Participant population
Serum samples from 108 participants (53 males and 55 females) between the ages of 12 and 78 (mean=42 years) living in Denver, Colorado and the surrounding suburbs, were tested for the presence of IgA, IgG, and IgM antibodies against SARS-CoV-2 antigens, including S1, RBD, S2, and NP using a multiplex chemiluminescence immunoassay26. Thirty-one participants tested positive for at least one SARS-CoV-2 antibody/antigen combination (antibody titers > 1.0). Seventy-six participants tested negative for all antibody/antigen combinations (titers < 1.0). Sixty-five of the participants (28 COVID-19-positive and 36 COVID-19-negative; one that did not have serology tested) completed extensive health questionnaires to identify symptom severity and potential comorbidities. Twenty-one COVID-19-positive participants and 18 COVID-19-negative participants reported experiencing one or more symptoms associated with COVID-19. As assessed by patient responses to questionnaire items related to medical history, physical activity levels, and general lifestyle practices, this population was considered to be healthy and active. This population had an average body mass index (BMI) of 25, only 21 participants reported chronic illnesses, and only 10 participants had a history of smoking tobacco or marijuana (Table 1). There were no significant differences in age, sex, BMI, smoking history, or the presence of chronic illnesses between the COVID-19-positive and COVID-19-negative participants (Table 1).
Antibody titers against SARS-CoV-2 antigens
We first confirmed that all combinations of antibody titers against SARS-CoV-2 antigens were significantly elevated in COVID-19-positive participants (n=31) versus COVID-19-negative participants (n=76) using two-tailed Independent Samples T-Tests. Titers for all 12 antibody/antigen combinations were significantly increased in COVID-19-positive versus COVID-19-negative participants (Table 2). Titers were not significantly different between males and females (data not shown). The Vibrant assay is unique in its ability to detect a variety of antibody/antigen combinations26. Many of the other tests FDA approved for Emergency Use Authorization may be limited in their detection of COVID-19-positive participants because they only test one antibody against one antigen. Therefore, several of these other assays may incorrectly categorize COVID-19- positive patients as COVID-19-negative if that patient is not positive for the specific antibody/antigen combination used in that specific test. The number COVID-19-positive participants from our study sample (n=31) that would have been misidentified as COVID-19-negative are shown for each test based on the antibodies used against SARS-CoV-2 antigens (Table S1).
Loss of smell was more severe in COVID-19-positive participants
Several participants in both the COVID-19-positive and COVID-19-negative groups reported symptoms. Therefore, we analyzed differences in symptom presentation and severity between the 21 symptomatic participants that tested positive and the 18 symptomatic participants that tested negative using two-tailed Independent Samples T-Tests. All participants self-reported the first date they remembered experiencing symptoms, symptom severity, and the date on which the symptoms resolved. Days between symptom onset and symptom resolution were not significantly different between the COVID-19-positive symptomatic participants and COVID-19-negative symptomatic participants (Table S2). Eighteen different symptoms were reported with varying severities between the symptomatic COVID-19-positive and COVID-19-negative participants (Table S2). Severity of loss of smell was the only symptom significantly increased in positive participants (mean(SE)=4.50±0.50) when compared to negative participants (mean(SE)=2.50±0.66) (t(6)=-1.57, p<0.05, Table 1, Figure 1a). These data suggest that severe loss of smell may be uniquely associated with SARS-CoV-2 infection. We ran Pearson’s correlations with the total symptom severity scores for each symptom and the compiled total score and found that none of the 18 symptoms were significantly correlated with age or BMI (data not shown). There were no differences in symptom severity between males or females for any of the reported symptoms (data not shown).
