Seroreactivity of the Severe Acute Respiratory Syndrome Coronavirus 2 Recombinant S Protein, Receptor-Binding Domain, and Its Receptor-Binding Motif in COVID-19 Patients and Their Cross-Reactivity With Pre-COVID-19 Samples From Malaria-Endemic Areas

Despite the global interest and the unprecedented number of scientific studies triggered by the COVID-19 pandemic, few data are available from developing and low-income countries. In these regions, communities live under the threat of various transmissible diseases aside from COVID-19, including malaria. This study aims to determine the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroreactivity of antibodies from COVID-19 and pre-COVID-19 samples of individuals in Mali (West Africa). Blood samples from COVID-19 patients (n = 266) at Bamako Dermatology Hospital (HDB) and pre-COVID-19 donors (n = 283) from a previous malaria survey conducted in Dangassa village were tested by ELISA to assess IgG antibodies specific to the full-length spike (S) protein, the receptor-binding domain (RBD), and the receptor-binding motif (RBM436–507). Study participants were categorized by age, gender, treatment duration for COVID-19, and comorbidities. In addition, the cross-seroreactivity of samples from pre-COVID-19, malaria-positive patients against the three antigens was assessed. Recognition of the SARS-CoV-2 proteins by sera from COVID-19 patients was 80.5% for S, 71.1% for RBD, and 31.9% for RBM (p < 0.001). While antibody responses to S and RBD tended to be age-dependent, responses to RBM were not. Responses were not gender-dependent for any of the antigens. Higher antibody levels to S, RBD, and RBM at hospital entry were associated with shorter treatment durations, particularly for RBD (p < 0.01). In contrast, higher body weights negatively influenced the anti-S antibody response, and asthma and diabetes weakened the anti-RBM antibody responses. Although lower, a significant cross-reactive antibody response to S (21.9%), RBD (6.7%), and RBM (8.8%) was detected in the pre-COVID-19 and malaria samples. Cross-reactive antibody responses to RBM were mostly associated (p < 0.01) with the absence of current Plasmodium falciparum infection, warranting further study.


INTRODUCTION
Coronaviruses are a group of enveloped viruses containing a single-stranded RNA genome with positive polarity (1). They include severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome (MERS-CoV) (1)(2)(3), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or COVID-19 (4,5). COVID-19 affects people of all ages, but morbidity and mortality are more significant in the elderly and those with chronic diseases (6)(7)(8)(9). Emerging in China in 2019 (10), COVID-19 rapidly spread worldwide and was declared a pandemic by the WHO in March 2020 1,2 . More than 200 million cases and over 4 million deaths have been reported worldwide, affecting 220 countries and territories 3 , generating massive economic and social consequences. The first COVID-19 case diagnosed in Mali was reported on March 25, 2020, and Malian health authorities quickly established a strategy to control the disease 4 . In addition, the authorities have promoted the harmonization of research activities by leveraging research laboratory capacities and strengthening relationships among local and international stakeholders (11)(12)(13).
Despite considerable global efforts to study the immune responses elicited by SARS-CoV-2 and their role in clinical protection and pathogenesis (14)(15)(16)(17), the host factors leading to low or moderate clinical manifestations, as well as completely asymptomatic infections, are not well understood. Initial analysis indicates that certain populations have been exposed to other microorganisms, either pathogenic or non-pathogenic, which appear to induce immune responses against COVID-19 (i.e., antibodies or potentially other immune effectors that contribute to reducing or preventing COVID-19 clinical manifestations (18)(19)(20)(21)(22)(23)(24)(25)).
Specific antibody responses to COVID-19 have been reported in moderately and severely symptomatic SARS-CoV-2-positive individuals (26)(27)(28)(29)(30)(31)(32). However, there are few data available linking symptomatic disease and duration of hospitalization or treatment with specific antibodies to SARS-CoV-2 antigens. Such antibodies may be detected as early as the end of the first week of illness; however, they may also take weeks to appear, giving rise to different clinical outcomes (29,33). In addition, the presence or absence of protective immunity due to infection or vaccination may affect future transmission and disease severity (29).
Of notable importance, it has been observed that there are significantly lower COVID-19 clinical cases and fatalities in malaria-endemic regions than in non-endemic areas (19,22,34). Several host factors, including sociodemographic conditions, genetic background, and immune status, could be influencing the COVID-19 clinical evolution. Moreover, other SARS cases, induced by viruses potentially sharing common immunodominant antigens, might affect the outcome of the disease (18,20,35,36).
Considering the burden of malaria in Mali (37) and the potential for clinical overlap with COVID-19, efforts to both study diseases and understand the potential immunological interplay are ongoing (19,22). This potential relationship has tremendous epidemiological relevance not only for understanding clinical outcomes in malaria-endemic and non-endemic regions but also for COVID-19 vaccination efforts. In the absence of a specific anti-SARS-CoV-2 treatment, research into this area is of considerable importance.
The spike (S) protein is encoded by a systematic interplay between the SARS-CoV-2 genome, the nucleocapsid (N), the membrane (M), the envelope (E), and various additional structural proteins. It plays a crucial role in viral infection and pathogenesis of COVID-19 (38,39), as it is essential for the viral invasion of the host cell, mainly through its RBD domain (5,9,37). Both RBD and its ligand, the human angiotensin-converting enzyme-2 (ACE2), are crucial research targets for developing COVID-19 therapeutic antibodies, vaccines, and serological tests (2,(40)(41)(42)(43)(44)(45). Currently, most COVID-19 vaccines in use or development are based on the S protein; however, the different vaccine platforms have demonstrated a variety of strengths and weaknesses 5 . In addition to the commonly used S protein and its RBD, we designed (manuscript submitted) and studied the S protein's receptor binding motif (RBM 436-507 ) that interacts with ACE2.
Vaccine success is likely associated with the specificity and strength of the immune response it triggers against the S protein, specifically against its RBD. However, this immune response may also correlate with factors like age, gender, ethnicity, disease experience (i.e., disease evolution), treatment duration, and comorbidities, among others (6)(7)(8).
In light of all these issues, this study aimed to assess the natural antibody response specific to the full-length S protein, its functional domains RBD (protein), and RBM (peptide) using plasma collected from COVID-19-positive patients and pre-COVID-19 participants from a malaria-endemic region. The epidemiological paradox observed in COVID-19 and malaria patients in the initial phase, and in the dynamics of infection in malaria-endemic countries (19,22), promotes the need for further studies in this area to produce a better understanding of the genetic and immunological factors involved.

