Part of the adaptive immune system of sharks, VNARs are evolutionarily distinct from immunoglobulins despite sharing some structural similarity with mammalian heavy and light variable chains. VNARs are the smallest (~11 kDa) naturally occurring independent heavy chain-only binding domains in the vertebrate kingdom. Variable New Antigen Receptors (VNARs) represent an unexplored technology for the development of next-generation NAbs for SARS-CoV-2. Thus, there is a need for NAbs that can recognize cryptic epitopes inaccessible to human antibodies that are impervious to mutational drift. These mutations can result in the modification of NAb epitopes leading to attenuated or abrogated neutralization of the virus by antibodies. Studies have shown that mutational changes in the RBD observed in the variants correspond to surface-exposed residues within or proximal to the ACE2 binding interface. Complicating the development of effective NAbs is the emergence of new SARS-CoV-2 variants with highly mutated S proteins. In general, NAbs act by blocking the ACE2 binding interface or by trapping the RBD in the unstable “up” conformation. Studies with NAbs that target the RBD have revealed mechanisms of viral neutralization based on changes in the “up” and “down” conformations. The RBD exists in two different conformations the closed “down” conformation and the open “up” conformation which is highly accessible to ACE2. The receptor-binding domain (RBD) on the S1 subunit is responsible for engaging angiotensin-converting enzyme 2 (ACE2)-the cognate receptor required for membrane fusion. The S protein has two distinct functional subunits that facilitate cell attachment (S1) and fusion of the viral and host cell membranes (S2). SARS-CoV-2 NAbs target the trimeric spike (S) protein on the viral surface that mediates cell entry. Two NAb therapeutics (LY3819253 and REGN-COV2) received emergency use authorization status from the Food and Drug Administration for use in the clinic 7. Neutralizing antibody (NAb) therapeutics that block virus entry into the host cell have demonstrated efficacy at treating COVID-19 infection. As we enter the next key stage in our global escape plan from this pandemic, it is vital to develop alternative therapeutic approaches and, concurrently, expand our knowledge of this virus. It has been documented that people with compromised immune systems respond poorly to COVID-19 vaccines, thus necessitating the development of additional antiviral therapeutics 5, 6. Researchers have reported that the new SARS-CoV-2 variants can result in reduced sensitivity to antibody therapies, convalescent plasma, and vaccine sera 1– 4. The rapid evolution of SARS-CoV-2 into highly infectious variants across the globe also has the potential to impact vaccine efficacy. The widely implemented two-dose requirement to achieve efficacy, leaves the possibility of non-compliance for the second dose, a situation that may be exacerbated further by the decision in certain areas to extend the time interval between dosing. Though vaccines are the centrepiece for controlling the pandemic, the benefits of vaccines depend upon complex population vaccination strategies that remain vulnerable to manufacturing or deployment delays. The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a devastating global health crisis. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature.
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