key: cord-0713913-zi6ipzt3 authors: Wang, Xiaoli; Guo, Xianghua; Xin, Qianqian; Pan, Yang; Hu, Yaling; Li, Jing; Chu, Yanhui; Feng, Yingmei; Wang, Quanyi title: Neutralizing Antibodies Responses to SARS-CoV-2 in COVID-19 Inpatients and Convalescent Patients date: 2020-06-04 journal: Clin Infect Dis DOI: 10.1093/cid/ciaa721 sha: d17e70454dfb0b053009a3d20ec44a700a109e16 doc_id: 713913 cord_uid: zi6ipzt3 BACKGROUND: COVID-19 is a pandemic with no specific antiviral treatments or vaccines. The urgent needs for exploring the neutralizing antibodies from patients with different clinical characteristics are emerging. METHODS: A total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. Antibodies were determined with a modified cytopathogenic neutralization assay (NA) based on live SARS-CoV-2 and enzyme linked immunosorbent assay (ELISA). The dynamics of neutralizing antibody levels at different time points with different clinical characteristics were analyzed. RESULTS: The seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till day 41-53. The total GMT was 1:163.7 (95% CI, 128.5 to 208.6) by NA and 1:12441.7 (95% CI, 9754.5 to 15869.2) by ELISA. The antibody level by NA and ELISA peaked on day 31-40 since onset, and then decreased slightly. In multivariate GEE analysis, patients at age of 31-45, 46-60, and 61-84 had a higher neutralizing antibody level than those at age of 16-30 (β=1.0470, P=0.0125; β=1.0613, P=0.0307; β=1.3713, P=0.0020). Patients with a worse clinical classification had a higher neutralizing antibody titer (β=0.4639, P=0.0227). CONCLUSIONS: The neutralizing antibodies were detected even at the early stage of disease, and a significant response showed in convalescent patients. The family Coronaviridae is comprised of large, enveloped, single-stranded, and positive-sense RNA viruses that can infect a wide range of animals and human [1] . Two coronavirus pandemics in human have emerged in the past two decades. Severe acute respiratory syndrome coronavirus (SARS-CoV) was first recognized in 2003, causing a global outbreak [2] . It was followed by another pandemic event in 2012 designated as Middle East respiratory syndrome coronavirus (MERS-CoV) [3] . In December 2019, emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) originating in Wuhan, China, has rapidly spread worldwide, and the World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19) a pandemic. As of April 12, 2020 , the cases of COVID-19 have been reported in 211 countries and territories worldwide, with a total of 1,696,588 confirmed cases and 105,952 deaths [4] . Moreover, the number of confirmed cases continues to grow at a rapid rate, including United States [5] . To date, the outbreak in China has been effectively controlled by widespread testing, quarantine of cases, contact tracing and social distancing [6] . As of April 12, 2020, a total of 82,160 of COVID-19 patients were confirmed in China, of which 1,156 remained hospitalized for treatment [7] . Despite supportive care and conventional anti-virus therapies, neither antiviral treatments nor vaccines that could specifically target against COVID-19 have been achieved [8] . Neutralizing antibodies play an important role in virus clearance and have been considered as a key immune product for protection or treatment against viral diseases. The results from some researches indicated that using convalescent plasma on Ebola, SARS-CoV and H5N1 avian influenza patients were proved to be effective [9] , moreover, COVID-19 Joint Investigation Report A c c e p t e d M a n u s c r i p t by China-WHO pointed out that serum collected from COVID-19 convalescent patients can fully neutralize the cellular infectivity of the isolated virus [10] . In addition, Shen et al [11] pointed out that 5 critically ill patients with COVID-19, administration of convalescent plasma containing neutralizing antibody was followed by improvement in their clinical status. These findings raise the hypothesis that using convalescent plasma transfusion could also be beneficial in COVID-19 patients. However, immunity duration and changes on immunity levels of patients in convalescent period remains largely unknown. Given the knowledge gap of this field, we determined that an updated analysis of antibody levels of COVID-19 patients at different time points and severity of illness might help develop rapid diagnostic reagents, vaccines, drugs, and other treatments. It's of great significance for the long-term control and treatment