Evaluating the Association of Clinical Characteristics With Neutralizing Antibody Levels in Patients Who Have Recovered From Mild COVID-19 in Shanghai, China

Key Points Question Are clinical characteristics of patients who recovered from mild coronavirus disease 2019 (COVID-19) associated with levels of neutralizing antibodies? Findings In this cohort study of 175 patients who recovered from mild COVID-19, neutralizing antibody titers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) varied substantially at the time of discharge. In addition, neutralizing antibodies were not detected in 10 patients. Meaning Further research is needed to understand the implications of variable levels of SARS-CoV-2–specific neutralizing antibodies for protection against future infections with SARS-CoV-2.

A s of July 30, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused a total of 16 812 755 infections and resulted in 662 095 deaths worldwide. 1 In a study of 72 314 patients with SARS-CoV-2 infection in China, about 81% of the patients showed mild symptoms, but 14% had severe symptoms, such as dyspnea, high respiratory rate, and low blood oxygen saturation. 2 Approximately 6.3% of patients died from respiratory or multiple organ failure. 1 Currently, no licensed vaccine is available; there are limited drugs available for treatment (eg, remdesivir and dexamethasone), but most treatment is based on supportive care.
Neutralizing antibodies (NAbs) are important for viral clearance and are considered key to recovery and protection against viral diseases. The level of NAbs has been used as a standard to evaluate the efficacy of vaccines against smallpox, polio, and influenza viruses. 3 Passive antibody therapy has been successfully used to treat infectious viral diseases, including those caused by SARS-associated CoV, 4 influenza viruses, 5 and Ebola virus. 6 The efficacy of passive antibody therapy was associated with the concentration of NAbs in the plasma of recovered donors. 6 Transfusion of convalescent plasma or serum from patients who have recovered from coronavirus disease 2019 (COVID-19) has been used for treatment and prophylaxis of infection with SARS-CoV-2. 7,8 Consistent with other viral diseases, it has been assumed that patients who have recovered from COVID-19 would develop NAbs and may be protected from reinfection with SARS-CoV-2. To better understand the development of NAbs, we measured SARS-Cov-2-specific NAbs in plasma from patients with mild symptoms and examined the association between clinical characteristics and the level of NAbs.

Participants and Design
All patients admitted to Shanghai Public Health Clinical Center, Shanghai, China, from January 24 to February 26, 2020, who were diagnosed with laboratory-confirmed SARS-CoV-2 infection by positive results of reverse transcriptase-polymerase chain reaction testing of nasopharyngeal samples were isolated and hospitalized. We included in this study patients who were categorized as having mild symptoms according to the Guidelines on the Diagnosis and Treatment of Novel Coronavirus issued by the National Health Commission, China. Mild symptoms were defined as fever, respiratory symptoms, and radiologic evidence of pneumonia but not meeting any of the following manifestations: respiratory rate greater than 30/min, oxygen saturation levels less than 93%, ratio between arterial partial pressure of oxygen and fraction of inspired oxygen 300 mm Hg or less, or pulmonary imaging showing multilobular lesions or lesion progression exceeding 50% within 48 hours, as previously described. 9 Patients with severe, critical COVID-19 were excluded from the study because they received passive antibody treatment before sample collection. Patients were discharged after meeting national treatment standards, including testing as afebrile for more than 3 days, improved respiratory symptoms, pulmonary imaging showing lessening of inflammation, and 2 sequential negative tests for nucleic acid in nasopharyngeal samples. Patients were followed up at 2 weeks post discharge until March 16, 2020. Healthy volunteers in Shanghai Public Health Clinical Center who did not have a history of exposure to SARS-CoV-2 and had negative tests on 2 occasions for SARS-CoV-2 viral RNA, were recruited as controls. This study was conducted under a clinical protocol approved by the investigational review board of Shanghai Public Health Clinical Center. All participants signed an informed consent form approved by the investigational review board; participants did not receive financial compensation. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies.
Plasma was collected from the patients at the time of discharge. NAb titers were measured using a single-round pseudovirus infection assay. The reliability of single-round pseudovirus infection assay was validated by comparison with a viral cytopathology neutralization assay against live SARS-CoV-2 virus. Binding antibodies to SARS-CoV-2 spike (S) proteins, S1, receptor binding domain (RBD), and S2 were measured by enzyme-linked immunosorbent assay (ELISA). Plasma with high titers of NAbs was measured for cross-reactivity against SARSassociated CoV (SARS-CoV). To evaluate the kinetics of NAb development, sequential plasma samples were collected from admission to discharge at intervals of 2 to 4 days. NAb titers were also measured for patients who were followed up at 2 weeks post discharge and evaluated in pairwise comparison with NAb titers at the time of discharge. Clinical information, including age, sex, complete blood cell counts, blood biochemistry tests on admission, disease duration, and length of stay, were collected to explore the clinical characteristics associated with NAb levels.

