Though the current situation regarding COVID-19 is still severe, since July, many successes have been achieved on SARS-CoV-2 neutralizing antibody and Phase II clinical trials of SARS-CoV-2 vaccines. With the effort of scientists worldwide, results indicate that research will continue to make progress in the fight against COVID-19.
Herein, we discuss several frequently asked questions regarding both the design of the neutralizing antibody and the progress of vaccines for SARS-CoV-2. What is meant by neutralizing antibody research? What is current progress of SARS-CoV-2 vaccines? How do we verify the safety and efficiency of vaccines with animal models?
When a virus invades a human host, the body will produce corresponding antibodies to neutralize the virus in order to reduce the virus's damage to the body and prevent it from invading cells by interacting with extracellular receptors.
Usually speaking, research on Class A infectious diseases like COVID-19 must be performed in a biosafety level 3 laboratory. However, virologists have found that pseudoviruses can be of great utility in the investigation of virus neutralizing antibody (NAb) and vaccine. Since pseudoviruses have no transmission capabilities, the experimental environment for corresponding research is not as strict as it for the real virus. A biosafety level 2 facility is sufficient for research using pseudoviruses.
Research on the pseudo-coronavirus’ neutralizing antibody can be traced back to the severe acute respiratory syndrome (SARS) outbreak of 2004. Although the sudden breakout of SARS in 2004 lead to a global panic, the threat also receded within several months. As the SARS crisis was quickly eliminated, many research projects on it were canceled as funding ceased. However, many of these studies provided significant references for current research on SARS-CoV-2, jump-starting our progress on vaccine and treatment strategies.
One important case is "Longitudinally profiling neutralizing antibody response to SARS coronavirus with pseudotypes." by the research group leading by Professor.Robin A. Weiss from the University of London, which is published in Emerging infectious diseases. This paper indicated that SARS-CoV spike protein S is the main target of neutralizing antibodies (NAbs). By constructing a retroviral S-related pseudovirus, a neutralizing antibody against SARS-CoV was successfully prepared. This antibody was confirmed to have a neutralization effect on the SARS-S protein among patients. This research is of great significance for evaluating the potency of NAbs during natural infection and preclinical evaluation of vaccines. Meanwhile, preclinical studies conducted by researchers in mice models and hamster models have found that NAbs can significantly resist the invasion of live viruses. These findings indicate that NAb drugs demonstrate promise in helping humans resist virus infections, with data supporting the specific implementation of NAbs against the SARS-S protein.
Another case is more recent. In March 2020, The Institute of Pathogenic Microbiology of the Chinese Academy of Medical Sciences published a paper in Nature Communications. In this research, a pseudovirus system was used to successfully identify the characteristics exhibited by SARS-CoV-2-S protein when the virus invades cells, and elucidated the immune cross-reactivity between SARS-CoV-2 and SARS-CoV. By using the lentivirus SARS-CoV-2-S pseudovirus system is used in this system, the study confirmed that Huh7, Vero81, LLCMK2, Calu3 and other cells are susceptible to this pseudovirus system. The article also details the pathway by which the virus enters the cell upon binding to the ACE2 receptor. The SARS-S protein is a typical type I virus fusion protein, which requires protease cleavage between S1 and S2 subunits of the virus S protein to activate its fusion potential. Depending on the virus strain and cell type, the SARS-S protein may be cleaved by one or more host proteases, including furin, trypsin, cathepsin, transmembrane serine protease 2 (TMPRSS-2), TMPRSS-4, or human airway Trypsin-like protease (HAT). The presence of these proteases on target cells largely determines whether the coronavirus enters the cell through the plasma membrane or through endocytosis.
Figure 1: SARS-CoV-2-S pseudovirus invading cells. VSV-G indicates pseudovirions used as a positive control.
(Xiuyuan Ou et al. 2020)
Moreover, with soluble hACE2 flow cytometry and competitive inhibition experimental analysis, this study verified that the S proteins of SARS-CoV-2 and SARS-CoV both use hACE2 as the receptor for virus binding.
Coronavirus S protein is a key factor which determines virus virulence, tissue tropism, and host range – which have made it the main target of neutralizing antibody and vaccine design.
Figure 2: Limited cross-neutralization reaction of SARS and COVID-19 patient serum
(Xiuyuan Ou et al. 2020)
Additionally, this study explores the immune cross-reactivity between SARS-CoV-2-S and SARS-CoV-S. It was found that the sera of recovered SARS patients showed a strong inhibitory effect on the invasion of SARS-CoV S pseudovirus. Given that the sera only exhibited moderate cross-neutralization activity on SARS-CoV-2 S pseudovirus particles, results suggests that recovery from SARS-CoV infection does not provide protection from SARS-CoV-2 infection.
