A new study published on the online preprint server bioRxiv* in May 2020 reports the construction and testing of a new ferritin-based antigen-bearing protein that has biological activity towards the ACE2 receptor. This could indicate its potential for development as a vaccine or antiviral.
While the current COVID-19 pandemic continues to cause new cases and deaths by the thousands, researchers are trying to understand the virus, its mechanism of pathogenesis, and how it induces an immune response in the host. The results could inform a more effective strategy towards the development of a vaccine or therapeutic drug, helping to control the outbreak.
The Structure of SARS-CoV-2
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the etiological agent of the current pandemic, is an RNA virus with a large genome, four structural, and 16 non-structural proteins. Of the four structural proteins, one is a spike protein, responsible for virus attachment to and entry into the host cells. The others comprise the envelope, membrane, and nucleocapsid proteins.
The spike protein is being investigated as a potential vaccine target. Antibodies binding to the spike glycoprotein, specifically its receptor-binding domain (RBD), can efficiently neutralize virus infection. However, there is little cross-reactivity between SARS-CoV-2 and SARS-CoV antibodies.
SARS-CoV-2 viral entry into the host cells depends on the existence of a functional ACE2 receptor on the host cell. The viral spike protein binds to the receptor via its receptor-binding domain, as revealed by cryo-electron microscopy. The spike protein has two domains, S1 and S2.
The receptor-binding site on the S1 domain changes conformation, through a hinge-like site, from the flip-down to the flip-up conformation, before it can bind to the ACE2 receptor. If a neutralizing antibody can target this, the entry of the virus could be prevented. This, in turn, has led to intensive research into the atomic disposition of this state, along with the identification of the receptor-binding motif (RBM).
Ferritin – A New Avenue
Ferritin is a polymeric protein made of 24 units, comprising both heavy and light chains, forming a cage-like structure similar to that of SARS-CoV-2. This has attracted the attention of many researchers as an attractive platform for immunity induction. It is 12 nm in diameter, which allows for rapid penetration into tissues as well as its entry into the regional lymph nodes.
The heavy chain of ferritin is of low immunogenicity and has found multiple applications in nanomedical and molecular diagnostics, including antiviral and anticancer vaccines.
The current study thus examines the biological activity and immunogenicity of a fused ferritin-RBM protein as a potential vaccine precursor.
The Building of the Fusion Protein
The researchers incorporated the RBM of the virus, containing 72 amino acids, into the heavy chain of the human ferritin molecule (HFtn) to form a fusion protein termed RBM-HFtn.
The resulting fusion protein displays eight fused protein trimers with increased potential for immune response induction. The RBM subunits are placed distant enough from each other that they still allow the formation of the polymeric structure. A small RBM peptide was chosen for the same reason, so that it may be well displayed on the surface of the ferritin heavy chain.
The fusion protein was transfected into the E. coli BL21 strain for cloning and refolding. It was then characterized by physical and chemical methods, as well as transmission electron microscopy (TEM). The binding affinity of RBM-HFtn for the ACE2 receptor was also analyzed by ELISA techniques.
The researchers found that the protein harvested from the bacterial culture was mainly in the form of inclusion bodies instead of in a soluble form as expected. When it was induced to refold, the protein was found to be about 15 nm, with a uniform round shape.
The binding affinity assay showed that the viral RBD has a lower binding affinity than the fusion protein’s RBM-HFtn. The higher binding affinity of the latter may be due to the cage-like surface of the fusion protein, which can bear 24 copies of the RBM on its surface.
Schematic representation of RBM-HFtn structure with ferritin as scaffold (blue) fused with RBM of SARS-CoV-2 spike glycoprotein (red); The RBM-HFtn is capable of complexing with ACE2 (green) (A). Total 24 copies of RBM are presented on the ferritin surface, here only 14 copies were shown for symbolic representation. Two hypothetical antivirus pathways of RBM-HFtn by inducing antibodies against SARS-CoV-2 (B, upper pathway) and/or by blocking virus entry through competitive binding to ACE2 (B, lower pathway).
*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.
The Mechanism of Action of The Fusion Protein
The fusion protein RBM-HFtn may act against SARS-CoV-2 in two different ways. One, it may stimulate neutralizing antibody formation directed against the RBM of the spike protein, which may allow its use as a vaccine. The neutralized virus will then be cleared from the body by the immune system.
Research on COVID-19 patient samples has shown the presence of two antibodies that inhibit viral RBD-ACE2 binding. Animal experiments have shown that these could reduce the viral load in the lungs of infected mice. This leads the researchers to theorize that “by properly presenting S-protein RBD/RBM, the designed RBM-HFtn has great potential as an anti-SARS-CoV-2 vaccine.”
Another possible mechanism of action is blocking the cell entry of the virus by the binding of the RBM-HFtn fusion protein to the ACE2 receptor, thus blocking viral proliferation. While the first mechanism could prevent infection, the second may reduce the severity of infection by antagonizing its action at the receptor.
The study thus reports the construction of a fusion protein, which could be isolated at high purity, with the size and shape as expected. It was also active against the ACE2 receptor, as confirmed by ELISA.
The results suggest that “the RBM is properly presented on the surface of heavy chain human ferritin and is recognizable by the ACE2 receptor. Its potential as SARS-CoV-2 vaccine and as an antagonist of ACE2 receptor is further studied in animal experiments.”
*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.
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