Neutralizing antibody response and associated factors in Coronavirus-19 disease (COVID-19) up to one month

Ethical statement

Official permission was obtained from the Ministry of Health, and the Ethical Committee approved the study protocol of Ankara City Hospital (E1-20-532,7.5.2020).

Study design and participants

Data from 129 adult COVID-19 patients admitted to Ankara City Hospital. All participants met the diagnostic criteria according to WHO interim guidance [6] (Figure 1).

Figure 1:

Flowchart of the study.

Demographic, clinical, chest computer tomography (CT) findings, oro/nasopharyngeal swab sample RT-PCR results for SARS-CoV-2 were extracted from electronic medical records and case follow-up forms.

Two patients were asymptomatic at admission and were excluded in analyses based on the time from symptoms to testing. Patients were classified as severe and non-severe cases according to the National Institutes of Health (NIH) classification, in assessing whether severity was associated with immunological response [7].

Serum samples were obtained from patients on the day of hospital discharge to analyze antibody response. SARS-CoV-2 IgG was analyzed in sera samples using ELISA. The presence and titers of NAbs were analyzed with VNA. The cut-off for NAb positivity was 1:5, and it was used for comparing NAb positive and negative patients. The median NAb titer detected in this study (1:25) was used to compare patients with higher and lower titers (patients with 1:25 or higher vs. lower titers) to determine the potential factors that may elicit higher NAb titers.

The time from symptoms onset to collection of serum samples was recorded for each patient for statistical analyses. Before performing the tests, the serum samples were heat inactivated at 56 °C for 30 min and stored at −20 °C until used.

To determine the factors affecting the presence of antibodies in the early convalescent period, NAbs-positive patients were compared with NAbs-negative ones. Additionally, patients with higher and lower titers of NAbs were compared to elucidate the factors that may have an impact on eliciting higher NAb titers.

Production of in-house ELISA for detection of SARS-CoV-2 IgG and IgM

The receptor-binding domain (RBD) of SARS-CoV-2 (MN908947) spike protein was expressed in the HEK-293T cells (ATCC^® CRL-3216), purified, and used as the ELISA antigen. The details about the production method of recombinant RBD-antigen for in-house ELISA tests are summarized in Supplementary file 1 [8]. For negative controls (NCs) in tests, serum samples from nine healthy volunteers were randomized into 3 groups and pooled to obtain 3 different NC samples. Each patient sample and the NC material were assayed in triplicates, and means of absorbance values were used for interpretation. The samples with mean optical densities (ODs) equal to or greater than 0.22 were evaluated positive, and negative were equal to or less than 0.18. OD values between these values were deemed indeterminate.

Virus neutralization assay (VNA)

VNA was performed in microtiter plates as described by Hanifehnezhad et al. [9]. Briefly, two-fold diluted serum samples, starting from 1:5 (the threshold dilution for positivity), were mixed with an equal volume of 100TCID₅₀ SARS-CoV-2 Ank1 isolate (1:10,000) in quadruplicates and incubated for 1h at 37 °C for neutralization. The serum–virus mixtures were subsequently inoculated onto 90% confluent Vero E6 cells grown in 96-well plates. VNA was evaluated via an inverted microscope when 100% cytopathic effect (CPE) was observed in virus control wells. Seroconversion detected in 1:5 dilution of test serums was accepted as the threshold. The positive serum samples were subsequently five-fold diluted starting from 1:10 dilution. The mean NAb titer was taken as the dilution factor in which 50% of the infection of the 100TCID50 virus was blocked. For instance, if the virus-neutralization was recorded 0/4 in 1:5 dilution and 4/4 in 1:10 dilution, the mean NAb titer (SN50) of the given virus was accepted as 1:7.5.

Statistically calculated median NAb titer of the patients detected in this study (1:25) was used in the comparison of the patients. To determine the “median” NAb titer, NAb titers of the individuals were ranked from the lowest to the highest. The value of the middle observation was taken in cases for odd number of observations, and the arithmetic average of the two observations in the middle was taken for an even number of observations.

VNA was accepted as a reference test for validity analysis. Sensitivities and specificities of lgM and lgG ELISA tests were calculated with the following formulas: sensitivity=(number of lgG or IgM, and NAbs positives)/number of NAbs positives with VNA) × 100, specificity=(number of lgG or IgM, and NAbs negatives/number of NAbs positives with VNA) × 100.

