Immunological changes in a cohort of COVID-19 survivors: Mansoura University experience

Background: COVID-19 is a global pandemic that has affected millions of people all over the world since 2019. Infection with COVID-19 initiates a humoral immune response that produces antibodies against specific viral antigens, which in turn is supposed to provide immunity against reinfection for a period of time. The aim of this research was to study the kinetics of IgM and IgG antibodies against SARS-CoV-2. Methods: One hundred and seventeen post-COVID-19 participants were enrolled in the study. Qualitative assessment of IgM and IgG antibodies over six months (three visits) post recovery was conducted. Results: The current study revealed a significant reduction in IgM and IgG titers between the first and second visits (p <0.001). After six months, the antibody titer had declined by 78.8% from the first visit for IgM and by 49.2% for IgG antibodies. Regarding younger age and male sex, statistically significant persistence of IgM antibodies was noticed at the six months follow up. Also, statistically significant persistent IgG immunity was found in male patients and diabetics by the end of the six months follow up. Conclusions: We observed a significant waning of IgM and IgG titers over a period of six months follow up.. The persistence of positive IgM and IgG antibodies by the end of six months was variable due to differences in age, gender and presence of diabetes mellitus.

Introduction COVID-19 is considered the most catastrophic pandemic in the 21 st century until now.Globally, as of 3 June 2022, 5:37pm CEST, there have been 528,816,317 confirmed cases of COVID-19, including 6,294,969 deaths, reported to WHO (WHO, 2022).
Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, initiates a humoral immune response that produces antibodies against specific viral antigens.These include anti-S protein antibodies that target the virus spike S1 protein subunit and receptor binding domain (RBD) (CDC, 2022).
Antibodies are detected in the blood of people who have been previously infected with a virus that causes a disease; they show the body's efforts to fight off a specific virus.Antibodies may protect people from reinfection or becoming severely ill for some time afterward (Qu et al., 2020).However, antibodies wane over time and how quickly antibodies wane is different for each disease and each person.
The aim of this study was to identify the kinetics of IgM and IgG antibodies in a cohort of COVID-19 survivors over six months' follow up and evaluate the longevity of immunity provided by COVID-19 infection.

Study design and setting
This longitudinal follow up study was conducted between May 2020 to April 2021 at post-COVID-19 outpatient clinics at several Mansoura University Main Hospital.Ethical approval was obtained from the Mansoura University Institutional Research Board (IRB) (approval number: RP.20.05.67).Written informed consent was taken from all participants and all study participants were assured of the confidentiality and anonymity of the data.

Study participants
A convenience sample of 123 post-COVID-19 patients who attended the outpatient's clinic were recruited.The follow up schedule was explained to them and they were free to leave the study at any time.Six participants were excluded because they refused follow up.Consequently, 117 post-COVID-19 participants were studied.Follow up was conducted at one month (first visit), three months (second visit) and six months (third visit) after recovery from COVID-19.Of 117 survivors who attended the first visit, only 98 attended the second visit and 76 completed the third visit (Figure 1).Demographics, comorbidities, severity of previous COVID-19 and laboratory data of the participants were recorded.Severity of previous COVID-19 infection was classified according to WHO severity definitions (WHO, 2021).

Data collection tool
A clinical sheet was used including demographic data such as age, gender, marital status, smoking status, history relevant to clinical presentation and medical comorbidity, pulmonary function testing, electrocardiogram, necessary laboratory analysis such as complete blood picture (CBC), COVID-19 IgG and IgM antibodies titer and computed tomography (CT) chest (see Extended data (Khashaba, 2023)).

REVISED Amendments from Version 1
Limitations of study are now added.Few modifications in discussion to illustrate the significant difference in Antibody levels according to age according to reviewer comments.One Keyword is added.Changing the word neutralising to neutralizing throughout the article.
Any further responses from the reviewers can be found at the end of the article

Statistical analysis
The collected data was revised, coded, tabulated and introduced to a PC using Statistical Package for Social Science software (IBM SPSS Statistics for Windows, Version 23.0.Armonk, NY: IBM Corp).Descriptive statistics in the form of means and standard deviation (AESD) was used for normally distributed numerical data, while median and range was used for non-normally distributed numerical data.The frequency and percentage of qualitative data was recorded.The Wilcoxon signed rank test was used to compare quantitative data at different times, which was non-normally distributed.A Chi square test was used to compare qualitative data in two groups.The McNemar test was used to compare immune status at different points of time.Logistic regression analysis was used for predictors of positive immunity among the studied group using a forward Wald model.Adjusted odds ratios and their 95% CIs were calculated.A p-value less than 0.05 was considered as statistically significant.