Study Type, Periods, and Sites
A cross-sectional study was conducted to assess the seroreactivity of COVID-19 patients and pre-COVID-19 donors against the SARS-CoV-2 full-length recombinant S protein and its binding domains RBD and RBM. Samples were collected from the Dermatology Hospital of Bamako (HDB) in Mali (West Africa); sociodemographic and epidemiological surveys were also carried out. While all COVID-19 blood samples were collected from patients confirmed to harbor SARS-CoV-2 by RT-PCR test, pre-COVID-19 plasma samples were gathered in 2019-before the onset of the COVID-19 pandemic-and therefore were not tested by COVID-19 RT-PCR. The latter were collected from donors living in the Village of Dangassa in Mali, a malaria-endemic zone, and were stored frozen at −20°C. All laboratory tests were performed at the Laboratory of Immunogenetic and Parasitology, at the International Centre of Excellence in Research (ICER-Mali) of the University of Sciences, Techniques and Technologies of Bamako (Mali). The data management and sample processing were carried out from May 2021 to September 2021.

Study Population
The study population included COVID-19-infected patients (n = 266; sex ratio = 1.2 in favor of men) with SARS-CoV-2 confirmed by RT-PCR and admitted to the HDB for inpatient care. The pre-COVID-19 population consisted of volunteers (n = 283; sex ratio = 1.1 in favor of women) who had participated in a previous malaria survey study in 2019, before the onset of COVID-19 in Mali. The study population (COVID-19 and pre-COVID-19 participants) were stratified by age groups 1-4, 5-9, 10-14, 15-19, 20-29, 30-39, 40-49, 50-59, 60-69, and 70+ years. This adjusted for the age structure of the population as recommended by the WHO guidelines on population-based sero-surveys of SARS-CoV-2 infection 6 . COVID-19 participants provided sociodemographic and epidemiological data, including comorbidities and length of treatment duration. Pre-COVID-19 participants had records of sociodemographic and epidemiological data, and current Plasmodium falciparum infection (parasitemia) was confirmed by microscopic examination after Giemsa staining of blood smear (BS) slides. None of the participants had a history of COVID-19 vaccination.