of COVID-19. The purpose of this current study was to analyze the dynamics of neutralizing antibody levels at different time since onset from different severity COVID-19 inpatients and convalescent patients, and to provide information for the scientific community to understand, detect, and treat COVID-19. c c e p t e d M a n u s c r i p t study the dynamics of neutralizing antibody response, blood samples of patients were collected successively. Among 70 patients, only 8 were followed up and tested for another time after discharging from hospital. The 8 convalescent patients were selected to study longitudinal changes of antibody titers, including 4 in mild group and 4 in moderate group. Two patients were tested for twice, 2 patients were tested for three time, and 4 patients for four times. Together with 39 patients with only one blood sample collection, a total of 117 blood samples were analyzed in the study. The protocol of the study was reviewed and approved by the Medical Ethical Committee of Beijing Youan Hospital, Capital Medical University (approval number LL-2020-041-K). Before enrollment, written informed consent was obtained from each enrolled patient. The demographic characteristics, clinical manifestations, and underlying conditions of patients were collected. In addition, the history of residence in or traveling to Wuhan within recent weeks was obtained. The indicators for immunogenicity assessment included seropositivity rate and the geometric mean titer (GMT). We conducted neutralizing assay (NA) to evaluate antibody level according to Reed-Muench method on day 5. The presence of neutralizing antibody was determined by a modified cytopathogenic assay. Serum samples were inactivated at 56°C for 30 minutes and serially diluted with cell culture medium in two-fold steps. The diluted serums were mixed with a virus suspension of 100 TCID50 (50 tissue culture infective dose) in 96-well plates at a ratio of A c c e p t e d M a n u s c r i p t 1:1, followed by 2 hours incubation at 36.5°C in a 5% CO2 incubator. 1-2×10 4 Vero cells were then added to the serum-virus mixture, and the plates were incubated for 5 days at 36.5°C in a 5% CO2 incubator. Cytopathic effect (CPE) of each well was recorded under microscopes, and the neutralizing titer was calculated by the dilution number of 50% protective condition. A titer of 1:4 or higher indicated seropositivity. For calculation of GMT, antibody titers of <1:8, >1:512, and >1:1024 were assigned values of 1:4, 1:(512+512/2), and 1:(1024+1024/2), respectively. To double check seropositivity rate and the geometric mean titer (GMT), enzyme linked immunosorbent assay (ELISA) was additionally conducted. 96-well micro plates were coated with 1μg/ml purified SARS-CoV-2 virus solution at 2-8℃ overnight, and blocked with 1% BSA for 2～4h at 37 ℃. Diluted sera(1:100) were applied to each well for 1h at 37℃ followed by incubation with goat anti-human antibodies conjugated with HRP for 1h at 37℃ after 3 times PBS wash. The plate was developed using TMB, followed by 2M H2SO4 addition to stop the reaction. To determine the final result, ELISA plate was read at 450/630 nm by ELISA plate reader. Mean with standard deviation was used for continuous variables description, and number with percentage was used for categorical variables description. Median with minimum and maximum was used to describe days for antibody testing of 1st sample since onset. Kruskal-Wallis rank-sum nonparametric method was used to compare log-transformed neutralizing antibody values. The comparison of categorical data was performed using Chi-square test or Fisher's exact test. The association between antibody levels and potential factors, i.e., gender, age, clinical classification, A c c e p t e d M a n u s c r i p t and time since onset of symptoms, were estimated by Generalized Estimating Equations (GEE) model with logit link function, which took into account the correlation between repeated measurements of each patient. Hypothesis testing was two-sided with an alpha value of 0.05. Analyses were conducted by SAS 9.4 (SAS Institute, Cary, NC, USA). The funders had no role in study design, data collection, data analysis, data interpretation, or writing of the report. Of the 70 patients enrolled into this study, 58 were recovered and discharged from hospital, 12 were inpatients. The average age of the patients was 45.1 years (range 16.0-84.0). A total of 58.6% were female. Thirty eight (54.2%) patients were residents or ever travelled in Wuhan, Hubei. The number of patients with a history of cardiovascular disease, diabetes, and hypertension was 2 (2.8%), 5 (7.1%) and 9 (12.9%), respectively. One (1.4%) patient was asymptomatic infection, 22 (Table 1) . A c c e p t e d M a n u s c r i p t The seropositivity rate reached up to 100.0% for 117 blood samples at different stages of illness both by NA and ELISA. The total GMT by NA was 1:163.7 (95% CI, 128.5 to 208. 