The receptor binding domain (RBD) of the SARS-CoV-2 spike is the major target for potent neutralising antibodies elicited by SARS-CoV-2 vaccines today. In contrast to whole spike antigen utilised in these vaccines, the RBD has been explored as an alternative subunit antigen to focus neutralising responses. However, RBD alone exhibits poor immunogenicity compared to full-length spike, in part due to deficient availability of CD4 T helper cell epitopes, consequently driving poor germinal centre and antibody responses.
We engineered a fold-on stabilised chimeric trimer-RBD (CTR) glycoprotein, replacing the RBD of human coronavirus HKU-1 spike with SARS-CoV-2 RBD (Wuhan-Hu-1 or BA.2). This approach aims to: 1) focus RBD-specific antibodies through selective RBD presentation via an HKU-1 scaffold, while 2) enhancing RBD immunogenicity via supplementation of HKU-1-specific CD4 epitopes. We validated the antigenicity and trimeric conformation of these CTR antigens using RBD-specific monoclonal antibodies and TEM, respectively.
In C57BL/6 mice, intramuscular prime-boost vaccination with the CTR-BA.2 immunogen elicited high anti-BA.2-RBD titres (~104) and BA.2 virus neutralisation (IC50: 4.0x102), comparable to native Wuhan-Hu-1 or BA.2 spike-vaccinated animals. Germinal centre and memory B cells in draining lymph nodes elicited by the CTR-BA.2 antigen displayed predominantly cross-reactive Wuhan-Hu-1+/BA.2+ specificities. HKU-1-specific CD4 T cell helper responses, measured by CD154+ and CD25+OX40++, were also elevated in CTR-BA.2 immunised animals. Prime-boost immunisation of pigtail macaques with CTR-Wuhan-Hu-1 elicited high Wuhan-Hu-1 anti-spike or anti-RBD IgG titres (~104-105), equivalent to animals vaccinated with native Wuhan-Hu-1 spike. Neutralisation of live ancestral SARS-CoV-2 virus was comparable in plasma of CTR-Wuhan-Hu-1 or native Wuhan-Hu-1 spike vaccinated macaques (IC50: 7.5x102 vs 1.24x103, respectively).
Our findings demonstrate that a chimeric design for coronavirus glycoproteins offers a pathway to enhance intrinsically poor immunogenicity of subunit antigens, such as the SARS-CoV-2 RBD, by simultaneously engaging CD4 T cell helper responses and immune-focusing of antibody responses.