comment 2: The study's gaps should be clearly defined in the introduction section with the applicable references.

Response: the biggest gap of this field is that seroconversion and duration of immune response among patients with COVID-19 in Indonesia, particularly in a cohort setting, has never been reported (line 85-86, highlighted)

“Seroconversion and duration of immune response among patients with COVID-19 in Indonesia, particularly in a cohort setting, has never been reported.”

Comment 3: The flow of the introduction is not perfect and unspecific. My advice is to make the sentences more lucid and legible for more productive comprehension.

Response: Thank you for your constructive comment. We revised the introduction section majorly.

Comment 4: In the introduction section, there are many redundant sentences repeating in the whole paper, making me feel the paper like a "cut and paste" from several other resources.

Response: Thank you. We re-structured the introduction and made the better flow of the story as a background of the study

Comment 5: The paragraphs are not logically arranged; there are unnecessary repeats.

Response: Thank you, we deleted any repetition and re-arranged the whole introduction.

Comment 6: Arrange the sentences: “There is an urgent need for assays that…”.

Response: We deleted that sentence and replaced it with a new paragraph (line 211-223, highlighted, tracked changes)

Comment 7: Many research programs still use paper-based…Not clear.

Response: We deleted that sentence and replaced it with a new Introduction section (line 56-93).

Comment 8: Delete I, we, our throughout the manuscript.

Response: Thank you for your suggestion. We deleted I, we and our throughout the manuscript.

Comment 9: The first four paragraphs of the introduction section is not good. Need to merge and concise the whole introduction into four paragraphs.

Response: Thank you for your suggestion. We revised the Introduction section thoroughly (line 56-93).

Comment 10: The introduction section seems missing important information. This section needs profound modification with more Up-to-date references.

Response: We revised the introduction thoroughly, and we added up-to-date references. (line 152-231)

Comment 11: Aim of the study not clear.

Response: Thank you so much for your input. Please refer to our response to your general comments, comment#4.

Comment 12: The information which authors provided is old.

Response: Thank you for your comment. We deleted several references and replaced them with recent references.

Materials and Methods:

Comment 1: Exclusion criteria…what was the purposes?

Response: Thank you for the question. We revised the exclusion criteria (line 155-157), Those who have had immunosuppressive conditions, or immunodeficiency disease, or is currently receiving any immunosuppressive therapy were not our exclusion criteria.

“The exclusion criteria are any eligible person who shows a flu-like syndrome on the screening day.”

Comment 2: Blood samples collection and storage. Need details.

Response: Thank you for your suggestion. We revised the blood samples collection and storage section (line 197-207, highlighted).

Comment 3: Data management plan: The writing is not good.

Response: Thank you for your comment. We revised data management plan section (line 227-242, highlighted).

“Data management plan

All respondents will have unique identification. Research nurse/field officers will enter data into eSynthesis (version 1.9.8 - A survey tool. Sleman, Yogyakarta, Indonesia), an application, that have been installed on their mobile gadget tablet. Supervisors and data managers will check the suitability of the data to ensure data quality. Maintaining data quality will include calibrating instruments regularly, and having a validation feature in the application. A red warning sign will appear if the input format does not match or if there are data that have not been filled in. The data will be cleaned several times by the data manager until ready for analysis. Data types consist of text, number, audio, and image data. The data consist of primary and secondary data. Primary data will be obtained by measuring directly and interviewing respondents in the field. Secondary data will be taken from the respondents' medical records. Data will be stored on Google Drive and institutional servers which are restricted to a particular research team. Data processing (collection, storage, use) in the study is based on the UGM Academic Hospital and the Health and Demographic Surveillance System Sleman (HDSS Sleman) data protection concepts.”

Comment 4: Safety considerations. Is this necessary?

Response: Thank you for pointing this out. We agree with your comment, and we deleted that sub section of safety consideration, accordingly.

Comment 5: Very few references are in the material and methods.

Response: Thank you. We revised the Material and methods section majorly, and added supportive references.

Comment 6: The type of data and statistical analyses planned: Not clear.

Response: Thank you. We revised the type of data and statistical analyses planned section. Please refer to line 252-263 (highlighted).

“The type of data and statistical analyses planned

Continuous variables with normal distribution will be reported as mean with standard deviation (SD), and non-normal distributed variables as median with interquartile range (IQR). The Kaplan-Meier method will be used to calculate cumulative seroconversion rates. The association between antibody level and disease severity will be estimated by logistic regression. The characteristics of the case and control groups will be described as mean and SD or frequency and percentage. The factors (risk and protective) associated with the COVID-19 infection will be determined using conditional (matched) logistic regression and presented as OR and its 95% CI. This study will be reported in accordance with the Strengthening the Reporting Observational Studies in Epidemiology (STROBE) checklist.36 Missing data and lost to follow-up patients will be reported. Missing data will be handled by using multiple imputation models.37 “

Comment 7: Patient and public involvement and ethics approval. Number is missing.