Results
Baseline characteristics of the cohort (117 post-COVID-19 participants) were as follow: mean age was 54 years old, about 54.7% of the participants were male, diabetes mellitus was detected in 38.5% while hypertension was detected in 34.5%.In addition, 46.2% and 47.9% of participants previously had non-severe and severe COVID-19 infection respectively (Underlying data (Khashaba, 2023)).Of 117 participants, only 98 were enrolled in the second visit (three months post COVID-19) of which one IgM result was not recorded.Only 76 participants completed the third visit (six months post COVID-19) (Table 1).
Regarding the COVID-19 IgM antibody titer, there was significant reduction in IgM titer between the first and second visits (percentage change 68.8%; p<0.001).Moreover, a reduction in the IgM titer was observed between the second and third visit (percentage change 32.3%).After 6 months follow up (third visit) COVID-19 IgM titer declined by 78.8% from the first visit (Underlying data (Khashaba, 2023)).
A total of 88% (103 out of 117) of participants had a positive COVID-19 IgM in the first visit and after six months follow up 53.9% (41 out of 76) participants had a positive COVID-19 IgM and the difference was statistically significant (p<0.05)(Table 1).
For the COVID-19 IgG antibody titer, there was a significant reduction in IgG titer between the first and second visits (p<0.001).Also, a significant reduction in IgG titer was observed between the first and third visit.After six months follow up, at the third visit, the COVID-19 IgG titer had declined by 49.2% from the first visit (Table 1).
A total of 93.2% (109 out of 117) participants had positive COVID-19 IgG at the first visit and after six months follow up 60.5% (46 out of 76) of participants had positive COVID-19 IgG (Table 1).
The existence of a positive IgM test at the third visit was significantly associated with the age of the studied participants (mean age was 52 in participants with a positive test compared to 57.4 in participants with a negative test (p=0.04).Also, a positive test was more frequent among males than females (p=0.04).Moreover, positivity was significant among healthcare workers compared to non-healthcare workers (p=0.04).Although a non-significant association was detected between a positive IgM test and diabetes mellitus, a positive IgM test was frequent among non-diabetic participants (60.9% versus 43.3%).In addition, a positive IgM test was frequent among non-smokers and participants who had previously had severe and critical COVID-19 despite no statistical significance (Table 2).
On studying persistent IgG positivity at the third visit, it was slightly associated with older age of studied participants (mean age was 56.7 in participants with a positive test compared to 51.4 in participants with a negative test (p=0.05)).Also, a positive test was more frequent among males than females (p=0.008).A significant association was detected between a positive IgG test and diabetes mellitus as it was more frequent among diabetic than non-diabetic participants (83.3% versus 45.7%).Finally, a positive IgG test was more frequent among non-smokers, and participants previously having had severe and critical COVID-19 despite the absence of statistical significance (Table 3).
There was an overall reduction in the level of IgM from 10.9 units at one month to 3.4 units after three months and 2.3 units at six months (Figure 2).There was an overall reduction in the level of IgG from 6.7 units at one month to 4.3 units after three months and 3.4 units at six months (Figure 3).When relating this positivity with different factors, there was significant association with younger mean age of 52 years old and male sex.Although not statistically significant, there was persistently more positive IgM in non-smokers, nondiabetics, non-hypertensive and in more severe and critical cases.Forty-six out of 76 cases remained IgG positive by the end of the sixth month.Positive IgG immunity is found more frequently and statistically significant in older participants, male sex and diabetics.Although not statistically significant, there was persistently more positive IgG in non-smokers, hypertensive, and in more severe and critical cases.Based on logistic regression analysis, age, sex and occupation are significant predictors for positive IgM immunity; older age is associated with a decreased chance of positive IgM.
The persistence of IgG antibodies in diabetics compared to non-diabetic participants may be explained by more severe disease in diabetic patients.This is supported by several studies which stated that ICU patients showed a faster and increased neutralising antibody response compared to non-ICU patients (Lau et al., 2021;Liu et al., 2020;Qu et al., 2020;Williamson et al., 2020).The association between serum IgA, IgG, and IgM levels and glycated haemoglobin, an indicator of long-term diabetes control, fructosamine, and 111 healthy non-diabetic people was examined in 110 diabetic patients.While the concentration of IgM was considerably lower (by 46.7%, p<0.001) in diabetic patients compared to non-diabetic participants, there were significant increases in serum IgA (by 82.7%, p 0.001) and IgG (by 35.2%, p 0.001) concentrations (Ardawi et al., 1994).

Major concerns.
Have all study participants not received any COVID-19 vaccination before the study termination?Vaccination during the study followed-up may alter the immunologic outcome and distort the results. 1.
Have all study participants got re-infection before the study termination?Infection during the study followed-up may alter the immunologic outcome and distort the results. 2.

