Specific COVID-19 Symptoms Correlate with High Antibody Levels against SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), isolated January 2020 (1), causes coronavirus disease 2019 (COVID-19), which ranges from no symptoms to a flu-like illness to death (2). As of May 2021, there have been over 156 million cases worldwide and over 3.2 million deaths (3), with devastating effects on health, economies, and societies (4).

Lasting immunity, often estimated by persistent Abs, will be critical for overcoming the COVID-19 pandemic, but our understanding of the development of persistent anti–SARS-CoV-2 Abs is still emerging. In severe acute respiratory syndrome (SARS), caused by related SARS-CoV, Abs typically persist at least 3 y (5–7). SARS-CoV-2 has not existed for 3 y yet, but some reports suggest that immunity may last at least 3 to 6 mo (8–11). However, other reports suggest that anti–SARS-CoV-2 neutralizing and IgG Abs can decline within a few months, with some patients becoming seronegative (12–20, K. Röltgen, O.F. Wirz, B.A. Stevens, A.E. Powell, C.A. Hogan, J. Najeeb, M. Hunter, M.K. Sahoo, C. Huang, F. Yamamoto, et al., manuscript posted on medRxiv, DOI: 10.1101/2020.08.15.20175794). These discrepant findings may be due to small sample sizes, use of variable or loosely defined time points, differing disease severity [a known correlate of Ab levels and persistence (15, 20–22)], and the use of different Ab detection methods, with neutralizing titers more likely to be low (23, 24). Also, many studies do not evaluate clinical correlates of Ab titers, and none have systematically evaluated COVID-19 symptoms. A standardized approach to evaluating anti–SARS-CoV-2 Abs with uniform time points defined by the resolution of disease, multiple Ab tests, and incorporation of clinical and demographic factors including COVID-19 symptoms would shed light on the development of Ab-based immunity in COVID-19.

Thus, we broadly evaluated the Ab response against SARS-CoV-2 in a clinically diverse COVID-19 convalescent population at 5 wk and 3 mo after symptom resolution using three different assays and then correlated Ab levels with clinical and demographic factors including COVID-19 symptoms. We found that greater disease severity, older age, male sex, higher body mass index (BMI), and higher Charlson Comorbidity Index score correlate with higher anti–SARS-CoV-2 Ab levels. We also identified fever, body aches, and low appetite as symptoms that consistently correlate with higher anti–SARS-CoV-2 Ab levels and demonstrate Ab persistence 3 mo after symptom resolution.

Human studies were performed according to the Declaration of Helsinki and were approved by the University of Wisconsin (UW) Institutional Review Board. All subjects provided written informed consent. COVID-19 convalescent sera and data were obtained from the UW COVID-19 Convalescent Biorepository, and control sera collected prior to 2019 were obtained from the UW Rheumatology Biorepository (25) and the National Institutes of Health clinical protocol VRC200. For the COVID-19 Convalescent Biorepository, all individuals 18+ y old who tested positive for SARS-CoV-2 by PCR at UW Health were invited to participate until 120 subjects were recruited. Clinical and demographic data were collected by survey upon recruitment. Additional data and blood were collected 5 wk and 3 mo ±10 d postsymptom resolution. Age, sex, address [for area of deprivation index (ADI) (26)], medications, laboratory values, height and weight (for BMI), medical problems, and the date of the most-recent primary care appointment were abstracted from the UW Health electronic medical record. Race, ethnicity, tobacco use, COVID-19 symptoms, and date of symptom resolution were self-reported by questionnaire. Hospitalization and intubation for COVID-19 were obtained by questionnaire and electronic medical record abstraction. COVID-19 severity was scored as critical (4, intubated), severe (3, hospitalized but not intubated), moderate (2, fever defined as temperature >100°F, chills, productive cough, or shortness of breath but not hospitalized), or mild (1, none of the above). Charlson Comorbidity Index scores were calculated (27). Subjects were excluded from this study if they received convalescent plasma, if blood was collected >14 d from the intended time point, or if they did not provide consent for all aspects of the study.