Response: Than you, the number of ethics approval was mentioned in line 266-269 (highlighted).

“This study was reviewed and approved by the Medical and Health Research Ethics Committee (MHREC) Faculty of Medicine, Public Health, and Nursing UGM (KE/FK/0882/EC/2020). Written informed consent will be from all respondents prior to enrolment into the study.”

Comment 8: Need to add references for different sections.

Response: Thank you for your suggestion. We added supporting references in every sub-section in the Methods.

Discussion:

Comment 1: The current discussion is poor.

Response: Thank you for your valuable comment. We revised the Discussion section majorly as you can refer to the line 279-362.

Comment 2: unknown of sero-conversion and sero-reversion of antibodies: Need to elaborate.

Response: Thank you. We elaborated this in the line 288-297 (highlighted).

“Potential key points relevant to the current pandemic include unknown seroconversion and seroreversion in hospitalized patients. Time to seroconversion may be related to the severity of disease. A study from North America showed that in symptomatic COVID-19 patients, the median time to seroconversion of SARS-CoV-2 RBD (IgG and IgM) among hospitalized patients was 4 days earlier compared to non-hospitalized patients, proposing an association between antibody kinetics and disease severity.8 IgG responses to the SARS-CoV-2 spike RBD showed minimal seroreversion, which were sustained for more than 3 months. Meanwhile, the IgM responses were short-term, and most patients seroreverted within 2.5 months following symptom onset.8”

Comment 3: Many text repetitions are found in the discussion section. It is highly recommended to emphasize findings and assumptions that support or disagree with other work(s).

Response: Thank you so much for your input. We revised the Discussion section majorly, and we added previous reports those support our study, since our current study is still on going.

Comment 4: Addendum, repetition of the results should be avoided in the discussion section.

Response: Thank you for your valuable input. We revised and deleted any repetition. Our study is still on going, so we still do not report any result.

Comment 5: It is still unclear how long the immunity lasts after patients recover from SARS-CoV2 infection…Is this okay?

Response: Thank you for your comment. We deleted that sentence and moved to that idea in introduction section (line 57-86).

Comment 6: Understanding about long-term antibody responses is currently limited…What’s the basis?

Response: Thank you. We deleted that sentence and moved that idea into the introduction section (line 73-86, highlighted), especially long-term antibody response in correlation with severity.

Comment 7: There had also been frequent adaptations…need more insights with references.

Response: Thank you for your suggestions. We added new references to support our discussion.

Comment 8: Challenges include a nationwide restriction: poor writings.

Response: Thank you for your comment. We revised the paragraph as you can find in line 299-302 (highlighted).

“As the SARS-CoV-2 delta variant surge occurred in mid-2021, several hamlets and sub-districts in the study area implemented lockdown.39 During the high transmission months, obstacles were encountered, that included patients’ reluctance to enroll because of fear that their infection status could be exposed”

Comment 9: Our study targets a large sampling…. not justified.

Response: Thank you, we revised the whole paragraph (line 358-362, highlighted).

“The study presented here will provide current and local evidence, even though it may not be generalizable on a global scale. The study outcome will provide data for better understanding in serological measurement of SARS-CoV-2 infection which will improve insights into at-risk population and post-infection long-term follow-up for the kinetics of anti-SARS-CoV-2 antibody.”

Figures:

Comment 1: The resolution of figures can be improved.

Response: Thank you for your suggestion. We uploaded the figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool to ensure that figure meets PLOS requirements.

References:

Comment 1: For better understanding, I feel this manuscript needs more detailed background information and a precise explanation of their study in the Introduction and Discussion section.

Response: Thank you for your valuable suggestion. We agree with your comment, therefore we revised the introduction and discussion section majorly.

Comment 2: Several published articles related to antibody responses following SARS-CoV-2 infection in humans must be included within the relevant text part of the manuscript.