6 (Table 2) . Univariate GEE analysis showed that the neutralizing antibody level during day 31-40 was significantly higher than other phases. However, multivariate GEE analysis showed that the antibody level during day 31-40 was only higher than day 10-20 (β= -0.6276, P=0.0201) ( Table 3) . Blood samples at different time since onset also showed differences in the distribution of neutralizing antibody titers ( Table 2 and Figure 1 ). The proportion with a titer less than 1:64 decreased with days since onset (Ptrend=0.0061), and the lowest was found during day 41-53. The neutralizing antibody titers were similar in the two gender groups, of which 1:168.6 (95% CI, Table 3 and 4. Due to the COVID-19 widely spreading around the world, the specific therapeutic agents or vaccines for COVID-19 are urgently needed. Neutralizing antibodies have been expected as an effective measure to treat or prevent SARS-CoV-2 infection. Recent researches demonstrated a complete protection against SARS-CoV-2 with purified inactivated SARS-CoV-2 virus vaccine in macaques [12, 13] . Some studies used pseudovirus (PsV) neutralization assay to evaluate the neutralizing antibody for SARS-CoV-2 [14] [15] [16] [17] . To improve the performance of the test, we used neutralization assay based on live SARS-CoV-2. The results indicated a significant neutralizing antibody response in convalescent patients. patients regardless the stage of the disease. Moreover, the seropositivity rate can reach up to 100.0% on day 10. The GMT peaked between day 31-40 after onset of symptoms. Even though the GMT had a slight decrease at day 41-53, the seropositivity rate remained 100.0%. The result was A c c e p t e d M a n u s c r i p t different from another study which indicated the titers of antibodies peaked between 10 to 15 days after disease onset [17] . After adjusting confounding factors, multivariate GEE analysis demonstrated that the antibody levels were comparable between day 31-40 and day 41-53 since disease onset. However, the proportion with a titer of 1:512 or above decreased from 52.8% on day 31-40 to 27.6% on day 41-53. How long will antibody levels last is a key concern for safe and effective antiviral treatments and vaccines in the future [18] . It is worthy of further study to analyze antibodies after COVID-19 patients recovered for a longer time. For other coronaviruses, immunity after an infection was strong for several months [19] . Liu [20] found that the neutralizing activity infected by SARS pseudovirus declined from 96% at month 3 to 48% at month 36. Cao [21] showed that IgG and neutralizing antibodies were undetectable in 19.4% and 11.1% of serum samples at month 30 after onset, and in 25.8% and 16.1% at month 36. It's uncertain whether the presence of antibodies against SARS-CoV-2, lower or even undetectable levels of specific neutralizing antibodies could protect them from re-infection. Longitudinal observations in addition to stringent clinical and immunological characterization are needed to further assess the specificity and relative contribution to protection of neutralizing antibodies against SARS-CoV-2. We found that the neutralizing antibody titers significantly increased along with age. Wu [17] also showed that elderly and middle-age COVID-19 patients had significantly higher plasma antibody titers and spike-binding antibodies than young patients. This indicated that elderly patients might have stronger immune response against SARS-CoV-2 than young patients. Whether high antibody levels protect these patients from progression into severe or critical conditions needs further studies. A c c e p t e d M a n u s c r i p t Our results indicated that convalescent patients had a higher antibody level than inpatients, which highlight the positive correlation between recovery and days since onset (Spearman correlation coefficient=0.5426, P<0.0001). However, 3 of the 58 patients recovered with a low level of GMT (1:8), not significantly higher than the lowest titer of inpatients (1:6), suggesting that besides neutralizing