Plates (96 well) printed with SARS-CoV-2 spike protein, receptor binding domain (RBD) of spike, N-terminal domain (NTD) of spike, and nucleocapsid protein as well as spike from SARS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63, and HCoV-229E in addition to BSA were supplied by Meso Scale Discovery (MSD, Rockville, MD). Plates were blocked for 60 min with MSD Blocker A (5% BSA) followed by washing. Then, sera were applied to the wells at four dilutions (1:100, 1:800, 1:3200, and 1:12,800) and incubated with shaking for 2 h. Plates were washed, and SULFO-TAG–labeled anti-IgG (MSD) was applied to the wells for 1 h. Plates were washed, ECL substrate (MSD) was applied, and light emission (as a measure of bound IgG) was read with the MSD Sector instrument. BSA readings were subtracted from CoV Ag readings. Area under the curve values for each sample were used for statistical analysis with zero values (three samples for anti-NTD) depicted as 10 in graphs for optimal visualization using the log scale.

Plates (384 well) precoated with RBD were supplied by MSD. Plates were blocked for 30 min with MSD Blocker A and washed, sera were applied at 1:10 dilution, and plates were incubated with shaking for 1 h. SULFO-TAG–labeled angiotensin converting enzyme (ACE) 2 was applied to the wells, incubated for 1 h, and washed. ECL substrate was applied, and light emission (a measure of RBD-ACE2 complex) was read by the MSD Sector instrument. The amount of light emitted in wells containing no sample (assay diluent only) was considered the maximal binding response. Reduction of ECL response from the maximal binding response was directly proportional to the extent of competitive binding activity.

Vero E6/TMPRSS2 cells were grown in DMEM supplemented with 5% FCS, HEPES, amphotericin B, and gentamicin sulfate. Sera (100 µl) were diluted in cell culture solution with 2-fold serial dilutions from 5× to 2560×. Virus (SARS-CoV-2/UW-001/Human/2020/Wisconsin) was diluted in cell culture solution to an adjusted titer of 100 PFU per 60 µl. Diluted sera (60 µl) and diluted virus (60 µl) were mixed in wells of 96-well U-bottom plates in duplicate. Plates were incubated at 37°C for 30 min. Culture supernatant was aspirated from Vero E6/TMPRSS2 cells plated in 96-well dishes and replaced with the mixtures of serially diluted sera and virus (100 µl/well, in duplicate) followed by incubation at 37°C with 5% CO₂ for 3 d. Crystal violet stain was added to wells to stain for living cells. Neutralization titers were determined by the maximum fold dilution at which the serum samples could completely prevent cell death as determined by eye. Some duplicates diverged by a single-fold dilution in their neutralization titer. In this situation, the lower dilution was used as the neutralization titer. Sera with cell death at all dilutions were assigned a dilution value of 1 for analysis purposes.

Ab levels were compared between COVID-19 convalescent and control sera using a t test or among subsets of COVID-19 convalescent sera by one-way ANOVA with Tukey, Dunnett, or Dunn multiple comparisons tests. Welch correction was used for unequal variance. Anti–SARS-CoV-2 Ab levels from different time points in the same subject were compared by paired t test. Correlations between Ab levels from different tests were estimated by Spearman rank correlation. The relationship between clinical and demographic factors and COVID-19 hospitalization or Ab levels were examined using the Pearson χ² test for categorical variables and Kruskal–Wallis test for nonnormally distributed continuous data. Multiple linear regression analysis was performed to determine if the presence of specific symptoms was associated with transformed Ab levels (square root transformed for spike, RBD, and nucleocapsid and log transformed for NTD, ACE2 inhibition, and neutralizing titers) adjusted for age, BMI, Charlson Comorbidity Index score, and sex. We also modeled the combination of symptoms indicative of moderate disease (severity score 2) and the total number of symptoms to determine their associations with Ab titers. We examined adjusted R² values to determine if modeling symptoms in these ways explained more of the variability in Ab titer as compared with symptom-specific regressions. Analyses were performed using GraphPad Prism software (San Diego, CA) and STATA version 16 (College Station, TX). For all analyses, p < 0.05 was considered statistically significant.

