While global immunity to SARS-CoV-2 is increasingly widespread, continued manufacture and distribution of vaccines is necessary due to waning immunity after vaccination or infection1. Booster mRNA vaccines have been approved and distributed in high-income countries to protect against Omicron and other variants of concern (VOCs) that evade approved vaccines for COVID-19 matched to the original Wuhan-Hu-1 strain2,3. mRNA vaccines, however, are still not widely available in low- and middle-income countries (LMICs) due to high prices and limited supply4. Several protein subunit vaccines for COVID-19 based on the SARS-CoV-2 receptor binding domain (RBD) have been manufactured, approved, and widely distributed in LMICs5. Protein vaccines may be stored at higher temperatures for distribution in rural areas and manufactured in LMICs at low-cost using microbial hosts such as yeasts. Additional candidates for accessible booster vaccines could augment the expected need for new boosters to address the continued evolution of SARS-CoV-2.
Virus-like particles (VLPs) that multivalently display the RBD antigen can increase immunogenicity and protection compared to monomeric RBD in animal models6,7 and humans8. Manufacturing of VLPs, however, is currently performed in mammalian cell hosts which increase production costs9. Indeed, the only currently approved RBD-VLP vaccine is produced with HEK-293 cells in Korea8. Several studies report that covalent or genetic multimerization of the RBD, like dimers and trimers, may improve immunogenicity relative to the monomeric RBD10–13. Some of these simple RBD multimers are currently under clinical evaluation14,15. Dimer- and trimer-based antigens could provide a modular platform for new variants. These could be designed and manufactured with updated serotypes to match existing Wuhan-Omicron boosters, for example. Moreover, they can leverage the low-cost manufacturing approaches for simpler antigens like monomeric RBDs. To our knowledge, there has not been a direct comparison between multimeric RBD vaccines and ones using VLP scaffolds. Here, we designed simple covalent multimers of the SARS-CoV-2 RBD protein that can be modularly manufactured in yeast16,17. We demonstrate that the immunogenicity of these multimers was improved related to the monomeric RBD, and was not significantly different from a previously reported RBD-VLP clinical candidate.
We sought to design simple covalent multimers of the SARS-CoV-2 RBD. We previously reported an RBD-VLP-based vaccine candidate conductive for manufacturing modularly in yeast6, and we reasoned that a similar approach for covalent transpeptidation using SpyTag-SpyCatcher18 could construct RBD dimers and trimers. We expressed genetic fusions of the RBD with SpyTag or SpyCatcher in yeast, and then covalently linked two or more complementary components by spontaneous overnight conjugation (Fig. 1a). This process yielded dimers and trimers of RBD or VLP’s bearing RBD. All RBD molecules contained two mutations (L452K+F490W) that, in our previous work, improved both the manufacturability and immunogenicity of the ancestral RBD molecule19; this engineered RBD also exhibited increased potency when displayed at high valency on a protein nanoparticle. We also constructed a heterotypic dimer of the RBD-L452K-F490W (RBD-J) and the Omicron (BA.1) RBD. Multimeric protein vaccines that include multiple RBD serotypes have exhibited increased breadth against SARS-CoV-2 VOCs20,21. We verified correct assembly of dimers, trimers, and VLPs by SDS-PAGE and biolayer interferometry (supplementary fig. S1). As expected, the dimers, trimers and VLPs showed increased avidity compared to the monomeric RBD. (supplementary fig. S1)
We evaluated the immunogenicity of each design in BALB/c mice by immunizing groups of five mice intramuscularly with 10µg of each RBD construct (Fig. 1a) formulated with 40µg of alum hydroxide. Separate groups of mice were immunized with either two doses at weeks 0 and 6, or three doses at weeks 0, 3, and 6. We collected and analyzed sera at weeks 0, 3, 6, 8, 10, and 24 (Fig. 1b).
After three weeks, we observed that a prime dose of the RBD-VLP elicited a potent response and high titers of antibodies against the WA1/2020 RBD. Mice immunized with a prime dose of RBD dimers (both homo- and hetero-dimer) and trimer showed lower, though not statistically different, levels of Spike-specific antibodies, compared to the RBD-VLP (Fig. 1c-d). The levels of Spike antibodies were significantly lower in mice immunized with a prime dose of either the RBD-monomer or SpyTag-RBD compared to the RBD-VLP at week 3.
A second dose of RBD led to seroconversion in all groups. At week 8 (after all doses), levels of Spike-specific antibodies were not significantly inferior between mice immunized with the RBD dimers or RBD trimer compared to mice immunized with the RBD-VLP, for mice that received either two or three doses (supplementary Table 1-2). The levels of Spike-specific antibodies in mice immunized with two doses of the RBD monomer or SpyTag-RBD monomer, however, remained significantly lower than the levels of antibodies in mice immunized with the RBD-VLP (supplementary Fig. 2, supplementary Table 1). When immunized with three doses, titers of Spike-specific antibodies in mice immunized with the RBD monomer were statistically comparable to the RBD-VLP, although we observed a higher variance in response in this group.
