Correlating physicochemical and biological properties to define critical quality attributes of a recombinant AAV vaccine candidate (preprint)

Recombinant adeno-associated viruses (rAAVs) are a preferred vector system in clinical gene transfer. A fundamental challenge to formulate and deliver rAAVs as stable and efficacious vaccines is to elucidate interrelationships between the vectors physicochemical properties and biological potency. To this end, we evaluated an rAAV-based COVID-19 vaccine candidate which encodes the Spike antigen (AC3) and is produced by an industrially-compatible process. First, state-of-the-art analytical techniques were employed to determine key structural attributes of AC3 including primary and higher-order structures, particle size, empty/full capsid ratios, aggregates and multi-step thermal degradation pathway analysis. Next, several quantitative potency measures for AC3 were implemented and data were correlated with the physicochemical analyses on thermal-stressed and control samples. Results demonstrate links between decreasing AC3 physical stability profiles, in vitro transduction efficiency in a cell-based assay, and importantly, in vivo immunogenicity in a mouse model. These findings are discussed in the general context of future development of rAAV-based vaccines candidates as well as specifically for the rAAV vaccine application under study.

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.