We recruited 120 COVID-19 convalescent subjects into the UW COVID-19 Convalescent Biorepository. Seven subjects were excluded from this study because of erroneous blood collection timing (n = 1), receipt of convalescent plasma (n = 3), or partial consent (n = 3). Two additional subjects were excluded from longitudinal evaluation because of a blood draw >14 d from the 3 mo time point. Of the included subjects, blood was collected from 113 at 5 wk (range 29–48 d, median 36 d, interquartile range [IQR] 35–39 d) and from 79 at 3 mo (range 85–102 d, median 91 d, IQR 90–93 d) postsymptom resolution. Eighty-one percent of COVID-19 convalescent subjects had a primary care appointment within 2 y of the first blood draw and/or a hospital admission note with past medical history and medications. Subjects ranged in age from 19 to 83 y and had a variety of COVID-19 manifestations (Supplemental Table I, Supplemental Fig. 1). One subject was a current smoker. As expected (21, 28), hospitalized subjects were more likely to be older and male with more comorbidities, like vascular disease, but less likely to have asthma (Supplemental Table I). Additionally, hospitalized subjects were more likely to have fever and less likely to have chest tightness, sore throat, or headache than nonhospitalized patients. We detected no correlation between race, ethnicity, ADI, BMI, cancer, immunosuppressing medications, or other COVID-19 symptoms and hospitalization, potentially because of the relative uniformity of race and ethnicity and the low number of subjects with cancer or immunosuppressing medications in our cohort.

We used a multiplex approach to evaluate IgG levels against four SARS-CoV-2 Ags (spike, RBD of spike, NTD of spike, and nucleocapsid) as well as IgG against the spike protein of SARS-CoV and four seasonal coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E) in subjects 5 wk post–COVID-19 symptom resolution ((Fig. 1A). COVID-19 convalescent subjects had higher IgG levels against all four SARS-CoV-2 Ags compared with naive subjects. Furthermore, 98% of convalescent subjects had higher binding than any naive subject in at least one test. Finally, IgG levels against spike from SARS-CoV, HCoV-OC43, and HCoV-HKU1, but not HCoV-NL63 and HCoV-229E, were higher in convalescent subjects compared with controls.

We then evaluated the Ab response against SARS-CoV-2 in a more-functional manner. Because the RBD of spike binds to ACE2, enabling viral entry (29), we quantified the ability of sera to inhibit RBD binding to ACE2. Compared with naive sera, 5 wk convalescent sera demonstrated much higher inhibition of RBD-ACE2 binding ((Fig. 1B). Furthermore, in a neutralizing assay using live SARS-CoV-2, COVID-19 convalescent sera had higher titers compared with naive sera, although 15% of convalescent subjects did not have neutralizing titers above controls ((Fig. 1C). Overall, neutralizing titers correlated well with IgG levels against SARS-CoV-2 and RBD-ACE2 binding inhibition ((Fig. 1D).

Next, we evaluated if Ab titers at 5 wk postsymptom resolution varied with disease severity in our cohort. Hospitalized subjects had higher Ab levels than nonhospitalized subjects according to all of our tests, and, in general, Ab titers increased with COVID-19 severity ((Fig. 2). To determine if clinical and demographic factors apart from severe disease correlate with Ab levels, we analyzed nonhospitalized subjects alone. Older age, male sex, higher BMI, and a Charlson Comorbidity Index score >2 correlated with higher Ab titers in nonhospitalized subjects for all or most tests (Table I). Race, ethnicity, ADI, cancer, diabetes, vascular disease, asthma, immunosuppressive medications, and inhaled/intranasal steroids did not correlate with Ab levels in general (Supplemental Table II).