Statistical analysis

Statistical analyses were performed using SPSS v.21.0 (Armonk, NY, USA). The Kolmogorov–Smirnov test was performed to check the normality of the variables. Descriptive analysis was presented using mean ± SD for normally distributed variables and median (minimum–maximum) for non-normally distributed variables. Demographic and laboratory data were compared with the student’s t-test for parametric and the Mann–Whitney U test for non-parametric variables. Comparisons for categorical variables were executed using the Chi-square test or Fisher’s exact test. Kruskal Wallis test was used for comparisons of more than two groups.

The odds ratio is defined as the odd that an outcome may occur given a particular exposure compared to the odds of the outcome occurring in the absence of that exposure.

The rate ratios and confidence intervals were calculated using the “rate ratio. test” package in the R software (version 3.6.1).

Potential associations between disease severity and the NAb status were studied using the Kaplan–Meier analysis via the time through the date of hospital discharge (i.e., time of testing).

Distributions of NAb titers among different patient sub-categories were analyzed with Mann–Whitney U test and plotted by GraphPad Prism version 9 (GraphPad Software, San Diego, California, USA). Statistical significance was defined as p<0.05.

Clinical and radiological characteristics of COVID-19 patients

All patients had chest CT findings and/or concordant symptoms for SARS-CoV-2 infection, in addition to laboratory confirmation with a positive PCR (n=97, 75.2%) and/or antibody tests (n=111, 86%). The mean age of the patients was 46.4 ± 15.8 years, and 54.3% of them were male. The median length of hospital stay was 7 days (min–max: 1–24 days). NAb positivity was significantly higher on admission in patients with fever (69.5%, Chi-Square test, p=0.05) and dyspnea (74.3%, chi-square test, p=0.05) (Table1).

Serum samples were obtained to test for antibodies against SARS-CoV-2. Patients were grouped for analyses into three, as 5–9, 10–14, and 15–28 days after symptom onset. The median time from onset of symptoms to antibody test was 11 days (5–28 days). It was median 9 days (5–23; min–max) in mild patients and median 15 days (7–28; min-max) in severe ones (Table2).

NAb positivity was significantly higher in patients with abnormalities on chest CT (66.7% vs. 33.3%, chi-square test, p<0.001). Bilateral multi-lobar ground-glass opacities and consolidation on admission were determined in 35.2% of the patients. Patients with advanced stage abnormalities on chest CT (such as bilateral multi-lobar ground glass opacities and consolidation) had significantly higher NAb titers at hospital discharge (chi-square test, p=0.012) (Table1).

The distribution of NAb titers obtained in the discharge serum of the patients were plotted by age, gender, disease severity, and chest CT findings at the time of admission (Figure 2). Age and gender had no significant impact on NAb titers (Figure 2A and B). The median NAb titer (SN₅₀) was 1:25 (interquartile range (IQR) 1:42.5) in severe patients and 1:7.5 (IQR 1:25) in non-severe counterparts (Mann–Whitney U test, p=0.008) (Figure 2C). The median NAb titers were 1:17.5 (IQR 1:20) and 1:7.5 (IQR 1:17.5) in patients with advanced and early stage findings on thorax CT, respectively (Figure 2D).

Figure 2:

Distribution of neutralizing antibody titers against severe acute respiratory syndrome-Coronavirus-2 in different patient groups.

CT, computed tomography, NAb: neutralizing antibody.

Severe patients constituted 17.8% (n=23) of the total patients, of whom nine (39.1%) required ICU support during the follow up. The mean age of the severe and non-severe patients was 60.9 ± 13.5 and 43.4 ± 14.7 years, respectively (Student’s t-test, p<0.001) (Table2).

Seroconversion characteristics of NAbs, lgM, and IgG against SARS-CoV-2

The seroconversion rates at the time of hospital discharge were 60.5%, 30.2%, and 51.9% for NAb, lgM, and IgG, respectively. Using the VNA as the reference test, the sensitivity and the specificity of the IgG test was determined to be 67.9% and 72.5%, respectively. The median time to testing (discharge) were longer (12 vs. 8 days, Mann–Whitney U test, p<0.001) in the group with NAb positivity at discharge (Table 1). NAb positivity was significantly higher in samples obtained after ten days from symptom onset (10–14 days and 15–28 days were combined) than in samples obtained within ten days after onset (75% vs. 42.4%, respectively. chi-square test, p<0.001).NAb positivity was the highest (79.5%) among those who were tested 10–14 days after symptom onset (chi-Square test, p<0.001) (Figure 3).

