The entry of new coronaviruses into cells depends on the viral S protein (spike protein) and human cell receptor binding domain (RBD). Although there is no new crown vaccine, antibodies are essential for global anti-epidemic. At present, a top AIDS research team has extracted and identified antibodies from the plasma of the newly rehabilitated people, and discovered high-neutral and active, rare but recurring RBD-specific antibodies. This suggests that the vaccine developed based on this antibody may be generally effective for the public.
The above findings are from the new research on the new crown antibody of Rockefeller University, California Institute of Technology, Howard Hughes Medical Institute, and Chan Zuckerberg Biohub published on the medical preprint website bioRxiv: Convergent Antibody Responses to SARS-CoV-2 Infection in Convalescent Individuals. The paper was published on May 15th local time. Corresponding authors include Michel Nussenzweig and Professor Paul Bieniasz, director of the Laboratory of Molecular Immunology at Rockefeller University.
As a top scholar in the field of AIDS, Nussenzweig's research focuses on the molecular mechanism of the immune system's inherent and adaptive responses. The research method combines biochemistry, molecular biology and genetics. He specializes in B lymphocyte and HIV-1 antibody research. In response to the COVID-19 outbreak, Nussenzweig has extended its research to SARS-CoV-2, and isolated and identified highly efficient neutralizing antibodies from recovered patients.
Prof. Paul Bieniasz, another corresponding author, is also a top scholar in the field of AIDS. His research attempts to define how host genes affect virus replication, focusing on human and primate immunodeficiency viruses. Its laboratory is dedicated to characterizing the host functions of virus imitation, manipulation, and other uses, as well as the characteristics of defense cells that have evolved in response to viral infections.
On May 13, local time, the head of the WHO (WHO) health emergency project, Mike Ryan, said that the new coronavirus may become a long-term problem, it is difficult to predict when the virus can be defeated, it may become that it will never disappear Epidemic virus. Ryan, taking AIDS as an example, pointed out that although HIV has not disappeared, humans have found treatments and prevention methods, and people no longer fear AIDS as before.
The S protein of the new coronavirus is responsible for binding and invasion with human receptors, which is usually an important target for drug development. During infection, the S protein is cleaved into N-terminal S1 subunit and C-terminal S2 subunit by host proteases (such as TMPRSS2). S1 and S2 mediate receptor binding and membrane fusion, respectively. Among them, S1 contains an N-terminal domain (NTD) and a receptor binding domain (RBD), which is crucial in determining tissue orientation and host range. When the virus invades the human body, RBD will bind to the human receptor ACE2 (angiotensin converting enzyme 2).
But at present, little is known about human antibody responses to SARS-CoV-2. In this study, the research team reported 68 COVID-19 recoverers and none were hospitalized. The distribution of the half-maximum neutralizing titer range of the plasma collected by these recovered persons 30 days after the onset of symptoms varied from undetectable (18% of the sample) to less than 1: 1000 (78% of the sample), only 3% of the recovered persons are greater than 1: 5000.
Antibody clones show expanded clones of RBD-specific memory B cells (expressing closely related antibodies) in different individuals. Despite the low plasma titer, the half-inhibitory concentration of antibodies against unique epitopes on RBD can be neutralized at levels as low as ng / mL. In this way, most individuals who recover from COVID-19 without hospitalization will not have high levels of neutralizing activity in their restorative plasma. However, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that vaccines developed based on such antibodies may be generally effective.
Between April 1 and April 17, 2020, 73 eligible participants entered the study. Of these, 48 (65.8%) were new crown patients diagnosed by RT-PCR (hereinafter referred to as "cases"), and 25 (34.2%) were close contacts of diagnosed cases (hereinafter referred to as "contacts"). Five close contacts without symptoms were excluded from further analysis.
At the time the samples were collected, the 68 people had no symptoms for at least 14 days. There was only one asymptomatic person who tested positive for SARS-CoV-2 nucleic acid, and the average time of the other 67 participants was about 30 days (17 to 48 days) before the sample was collected. In this cohort, symptoms lasted an average of 10 days (0-28 days), and none were admitted. The most common symptoms are fever (82.4%), cough (64.7%), myalgia (55.9%) and fatigue (54.4%), and comorbidities are rare (8.8%). There were no significant differences between genders, between cases and contacts, the duration or severity of symptoms, or the time from the onset of symptoms to the collection of samples.
Of the plasma samples tested, 88% and 66% showed IgG and IgM antibodies specific for RBD (at least 2 standard deviations above the control group). However, the IgG and IgM antibody responses of anti-trimer S protein in plasma samples were only 40% and 21% (at least 2 standard deviations higher than the control group). There was no significant difference between IgG and IgM antibody levels and how long the samples were collected after the onset, age, gender, case, or contacts. In contrast, the binding of IgG antibodies to RBD and S proteins is directly related to the duration of symptoms, but the binding of IgM antibodies to RBD and S proteins is not related to the duration of symptoms. Finally, there is little difference in the level of antibody against S protein, but the effect of women on RBD IgG binding antibodies is lower than that of men.