We then determined if symptoms might correlate with Ab levels. To this end, we first performed a univariate analysis to determine if any symptoms correlated with age, sex, BMI, or Charlson Comorbidity Index score (i.e., characteristics associated with Ab levels) (Table I). Because some symptoms correlated with hospitalization, again we evaluated only nonhospitalized patients. As shown in detail in Table II and in a summarized form in (Fig. 3, we found that most symptoms did not associate with these characteristics. However, abdominal pain was associated with older age and higher Charlson Comorbidity Index score, diarrhea was associated with higher BMI and male sex, vomiting was associated with higher BMI, fever was associated with higher Charlson Comorbidity Index score, and body aches and productive cough were associated with male sex.

We then performed a univariate analysis to determine if Ab titers at 5 wk postsymptom resolution varied with symptoms in our nonhospitalized subjects. As shown in (Fig. 3 and Table III, fever, low appetite, abdominal pain, and diarrhea correlated with higher Ab levels measured by every test. Cough, body aches, headache, nausea, and vomiting correlated with some Ab tests. Chills, shortness of breath, chest tightness, sore throat, loss of taste or smell, and runny or stuffed nose correlated with no or almost no Ab tests.

Concerned that age, sex, BMI, or Charlson Comorbidity Index score, which correlate with anti-SARS-CoV-2 Abs and some symptoms (Tables I, II), could be confounding our univariate analysis, we performed a multiple linear regression analysis controlling for these variables ((Fig. 3, Table IV). We found that fever and low appetite remained strong correlates and that chills, shortness of breath, chest tightness, sore throat, and runny/stuffed nose remained weak correlates of anti–SARS-CoV-2 Ab levels when accounting for these characteristics. However, gastrointestinal symptoms had little correlation with Ab levels in the multivariable analysis. Furthermore, the strength of association between Ab levels and cough, particularly productive cough, was reduced, whereas the correlation between body aches and headaches increased. Interestingly, when adjusting for age, BMI, comorbidities, and sex, loss of taste or smell was strongly associated with almost all Ab tests.

Because almost all subjects experienced more than one symptom, we also evaluated if the combination of symptoms included in moderate disease (severity score 2: fever, chills, productive cough, or shortness of breath but not hospitalized) or the total number of symptoms correlated with Ab levels. As shown in Table IV, having a combination of symptoms indicative of moderate disease correlated with some Ab levels, but the explanatory power was not enhanced because the adjusted R² value fell within the range of adjusted R² values from symptom-specific regression models. This was unsurprising because most people experienced multiple symptoms. However, there was evidence that number of symptoms experienced had greater explanatory power (i.e., higher adjusted R² values) of the variability in each of the Ab levels after adjusting for age, sex, BMI, and Charlson Comorbidity Score.

Finally, we evaluated the Ab response against SARS-CoV-2 and other coronaviruses in COVID-19 convalescent subjects 3 mo after symptom resolution. In our cohort as a whole, anti–SARS-CoV-2 IgG, RBD-ACE2 binding inhibition, and neutralizing titers did not decline from 5 wk to 3 mo postsymptom resolution ((Fig. 4). However, when hospitalized and nonhospitalized subjects were analyzed separately ((Fig. 4), anti-spike and anti-nucleocapsid IgG rose slightly in hospitalized subjects (area under the curve: 4,525,184 ± 796,452 versus 5,247,312 ± 811,346 and 5,765,064 ± 588,170 versus 7,004,295 ± 654,857, respectively), and neutralizing titers decreased slightly in nonhospitalized subjects (titers: 49 ± 11 versus 37 ±10) over time. Titers against SARS-CoV and seasonal coronaviruses also did not fall regardless of hospitalization status and rose slightly for seasonal coronaviruses (Supplemental Fig. 2).