Response: Several supporting and up-to-date references already added to the manuscript. Those references are as follow:

Crawford KH, Dingens AS, Eguia R, Wolf CR, Wilcox N, Logue JK, et al. Dynamics of neutralizing antibody titers in the months after severe acute respiratory syndrome Coronavirus 2 infection. J Infect Dis. 2021;223(2):197-205. doi: 10.1093/infdis/jiaa618

Wang H, Yuan Y, Xiao M, Chen L, Zhao Y, Zhang H, et al. Dynamics of the SARS-CoV-2 antibody response up to 10 months after infection. Cell Mol Immunol. 2021;18(7):1832-1834. doi: 10.1038/s41423-021-00708-6

Wang K, Long QX, Deng HJ, Hu J, Gao QZ, Zhang GJ. et al. Longitudinal dynamics of the neutralizing antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Clin Infecti Dis. 2021;73(3):e531-9. doi: 10.1093/cid/ciaa1143

Liu C, Yu X, Gao C, Zhang L, Zhai H, Hu Y, et al. Characterization of antibody responses to SARS‐CoV‐2 in convalescent COVID‐19 patients. J Med Virol. 2021;93(4):2227-2233. doi: 10.1002/jmv.26646

Feng C, Shi J, Fan Q, Wang Y, Huang H, Chen F, et al. Protective humoral and cellular immune responses to SARS-CoV-2 persist up to 1 year after recovery. Nat Commun. 2021;12(1):1-7. doi: 10.1038/s41467-021-25312-0

Gallais F, Gantner P, Bruel T, Velay A, Planas D, Wendling MJ, et al. Evolution of antibody responses up to 13 months after SARS-CoV-2 infection and risk of reinfection. EBioMedicine. 2021;71:103561. doi: 10.1016/j.ebiom.2021.103561

Lumley SF, Wei J, O’Donnell D, Stoesser NE, Matthews PC, Howarth A, et al. The duration, dynamics, and determinants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody responses in individual healthcare workers. Clin Infect Dis. 2021;73(3):e699-709. doi: 10.1093/cid/ciab004

Petersen MS, Hansen CB, Kristiansen MF, Fjallsbak JP, Larsen S, Hansen JL, et al. SARS-CoV-2 natural antibody response persists for at least 12 months in a nationwide study from the Faroe Islands. Open Forum Infect Dis. 2021;8(8):ofab378. doi: 10.1093/ofid/ofab378

Varona JF, Madurga R, Peñalver F, Abarca E, Almirall C, Cruz M, et al. Kinetics of anti-SARS-CoV-2 antibodies over time: results of 10 month follow up in over 300 seropositive health care workers. Eur J Intern Med. 2021;89:97-103. doi: 10.1016/j.ejim.2021.05.028

Dan JM, Mateus J, Kato Y, Hastie KM, Yu ED, Faliti CE, et al. Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science. 2021;371(6529):eabf4063. doi: 10.1126/science.abf4063

Anand SP, Prévost J, Nayrac M, Beaudoin-Bussières G, Benlarbi M, Gasser R, et al. Longitudinal analysis of humoral immunity against SARS-CoV-2 spike in convalescent individuals up to 8 months post-symptom onset. Cell Rep Med. 2021;2(6):100290. doi: 10.1016/j.xcrm.2021.100290

In addition to the above comments, all spelling and grammatical errors pointed out by the reviewers have been corrected.

We look forward to hearing from you in due time regarding our submission and to respond to any further questions and comments you may have.

Sincerely,

Lutfan Lazuardi

On behalf of all authors

Submitted filename: Response to reviewers.docx

Click here for additional data file.

8 Jul 2022

25 Jul 2022

Dear Academic Editor

PLOS ONE

Thank you for giving us the opportunity to submit a second round revised draft of our manuscript titled “Two years antibody responses following SARS-CoV-2 infection in humans: a study protocol” to PLOS ONE.

Again, we appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback after our first response. We have highlighted the changes within the manuscript and you can identify the changes from track changes. Here is a point-by-point response to the reviewers’ comments and concerns.

Reviewer #1: Reviewer #1: Thank you for revising the manuscript according to my comments. The revision has made significant improvements to the overall quality of the manuscript, especially in the Methodology section. I think the manuscript can now be accepted for publication.

Response: Thank you so much for your positive comment.

Reviewer #2: Although the article has scientific rigor, several minor flows need to be corrected before publication.

Minor comments:

Comment 1:

Introduction: The introduction section seems missing important information. This section needs profound modification with more Up-to-date references.

Response: Thank you for your valuable feedback. We revised the introduction section (highlighted)

Comment 2:

Discussion: The discussion section can improve.

Response: Thank you so much. We appreciate your comment. We revised the discussion section (highlighted)

We look forward to hearing from you in due time regarding our re-submission.

Sincerely,

Lutfan Lazuardi, MD, PhD

Corresponding author

On behalf of all authors

Juli 16, 2022

Submitted filename: Response to Reviewers-round2.docx

Click here for additional data file.

26 Jul 2022

Two years antibody responses following SARS-CoV-2 infection in humans: a study protocol

PONE-D-21-34739R2

Dear Dr. Lazuardi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Harapan Harapan, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

4 Aug 2022

PONE-D-21-34739R2

Two-years antibody responses following SARS-CoV-2 infection in humans: a study protocol

Dear Dr. Lazuardi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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