On April 14, the Wuhan Municipal Health and Health Commission launched a new epidemiological survey of asymptomatic infections of new coronaviruses. This survey adopted the principle of fixed-point random sampling. A total of 100 communities were used as the survey site, with a total of about 11,000 samples. Before the results of this serum rheology came out, there have been more and more discussions about antibodies in the serum of patients with new coronavirus.
Recently, the research team of the Shanghai Public Health Clinical Center affiliated to Fudan University, the Department of Pathogens of the School of Basic Medical Sciences of Fudan University, and the Key Room of the Ministry of Education / Ministry of Health of the Medical Molecular Virology published on the preprinted website medical preprinted website medRxiv the title ” Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications. The study has not been peer-reviewed.
Their results showed that analysis of serum samples from 175 rehabilitation patients found that neutralizing antibodies (Nabs) peaked 10-15 days after the onset of the patient and then remained stable. Thirty percent of patients produced very low neutralizing antibody titers. Among them, the antibody titers of 10 rehabilitation patients did not reach the detectable limit. Two patients had very high neutralizing antibody titers, but no antibody-related adverse reactions occurred.
The research team pointed out that these results indicate that some of the patients infected with the new coronavirus can recover without high-titer virus-specific antibodies. How these patients recover without the help of neutralizing antibodies, and whether there is a higher risk of repeated illness or reinfection, remains to be further studied.
It is worth noting that this study found that the neutralizing antibody titer of COVID-19 patients is also related to the age of the patients. The neutralizing antibody titer produced by middle-aged and elderly patients is much higher than that of young patients.
Corresponding authors of the study are Huang Jinghe, a professor at the School of Basic Medicine of Fudan University, and Wu Fan, an associate researcher at the Shanghai Public Health Clinical Center affiliated to Fudan University. Huang Jinghe has long been engaged in the research of AIDS broad-spectrum neutralizing antibodies and influenza vaccines. She pioneered the development of a new technology to isolate antibodies directly from B lymphocytes (Nature Protocols 2013). In her previous research work, a series of new high-efficiency broad-spectrum neutralizing antibodies 10E8, 35O22 and N6 have been isolated from AIDS patients.
This latest study is the first research report on the extraction of neutralizing antibodies from the plasma of the COVID-19 rehabilitation patient cohort. It may provide useful information for passive antibody therapy such as plasma therapy and the development of vaccines against new coronaviruses. The correlation between the neutralizing antibody titer and the patient’s age, lymphocyte count and blood CRP level also laid the foundation for further discussion of the mechanism of COVID-19 patients’ neutralizing antibody.
It is currently known that neutralizing antibodies play an important role in virus clearance and can be used to protect or treat viral diseases. Virus-specific neutralizing antibodies induced by infection or vaccination have the ability to block viral infections. Neutralizing antibody levels have also been used as the gold standard for evaluating the effectiveness of smallpox, polio and influenza virus vaccines.
At the same time, passive antibody therapy such as plasma therapy has been successfully used to treat infectious viral diseases, including SARS-CoV, influenza virus and Ebola virus. The effectiveness of passive antibody therapy is related to the concentration of neutralizing antibodies in plasma or the concentration of antibodies in rehabilitation patients. In the context of the COVID-19 global pandemic, plasma therapy is also considered a promising method of infection prevention or disease treatment.
However, the research team mentioned that the current level and role of new coronavirus-specific neutralizing antibodies in COVID-19 patients have not been reported. Therefore, they used a safe and sensitive neutralization analysis method based on pseudovirus-lentiviral vectors to screen and determine the plasma of 175 patients with mild symptom recovery from the Shanghai Public Health Clinical Center.
The PsV neutralization test is a sensitive and repeatable method that does not produce any highly pathogenic viruses and can be safely handled in a biosafety secondary facility. This method has been widely used for the detection of neutralizing antibodies in a variety of highly pathogenic viruses (including Ebola virus, highly pathogenic influenza virus, SARS-CoV, MERS-CoV).