antibodies, other immune response, including T cell or cytokines might contribute to the convalescence [17] . Besides, we also found that neutralizing antibody levels in asymptomatic or mild patients were slightly lower than moderate or severe patients, which matches with other previous studies [22, 23] . Zhang [23] concluded that severe cases were more frequently found in COVID-19 patients with high IgG levels, compared to those with low IgG levels. Previous data showed that severe SARS-CoV was also associated with more robust serological responses including early seroconversion and higher IgG levels [24, 25] . The GMT was 1:4 from blood sample of the only one asymptomatic infection, lower than the lowest antibody level of symptomatic patients. However, the evidence that the antibody level of the asymptomatic infections are lower than that of symptomatic patients is not strong due to the small sample size in our study. Several limitations of this study should be noted. First, the involved patients were selected by convenient sampling instead of random sampling. So the representativeness is relatively insufficient, and the samples could only represent the general situation to a certain extent. Second, among 70 patients, only 12 of them were followed up more than twice, and the average follow-up A c c e p t e d M a n u s c r i p t period was relatively short, about only 14.3 days (range 3.0-36.0). Third, the subjects were mainly mild or moderate by illness severity, and only 1 asymptomatic patient and 4 severe patients were included. The neutralizing antibody response in asymptomatic infection and critical patients needs further explored in future. In conclusion, this study showed that all COVID-19 patients were seropositive to SARS-CoV-2 even at the early stage of illness, and a significant neutralizing antibody response was observed in convalescent patients. Neutralizing antibody levels depends on time after onset of symptoms, age and the severity of disease. Authors certify no potential conflicts of interest. A c c e p t e d M a n u s c r i p t M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Dynamics of neutralizing antibody titers in 8 convalescent COVID-19 patients since onset. In the figure, a, b , c, d, e, f, g and h represented the 8 convalescent patients. In Figure 2 -A, four patients whose antibody titers showed the obvious increasing trend were included. Figure 2 -B included the other four patients whose antibody titers showed the decreasing trend. A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t Evidence of the Recombinant Origin and Ongoing Mutations in Severe Acute Respiratory Syndrome 2 (SARS-COV-2) Management and Prevention of SARS in China Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Challenges in Identifying Its Source and Controlling Its Spread WHO. Coronavirus Disease (COVID-2019) Situation Reports Assessing the Global Tendency of COVID-19 Outbreak COVID-19: Learning from Experience National Health Commission of China. Do Our Best to Prevent and Control the Outbreak of the New Type of Tubular Virus Pneumonia: Report on Situation The Outbreak of SARS-CoV-2 Pneumonia Calls for Viral Vaccines. npj Vaccines Convalescent Plasma as a Potential Therapy for COVID-19 Bureau of Disease Control and Prevention, National Health Commission of China Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma Rapid Development of an Inactivated Vaccine Candidate for SARS-CoV-2 ChAdOx1 nCoV-19 Vaccination Prevents SARS-CoV-2 Pneumonia in Rhesus Macaques SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Inefficiency of Sera from Mice Treated With Pseudotyped SARS-CoV to Neutralize 2019-nCoV Infection Neutralizing Antibody Responses to SARS-CoV-2 in a COVID-19 Recovered Patient Cohort and their Implications Coronavirus Vaccines: Five Key Questions as Trials Begin Longitudinal Profiles of Immunoglobulin G Antibodies against Severe Acute Respiratory Syndrome Coronavirus Components and Neutralizing Activities in Recovered Patients Disappearance of Antibodies to SARS-Associated Coronavirus after Recovery Antibody Responses to SARS-CoV-2 in Patients of Novel Coronavirus Disease Immune Phenotyping Based on Neutrophil-to-lymphocyte Ratio and IgG Predicts Disease Severity and Outcome for Patients with COVID-19 Anti-SARS-CoV IgG Response in Relation to Disease Severity of Severe Acute Respiratory Syndrome GMT(1:) by NA, value(95% CI) GMT(1:) by ELISA , value(95% CI) A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t