We observed a similar trend for Omicron BA.1 Spike-specific antibodies. Levels of Omicron BA.1 Spike antibodies in mice immunized with the RBD dimers or RBD trimer were not significantly inferior compared to mice immunized with the RBD-VLP, for mice that received either two or three doses (Fig. 1c-d). Levels of antibodies against the Omicron BA.1 Spike in mice immunized with two doses of either RBD monomer were significantly lower compared to the RBD-VLP at week 8 (supplementary Fig. 2a). Mice immunized with three doses of the RBD monomers exhibited statistically comparable levels of antibodies against Omicron BA.1 Spike compared to the RBD-VLP.
We then measured the titers of antibodies that neutralized the WA1/2020 or Omicron BA.1 SARS-CoV-2 pseudoviruses. When immunized with two doses of the RBD dimers and trimer, nAb titers elicited against the WA1/2020 or Omicron BA.1 pseudovirus were not significantly inferior compared to the RBD-VLP group at week 8 (Fig. 1e). In contrast, nAb titers against the WA1/2020 or Omicron BA.1 pseudoviruses elicited by two doses of either RBD monomer were significantly lower compared to the RBD-VLP, as expected (Fig. 1e). When immunized with three doses, nAb titers against the WA1/2020 pseudovirus elicited by the RBD monomers, dimers, and trimer were all statistically similar to the RBD-VLP at week 8 (Fig.1f). In mice immunized with three doses, nAb titers against the Omicron BA.1 were only significantly different from the RBD-VLP in mice immunized with the SpyTag-RBD (Fig. 1f). These results are consistent with reports suggesting that two vaccine doses elicit poor neutralization against Omicron BA.1, and that a third dose is required to have neutralizing activity in humans22.
For all groups, Spike-binding antibodies were sustained through the end of the study at 24 weeks (Fig. 1c-d). Notably, neutralizing titers against the WA1/2020 and Omicron BA.1 pseudoviruses were also sustained for at least 6 months (Fig. 1g-h). The antibody levels and neutralizing antibody titers sustained in mice immunized with the RBD dimers and trimer were not significantly inferior to the RBD-VLP.
Overall, Spike-specific IgG levels had strong correlations with RBD-specific IgG levels for both WA1/2020 (Spearman R=0.9922, p<0.0001) and Omicron BA.1 (Spearman R=0.9904, p<0.0001) binding antibodies (Supplementary fig. 2a-b). Likewise, IgG levels had strong correlations with neutralizing titers (NT50) against the WA1/2020 (Spearman R=0.9281, p<0.0001) and Omicron BA.1 (Spearman R=0.7463, p<0.0001) pseudoviruses (Supplementary fig. 2c-d).
Lastly, we evaluated breadth against several SARS-CoV-2 VOCs. We observed that levels of Spike binding antibodies against VOCs from two doses of the RBD dimers and trimer were statistically comparable to the RBD-VLP (Fig. 2a, Supplementary Table 5). Two doses of the monomeric RBD groups exhibited substantially lower coverage to VOCs (Fig. 2a). After three doses, we observed that the RBD dimers and trimer elicited similar coverage to VOCs compared to the RBD-VLP (Fig. 2b, Supplementary Table 6). In this case, the Omicron (BA.1) Spike-specific IgG response elicited by the hetero-dimer was highest among the three dose groups, although not significantly higher. Finally, we evaluated the durability of this response to more novel VOCs, such as Omicron sub-variants. We observed that RBD dimers, trimer and VLP sustained comparable levels of Spike protein specific antibodies at week 24 (Fig. 2c-d). Notably, the RBD hetero-dimer sustained the highest median IgG levels to Omicron sub-variants (Fig. 2d), although not significantly different from the RBD homo-dimer, trimer and VLP.
In this study, we have demonstrated that RBD dimers can elicit comparable responses to RBD-VLPs in mice after two or three doses with a conventional adjuvant (alum). These results support the utility of RBD dimers to elicit strong responses and breadth against SARS-CoV-2 variants. In particular, a hetero-dimeric presentation of RBDs including the Omicron BA.1 RBD, showed a slightly enhanced response against VOCs. Given the evolving viral landscape23, future studies should focus on evaluating these RBD dimers as booster-only alternatives in animal models with preexisting immunity. These RBD dimers can be modified or mixed to present RBD combinations that may lead to increased coverage to VOCs. Furthermore, additional studies with novel adjuvants24 could inform approaches to further potentiate the neutralizing response and durability of dimer-based antigens.
Additionally, questions remain about the durability of neutralizing responses to novel variants2 afforded by currently approved vaccines to SARS-CoV-2. Alternative formulations for boosters could rely on candidates amenable to low-cost, scalable and transferable manufacturing that is accessible globally. RBD dimers could also serve as a multivalent display platform for novel RBD variants either in heterotypic or cocktail forms21. Such candidates may also have utility for a low-cost seasonal25 solution to decaying immunity and emerging variants.