Ethical Considerations
This study was approved by the Institutional Review Board (Ethics Committee, EC) of the Faculties of Medicine and Odontostomatology and of the Pharmacy of Bamako (with reference N°2021/25/CE/USTTB). Written informed consent (IC) was obtained from each COVID-19 patient for the collection of blood samples, sociodemographic information, and clinical data for future investigative purposes. The authorization of the use of pre-COVID-19 samples and data was also obtained from the same EC and under the reference cited above. The current study was based on available data from participants whose plasma samples and related data were available and accessible. The confidentiality of the participants' data was preserved throughout this study.

Variables, Data, and Sample Collections
Data analysis was carried out using medical records from the HDB data register. Data were collected at the time of hospital admission (on week 1) and during hospitalization at HDB in 2020. Data were collected using a paper questionnaire developed for this purpose, including 1) sociodemographic information; 2) symptoms and severity of disease; 3) comorbidities or factors such as diabetes, hypertension, asthma, and body weight; 4) clinical evolution of the disease's form; and 5) duration of hospital stay or treatment. The pre-COVID-19 participant samples were collected from the village of Dangassa in 2019 before the onset of COVID-19 in Mali. The variables in the pre-COVID-19 group included sociodemographic (age and gender) and epidemiological data such as the presence and density of current P. falciparum infection. A BS slide was performed and examined by microscopy for the presence and density of P. falciparum [positive (BS+) or negative (BS−) for each pre-COVID-19 sample].
Whole blood (5-10 ml) was collected from each COVID-19 patient by venipuncture upon admission to HDB, and the sample transportation to the laboratory was carried out following the WHO guidelines for Infectious Substances 2019-2020 (46). Trained biologists were responsible for ensuring compliance with these guidelines.

Protein Sequence Analysis, Design, and Antigen Production
Sequences of the S protein were downloaded from the National Center for Biotechnology Information (NCBI) SARS-CoV-2 Resources 7 . Recombinant proteins from the full-length S and RBD were provided by ExcellGene SA (Monthey, Switzerland) and Protein Production and Structure Core Facility, EPFL (Lausanne, Switzerland) 8 . Proteins were produced according to the manufacturer's recommendations 9 . A peptide covering the receptor-binding interface (receptor binding motif, RBM 436-507 ) of the S protein was synthesized at the Chemistry Department, Florence University, Florence, Italy. RBM is known to undergo some post-translational modifications (PTMs) such as glycosylation, but this does not directly contribute to the binding affinity between SARS-CoV-2 S and ACE-2 (47). In addition, as it is a synthetic product used in ELISA, RBM is not expected to undergo any further modification. The 3D images were generated using PyMol software, an open-source molecular graphics tool (48) using the atomic coordinates from PDB entry 6ZOY (49). The illustrative diagram of domains, amino acid sequences, and the 3D structure of the S protein displaying both the RBD and RBM sequences are all shown in Supplementary Figure 1.

Enzyme-Linked Immunosorbent Assay
Sample seroreactivity was studied using an ELISA with 96-well plates (type of plate, Ref 442404). Plates were coated with 1 mg/ ml of S, RBD, or RBM (antigen coating) or not coated with an antigen (non-antigen coating) and then incubated overnight (O/ N) at 4°C. The plates were then blocked for 1 h at room temperature (RT) with phosphate-buffered saline (PBS) 1× (3% milk) before being incubated for 2 h at RT with COVID-19 and pre-COVID-19 plasma samples at a dilution of 1:100. Goat antihuman IgGs, conjugated to horseradish peroxidase (HRP), were used as secondary antibodies, diluted to 1:5,000 (Life Technologies, Carlsbad, CA, USA; Ref H10307), and incubated for 1 h at RT. Signals were revealed using TMB substrate reagent (BD OptEIA, cat 555214; BD Biosciences, San Jose, CA, USA) for 20 min in the dark at RT, and the reaction was stopped using 1 M of sulfuric acid (Merck, Darmstadt, Germany; 1.00731.1000). Optical density (OD) was measured at 450/630 nm in a microplate ELISA-Reader (SoftMax ® Pro Software). Samples were considered positive when their mean OD was ≥mean OD + 3SD of the negative control samples (indicated as the cutoff). The cross-reactivity of pre-COVID-19 samples was considered significant for the samples with a mean OD ≥ mean OD + 3SD of the negative controls with a dilution of 1:100 (indicated as the cutoff). Non-specific binding samples (i.e., samples with antibody responses in non-antigen-coated plates), were determined to be samples with an OD against non-coated plates greater or equal to the same sample's response against antigen-coated plates (i.e., responder sample).