Key Points
Question Are clinical characteristics of patients who recovered from mild coronavirus disease 2019 (COVID-19) associated with levels of neutralizing antibodies?
Findings In this cohort study of 175 patients who recovered from mild COVID-19, neutralizing antibody titers to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) varied substantially at the time of discharge. In addition, neutralizing antibodies were not detected in 10 patients.
Meaning Further research is needed to understand the implications of variable levels of SARS-CoV-2-specific neutralizing antibodies for protection against future infections with SARS-CoV-2.
Pseudovirus samples of SARS-CoV-2, SARS-CoV, and VSV-G virus were generated by cotransfection of 293T cells with pNL4-3.Luc.R-E-backbone and viral envelope protein expression plasmids, pcDNA3.1-SARS-CoV-2-S, pcDNA3.1-SARS-S, or pHEF-VSVG as previously described. 11 The neutralization assay was performed in accordance with the following steps. First, 293 T/ACE2 cells were seeded in a 96-well plate at a concentration of 10 4 cells per well in 100 μL of DMEM with FBS, 10%, and cultured for 12 hours. Then, 10 μL of heat-inactivated plasma was 5-fold serially diluted with DMEM with FBS, 10%, and mixed with 40 μL of pseudovirus. After incubation at 37°C for 30 minutes, the mixture was added to cultured 293 T/ACE2 for infection. The culture medium was refreshed with 200 μL of DMEM with FBS, 10%, after 12 hours and incubated for an additional 48 hours. Assays were developed with a luciferase assay system (Promega), and the relative light units were read on a luminometer (Perkin Elmer, EnSight).
Viral cytopathology neutralization assay was performed in a biosafety level 3 facility in the School of Basic Medical Sciences, Fudan University. Briefly, the plasma samples were serially diluted using DMEM with FBS, 2%, and mixed with 200 plaque-formed units of SARS-CoV-2 SH01 isolate (GenBank accession MT121215.1). The mixtures were incubated at 37°C for 1 hour before adding to 2 × 10 4 Vero-E6 cells seeded in a 96well plate. The cytopathologic changes of Vero-E6 cells was evaluated 3 days later and recorded by microscope.

Outcomes
The primary outcome was the titers of SARS-CoV-2-specific NAbs, which was calculated as a 50% inhibitory dose (ID50) and expressed as the dilution of plasma that resulted in a 50% reduction of luciferase luminescence compared with virus control in single-round pseudovirus infection assay, with higher values indicating higher levels of NAbs. The NAb titers were defined as low (ID50, <500), medium-low (ID50, 500-999), medium-high (ID50, 1000-2500), and high (ID50, >2500), and the detection limit was 40. The secondary outcomes included spike-binding antibodies, which were expressed as absorbance (optical density [OD]) at 405 nm (OD 405) measured by ELISA, ranging from 0 to 5, with higher values indicating higher levels of binding antibodies; cross-reactivity against pseudotyped SARS-CoV virus; kinetics of NAbs development during disease duration; clinical characteristics, including age, sex, lymphocyte counts, blood C-reactive protein (CRP) level on admission, disease duration, and length of stay.

Statistical Analysis
Statistical analyses were carried out using Prism, version 7.0 (GraphPad). The data are expressed as median (interquartile range [IQR]). All of the patients were included to analyze the NAb titers at time of discharge. The patients were numbered in the order of low to high ID50 values of NAbs. The nonparametric Mann-Whitney t test was used to compare the differences between 2 unpaired groups. The Kruskal-Wallis test was used to compare the differences between 3 or more groups and the Dunn multiple comparisons test was used to correct for multiple comparisons. Correlation coefficients with 95% CIs were calculated by the Spearman correlation coefficient test. The Wilcoxon matched-pairs signed-rank test was used to compare the NAbs difference between discharge and follow-up, with the exclusion of the patients who were lost to follow-up. All of the tests were 2-tailed, median difference with 95% CI was calculated, and P < .05 was considered statistically significant.