Antibody titers against SARS-CoV-2 antigens in mild versus severe symptoms
Although no participants in the present study required hospitalization, both COVID-19-negative and COVID-19-positive participants reported experiencing symptoms that were severe (defined as a symptom that could not be ignored and was the worst the patient had ever felt) or moderate (defined as a symptom that could not be ignored and limited daily activities). To further investigate whether titer levels were elevated in participants with more severe symptomology overall, we compared antibody titers against SARS-CoV-2 antigens in COVID-19-positive participants (n=21) who reported having mild-moderate symptoms (n=13) and those who reported moderate-severe symptoms (n=8) on the questionnaire using two-tailed Independent Samples T-Tests. Antibody titers against SARS- CoV-2 antigens were compared in the 21 symptomatic COVID-19-positive participants (Figure 1b). Titers of most antibody/antigen combinations were elevated in COVID-19-positive participants with moderate-severe symptoms when compared to those with mild-moderate symptoms (data not shown). However, only titers of IgA antibodies against SARS-CoV-2 RBD were significantly higher in COVID-19-positive participants with moderate-severe symptoms (mean(SE)=1.00±0.23) when compared to participants with mild-moderate symptoms (mean(SE)=0.48± 0.05), (t(19)=-2.72, p<0.01, Figure 1b).
IgM and IgG timelines
Next, we assessed the variation between reported symptom onset and serology test date. There were no significant correlations between titers and days between symptom onset and resolution or days between symptom onset and the initial test (Pearson’s correlations, data not shown). However, within the COVID-19-positive population, subgroups of participants with high titers of antibodies stratified to distinct intervals after symptom onset (Figure 2a-b). COVID-19-positive participants displayed elevated levels of IgM and IgG (each immunoglobulin averaged for the four antigens tested) between 0-30 and 0-60 days, respectively, after symptom onset. IgM levels appeared to decline 30 days past symptom onset.
Based on the findings described above, we next compared all combinations of antibody titers against SARS-CoV-2 antigens in the 21 symptomatic COVID-19-positive participants at 0-30, 30-60, 60-90, and 90-120 days between symptom onset and the serology test date using one-way ANOVA tests. Average IgG antibody concentration was not significantly different between each interval, but was significantly higher between 0-30 days (mean=1.14) compared to 30-60 days (mean=1.03) (Figure 2a). The average levels of IgM antibodies against the S1 (F(3,17)=10.89, p<0.001; Figure 2c) and RBD (F(3,17)=4.584, p<0.01; Figure 2d) antigens were significantly different between the four intervals described above (Figure 2c-d). Specifically, there were significant differences between the 0-30 and 30-60 day intervals (p<0.0001) and the 0-30 and 60-90 day intervals (p<0.001) for the S1 antigen and the RBD. Furthermore, average IgM titers were significantly different between the 0- 30 and 30-60 day intervals and between the 0-30 and 60-90 day intervals between symptom onset and the initial test (F(3,17)=4.42, p<0.01, Figure 2b). The 0-30 day interval had the highest average IgM concentration (mean(SE)=1.08±0.11), compared to the other three intervals.
Antibody titers in COVID-19-positive symptomatic versus asymptomatic participants
Differences in demographics and clinical characteristics between asymptomatic and symptomatic COVID-19-positive participants were examined next. There were no significant differences in sex, age, BMI, smoking history, or the presence of chronic illnesses between these two groups when analyzed by two-tailed Independent Samples T-Tests (Table 3 and data not shown). Fisher’s Exact Tests were used to compare the proportion of asymptomatic and symptomatic participants who tested positive for each antibody/antigen combination. We found that a greater number of symptomatic participants tested positive for IgG against the S1 antigen (n=10, p<0.05) and the NP antigen (n=10, p<0.05) when compared to asymptomatic COVID-19-positive participants (n=0 for both S1 and NP; Table 3). Similarly, more symptomatic participants tested positive for IgG against S2 (n=12) than asymptomatic participants (n=1), and this result trended towards significance (p=0.08, Table 3). The titers for the remaining antibody/antigen combinations were compared via two-tailed Independent Samples T-Tests and there were no significant differences between the two groups (Table 3).