Data Management and Statistical Analysis
Data from the coded questionnaires were directly entered into the electronic data entry system during data and sample collection. Each participant was assigned a number that was known only to the investigators. The information was entered in Excel 2013, and ELISA data were imported directly into Excel and associated with the participants' sociodemographic and epidemiological data. The analysis and generation of figures were done with Stata and Prism 5 software. The unpaired ttest, chi-squared test, and Fisher's exact test were used to compare groups with a significance threshold of 5%.

Sequences and 3D Structures of S Protein, and the Receptor-Binding Domain and Receptor-Binding Motif Domains
Three antigens, namely, the full-length S protein (1250 aa), its RBD (211 aa), and a synthetic peptide covering the binding interface (RBM; 72 aa) of RBD, were used in this study (Supplementary Figure 1). The S protein plays a crucial role in viral infection and pathogenesis, as it mediates the SARS-CoV-2 binding to human ACE2. It comprises two functional subunits: S1, which harbors the N-terminal domain (NTD) and the receptorbinding domain (RBD), responsible for binding to the host cell receptor; and the S2, which harbors the heptad repeat 1 (HR1) and 2 (HR2), responsible for the fusion of viral and cell membranes (39) (Supplementary Figure 1A). The full-length sequence of the S protein of SARS-CoV-2 was obtained using the BLASTP search program (50,51). The SARS-CoV-2 RBD shows significant sequence homology (~73%) with seasonal phylogenetically related coronaviruses (25, 52-54) (Supplementary Figure 1B). The RBM is a segment representing approximately 6% of the S protein's length, located within the RBD domain. It is recognized by the ACE2 protein and not only represents the most variable region of the protein but is also highly specific to SARS-CoV-2 (Supplementary Figure 1C). The 3D image of the SARS-CoV-2 S protein structure was made while displaying the RBD and RBM locations (48, 49) (Supplementary Figure 1D).
When only the reactive samples (responders) were assayed, the S protein showed a higher median OD for Q1 and Q3 This reactivity would be relevant in selecting antibody donors and antigens for further analysis. The recognition of S correlated with recognition of RBD (r = 0.63, p = 0.001; Figure 2A), and recognition of RBD correlated with recognition of RBM (r = 0.45, p = 0.001; Figure 2B). In contrast, there was little correlation between the recognition of S and the recognition of RBM (r = 0.003, p = 0.9; Figure 2C). Although samples from pre-COVID-19 volunteers (n = 283) presented lower reactivity frequencies and ODs than the COVID-19 samples (p < 0.05; Figures 1, 2; Table 1), they still displayed a significant level of cross-reactivity against the three antigens (see Figure 3 and Supplementary Figure 4).
The analysis of IgG antibody levels by gender (male (M) and female (F)) in the COVID-19 patient group indicated comparable results between the two genders for each antigen   Table 2). Furthermore, the non-specific binding of antibody samples in COVID-19 patients accounted for 8.9% (17 out of 189), and 14.1% (12 out of 85) of the seroreactive samples for S, RBD, and RBM, respectively (Table 3).
Overall, antibody levels increased as a function of ageparticularly for S and RBD-but not for the RBM fragment ( Figure 4). Furthermore, antibody levels to S and RBD were comparable at the earlier ages under 19 and above 59 years and were significantly greater than those against RBM. Here, we analyze the association between antibody levels toward S, RBD, and RBM at the time of hospital admission and duration of treatment (i.e., the remission of symptomatic forms). Duration of remission was thus defined as the estimated time in days (≤30 or >30 days) from hospital admission to recovery from symptomatic SARS-CoV-2 infections, as confirmed by at least two negative RT-PCRs. Overall, the duration of treatment was shorter for participants who had higher antibody levels at admission for all three antigens, especially for RBD (p < 0.01) ( Figure 5). In addition, for the patient group with treatment periods ≤30 days, Ab levels for S, RBD, and RBM varied more significantly from each other (p < 0.0001) than among those hospitalized for longer periods (p = 0.037) ( Figure 5). However, the proportion of responder samples for S, RBD, or RBM was comparable between the ≤30and >30-day treatment groups ( Figure 5).

