Researchers have developed small proteins from sequences in SARS-CoV-2's RBD that bind to ACE2. These were as effective in binding to neutralizing antibodies as larger RBD proteins.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely related to SARS-CoV. While the fatality rate of the SARS-CoV-2 is lower, its transmissibility is much higher. This is likely due to the presence of a furin cleavage site around position 701 on the viral spike protein.
SARS-CoV-2 has been mutating since it first appeared, with changes in the furin cleavage site and the receptor-binding domain (RBD). Hence, it is important to have sensitive and specific diagnostic tools to detect these variants.
Techniques to detect prior SARS-CoV-2 infection generally detect antibodies to the spike protein in blood sera. The methods used to express the SARS spike protein have been adapted for SARS-CoV-2. But, it is possible that future variants of the spike protein will not be easily made using recombinant techniques.
So, researchers have tried reducing the size of the protein needed to identify COVID-19 by using the area of maximum variation in sequence between SARS-CoV and SARS-CoV-2. This region is also a target of many SARS-CoV-2 neutralizing antibodies.
Small proteins for identifying SARS-CoV-2
In a study published on the medRxiv* preprint server, researchers report small synthetic proteins similar to this region of the virus that bind antibodies from convalescent sera.
The authors synthesized two proteins about 7 and 10 kD in size, JS7 and JS10, respectively, similar to a portion of the RBD that complexes with the angiotensin-converting enzyme 2 (ACE2). They tested sera obtained from nine COVID-19 patients, who had disease severity varying from mild to fatal.
The team found the antibodies in all the nine sera recognized the 7 kD protein in a dotspot assay, whereas recognition of SARS-CoV-2 recombinant RBD varied. ELISA assays showed similar binding for both. Antibodies from all the patients recognized SARS-CoV-2 RBD protein, but antibodies obtained from one patient with mild disease did not recognize the SARS-CoV full-length spike protein.
ELISA assays showed antibody binding was proportional to the infection severity. When the team added some peptides from the RBD/ACE2 complex, the binding reduced for some sera. For the 7 kD protein synthesized, adding only one peptide from this region reduced binding almost completely. Thus, using a smaller protein to detect previous SARS-CoV-2 infection was as effective as using a larger RBD protein.
Smaller proteins are more advantageous
One of the issues with larger recombinant proteins is the need for protein tags, for solubility, easy purification, and the like. The team found that when tested with COVID-19 negative sera and patient sera, both bound to the protein, producing artifacts in the results. Two control sera also bound to three recombinant proteins to the same level as patients with mild COVID-19. This may be because of the previous infection with other coronaviruses or germline antibody recognition.
One of the big advantages of the synthesized 7 kD protein is that it can be modified quickly to account for the emerging new variants of SARS-CoV-2. Mutations in the ACE2 binding regions of the RBD may reduce the effectiveness of treatments like convalescent plasma and monoclonal antibodies.
There is evidence that any variation in the protein sequence of the JS7 region of SARS viruses limits neutralization by antibodies. Just a single change in an amino acid between SARS-CoV and SARS-CoV-2 in an area close to the JS7 region greatly reduced the affinity of the cross-reactive SARS-CoV antibody CR3022.
Although all the patient sera antibodies recognized the SARS-CoV-2 RBD, antibodies from one patient who only had mild disease did not recognize the entire spike protein of SARS-CoV, even though 80% of the sequence is identical. Some mutations in this region, which were likely to escape mutants of monoclonal antibodies, were insensitive to neutralization by convalescent sera, suggesting variations arising in different parts of the world may be selected because of immune pressure.
Thus, it is necessary to have a wide variety of antigens representing regions of variation in the RBD to enable quick identification of different variants. Modifications can be made to the JS7 protein to add any reagents or linkers for assay, and it may also be used to develop vaccines.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.