Finally, we utilized average titers for IgA, IgG, and IgM against all antigens and examined differences in COVID-19-positive asymptomatic and symptomatic participants using two-tailed Independent Samples T-Tests. We found that the average IgG antibody titers against all antigens was significantly greater in COVID-19-positive symptomatic participants (mean(SE)=1.04±0.11) than COVID-19-positive asymptomatic participants (mean(SE)=0.64±0.10), (t(26)=-2.00, p<0.05). These data suggest that asymptomatic and symptomatic COVID-19-positive participants may experience different immune responses to SARS-CoV-2 infection. We utilized a Fisher’s Exact Test to explore associations between IgA, IgG, or IgM antibody positivity and the presence or absence of symptoms. We found that a larger number of asymptomatic COVID-19-positive participants had at least one positive IgM antigen compared to the number with at least one positive IgG antigen (p<0.05, Table 3). Collectively, the above findings suggest that COVID-19-positive symptomatic participants exhibited a greater IgG immune response and that asymptomatic participants had a greater IgM response. Intriguingly, none of the COVID-19-positive asymptomatic participants were positive for IgA against any viral antigen tested (Table 3).
Given the potential differences in disease progression in the asymptomatic and symptomatic participants positive for COVID-19, we wanted to understand whether there were differences in the antibody and antigen profile between asymptomatic participants, participants experiencing symptoms at the time of their serology test date (i.e., current symptomatic) and participants who had experienced symptoms before their test date (i.e., prior symptomatic). A Chi-square goodness of fit test showed that prior symptomatic COVID-19-positive participants were more likely to be positive for only IgG antibodies against any of the four SARS-CoV-2 antigens (n=7) than current symptomatic participants (n=1) or asymptomatic participants (n=2), (χ²(1,2)=6.2, p<0.05, Table S3). Although these sample sizes are quite small, these data further indicate that the majority of the participants in our cohort mounted a typical immune response to SARS-CoV-2 with IgM peaking early and IgG peaking later in the course of infection. Although the data were not significant, asymptomatic COVID- 19-positive participants were more likely to be positive for only IgM antibodies against any of the four SARS-CoV-2 antigens (n=5; Table S3), than current or prior symptomatic participants (n=2; Table S3) suggesting a distinct immunoglobulin profile relative to symptom onset.
Effects of influenza vaccine on symptomatology
We also examined effects of a recent influenza vaccination (between January 1, 2018 and May 26, 2020) on SARS-CoV-2 symptoms and serology. A Chi-square test was used to analyze the relationship between receiving a recent influenza vaccination and testing positive or negative for COVID-19. Influenza vaccination questionnaire responses from 28 COVID-19-positive participants and 36 COVID-19-negative participants were analyzed. There was a significantly larger fraction of participants who tested positive for COVID-19 and had a recent influenza vaccination (n=18) than participants who tested negative (n=13) (χ²(1,64)=5.01, p<0.05, Table 2). These findings indicate that receiving the influenza vaccination had no effect on susceptibility to SARS-CoV-2 infection.
Next, we examined whether a recent influenza vaccination could attenuate symptomatology in COVID-19-positive participants. A Chi-square test was used to discern the relationship between asymptomatic and symptomatic participants who tested positive for COVID-19 and who received a recent influenza vaccination. We found that 11 positive symptomatic participants had a recent influenza vaccination compared to 7 positive asymptomatic participants. The 7 positive asymptomatic participants who received the influenza vaccination represent 100% of that group, whereas the 11 positive symptomatic participants who received the influenza vaccination constitute only 52% of that group (χ²(1,28)=5.19, p<0.05, Table 3). This result may indicate that the influenza vaccination provides protection against presentation or severity of COVID-19 symptoms. No significant effect was found for the BCG vaccine (data not shown). There were no significant differences between participants who received the influenza vaccination and those who did not, regardless of test result or symptom presentation, in terms of titers, sex, age, BMI, or symptom severity (Table S4).