Figure 3:

Seroconversion characteristics of severe acute respiratory syndrome-Coronavirus-2 specific neutralizing antibody (NAb), immunoglobulin M (lgM), and immunoglobulin G (lgG).

IgM and IgG antibody response were 20.3% and 44.1% among patients who were sampled on days 5–9 since onset, 34.1% and 47.7% on days 10–14; 50% and 83.3% among patients who were sampled on days 15–28 since onset (Figure 3).

IgG response was determined in 82.6% of the severe patients and 45.3% of the non-severe patients as of their hospital discharge (chi square test, p=0.006) (Table2). NAbs occurred in eight of the nine patients (88.9%) who needed ICU care (Table 1). The median SN₅₀ level was determined as 1:25 in severe patients and it was 1:7.5 in the non-severe group (Mann–Whitney U test, p=0.009) (Table2). Only two male patients had the highest (>1:1250) titers of NAb; one of them was 36 years old with non-severe SARS-CoV-2 infection and tested on day 12 after symptom onset. The other was 50 years old, with severe COVID-19, and was tested on day 28.

Comparison of the VNA and ELISA

A total of 129 discharged serum samples of the patients were analyzed with both in-house ELISA kit and VNA assay. We found that 39 (30.2%), 67 (51.9%), and 78 (60.5%) samples were IgM, IgG, and VNA positive, respectively. In addition, out of all 129 serum samples of the patients, 53 (53/129: 41.1%) samples were positive, and 37 (37/129: 28.7%) were negative in both IgG ELISA and VNA assays. Fourteen samples were VNA-negative but IgG ELISA positive, whilst 22 samples were VNA-positive but IgG ELISA negative. Three samples were indeterminate in IgG ELISA while positive with VNA (Table1). Accordingly, sensitivity and specificity values for IgG ELISA were 67.9% and 72.5% and 47.4% and 96.1% for IgM ELISA, respectively.

Subgroup analyses of patients sampled at least ten days after symptom onset showed that both sensitivity and specificity values for IgG ELISA were 70.6%. These values were 49.0% and 88.2% for lgM ELISA, respectively.

Association of disease severity with NAb presence and higher titers of NAb

In the study, the length of hospital stays of the patients varied largely, and so were the timing of antibody testing after the detection of the first symptoms. To adjust for follow-up times, incidence rate and rate ratios were calculated for individual groups and group comparisons. Incidence rate corresponded to the number of patients with NAbs positivity at hospital discharge divided by person-days. The rate ratio equals the relative incidence rate of the risk group of interest vs. the control group’s. The rate ratios for the presence of NAb (any vs. none) at hospital discharge were 1.16 (95% CI=0.67–2.01, p=0.68) and 0.66 (95% CI=0.33–1.27, p=0.24), comparing patients with discharge at days 10–14 and 15–28 after symptoms onset, respectively, considering the patients discharged within 10 days after symptoms onset as the reference group. In Kaplan–Meier analyses, the presence of NAb seems to get positive earlier in the follow-up compared to that in severe cases. Yet, NAb positivity among severe cases surpasses that in the non-severe cases as the length of stay increases. The study design hinders our ability to detect the initial time for NAb production; instead, NAbs were assessed only once in each patient at discharge. To control for the potential confounding effect of the timing of NAb measurement, Kaplan–Meier curves were further stratified on follow-up time periods, such as days 5–9, 10–14, and 15–28 after symptom-onset. These analyses revealed no statistically significant difference between severe and non-severe cases regarding time-to-NAb presence in any of the time periods (log rank test p-values were 0.32, 0.31 and 0.60, for days 5–9, 10–14, and 15–28 after symptom-onset, respectively) (Figure 4A–C). Overall, a significant difference was obtained for higher NAb titers (i.e., 1:25 or higher vs. lower titers) favoring severe cases (OR=2.89, 95% CI=1.15–7.28, chi square test, p=0.021) (Table 1). This difference disappeared in survival analysis when time-to-event (i.e., testing) was adjusted (log rank test p=0.15) (Figure 4D).

Figure 4:

Hazard curves of NAbs by the severity of patients.

(A) Association between NAbs presence and severity of disease was not statistically significant log rank test p-value=0.32 (strata 1=5–9 days). (B). Association between NAbs presence and severity of disease was not statistically significant log rank test p-value=0.31 (strata 2=10–14 days). (C). Association between NAbs presence and severity of disease was not statistically significant log rank test p-value=0.60 (strata 3=14–28 days). (D). Association between NAb titer (≥1:25 vs. <1:25) and severity of disease was not statistically significant log rank test p-value=0.15.