Preexisting Comorbid Conditions and Elicitation of Anti-S, Receptor-Binding Domain, and Receptor-Binding Motif Antibodies Among COVID-19 Patients
Comorbidities such as diabetes, hypertension and asthma, and high body weight were evaluated as factors that may impact the effective development of antibodies against S, RBD, and RBM in COVID-19 patients. The antibody levels (mean OD) for S, RBD, and RBM were similar between the patient groups with and without arterial hypertension (AHT) and were slightly higher in the patient groups not suffering from diabetes or asthma ( Figures 6A-C). Similarly, the prevalence of antibody responders for S and RBD remained similar between patient groups with or without comorbidity (p > 0.05; Table 4), whereas COVID-19 patient groups suffering from asthma and diabetes showed no positive antibody responses against RBM ( Table 4). In addition, increasing body weight was associated with a significant decrease in antibody responses to S and a slight decline in antibody response to RBD ( Figure 6D). The occurrence of two or more simultaneous comorbidities in a COVID-19 patient did not significantly impact the level of anti-S-and RBD-specific antibodies; however, there was no correlation between two comorbidities in COVID-19 patients and the response against RBM (Supplementary Figures 3A-C).

Level of Anti-S, Receptor-Binding Domain, and Receptor-Binding Motif Cross-Reacting Antibodies and Active Malaria Infection in the Pre-COVID-19 Malaria Infection Samples
The cross-reactivity of S, RBD, and RBM among the pre-COVID-19 samples from donors living in malaria-endemic areas (Dangassa village) was studied. The antibody OD FIGURE 3 | Cross-reactivity and non-specific binding against S, receptor-binding domain (RBD), and receptor-binding motif (RBM) in pre-COVID-19 and endemic malaria samples. The cross-reactive antibody levels (mean optical density (OD) shown as a horizontal black line in the dot plots) for S, RBD, and RBM were demonstrably higher than in non-specific binding antibody levels; this was significant for S (p < 0.01). The table shows the number and proportion (frequency) of samples showing cross-reactions or non-specific binding for S, RBD, and RBM. N, total number of pre-COVID-19 samples; n, number of cross-reactive or nonspecific binding samples; %, percent of cross-reactive or non-specific binding samples; Q1, quartile 1; Q3, quartile 3. The unpaired t-test and ANOVA were used to compare mean antibody ODs between different groups and within the groups themselves, respectively. **p ≤ 0.01; ns, not significant. distribution was similar among the S, RBD, and RBM (p > 0.05; Figure 3) with respective median antibody ODs (Q1; Q3) of 0.347 (0.269; 0.521), 0.324 (0.308; 0.351), and 0.391 (0.315; 0.467). There was a higher frequency of cross-reactive samples for S (21.9%) than for RBD (6.7%) or RBM (8.8%) (Figure 3). In addition, cross-reactive antibodies against all three antigens were present in all age groups; however, they were higher for S and RBM in most age ranges than they were for RBD (Supplementary Figure 4). No significant correlation was found between the density of malarial parasitemia and the level of antibodies cross-reacting with S (r = 0.10 p = 0.09; Figure 7A), RBD (r = 0.06, p = 0.35; Figure 7B), or RBM (r = −0.07 p = 0.27; Figure 7C). In contrast, cross-reacting antibodies appeared to be more common in samples without parasitemia (i.e., without active P. falciparum infection, or BS− samples), representing 77.4% (42 out of 62), 100% (19 out of 19), and 88% (22 out of 25) of the cross-reactive samples against S, RBD, and RBM, respectively ( Figure 7D). This correlation is made evident by the fact that BS− samples demonstrated significantly higher mean antibody ODs against RBM than BS+ samples ( Figure 7D).  patients. Antibody responses against S, RBD, and RBM were studied for each age group of COVID-19 patients. A correlation was observed between increasing antibody levels and increasing age. The average Ab response (mean optical density (OD)) against each antigen was calculated for each age group. Comparisons were made using an unpaired t-test to study the difference in responses against each antigen within each age group. NA, not applicable; *p < 0.05; **p < 0. 01; ***p < 0.001; ****p < 0.0001. ns, not significant; Age (year), age ranges in years.  antibody response to S, RBD, and RBM. The positive response of COVID-19 plasma against different sequence domains (RBD and RBM) of S protein highlights peptide synthesis as an effective vaccine approach, which could ultimately contribute to the mass production of crucial COVID-19 good manufacturing practice (GMP) products (55)(56)(57).