As of February 26, 2020, a total of 175 COVID-19 patients have been discharged from the Shanghai Public Health Clinical Center. Their symptoms are mild and no one has entered the ICU. The median age of patients was 50 years old (16-85 years old); female patients accounted for 53%. The median length of hospital stay was 16 days (7-30 days), and the median duration of illness was 21 days (9 to 34 days).
It is worth noting that Qin Chuan, director of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences and others, have also provided relevant information on rhesus monkeys in response to whether the new coronavirus patients will re-infect, that is, “reyang” phenomenon The research by Jinghuang Huang and others is relatively optimistic.
They analyzed that from the current longitudinal study of monkeys by the research team, if the monkeys produce neutralizing antibodies early after the initial infection, reinfection will not occur. This means that when the rehabilitation patients have established enough specific antibodies to generate immunity to the new coronavirus, they will no longer be infected. They believe that the “re-yang” of the previously discharged patients cannot be caused by reinfection, and more complicated issues need to be considered to find out the cause. “
The epitope or immunogenicity of New Coronavirus and SARS-CoV are different
The research team collected plasma samples from five patients with COVID-19 at the time of discharge and tested the neutralizing potency of 293T / ACE2 cells against new coronavirus infection. All five plasmas showed concentration-dependent inhibition of PsV infection of 293T / ACE2 cells. High-titer neutralizing antibody plasma showed higher titers of new coronavirus RBD, S1 and S2 specific binding antibodies.
In addition, the plasma of these patients also showed cross-binding with the RBD and S1 regions of the new coronavirus, but the binding to the SARS-CoV S protein and the new coronavirus S protein were not consistent. In addition, in the PsV neutralization test, COVID-19 patient plasma could not inhibit SARS-CoV infection.
Twenty-six plasma samples from patients with COVID-19 that showed strong neutralizing activity of the new coronavirus could neither neutralize SARS-CoV PsV infection nor inhibit VSV-G PsV infection.
There is a premise here that because the amino acid identity of S protein between New Coronavirus and SARS-CoV is 77.2%, a conserved epitope may exist between the two viruses. But the results of this study showed that although the plasma of COVID-19 patients showed cross-combination with SARS-CoV, it did not neutralize SARS-CoV. This suggests that the epitope or immunogenicity of New Coronavirus and SARS-CoV are different.
The research team mentioned that the evidence inferred from this study only shows that during COVID-19 infection, cross-neutralizing antibodies against the conserved epitopes of SARS-CoV and the new coronavirus may not be easily induced.
The patient produces neutralizing and binding antibodies on the 10-15th day after infection
The research team monitored the development kinetics of the specific neutralizing antibodies of the new coronavirus during the course of the disease, and measured the plasma of 6 patients at different time points after the onset of disease. The development kinetics of the neutralizing antibodies were similar between the patients. The titer of neutralizing antibody in all patients was low before the 10th day of onset (ID50: <200), and reached a peak on the 10-15th day of onset, and then remained stable.
The researchers also tested the antibodies binding to the different binding domains (RBD, S1, and S2) of the new coronavirus S protein (spike glycoprotein) in the plasma of these six patients. The kinetics of the neutralizing antibody (right Y axis) and the binding antibodies against the RBD (receptor binding domain), S1 and S2 (left Y axis) are consistent.
S protein is the most important surface protein of coronavirus. It usually determines the host range and specificity of the virus. It is an important site for neutralizing antibodies in the host and a key target for vaccine design. Previous studies have shown that coronavirus binds to receptor ACE2 through S protein, thereby mediating membrane fusion and virus invasion. S protein is divided into two functional units according to protein structure and function, namely S1 and S2 protein subunits.
The research team evaluated the titers of new coronavirus-specific neutralizing antibody and the level of S protein-binding antibody in the plasma of 175 rehabilitation patients on the day of discharge. They observed that the neutralizing antibody titer of the new coronavirus was moderately correlated with the S protein antibody targeting RBD, S1 and S2.