We have demonstrated that the vast majority of COVID-19 convalescent subjects generate Abs against SARS-CoV-2 that inhibit ACE2 binding, neutralize live SARS-CoV-2, and persist at least 3 mo post–COVID-19 symptom resolution. Furthermore, greater disease severity, older age, male sex, higher BMI, higher Charlson Comorbidity Index score, fever, body aches, and low appetite consistently correlate with higher Ab titers.

Although we detected IgG against SARS-CoV-2 in the majority of COVID-19 convalescent subjects, there was variability among tests. IgG levels against spike and RBD, but not NTD or nucleocapsid, showed an impressive difference between COVID-19 convalescent and control sera. Similar strong results were observed for RBD-ACE2 binding inhibition. Of note, two convalescent subjects had similar values to naive subjects in all three tests. It is unknown if these subjects had false-positive SARS-CoV-2 PCR tests, if they did not make Abs, or if our tests were insufficiently sensitive.

All of the Ab tests correlated well with neutralizing titers, a gold standard for protective Abs. This correlation is encouraging, suggesting that our Ab assays are measuring relevant Abs without the cost, hazards, time, and expertise needed for neutralizing assays. However, similar to related studies (23, 24), more COVID-19 convalescent subjects had Ab titers no higher than naive controls using the neutralizing assay as compared with other assays. It is unknown if these subjects truly lack protective Abs (in many cases despite the presence of anti–SARS-CoV-2 IgG) or if the neutralization assay is insufficiently sensitive.

In addition to the development of anti–SARS-CoV-2 Abs in COVID-19 convalescent sera 5 wk postsymptom resolution, we saw a small increase in Abs against seasonal betacoronaviruses (OC43 and HKU1), but not alphacoronaviruses (NL63 and 229E), likely because SARS-CoV-2 is a betacoronavirus (29). Moreover, Abs that bind to seasonal coronaviruses rose slightly from 5 wk to 3 mo postsymptom resolution. This “back boost” phenomenon (30) could represent cross-reactivity of newly developed anti–SARS-CoV-2 Abs and/or stimulation of memory B cells originally developed in response to circulating coronaviruses. In contrast, the high anti–SARS-CoV levels seen in COVID-19 convalescent subjects, which did not change over time, are probably due to cross-reactive anti–SARS-CoV-2 Abs because the Wisconsin cohort was almost certainly not exposed to SARS-CoV.

Similar to recent reports (20–22, 31–33), we demonstrated that anti–SARS-CoV-2 Abs were highest in patients with severe disease, perhaps because of different immunophenotypes in COVID-19 patients (34) with a stronger inflammatory response in severe disease driving higher Ab titers. We also found that in nonhospitalized patients, higher Ab levels correlated with older age, male sex, higher BMI, and higher Charlson Comorbidity Index score. These factors all correlated with severe disease in our cohort as measured by hospitalization except for BMI, which others have reported to be associated with severe disease (35). Thus, nonhospitalized subjects with these high-risk characteristics might have had relatively severe disease that our methods could not measure, driving higher Ab levels. Alternatively, these characteristics could contribute directly to increased Ab levels. However, other reports suggest that older age and obesity impair Ab responses (36), and males have no generalizable increased Ab response (37). Of note, the correlation between anti–SARS-CoV-2 Ab titers with age, sex, and obesity has been previously reported (22, 38), but we are the first to report (to our knowledge) a correlation with the Charlson Comorbidity Index score.

