Preclinical Efficacy of IMM-BCP-01, a Highly Active Patient-Derived Anti-SARS-CoV-2 Antibody Cocktail (preprint)

Using an unbiased interrogation of the anti-viral memory B cell repertoire of convalescent COVID-19 patients, we identified three human antibodies that when combined demonstrated both robust viral suppressive properties against all tested SARS-CoV-2 variants of concern in vitro and profound anti-viral efficacy in vivo. In this report, we describe the pre-clinical characterization of an antibody cocktail, IMM-BCP-01, that consists of three unique, patient-derived recombinant antibodies directed at non-overlapping surfaces on the Spike protein, each with particularly effective antiviral activity. One antibody has a composite epitope blocking ACE2 binding, one antibody bridges two Spike proteins, and one antibody neutralizes virus by binding to a conserved epitope outside of ACE2 binding site. These antibodies, when administered after viral infection, potently decreased viral load in lungs of infected Syrian golden hamsters in a dose-dependent manner, elicited broad anti-viral neutralizing activity against multiple SARS-CoV-2 variants, and induced a robust anti-viral effector function response, including phagocytosis, and activation of classical complement pathway. Our pre-clinical data demonstrate that the unique three antibody cocktail IMM-BCP-01 is a potent and dose-efficient approach to treat early viral infection and prevent SARS-CoV-2 in susceptible individuals.

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates (preprint)

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.

In-Vitro Fluorescence Microscopy Studies Show Retention of Spike-Protein (SARS-Cov-2) on Cell Membrane in the Presence of Amodiaquin Dihydrochloride Dihydrate Drug (preprint)

The ability of S-glycoprotein (S-protein) in SARS-Cov-2 to bind to the host cell receptor protein (angiotensinconverting enzyme 2 (ACE2)) leading to its entry in cellular system determines its contagious index and global spread. Three available drugs (Riboflavin, Amodiaquin dihydrochloride dihydrate (ADD) and Remidesivir) were investigated to understand the kinetics of S-protein and its entry inside a cellular environment. Optical microscopy and fluorescence-based assays on 293T cells (transfected with ACE2 plasmid) were used as the preamble for assessing the behaviour of S-protein in the presence of these drugs for the first 12 hours post S-protein - ACE2 binding. Preliminary results suggest relatively long retention of S-protein on the cell membrane in the presence of ADD drug. Evident from the %-overlap and colocalization of S-protein with endosome studies, a large fraction of S-protein entering the cell escape endosomal degradation process, suggesting S-protein takes non-endocytic mediated entry in the presence of ADD, whereas in the presence of Riboflavin, S-protein carry out normal endocytic pathway, comparable to control (no drug) group. Therefore, present study indicates ADD potentially affects S-protein's entry mechanism (endocytic pathway) in addition to its reported target action mechanism. Hence, ADD substantially interfere with S-protein cellular entrance mechanism. However, further detailed studies at molecular scale will clarify our understanding of exact intermediate molecular processes. The present study (based on limited data) reveal ADD could be potential candidate to manage Covid-19 functions through yet unknown molecular mechanism.

Molecular Mechanism of the N501Y Mutation for Enhanced Binding between SARS-CoV-2's Spike Protein and Human ACE2 Receptor (preprint)

Coronavirus disease 2019 (COVID-19) has been an ongoing global pandemic for over one year. Recently, an emergent SARS-CoV-2 variant (B.1.1.7) with an unusually large number of mutations had become highly contagious and wide-spreading in United Kingdom. From genome analysis, the N501Y mutation within the receptor binding domain (RBD) of the SARS-CoV-2's spike protein might have enhanced the viral protein's binding with the human angiotensin converting enzyme 2 (hACE2). The latter is the prelude for the virus' entry into host cells. So far, the molecular mechanism of this enhanced binding is still elusive, which prevents us from assessing its effects on existing therapeutic antibodies. Using all atom molecular dynamics simulations, we demonstrated that Y501 in mutated RBD can be well coordinated by Y41 and K353 in hACE2 through hydrophobic interactions, increasing the overall binding affinity between RBD and hACE2 by about 0.81 kcal/mol. We further explored how the N501Y mutation might affect the binding between a neutralizing antibody (CB6) and RBD. We expect that our work can help researchers design proper measures responding to this urgent virus mutation, such as adding a modified/new neutralizing antibody specifically targeting at this variant in the therapeutic antibody cocktail.

Molecular basis for a germline-biased neutralizing antibody response to SARS-CoV-2 (preprint)

The SARS-CoV-2 viral spike (S) protein mediates attachment and entry into host cells and is a major target of vaccine and drug design. Potent SARS-CoV-2 neutralizing antibodies derived from closely related antibody heavy chain genes (IGHV3-53 or 3-66) have been isolated from multiple COVID-19 convalescent individuals. These usually contain minimal somatic mutations and bind the S receptor-binding domain (RBD) to interfere with attachment to the cellular receptor angiotensin-converting enzyme 2 (ACE2). We used antigen-specific single B cell sorting to isolate S-reactive monoclonal antibodies from the blood of a COVID-19 convalescent individual. The seven most potent neutralizing antibodies were somatic variants of the same IGHV3-53-derived antibody and bind the RBD with varying affinity. We report X-ray crystal structures of four Fab variants bound to the RBD and use the structures to explain the basis for changes in RBD affinity. We show that a germline revertant antibody binds tightly to the SARS-CoV-2 RBD and neutralizes virus, and that gains in affinity for the RBD do not necessarily correlate with increased neutralization potency, suggesting that somatic mutation is not required to exert robust antiviral effect. Our studies clarify the molecular basis for a heavily germline-biased human antibody response to SARS-CoV-2.

A trimeric human angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro (preprint)

Effective intervention strategies are urgently needed to control the COVID-19 pandemic. Human angiotensin-converting enzyme 2 (ACE2) is a carboxypeptidase that forms a dimer and serves as the cellular receptor for SARS-CoV-2. It is also a key negative regulator of the renin-angiotensin system (RAS), conserved in mammals, which modulates vascular functions. We report here the properties of a trimeric ACE2 variant, created by a structure-based approach, with binding affinity of ~60 pM for the spike (S) protein of SARS-CoV-2, while preserving the wildtype peptidase activity as well as the ability to block activation of angiotensin II receptor type 1 in the RAS. Moreover, the engineered ACE2 potently inhibits infection of SARS-CoV-2 in cell culture. These results suggest that engineered, trimeric ACE2 may be a promising anti-SARS-CoV-2 agent for treating COVID-19.