The results of the neutralizing antibody research announced in July has greatly boosted confidence in research progress related to the COVID-19 pandemic. On July 9, Peking University Advanced Innovation Center for Genomics (ICG), Biomedical Pioneer Innovation Center (BIOPIC), and other units jointly published a paper in Cell. Through high-throughput single-cell sequencing of B cells from patients who have recovered from COVID-19, the effective neutralizing antibodies against SARS-CoV-2 were identified.
Figure 3: Generation of SARS-CoV-2 neutralizing antibody
(Yunlong Cao et al. 2020)
Through high-throughput scRNA/VDJ-seq of 60 recovered COVID-19 patients, this study identified 8,558 IgG1+ antigen-binding clonotypes and found 14 effective SARS-CoV-2 neutralizing antibodies. Further verification with SARS-CoV-2 and SARS-COV-2 VSV pseudovirus systems revealed that the most effective neutralizing antibody was BD-368-2, which showed strong therapeutic and preventive effects on hACE2 transgenic mice infected with SARS-COV-2.
At the same time, the study used bioinformatics analysis methods to predict the CDR3H structure of the monoclonal antibody (mAb). After experimental verification, it is found that the CDR3H structure in the mAb, which is highly similar to the SARS-CoV neutralizing antibody m396, has a very high neutralizing effect on SARS-CoV-2.
Figure 4: Strong therapeutic and preventive effects of BD-368-2 on hACE2 transgenic mice infected with SARS-COV-2
(Yunlong Cao et al. 2020)
Published on July 23, Wang Xiangxi from the Institute of Biophysics of the Chinese Academy of Sciences cooperated with multiple research groups to construct a coronavirus antibody library through phage display technology. High-throughput screening identified an antibody named H014 with broad-spectrum neutralization ability against β-coronaviruses.
Figure 5: Verification of Cross-neutralizing antibody H014
(Zhe Lv et al. 2020)
The humanized monoclonal antibody H014 can effectively neutralize the SARS-CoV-S pseudovirus (PSV) and the SARS-CoV-2 S PSV by binding to the S receptor binding domain (RBD). In hACE2 mouse models, the administration of H014 can simultaneously reduce the SARS-CoV-2 virus titer and mitigate pathological changes in the lungs.
The above research provides us with methods for screening neutralizing antibody candidates and delivers promising verification results. These findings have proven significant for guiding the development of antiviral drugs. It also outlines one of the potential directions for the ongoing exploration of vaccine targets.
As of July 21, 2020, there are 25 new coronavirus vaccines undergoing clinical trials across the world, of which, at least 4 vaccines have entered phase III clinical trials.
Table 1：Progress of some vaccines under development
Vaccines are biological preparations of artificially disabled pathogenic microorganisms, which are unable to cause infection but still elicit an immune response, that provide an active acquired immunity to a particular infectious disease. Artificial immune induction involves the administration of viral agents in a less dangerous form, which aims to reduce overall mortality of a disease. Traditional, artificial immunizations are divided into two types: active immunization, which includes vaccines, and passive immunization. Artificial active immunization vaccines are distributed into three categories: inactivated, attenuated, and toxoid. There are several ways to achieve passive immunizations through the transfer of antibodies, which includes administration of blood plasma or serum, human immunoglobulin preparation, cytokine preparation, and monoclonal antibody (MAb) preparation. Both methods of immunization can boost the body's immunity to resist diseases and reduce the occurrence of infectious diseases.
Advances in science and technology have brought about many innovations in vaccine preparation methods. According to different preparation methods, vaccine types now include subunit, conjugate, synthetic peptide, and genetic engineering (e.g. recombinant antigen, recombinant vector, DNA- RNA, transgenic plant) vaccines.
On July 14, 2020, the candidate vaccine mRNA-1273 developed by Moderna in the United States announced promising results in a new publication. It can stimulate a better neutralizing antibody response in the human body and a cellular immune response biased towards Th1 CD4 with relatively mild adverse reactions at a dose of 100 mg. Clinical trials are currently ongoing.
The Phase II clinical results of the adenovirus vector vaccine ChAdOx1 nCoV-19 (AZD1222) from the team of Academician Chen Wei from the University of Oxford and the Academy of Military Medical Sciences were announced on July 20. The study evaluated the adenovirus Ad5 vector candidate for COVID-19 and confirmed effective immunogenicity and acceptable safety profile of the vaccine, additionally determining the appropriate dosing of the candidate vaccine for the subsequent studies. ChAdOx1 nCoV-19 induced both humoral and cellular immune responses. The results support large-scale evaluation of this candidate vaccine in an ongoing phase 3 program.