Minor concerns.
"The assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG and IgM in human serum or plasma against the SARS-CoV-2 nucleoprotein."Are you sure that the IgM assessment in this study targeted anti-nucleocapsid? 1.
There was a lacks of information about the immunity trend (immunity waning).2.

Comments.
You can add a further keyword, such as; anti-nucleocapsid. 1.

2.
Suggest using one style of English.I found this manuscript contained both UK and US styles.For example, neutralising and neutralizing.

3.
The age group can be stratified (adolescent, young adult, middle-aged and elderly) to see more immunity waning trend in all Tables and Figures.Because a young age has higher immunity than an elder.If it works.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Clinical Trials, Vaccine Trials, Immunology, Infectious Diseases 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.

1.
In method of the Chemiluminescent immunoassay, the authors mention that they used human serum or plasma for IgG and IgM measurements.It should be noted that plasma was isolated from the vacutainer contained EDTA or heparin.There might be a difference in dilution factor depending on the isolation methods used.It is not clear if such dilution factor would affect the results with 2 times difference between IgG_1m and IgG_6m (Figure 3).The authors should clarify this.

3.
What's the detection limit of the IgM and IgG assay or how to define the positive and negative of the test?In Table 2 and Table 3, the authors separated the positive test and negative test for comparison.Can the authors explain? 4.
Human serum or plasma samples isolated from individuals without infection history should be included as cutoff values.Since NP is quite conserve between common cold coronavirus and SARS-CoV-2, the issue of antibody cross reactivity should be addressed.Reviewer Expertise: Immunity 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.
Author Response 20 Aug 2023

Eman Khashaba
Comment 1: The authors measured IgG and IgM titer against SARS-CoV-2 NP, not the spike.However, neither anti-NP IgM nor IgG have neutralizing activity.Although the results provide some information on humoral responses, the value for vaccine-mediated protection is limited.The authors should discuss this limitation of the study.Reply: Vaccine mediated immunity was not studied in current research as all study subjects were post-Covid and none of them received vaccination.This note will be added to limitations of study after discussion.
Comment 2: in method of chemiluminescent immunoassay, the authors mentioned that they used human serum or plasma IgG and IgM.It should be noted that plasma was isolated from vacutainer contained EDTA or heparin there might be difference in dilution factor There might be a difference in dilution factor depending on the isolation methods used.It is not clear if such dilution factor would affect the results with 2 times difference between IgG_1m and IgG_6m (Figure 3).The authors should clarify this.…….Reply: All samples included in the study were serum samples, the chemiluminescent microparticle immunoassay can detect IgG and IgM in human serum or plasma but only serum samples were included in this study as stated on page 3 so there was no need for a dilution factor …….
Comment 3: There were no standard assays used in this study for comparison with other studies.WHO already established the international standard for anti-SARS-CoV-2 immunoglobulin….. (https://cdn.who.int/media/docs/default-source/biologicals/bsdocuments-(ecbs)/2022-documents/new-2022-document-susan/bs-2022.2427_mattiuzzo-g._sars-cov-2_ab_2ndisandrpfor-voc_final.pdf?sfvrsn=90585abb_1&download=true).The authors may test some samples using a standard assay to increase the value of this study.Reply: We have used FDA approved standardized commercial kits from Abbott laboratories with provided calibrators used in calculation of antibody levels Comment 4: What is the detection limit of IgM and IgG assay or how to define the positive or negative …… In Table 2 and Table 3, the authors separated the positive test and negative test for comparison.Can the authors explain?Reply: The amount of IgG and IgM antibodies to SARS-CoV-2 in each sample is determined by its chemiluminescent relative light unit (RLU) to the calibrator RLU using an index S/C threshold of 1.4 for IgG and 1.0 for IgM as stated on page 3 Comment 5: Human serum or plasma samples isolated from individuals without infection history should be included as cutoff values.Since NP is quite conserve between common cold coronavirus and SARS-CoV-2, the issue of antibody cross reactivity should be addressed.Reply: We used FDA approved standardized commercial kits from Abbott laboratories with provided calibrators used in calculation of antibody levels.Cross reactivity could not be addressed in this study.This comment was added to limitations of study Competing Interests: No competing interests were disclosed.
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5 .
Is the work clearly and accurately presented and does it cite the current literature?PartlyIs the study design appropriate and is the work technically sound?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?Not applicableAre all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes Competing Interests: No competing interests were disclosed.

Table 2 .
Association between patients' characteristics and IgM level six months' post COVID-19.
Chi square test, row percent is considered.