Tiny shark-derived antibodies broadly neutralize most SARS-CoV-2 variants

A promising new Small Methods study discusses the ability of shark-derived antibodies to protect against multiple variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as the closely related bat and pangolin coronaviruses, following intranasal infection. This offers a valuable preventive and therapeutic option, while also increasing current knowledge of the immune evasion capabilities of SARS-CoV-2.

Study: A Class of Shark-Derived Single-Domain Antibodies can Broadly Neutralize SARS-Related Coronaviruses and the Structural Basis of Neutralization and Omicron Escape. Image Credit: ustas7777777 / Shutterstock.com

Introduction

Since the second year of the ongoing pandemic of coronavirus disease 2019 (COVID-19), vaccines have been deployed on a large scale. However, the emergence of numerous SARS-CoV-2 variants with increased immune escape capabilities reduced the efficacy of most vaccines.

Thus, effective therapeutics and preventatives are still urgently needed. Additionally, the constant threat of new zoonoses means also emphasizes the need to develop broadly protective agents, rather than those that are specific to a single threat.

The administration of antibodies is called passive immunization. Monoclonal antibodies, for example, have been particularly useful in preventing the development of severe COVID-19, especially in at-risk patient populations such as immunocompromised individuals who have a poor vaccine response, the elderly, and those with underlying illnesses. However, the cost, large doses required, and difficulty of storing and transporting these antibodies at very low temperatures restrict their universal use.

Another option is the use of nanobodies, which are single-domain antibodies that consist of the variable domain of the heavy chain of camelid antibodies. Notably, nanobodies have only one-tenth the molecular weight of human antibodies at 15 kilodaltons (kD), as compared to 150 kD. Currently, nanobodies are being investigated for their utility in treating cancer, infections, autoimmune diseases, and inflammatory conditions, with the first being approved in 2018.

A related class of single-domain antibodies is the variable new antigen receptor (VNAR), which are found in cartilaginous fish such as sharks and skates. These “vnarbodies” are slightly smaller than nanobodies at 13 kD.

Both of these types of single-domain antibodies show superiority to other types of antibodies, including their high target affinity, increased storage temperatures, small size, which allows them to bind to previously inaccessible epitopes, and lower production costs through the use of non-mammalian cells.

The researchers in the current study focused on vnarbodies in the hope that these would be more stable than human antibodies, as they are derived from animals with a high urea concentration in their bodies. To this end, anti-SARS-CoV-2 vnarbodies were isolated from bamboo sharks inoculated with the viral spike protein and screened for their picomolar binding affinities to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

The neutralizing antibodies, 20G6 and 17F6, were then selected as a result of their high neutralizing capability against multiple SARS-CoV-2 variants and ability to prevent infection and severe disease in mice exposed to the virus.

Study: A Class of Shark-Derived Single-Domain Antibodies can Broadly Neutralize SARS-Related Coronaviruses and the Structural Basis of Neutralization and Omicron Escape. Image Credit: Juvenile brown-banded Bamboo shark. Image Credit: SergeUWPhoto / Shutterstock

Study findings

The researchers dimerized isolated vnarbodies by attaching them to the human immunoglobulin (Ig) G1 Fc domain. These dimers bound to the SARS-CoV-2 Alpha, Beta, Kappa, Delta, Delta plus, Lambda variants, with similar binding affinities to the ancestral RBD variant, thereby indicating their broad reactivity to different SARS-CoV-2 RBDs.

The vnarbodies competitively inhibited the binding of the spike protein to the host cell angiotensin-converting enzyme 2 (ACE2) receptor at the RBD for all tested variants. Further study showed that pretreatment with the vnarbody 20G6 led to tight RBD binding that prevented human ACE2 (hACE2) binding. This was slightly lower with 17F6.

Pseudovirus neutralization assays expressing all of the aforementioned SARS-CoV-2 variants showed nanomolar half-maximal inhibitory concentrations (IC50). This was confirmed using the original SARS-CoV-2 strain, which showed a dose-dependent neutralization of all variants by both vnarbodies in both monomer and dimer form, but up to ten times more potently with the latter.

Subsequently, an in vivo study in mice showed that the prophylactic intranasal administration of 20G6-Fc three hours before intranasal inoculation of the Wuhan and the Beta strains reduced the viral load by 1.5 logs with the ancestral variant. With the Beta variant, which has shown a seven-fold increase in neutralizing titers with most human antibodies, an approximately 4.5 log reduction in the viral load was observed.  

When infected mice were treated with these antibodies, the viral load was reduced by two to three logs. Furthermore, no significant weight loss was found in the treated group, whereas untreated mice showed 10-20% weight loss by day three. Lung pathology was also reduced, as demonstrated by the lack of severe bronchopneumonia and lymphocyte infiltration.

Mechanism of inhibition

These vnarbodies bind to conserved RBD epitopes that are exposed in the RBD ‘up’ conformation. Thus, these antibodies can compete with the ACE2 receptor for RBD binding. The 20G6-RBD complex interacts to form a continuous β-sheet, thereby explaining its high stability and binding affinity.

Both 17F6 and 20G6 contain a “WXGY motif” in the CDR3 sequence where RBD-vnarbodies interact by forming the β-strand that prevents RBD-ACE2 binding. This is a mechanism of protective binding that appears to be common to shark vnarbodies.

The WXGY motif is responsible for the strong RBD-vnarbody binding interactions. This unique binding motif gains added binding strength from other amino acids in the same region. These additional interactions are responsible for the different activities observed between 20G6 and 17F6.

The SARS-CoV-2 Omicron variant is not neutralized by 20G6 and related antibodies, despite the presence of the highly conserved 365–380 region of the RBD that acts as the binding epitope for both vnarbodies. This accounts for the binding and neutralization of not only SARS-CoV-2 and its variants except Omicron, but other Sarbecoviruses including the pangolin and bat coronaviruses (CoVs), but not the Middle East respiratory syndrome (MERS)-CoV or SARS-CoV.

This could be due to the S375F mutation on the Omicron RBD. This mutation changes the conformation of the RBD, which impairs the formation of the β-strand structure in the RBD and prevents 20G6 binding.  

Implications

The findings of this report point to the discovery of SARS-related antibodies with broad neutralization and a new mechanism of binding. Furthermore, both 20G6 and 17F6 antibodies are capable of neutralizing most SARS-CoV-2 variants except Omicron and protect against, as well as treat intranasal SARS-CoV-2 infection in vivo.

By countering the virus at the point of first entry, vnarbodies are promising for their potential development into a nasal spray that can be used to protect front-line workers and travelers in high-risk areas. Added to their low manufacturing costs and thermal stability, these antibodies could be potentially useful in the current pandemic and other future outbreaks.

Moreover, these vnarbodies appear to be broadly neutralizing against a range of Sarbecoviruses. The epitope is masked, except in the ‘up’ conformation, and does not overlap with ACE2-RBD binding. Unlike other class 4 epitopes like CR3022, the vnarbodies discussed in this study have strong neutralizing capabilities because of their steric hindrance with a sugar moiety on ACE2, thus preventing their binding.

The unprecedented immune evasion characteristics of the SARS-CoV-2 Omicron variant defies these vnarbodies as well, as with almost all human neutralizing antibodies reported to date. This is due to the presence of the RBD S375F mutation that disrupts the β-sheet structure of the RBD.

Currently, the researchers are working to broaden the action of this class of molecules by affinity maturation techniques. The possibility of immunogenicity is low because of the shared immunoglobulin structure of both human antibody variable heavy and light chains with vnarbodies. Future humanized intranasal or topical vnarbodies may be capable of avoiding this risk.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.