DISCUSSION
Overall, our data demonstrate the importance of RBDwhich showed comparable antibody responses (71.9%) to the full-length S protein (80.5%)-as an alternative target for vaccinations and antiviral therapies (58,59). However it should be noted that we observed a relatively low prevalence of S antibodies (the most prevalent antigen); various other studies observed an antibody response of 95% from their COVID-19 patients (31,(60)(61)(62)(63)(64), indicating that our value of 80.5% is lower than expected. This may have been caused by a lack of seroconversion in some patients, as plasma was collected within the first week after hospital admission. According to the literature, at least 11-14 days after the onset of the disease is reported to be necessary to observe an average seroconversion rate of approximately 90%-100% for antibodies (IgM or IgG) against the SARS-CoV-2 S and N proteins (31,(60)(61)(62)(63)(64)(65)(66)(67)(68)(69). Future investigations of the antibody dynamics, including in the early (acute) and late (convalescent) phases of COVID-19 infection, may provide more insight into this issue.
Antibody responses to SARS-CoV-2 antigens increased with age but were not associated with gender. Indeed, the S antigen showed a higher antibody level than RBD or RBM across all age groups. The same was observed for RBD as compared to RBM. Some studies have indicated that immunity and COVID-19 infection correlate positively with age (27,70,71), while others have suggested that aged patients are more prone to developing an uncontrolled and ineffective immune response, thus increasing disease severity (27,70,71). Our data strengthen the argument for inadequate antibody immunity as the cause of higher incidence of hospitalization in elderly patients despite high antibody levels in such groups. Regarding gender, it has been suggested that an immune response to COVID-19 may differ between men and women, thus influencing their ability to recover from a severe infection (72)(73)(74)(75)(76)(77). Indeed, in women, higher IgG levels in the early phase and during COVID-19 (72)(73)(74)(75)(76)(77) appear to play an essential role in reducing severe disease and mortality (78). However, this study analyzed samples only once, enabling the comparison of antibody levels in mild, severe, and convalescent cases. Still, studies on the dynamics of antibody responses to S, RBD, and RBM-controlling for variables like age and gender-are now necessary. Moreover, it was not possible to determine whether the SARS-CoV-2 antibody levels at hospital admission were correlated with recent exposure to COVID-19, which might explain the benign outcome of the disease in this group of patients.
Concerning treatment duration, patients with stronger responses to S, RBD, or RBM experienced remission in a shorter time period (≤30 days), supporting the idea that S-and RBDspecific antibodies play a crucial role in controlling the severity of SARS-CoV-2 infections. These findings are consistent with other studies that showed that the failure to develop antibodies against SARS-CoV-2 was an essential factor in worsening the disease (79) and was problematic for serodiagnosis tests (30).
This study shows that an accurate assessment of the interactions between preexisting comorbidities and antibody elicitation in the onset of SARS-CoV-2 is essential for existing vaccination strategies and especially to protect those at higher risk from severe forms of COVID-19. Preexisting comorbidities such as diabetes, hypertension, and asthma did not appear to influence antibody response against S and RBD. However, it is interesting that asthma and diabetes seemed to impede the elicitation of antibodies against RBM (the more specific domain for SARS-CoV-2) and that higher body weights appeared to weaken the antibody responses against S in COVID-19 patients. Altogether, these data suggest that preexisting comorbidities-which are associated with disease severity-may be directly impacting the immune responses to SARS-CoV-2 (80-83). (i.e., >30 days) as shown in the graph. The correlation was strongest with RBD recognition. The table shows the proportions of S, RBD, and RBM responders as a function of their treatment duration, but no significant difference was observed between the three antigens and the treatment duration time. The unpaired t-test and ANOVA were used to compare the mean Ab optical density (OD) between the two treatment duration groups and between antigens, respectively, and Fisher's exact test was used to determine the proportion of responders with a treatment duration of ≤30 or >30 days. *p < 0.05; **p < 0.01; ****p < 0.0001. ns, not significant.
Additionally, our findings imply that even with a lack of specific binding, there is still a high degree of crossrecognition for the SARS-CoV-2 antigens among populations not infected with SARS-CoV-2 living in malaria-endemic areas. Cross-reactive as high as 21.9% against S (highest) is consistent with previous studies, where it reached 17% or even upwards of 20% in malaria-endemic areas (23,24). This cross-reactivity between malaria and SARS-CoV-2 raises the question of whether other SARS or malaria infections can produce similarly cross-reactive antibodies, playing a role in SARS-CoV-2 infection. In this regard, there is evidence for a crossneutralization reaction between SARS-CoV and SARS-CoV-2, albeit controversial (25,84). Malarial infections may also elicit a wide range of immune responses that could also be cross-reactive for COVID-19 antigens (18)(19)(20)(21)(22). In addition, antigen crossreactivity (85,86) may be due to a non-specific, antigenindependent antibody binding. In pre-COVID-19 volunteers, we observed false positivity against the three antigens in 9.6% to 20.0% of the cross-reactive samples, potentially indicating a nonspecific antibody binding. These findings further confirm that anti-SARS-CoV-2 antibody tests may exhibit some false positives, as revealed by ELISA after removing the antigen coating (87,88). Also, several proteins, present in human plasma at high concentrations-such as albumin (89)-can interfere with the detection of low abundance analytes (90) by increasing background signals and non-specific antibody binding (91).
Moreover, no correlation was found between the crossrecognition of SARS-CoV-2 antigens and current malaria infection. In contrast, the most cross-reactive antibodies were mainly associated with the absence of acute malarial infections, indirectly indicating a protective antibody response to malaria that cross-reacts with SARS-CoV-2. The cross-reactivity is more than likely to occur, since non-specific or poly-specific activation of B cells may occur during or before the process of induction of etiologic antibodies (92)(93)(94)(95). Therefore, the coinfection of malaria and COVID-19, their impact on each other (in terms of clinical issues), and the cross-reactivity of COVID-19 antigens with malaria-endemic samples may help to explain the paradox in the incidence of COVID-19 in malaria-endemic areas (20)(21)(22)(96)(97)(98). Further study is necessary to assess how the coinfection of malaria and SARS-CoV-2 can impact the clinical outcomes of each disease.
In conclusion, the characterization of the individual antibody target domains/epitopes (like RBD and RBM) present in the SARS-CoV-2 S-in both naturally COVID-19 exposed patients and malaria exposed donors without COVID-19 infection-not only would contribute to our understanding of the fine specificity of SARS-CoV-2 antigens and their cross-reactivity observed in these populations but also may offer strategies for designing a second-generation of vaccines. The cross-reactivity of the SARS-CoV-2 antigens was evident in pre-COVID-19 infected samples, as was the impact of protective malarial infection on said cross-reactivity. It can be noted that the early development of high antibody levels against RBD was essential in shortening treatment durations for SARS-CoV-2 infections. Furthermore, factors such as asthma, diabetes, and weight may adversely affect antibody responses to SARS-CoV-2.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories

ACKNOWLEDGMENTS
We are grateful to the volunteers who agreed to participate in this study and would like to acknowledge the Dermatology Hospital of Bamako (HDB) for the participants' recruitment, data collection, and sample procurement, and the Immunogenetics Laboratory and Parasitology and the Clinical Laboratory of ICER-Mali at USTTB, Mali, for sample processing and technical support. We thank Prof. Florian Wurn and Dr Maria Wurm at ExcellGene SA, Monthey, Switzerland, and Dr Florence Pojer at Protein Production and Structure Core Facility, EPFL, Lausanne, Switzerland, for providing S and RBD antigens.

SUPPLEMENTARY MATERIALS
The Show not significant variation of antibody responses against S, RBD and RBM according to the presence or absense of various comorbidities in COVID-19 patients, respectively. Unlike S and RBD, no association was found between two comorbidities and response to MBR (C). CMB, comorbidity; ns, not significant.
Supplementary Figure 4 | Cross-reactivity of S, RBD and RBM according to age group in pre-COVID-19 samples. Cross-reactive antibody levels (in pre-COVID-19 samples) for spike (S) and MBR were comparable, but significantly higher than for MBR in most of the different age groups. Comparison of antibody levels between different antigens in the same age group was determined in unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001; NA, not applicable; n, not significant; Age (year), age ranges.