RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants

By Bhavana KunkalikarReviewed by Danielle Ellis, B.Sc.Feb 22 2023

In a recent study published in iScience, researchers assessed the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike N-terminal domain (NTD) incorporation into the receptor-binding domain (RBD) protein vaccine.

Background

Recent evidence indicates that neutralizing antibody titers decline after two coronavirus disease 2019 (COVID-19) vaccinations and that protection against highly altered SARS-CoV-2 Omicron variants declines dramatically three months after booster vaccination. Thus, strategies involving immunization boosters are essential to curb the pandemic.

Future vaccine designs should concentrate on the humoral response against the RBD to generate high quantities of neutralizing antibodies (NAbs) and prevent the possibility of repriming immunological responses against non-neutralizing epitopes.

Unfortunately, the RBD by itself is a weak immunogen, failing to produce substantial amounts of NAbs in most cases. Multimerization of the RBD can enhance its immunogenicity, resulting in enhanced B cell receptor crosslinking and enhanced antigen-presenting cell phagocytosis.

About the study

In the present study, researchers developed a subunit vaccine consisting of an RBD tandem dimer linked to the N-terminal region of the SARS-CoV-2 spike protein NTD. By dimerizing the SARS-CoV-2 RBD and incorporating the NTD with the RBD-dimer construct, the team intended to improve the immunogenicity as well as the T cell response of an RBD-protein-based vaccine.

A variety of constructs, including tandem RBD sequences and a single NTD copy, were created by the researchers. The first generation of constructs was based on the SARS-CoV-2 Wuhan strains, while the second generation was created according to the SARS-CoV-2 Delta variant. The Wuhan residues comprised the monomeric RBD construct (W_RBD) and the RBD tandem dimer (W_RBD-RBD).

To assess the effect of design architecture on the expression, binding, and stability of angiotensin-converting enzyme-2 (ACE-2), the study generated controls comprising an RBD monomer as well as an RBD tandem dimer belonging to each strain and an RBD-NTD heterodimer.

Thermostability tests (TSA) were utilized to evaluate the stability and folding of the isolated proteins over several months. The initial protein candidate of the study was designed employing the RBD-RBD-NTD architecture and is subsequently designated VAANZ-W RRN. The ACE2 binding affinity for the W_RBD, W_RBD-RBD as well as the VAANZ-W RRN proteins was evaluated.

To assess whether VAANZ-Δ_RRN, which contains the essential S1 domain domains, was adequate in offering protection, its protective efficacy was evaluated in an animal model. This was achieved by vaccinating mice with 50 g of VAANZ-Δ_RRN AddaVax^TM on days zero and 21.

On day 14, a cohort of convalescent mice was established utilizing a sub-lethal dosage of 10² TCID₅₀ SARS-CoV-2 Wuhan strain. On day 35, the mice were inoculated with 1 x 10⁴ TCID50 with the Delta variant, and their body weight, as well as mortality, were recorded.

The team evaluated the antigen-specific response of the mice to Delta variant RBD dimer (Δ_RBD-RBD) vaccination to determine if adding NTD within VAANZ-Δ_RRN improved the breadth of the T cell response. By restimulating T cells with overlapping peptide pools from S1 or RBD, T cell responses to various sections of spike protein were evaluated.

Results

The study results showed that incorporating the NTD into the C-terminus of the RBD enhanced expression. After purification, the expression levels of the various constructs varied between approximately five and 30 mg/L of the medium. Notably, all proteins preserved identical TSA characteristics for at least five months at 4°C, demonstrating that RBD-based proteins are extremely stable at this temperature. ACE2 binding demonstrated that these proteins preserved the three-dimensional structure noted in the SARS-CoV-2 RBD.

According to affinity calculations, dimerizing the RBD quadrupled its binding affinity to ACE2. However, the Kd observed for all other constructs evaluated in this study was relatively similar at less than 2 nM, indicating that the presence of the NTD domain in the VAANZ-W RRN protein did not affect ACE2 interaction. All constructs were appropriately folded, and the presence of the NTD at the RBD dimer C terminus reported no effect on the RBD/ACE2 interaction. Thus, all domains remain free for antibody targeting.

Infecting unvaccinated mice led to fast weight loss, and by day 16, almost 61.5% of infected mice had perished. All mice inoculated with VAANZ-Δ_RRN + AddaVax^TM were protected against SARS-CoV-2 illness and did not experience weight loss. Similar viral titers were detected in the nasal and lung turbinates of SARS-CoV-2-infected immunized as well as convalescent mice.

VAANZ-W_RRN-vaccinated mice challenged with the SARS-CoV-2 Wuhan strain displayed a comparable level of protection against weight loss, with 100% of the mice surviving, compared to 7% surviving in the unvaccinated cohort.

There was no considerable variation between the number of RBD-specific and S1-specific interferon (IFN)-Ɣ-producing T cells in Δ_RBD-RBD-immunized mice. Vaccination with VAANZ- RRN produced a considerably greater number of S1-specific IFN-Ɣ-producing T cells than RBD-specific cells. By supplying additional T cell epitopes, incorporating NTD into the RBD dimer considerably enhanced the T cell response.

Conclusion

The study findings showed that VAANZ-Δ_RRN, an RBD-based protein vaccine, is highly immunogenic, robustly expressed, and has a strong stability profile. The study demonstrated that the inclusion of an NTD greatly broadened the T cell response, leading to increases IFN-Ɣ+ T cell responses. These observations imply that VAANZ- Δ_RRN proved to be a good candidate as a booster vaccine and may provide more effective protection against infection by SARS-CoV-2 variants of concern than RBD-based vaccines.