Results
A total of 175 patients who recovered from COVID-19 and were discharged from the Shanghai Public Health Clinical Center as of February 26, 2020, were included in the study. Their symptoms were mild, and none of them was admitted to the intensive care unit. The median age of the patients was 50 (IQR, 37-63) years; 93 patients (53%) were women and 82 patients (47%) were men. The median length of hospital stay was 16 (IQR, [13][14][15][16][17][18][19][20][21]  Since SARS-CoV shares 77.2% amino acid identity with SARS-CoV-2 in their S proteins, 12 the cross-reactivity of SARS-CoV-2 plasma in patients against SARS-CoV was evaluated. Plasma with high titers of NAbs showed higher binding abilities to the SARS-CoV-2 RBD, S1, and S2 domains (eFigure 1C in the Supplement). Moreover, plasma from these patients showed cross-binding to the SRAS-CoV RBD and S1 regions (eFigure 1D in the Supplement) but could not inhibit SARS-CoV in the pseudovirus neutralization assay. Twenty-six plasma samples from patients with COVID-19, which showed strong SARS-CoV-2-neutralizing activities, could neutralize neither SARS-CoV nor the control VSV-G (eFigure 1E in the Supplement).
Of the 11 patients for whom sequential plasma samples after admission were available, the kinetics of SARS-CoV-2specific NAbs development were evaluated. NAb titers started to increase at days 4 to 6 post disease onset and reached their peak levels at days 10 to 15 post disease onset ( Figure 2). The binding antibodies to the different domains (RBD, S1, and S2) of SARS-CoV-2 spike protein were also measured in these plasma samples. The kinetics of NAbs and binding antibodies targeting RBD, S1, and S2 domains were aligned for individual patients (eFigure 2A in the Supplement). The correlation of SARS-CoV-2-specific NAb titers and the spike-binding antibody levels were further evaluated in the plasma of the 175 recovered patients on the day of discharge. SARS-CoV-2-specific NAb titers correlated with spike-binding antibodies targeting RBD (r = 0.484; 95% CI, 0.358-0.592; P < .001), S1 (r = 0.451; 95% CI, 0.320-0.564; P < .001), and S2 (r = 0.346; 95% CI, 0.204-0.473; P < .001) (eFigure 2B in the Supplement). However, there were plasma samples from, for example, patients 3 and 8, that could not neutralize pseudovirus infection (ID50, <40) but developed high titers of spike-binding antibodies as measured by ELISA (eTable 1 and eFigure 2B in the Supplement).
The percentages of patients with different NAb titers are shown in Figure 3 and eTable 2 in the Supplement. Fifty-two patients (30%) who had recovered from COVID-19 generated low levels of NAbs (ID50, <500; median, 327; IQR, 189-404) (Figure 3; eTable 1 in the Supplement). NAb titers in 10 of these patients (19%) were below the limit of detection (ID50, <40), although SARS-CoV-2 was confirmed by polymerase chain reaction in all of these patients (eTable 1 in the Supplement). Those  We further explored the clinical manifestations associated with the NAb levels of the patients who recovered from COVID-19. We found that older patients developed higher titers of NAbs than younger patients. The 175 patients were divided into 3 groups based on their age: younger (15-  middle-aged patients also had significantly higher levels of spike-binding antibodies than those of younger patients in ELISA assay in either targeting RBD (older: It has been reported that older patients with COVID-19 are at higher risk of developing severe and critical disease than younger adults. 13 Low lymphocyte counts and high CRP levels were usually associated with poor outcome among patients with COVID-19 . 14 Consistent with the previous reports, the older and middle-aged patients in this cohort had significantly lower lymphocyte counts (r = −0.355; 95% CI, −0.482 to −0.214; P < .001) (eFigure 5A in the Supplement) and higher CRP levels (r = 0.439; 95% CI, 0.307-0.554; P < .001) (eFigure 5B in the Supplement) than younger patients at the time of admission. NAb titers at discharge negatively correlated with blood lymphocyte counts at admission (r = −0.427; 95% CI, −0.544 to −0.293; P<.001) (eFigure 5C in the Supplement) but positively correlated with blood CRP levels at admission (r = 0.508; 95% CI, 0.386-0.614; P < .001) (eFigure 5D in the Supplement).