Additionally, on July 20, the German BioNTech company and Pfizer and other institutions in the United States released the results of the phase 1/2 clinical trial of the mRNA vaccine BNT162b1 on the preprint website medRxiv. This research illustrated that the mRNA vaccine processed by lipid nanoparticles can stimulate the subject's cellular immune response and humoral immune response. However, the evaluations of the three other BNT162 RNA vaccine candidates by these companies revealed that BNT162b2 and BNT162b1 were comparably safe and effective. Ultimately, on July 27 Pfizer and BioNTech announced their selection of BNT162b2 as the candidate to progress to a Phase 2/3 study based on the comprehensive data from preclinical and clinical studies, including select immune response and tolerability parameters.
The quick succession of these announced results reflects the rapid pace of developments in science and technology. However, in the process of evaluating the safety and effectiveness of vaccines, we must take the potential effects of antibody-dependent enhancement (ADE) into consideration.
So, what is ADE? As early as 1964, Hawkes et al. mentioned the hypothesis of "antibody-dependent infection enhancement" in the study of arboviruses. ADE is mainly manifested when the replication of the virus in low-concentration immune serum is promoted rather than inhibited, which is also seen among the effects of dengue fever and dengue shock syndrome. People infected with a dengue subtype will develop an immune response that does not effectively inactivate the other subtypes of the virus upon binding, leading to more severe infections as the virus evades immune suppression. There are concerns that this phenomenon may also occur with COVID-19.
Figure 6: Potential ADE on SARS-CoV-2 infection
Studies have shown that in SARS-CoV infection, ADE occurs because the virus binds to Fc receptors (FcR) expressed on varies of immune cells (including monocytes, macrophages, and B cells). The pre-existing SARS-CoV specific antibodies may promote the virus to enter FcR-expressing cells. In fact, infection of macrophages by ADE does not result in effective virus replication and shedding. In contrast, the internalization of viral antibody immune complexes can promote inflammation and tissue damage through FcRs5 activation of myeloid cells. Viruses entering through this pathway may bind to pattern recognition receptors TLR3, TLR7 and TLR8. Macrophages and monocytes may have a higher infection rate from the SARS-CoV virus through the action of ADE, leading to increased production of TNF and IL-6. Therefore, in the process of vaccine development, we must take possible ADE effects into consideration. The way to avoid this phenomenon to the greatest extent is to verify its safety in model animals before vaccine application.
Pre-clinical in vivo safety and effectiveness evaluations are a necessary part of drug and vaccine development – testing with appropriate animal models is required before moving onto human studies.
Throughout the development of biological sciences, model animals have gone through a long process - from sea urchins to yeasts, nematodes, and mice – to reach our current level of capability for models to provide insights into human physiology and pathology. Since the mouse genome is both 90% homologous to humans and easy to manipulate with modern technology, mice have become the predominant animal model used to study human diseases.
The outbreak of COVID-19 has brought significant attention to mice with humanized ACE2, the host receptor that binds with the SARS-CoV-2 S protein and leads to onset of infection. Across both traditional transgenic and precise gene modification approaches, ACE2 gene modified mice have been developed and bred intensively to support rapid research results. Over recent months, mouse models developed with different gene-modification strategies have gradually been completed - encouraging news for researchers fighting against the COVID-19 pandemic.
In June, Prof. Zhao Jincun’s team developed an ACE2 mouse model for SARS-CoV-2 research using adenovirus vector (AAV) method. Experimental verification has shown that the mouse model prepared by this method can produce effective neutralizing antibodies (NAbs), which is of great importance for vaccine and drug evaluation. At the same time, highly effective neutralizing antibodies (NAbs) were prepared by the University of Washington Medical Department using the AdV-hACE2 mice model.
In July, the progress of vaccine research was successively reported. The mRNA-1273 developed by Moderna in the United States and the nanoparticle mRNA vaccine developed by BioNTech together with other institutions in Germany have made significant progress. These vaccines are confirmed to stimulate an immune response in the body, producing neutralizing antibodies, and exhibited acceptable safety profiles. The adenovirus vector vaccine ChAdOx1 nCoV-19 prepared by Prof. Chen Wei and the University of Oxford also produced promising results during July 2020.
These are just a handful of the numerous ongoing studies that continue to enable the wider application of model animals. In this COVID-19 pandemic, people have just begun to have a deeper understanding of virus. Through the advancement of science and technology, global medical health will enter a new phase, and people will have a more thorough understanding of the interconnected nature of life.
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