They believe that this suggests that the humoral immune response of COVID-19 patients to the new coronavirus occurs 10-15 days after infection. In addition to the RBD binding domain, the S2 region may be the target of the neocoronavirus neutralizing antibody.
However, since the bound antibody may also clear the virus through antibody-dependent phagocytosis or antibody-dependent cytotoxicity, the effect of neutralizing antibody and bound antibody on disease progression is worth further comprehensive evaluation.
Overall, most patients with COVID-19 developed new coronavirus-specific neutralizing antibodies during the recovery phase of infection. Neutralizing antibody titers peaked 10-15 days after the onset, and remained stable in patients thereafter. Antibodies against different domains of S protein (including S1, RBD, and S2) may all contribute to neutralization.
About 30% of rehabilitated patients produce very low titers of neutralizing antibodies
It is worth noting that the research team observed that there were differences in the plasma neutralizing antibody titers of 175 rehabilitation patients. ID50s range from below detection limit (<40) to 21567. Approximately 30% of rehabilitated patients produce a very low level of neutralizing antibody titer (ID50: <500). However, the course of these patients is similar to other patients.
Middle-aged and elderly rehabilitation patients have higher levels of neutralizing antibodies
The neutralizing antibody titer of patients with COVID-19 is also related to the patient’s age.
The research team observed that older patients are more likely to induce higher titers of neutralizing antibodies than younger patients. As shown in Figure 4A, they divided patients into three groups according to age, namely young people (15-39 years old), middle-aged people (40-59 years old) and elderly people (60-85 years old), the number of patients in each group Similar, 55 cases, 64 cases and 56 cases respectively.
The neutralizing antibody titers of middle-aged and elderly rehabilitation patients were significantly higher than those of young rehabilitation patients, and the corresponding median ID50s were 1537, 1255, and 488, respectively. Age and the neutralizing antibody titer showed a moderate positive correlation, confirming the important role of age in the production of neutralizing antibody. The RBD, S1 and S2 targeting antibodies of middle-aged and elderly rehabilitation patients were significantly higher than those of young rehabilitation patients. However, no difference was observed between the patient’s age and hospital stay.
The research team believes that these results suggest that high levels of neutralizing antibodies have a certain effect on the removal of the virus and have a certain help for the rehabilitation of middle-aged and elderly patients.
In addition, the age of COVID-19 rehabilitation patients and the specific neutralizing antibody of the new coronavirus were negatively correlated with lymphocyte count at admission and positively correlated with CRP levels.
It is worth noting that the current research shows that age is an important predictor of adverse disease prognosis after coronavirus infection (including SARS-CoV, MERS-CoV, and new coronavirus). Previous studies on SARS-CoV-infected rhesus monkeys have shown that older rhesus monkeys cause an increase in innate immune response, leading to a more serious pathological response than young adult rhesus monkeys.
In this study, middle-aged and elderly patients had lower lymphocyte counts and higher CRP levels than younger patients on admission. However, none of these patients developed a serious condition, and there was no significant difference in age and length of hospitalization among these patients.
The research team also observed that the neutralizing antibody titer was negatively correlated with blood lymphocyte counts and positively correlated with blood CRP levels, suggesting that humoral responses may play an important role when cell response dysfunction or damage is achieved. Elderly patients had higher blood CRP levels and lower lymphocyte counts upon admission, suggesting that the innate immune response was stronger than younger patients. In these elderly patients, high levels of neutralizing antibodies may be the result of a strong immune response.
However, whether a high level of neutralizing antibodies can protect these patients from progressing to a severe and critical state is worth a comprehensive evaluation. However, in this study, severe and critical patients were excluded from the study because they had received passive antibody treatment before collecting samples.
The paper reminds that in this study, the research team could not directly evaluate the effect of neutralizing antibodies on virus clearance or disease progression in COVID-19 patients. To solve this problem, further comprehensive research should be conducted.