Discussion
In this observational study, NAbs in 175 patients who recovered from mild COVID-19 were evaluated by pseudovirus neutralization assay. The titers of SARS-COV-2-specific NAbs   varied substantially, including 10 patients in whom NAbs were below the limit of detection. Most patients who recovered from mild COVID-19 developed SARS-CoV-2-specific NAbs at the convalescent phase of infection. The titers of NAbs reached their peak at 10 to 15 days after disease onset. Antibodies targeting different domains of S protein, including S1, RBD, and S2, may all contribute to the neutralization. Plasma from patients who recovered from mild COVID-19 showed cross-binding but did not neutralize SARS-CoV, suggesting that the antigenicity of SARS-CoV-2 is distinct from that of SARS-CoV. Conserved epitopes may exist between SARS-CoV-2 and SARS-CoV since they share 77.2% identical amino acids in their spike proteins. 12 Few reports have reported that SARS-CoV-specific monoclonal NAbs could crossneutralize SARS-CoV-2 pseudovirus infection. [15][16][17] Findings noted in this study suggest that cross-neutralizing antibodies targeting the conserved epitopes of SARS-CoV and SARS-CoV-2 may not be easily elicited during SARS-CoV-2 infection.
We noted variable levels of NAbs in patients. Thirty percent of the patients developed NAbs with titers less than 500 after COVID-19, and 10 patients had NAb titers under the detectable limit of the assay (ID50, <40). However, the disease duration of these 10 patients was not significantly different compared with the duration in the other patients. It is not clear how these patients recovered without developing detectable virus-specific NAbs. Whether other immune responses, including T cells or cytokines, contributed to the recovery of these patients and whether these patients are at risk for reinfection is not known. Two patients had very high titers of Nabs (ID50, 15 989 and 21 567). Studies on how these patients developed high titers of NAbs may provide useful information for the development of SARS-CoV-2 vaccines. In addition, the variability in NAb titers demonstrates the importance of titrating convalescent plasma before its use for prevention and treatment of COVID-19.
We found the NAb titers in patients appeared to be associated with age. Older patients had significantly higher titers of NAbs than younger patients in this cohort. Age has been reported to be an important predictor of adverse disease outcomes after infection with coronavirus, including SARS-CoV, 18 Middle East respiratory syndrome-CoV, 19 and SARS-CoV-2. 13 Previous studies in SARS-CoV-infected macaques revealed that aged macaques induced an elevated innate immune response, resulting in more severe pathologic changes than in younger adult macaques. 20 The older patients in this cohort also had higher blood CRP levels and lower lymphocyte counts at the time of admission; the higher blood CRP levels suggests induction of a stronger innate immune response than in younger patients. Furthermore, NAb titers at discharge positively correlated with blood CRP levels but negatively correlated with lymphocyte counts at admission, suggesting that high levels of NAbs may be a consequence of strong inflammation or innate immune response in these older patients in whom the lower lymphocyte count may reflect poorer T cell responses. Older patients developed higher NAb titers yet tend to have worse outcomes from COVID-19. This finding calls into question whether SARS-CoV-2 NAbs play protective roles in illness as assumed.

Limitations
This study has several limitations. First, the kinetics of NAb development were based on 11 of the 175 patients owing to the limited availability of sequential samples. Second, the patients were followed up for 2 weeks after discharge and only 117 patients were available for follow-up. Third, the disease duration was calculated from disease onset to discharge, which was longer than the symptom duration. Fourth, patients in severe and critical condition were excluded from the study because they received passive antibody treatment before sample collection.

Conclusions
The findings of this study noted that, among patients who recovered from mild COVID-19 in Shanghai, China, neutralizing antibody titers to SARS-CoV-2 appeared to vary substantially. The potential clinical implications of these findings for vaccine development and future protection from infection are unknown.