In addition to disease severity, we report for the first time (to our knowledge) that specific COVID-19 symptoms correlate with higher anti–SARS-CoV-2 Ab levels. Fever, body aches, and low appetite, which consistently correlated with higher Ab levels in both univariate and multivariable analyses, can be signs of a systemic inflammatory response, which is likely key for developing a strong anti–SARS-CoV-2 Ab response. Gastrointestinal symptoms correlated with older age, higher BMI, more comorbidities, and male sex as well as higher Ab levels. Thus, it is difficult to determine if disease severity, one of these clinical factors, or gastrointestinal symptoms might drive Ab development. Diarrhea, typically caused by enteric viral infection and damage, may directly exacerbate inflammation and COVID-19 severity (39), causing higher Ab titers. Alternatively, diarrhea may simply be more common in severe disease, which correlates with age, BMI, Charlson Comorbidity Index score, and male sex, although we did not see increased diarrhea in our hospitalized patients.

Interestingly, loss of taste or smell, a unique feature of COVID-19, is associated with higher Ab levels only in the multiple linear regression analysis, suggesting that it is a predictor of higher Ab titers independent of severe disease correlates, namely BMI, age, Charlson Comorbidity Index score, and male sex. The mechanism for loss of taste or smell remains unclear. It seems unlikely that anti–SARS-CoV-2 Abs cause this symptom because only a small percentage of subjects have Abs early in disease when loss of taste and smell occurs (40). Moreover, a mechanism by which this symptom could drive higher Ab levels is similarly hard to imagine. Thus, the relationship between loss of taste and smell and Ab titers is likely indirect, driven by factors that remain to be discovered.

Finally, many symptoms did not correlate consistently or at all with Ab levels, including shortness of breath, which was associated with higher Ab levels in COVID-19 convalescent plasma donors in a previous study (23). However, that study (23) had fewer subjects than our study, with plasma donated at various times post–COVID-19 and no other symptoms evaluated. Many symptoms that did not correlate with Ab levels also did not correlate with severe disease and may not be related to the inflammation that drives Ab production.

Finally, we found that Abs persist at least 3 mo after symptom resolution. Some Ab levels even continued to rise slightly during this time period, although neutralizing titers fell slightly in nonhospitalized subjects. Our findings are discrepant from some studies, which reported falling Ab levels against SARS-CoV-2 within a few months of disease (12–20, K. Röltgen et al., manuscript posted on medRxiv, DOI: 10.1101/2020.08.15.20175794). However, these studies had smaller sample sizes (particularly at later time points), variable or unclear time points as related to symptom resolution, Ab loss in subjects with mild or asymptomatic disease, or no clinical data. Our findings are consistent with reports of persistent Ab titers at least 3–6 mo after disease (8–11). As time goes on, additional studies will be needed at later time points that use a variety of Ab detection methods, defined collection time points, and extensive clinical data.

There are a few caveats to our studies. First, samples were not collected prior to 5 wk postsymptom resolution because of logistical issues, such as limited personal protective equipment early in the pandemic when the biorepository was established. Similarly, at the time that this study was performed, samples had not yet been collected later than 3 mo from symptom resolution, a task that is currently underway. Also, COVID-19 symptoms were self-reported up to a month after symptom resolution, which could lead to recall error. Additionally, some medical records were incomplete with no recent primary care or admission note for 19% of subjects and incomplete BMI data for 14%. This gap would be biased toward nonhospitalized patients. Also, our population was relatively racially and ethnically homogeneous. However, our study is strong in its wide breadth of COVID-19 severity in 113 subjects with consistent time points and multiple types of Ab tests.

In sum, we report that anti–SARS-CoV-2 Abs last at least 3 mo postsymptom resolution and that Ab titers consistently correlate with fever, body aches, low appetite, older age, male sex, higher COVID-19 severity, higher BMI, and higher Charlson Comorbidity Index score. Further work is needed to determine protective Ab levels against reinfection, how long protective titers last, and the mechanisms by which COVID-19 symptoms, demographics, and comorbidities may drive higher Ab levels.

We thank the UW Office of Clinical Trials and the UW Carbone Cancer Center Translational Science BioCore as well as the human subjects who participated in this study.

B.F. is a current employee of Amazon Com Services, LLC. Her contributions to the study occurred while she was employed by the National Institutes of Health. The other authors have no financial conflicts of interest.