SpyCatcher SpyTag technology to develop a recombinant protein-based intranasal vaccine against SARS-CoV-2

The results of a new study published on the bioRxiv* Preprint server demonstrated that the SpyCage intranasal vaccine platform can protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and could prove to be a versatile and adaptable modality for the formulation of intranasal vaccines targeting respiratory pathogens.

Study: Intranasal virus particle-mimicking vaccine improves SARS-CoV-2 clearance in Syrian hamster model.  Image Credit: WESTOCK PRODUCTIONS/Shutterstock
Study: Intranasal virus particle-mimicking vaccine improves SARS-CoV-2 clearance in Syrian hamster model. Image Credit: WESTOCK PRODUCTIONS/Shutterstock


The SARS-CoV-2 outbreak that spread into the global coronavirus disease 2019 (COVID-19) pandemic was partially controlled by widespread vaccination. COVID-19 vaccines prevent both disease transmission and infection.

In recent years there has been an avalanche of rapid vaccine developments that have proven highly effective. So far, most approved SARS-CoV-2 vaccines have to be administered intramuscularly (IM). IM vaccines induce high levels of circulating antibodies, memory B cells, and effector CD4+ and CD8+ T cells. However, evidence confirming the induction of mucosal immunity in the airways by these vaccines is lacking.

Intranasal vaccination may provide a solution to this shortcoming in current vaccination protocols. Recent studies have shown that SARS-CoV-2 vaccines administered intranasally provide mucosal immunity while preventing transmission of infection. These vaccines have been shown to reduce virus shedding in mice, hamsters and non-human primates.

About the study

The present study used SpyCatcher SpyTag technology to develop a recombinant protein-based intranasal vaccine against SARS-CoV-2. The results suggested an adaptable and flexible method to develop intranasal vaccines targeting respiratory infections.

This experimental study was conducted to fill the gap in the lack of mi=ucosal immunogenicity against SARS-CoV-2 by designing a self-assembling protein incorporated into a nanoparticle containing a SpyCatcher domain (SpyCage) for displaying SARS-CoV-2 RBD/SpyTag contains (RBD+SpyCage) for intranasal vaccination studies in hamsters.

Here, a virus particle emulating an intranasal vaccine against SARS-CoV-2 was used to perform preclinical vaccination and challenge testing. Based on the SpyCatcher SpyTag method, the vaccine candidate was self-assembled on a 60-subunit protein scaffold covalently coated with the SARS-CoV-2 receptor-binding domain (RBD).

In addition, researchers verified the expected antigen display properties by reconstructing the I3-01 to 3.4A framework using cryogenic electron microscopy (cryo-EM). Furthermore, RBD decoration was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy (TEM) with negative staining.

Researchers conducted two immunization trials using an intranasal prime and boost vaccination schedule followed by a SARS-CoV-2 challenge in Syrian hamsters. The RBD-grafted SpyCage scaffold (RBD+SpyCage) was used, which was initially evaluated for immunogenicity. A more comprehensive study was then performed to demonstrate the covalent attachment of RBD to the scaffold, necessary for antibody production.

Neutralizing antibody titers were determined by microneutralization experiments, and RBD-binding immunoglobulin (Ig)G and IgA antibody levels were determined by enzyme-linked immunosorbent assay (ELISA).

study results

Through cryo-EM reconstruction and refinement of an atomic model of the apo cage scaffold, researchers developed a robust, multimeric, spherical, protein-based scaffold that mimics the size of a virus particle for intranasal vaccinations. Covalent attachment of SARS-CoV-2 spike RBD to SpyCage was performed, which turned out to be a stable and saturable platform to present antigens of interest in a mix-and-go fashion.

The SpyCage scaffold was found to be immunogenic and engraftment of antigen enhanced antibody responses to both scaffold and antigen. Intranasal administration of RBD+SpyCage increased clearance of SARS-CoV-2 from the airways.

The results showed that SARS-CoV-2 was more effectively eliminated from the upper and lower airways of animals vaccinated with RBD+SpyCage. Histological analysis showed that animals vaccinated with RBD+SpyCage had less inflammation and lung damage.


Intranasal immunization with RBD grafted onto SpyCage elicited serum IgG responses in hamsters. As a result of this interaction, viral clearance from the upper and lower airways appeared to be faster after viral challenge.

While mice vaccinated with RBD+SpyCage showed a non-significant reduction in weight loss and lung damage – consistent with a non-neutralizing antibody response. It was determined that the immunogenicity and efficacy of the RBD+SpyCage vaccine required covalent attachment of the RBD to the SpyCage scaffold.

Therefore, it has been suggested that SpyCage scaffolded antigens can be used as a vaccine platform when administered intranasally. It should be further developed with the addition of intranasal adjuvants to increase immunogenicity.

An added benefit of SpyCage-derived intranasal vaccines is that they can be easily grafted onto scaffolds to induce mucosal immunity, making them ideal for combating other respiratory viruses. Therefore, this platform can be used as a fast-acting vaccine to target new pathogens or pandemics.


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