The onset of the coronavirus disease 2019 (COVID-19) pandemic has brought about an international effort to uncover the biology of its causative agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and to develop effective and safe antivirals to contain and treat the infection. A new study by a team of researchers in France and Sweden discusses one such study, exploring the relative immunogenicity of the three main structural proteins of the virus. The team has released their findings on the bioRxiv* preprint server.

SARS-CoV-2 infects the host cells via its surface spike glycoprotein. Once viral entry is accomplished, it uses the cell’s machinery to translate its RNA genome to structural and non-structural proteins, as well as accessory proteins, ensuring they have the proper secondary and tertiary structure to fulfill their function.

Among the chief viral proteins are the spike, envelope and membrane proteins, all of which are exposed to the host immune system. As a result, they trigger antibody responses, that in turn not only neutralize viral entry into the host cells, but activate other immune pathways including immune cells and complement-mediated cell destruction.

Antibodies to S, M and E proteins

Since not much is known about antibody responses to the M and E proteins, the current study aimed to understand the ability of these two viral proteins, along with the spike antigen, to stimulate antibody production, using a system based on human cells. This platform was designed to simulate viral insertion into the cell membrane and N-glycosylation, since these are considered essential for antibody recognition.

The researchers found that the serological responses showed a similar pattern in all COVID-19 patients, with mild, moderate or severe disease. The antibody profiles were comparable to those obtained with diagnostic assays, with around 90% concordance.

The control sera showed no binding to the S, E or M proteins, and were therefore used to set the reference for seropositivity.

Both COVID-19 patients and those with COVID-19-like symptoms showed immunoglobulin G (IgG), IgM and IgA antibodies to the spike protein. Patients with moderate to severe disease had higher titers of antibodies to the spike protein than with mild disease. Severe disease was also observed to be associated with anti-M antibodies.

Anti-M antibodies were always associated with anti-spike antibodies, but the converse was not true. Anti-E antibodies were not found in any patient.

D614G mutation alters spike structure

The researchers also found that the D614 spike, characteristic of the virus in the earlier part of the pandemic, was structurally different from the G614 mutant spike, which characterizes the current globally dominant variant. The earlier variant has a salt bridge connecting two spike protomers at the D614-K854 residue, as well as between R646 and E865.

With the G614 mutant, the K854 forms a hydrogen bond to the G614 backbone, which is a weaker and more energetic bond. There are changes in the non-polar region of spike protomer B, which projects towards protomer A in the D614 variant but is pushed away in the G614 mutant.

The only electrostatic interactions with the latter are with the second interaction. Additionally, the part around the K854 position in protomer B is moved around to point away from protomer A. More solvent exposure was seen with the polar residues around E685 of protomer B. Overall, therefore, the G614 mutation changes the structure of the spike protein, which in turn alters the immune response.

D614G mutation reduced antibody-spike binding

When the antibodies to the spike antigen were re-assessed with sera from COVID-19 patients, using both variants of the spike, they found that the mutation does not reduce the antigenicity of the spike antigen, but instead reduces IgG, IgM, and IgA binding to the G614 spike variant.

European patients were mostly infected by the G614 spike-bearing variant, rather than the D614 variant on which most commercial assays are based. No previous study has shown a difference in antibody binding, using ELISA testing.

This result shows the need for antigens used to explore antibody responses should reflect the viral proteins accurately.

What are the implications?

Our experimental system allows for discrimination between anti-S D614 vs. G614 Ig signals likely due to the advantage of using membrane inserted Spike following complex folding and post-translational modifications.”

The study also shows the advantages of using the full-length spike, rather than only the S1 subunit or the receptor-binding domain (RBD) alone. The former will undergo proper post-translational modification, allowing it to possess structural integrity. This makes it more likely to elicit the full range of the antibody response.

The researchers demonstrated the generation of antibodies against the full M protein rather than antigenic peptides, for the first time, validating earlier assays performed with microarrays. The absence of anti-E antibodies could be due to the lack of surface expression of this protein, or the lack of antigenicity. In fact, some research suggests that this protein is mainly found within the protein-processing compartments of the cell.

Since anti-M Ig responses were robust, and none of the new variants show M mutations, it is necessary to examine the potential of these antibodies to produce effective neutralization. If so, this protein, being conserved across lineages, could be the basis for more durable vaccines.

At present, with the vaccination campaigns in many countries being well underway, simultaneous with the spread of new variants, the researchers present their serological test as “a reliable test to verify the immunization efficiency during the vaccination and to analyze the impact of these Spike mutations on antibody responses.”

*Important Notice

bioRxiv 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.

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.