Without question, the global outbreak of the novel coronavirus 2019 (SARS-CoV-2) that causes COVID-19 has dominated scientific discussion and has been fodder for a wealth of publications as the world urgently races for a better understanding of the mechanism of action and designing an effective vaccine — a vaccine that would be key to easing the public health threat.
To date, we know that the SARS-CoV-2 genome is comprised of ∼30,000 nucleotides. It encodes four structural proteins: nucleocapsid (N) protein, membrane (M) protein, spike (S) protein, and envelope (E) protein, plus several non-structural proteins. Codex DNA recently described the on-demand production of the entire genome of SARS-CoV-2 in just over a week, with genome parts designed and synthesized using the BioXp™ system. Our application note reporting this work also described the joining of these parts to generate the SARS-CoV-2 genome as a bacterial artificial chromosome in E.coli. Given the number of SARS-CoV-2 genome variants that have now been identified — the use of automated cloning solutions may be important for achieving the rapid turnaround times demanded by vaccine, therapeutic, and diagnostic development pipelines.
Many research groups have elucidated the mechanisms of viral entry, replication, and RNA packing in the human cell. Briefly, the receptor-binding domain (RBD) of the coronavirus S protein attaches to angiotensin-converting enzyme 2 (ACE2) receptors found on the surface of many human cells, including those in the lungs. The S protein is then subjected to proteolytic cleavages by host proteases (i.e., trypsin and furin), triggering the membrane fusion mechanism’s activation. Membrane fusion allows the viral RNA to enter the host cell and undergo replication.
Most current therapeutic research strategies have relied on anti-SARS-CoV monoclonal antibodies to neutralize the virus by binding to epitopes on the RBD rather than blocking RBD-ACE2 binding. Recent work by MassBiologics, published in Nature, describes the discovery of a unique cross-reactive epitope within the core of the receptor binding interface of the S protein and the subsequent design of an effective cross-neutralizing human IgA monoclonal antibody, mAb362 IgA. This IgA antibody binds very efficiently to the SARS-CoV-2 RBD with high affinity, competing at the ACE2 binding interface by blocking interactions with the receptor.
As part of the overall workflow and methodology, scientists performed mutational scanning of the SARS-CoV-2 RBD residues in part by using DNA variant libraries built on the BioXp™ system to identify specific mAb362-binding components. Mutational scanning with a combination of alanine (to introduce a loss of interaction), tryptophan (to introduce a steric challenge), and lysine (to introduce charge mutations), was performed to delineate the binding surface better. The results showed that key residues — Y449A, Y453A, F456A, A475W, Y489A, and Q493W — were critical for the complex and, presumably, that alterations in the packing caused a marked loss of binding affinity. Of the mutants tested, A475W and Y489A also disrupted ACE2 binding. Interestingly, introduction of lysine mutations had little effect on binding, and some even showed enhanced binding, presumably owing to an overall more favorable charged interaction with mAb362.
The utility of the BioXp™ system and incorporation of a scanning library design was critical to quickly identifying these functional residues and affording insight into the ongoing design efforts of vaccine and prophylactic/therapeutic antibodies against future emerging infections caused by this viral family.