To measure the neutralizing activity of restoring plasma, the researchers used a pseudovirus assay, which uses HIV-1 based virus particles carrying the nanoluciferase reporter gene and SARS-CoV-2 S protein. The overall level of neutralizing activity in the cohort, measured by half-maximal neutralizing titer (NT50), is generally low, with 18% being undetectable and 78% for those below 1,000. The geometric mean value of NT50 is 212 (arithmetic mean value = 850), and the NT50 of only two individuals reaches more than 5000. Studies have found that neutralizing activity is related to the duration of symptoms and severity of symptoms, but not to the time of sample collection related to the onset of symptoms, age, gender, or case / contact status. It is worth noting that the level of IgG antibodies that bind RBD and S is closely related to NT50.
To determine the nature of the antibody caused by SARS-CoV-2 infection, the researchers used flow cytometry to isolate B lymphocytes with RBD receptors from the blood of 6 participants. Including 2 patients with the best antibody neutralizing activity. The frequency of antigen-specific B cells is determined by their ability to bind phycoerythrin (PE) and BV711-labeled RBD. The circulating B cells in the recovery period of COVID-19 range from 0.07 to 0.005%, but cannot be detected in the control group . Using reverse transcription and PCR, the researchers obtained 534 pairs of IgG heavy and light chain (IGH and IGL) sequences from a single RBD binding B cell of 6 individuals in recovery phase. Compared with the human antibody library, the IGH and IGL genes have several obvious over-standards. The average number of nucleotide mutations in the V gene of IGH and IGL is 4.2 and 2.8, respectively, which is lower than antibodies cloned from individuals with chronic infections, such as hepatitis B or HIV-1, and is similar to the original Antibodies to circulating IgG memory cells during malaria infection or non-antigen enrichment.
As with other human pathogens, there are expanded clones of antigen-binding B cells in all tested COVID-19 individuals. Overall, 32.2% of the reconstituted IGH and IGL sequences came from clonal expanded B cells (range 21.8-57.4%). Antibodies that share specific combinations of IGHV and IGLV genes in different individuals account for 14% of all cloned sequences. It is worth noting that the amino acid sequences of some antibodies found in different individuals are almost the same. For example, the amino acid sequences of cloned antibodies with IGHV1-58 / IGKV3-20 and IGHV3-30-3 / IGKV1-39 repeatedly found in different individuals have 99% and 92% identity. The researchers concluded that the IgG memory response to SARS-CoV-2RBD was highly enriched in the antibody sequences periodically cloned and amplified.
To examine the binding properties of anti-SARS-CoV-2 antibodies, the researchers expressed 34 representative antibodies, 24 of which were from clones and 10 from monomers (found in the plasma of 3 participants). ELISA analysis showed that 94% (32 of 34 antibodies) bound to SARS-CoV-2RBD at a half-maximal effective concentration (EC50) of 6.6 ng / mL. To determine whether these antibodies have neutralizing activity, the researchers tested the SARS-CoV2-Strunc pseudovirus. Of the 32 RBD-binding antibodies tested, 20 were found to have a small neutralization at a half-maximal inhibitory concentration (IC50) on the order of ng / ml, with concentrations ranging from 4.4 to 709 ng / ml. Effective neutralizing antibodies were found in individuals, independent of plasma NT50. For example, C002 and C121 were obtained from individuals with plasma NT50 values of 5053 and 298, COV21 and COV107, and IC50 was 8.9 and 6.7 ng / mL, respectively. Finally, clones with antibodies that share the IGHV and IGLV genes are one of the best neutralizers, such as antibody C002 with IGHV3-30 / IGKV1-39, and both donors have the best plasma neutralizing activity. The researchers concluded that even people with moderate plasma neutralizing activity have rare IgG memory B cells that produce effective SARS-CoV-2 neutralizing antibodies.
To determine whether a human anti-SARS-CoV-2 monoclonal antibody with neutralizing activity can bind to different domains on the RBD, the researchers conducted a two-layer interferometric experiment in which the preformed antibody-RBD immune complex was exposed to The second monoclonal antibody. The tested antibodies included 2 groups, while C002 and C105 bound to the pre-formed C121-RBD complex, while C104, C110 and C119 did not. The conclusion is that, like SARS-CoV, there are at least 2 different neutralizing epitopes on the RBD of SARS-CoV-2.
Through single-cell antibody cloning, individuals obtain human monoclonal antibodies that have neutralizing activity against pathogens such as viruses and parasites during natural infection. In model biology and early clinical research, several effective protection and treatment methods have been shown, but currently only one antiviral monoclonal drug is in clinical use. Compared to small molecule drugs, antibodies are relatively expensive and more difficult to produce. However, they differ from drugs in that they can participate in the host immune system through a constant domain that binds to Fcγ receptors on host immune cells. These interactions can boost immunity and help clear pathogens or infected cells, but they can also lead to increased dengue fever and coronavirus infection. This problem hinders the development of dengue fever vaccines, but does not interfere with the clinical application of effective neutralizing antibodies, which can be modified to prevent Fcγ receptor interactions and maintain protection against viral pathogens.
Antibodies are an essential element of most vaccines and may become a key component of an effective vaccine against SARS-CoV-2. Observation shows that most patients with convalescence have low plasma neutralization activity, but anti-SARS-CoV-2RBD recurrent antibodies with effective neutralization activity can be found in patients with abnormal plasma neutralization activity, indicating that humans have intrinsic Ability to produce anti-RBD antibodies that can effectively neutralize SARS-CoV-2. Therefore, vaccines that specifically and effectively induce antibodies targeting SARS-CoV-2RBD may be particularly effective.
Participants' demographic and clinical characteristics:
Surging news reporter He Liping