Objective:To investigate the efficacy of a SARS-CoV-2 recombinant protein vaccine as a booster dose.Methods:A new immunogen, namely RBD-sc-trimer, was designed by tandem repeating of single receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein to mimic the trimeric form of RBD presented by the virus. The RBD-sc-trimer protein was expressed as a His-tagged fusion protein using a baculovirus expression system and purified by nickel affinity column. The purified protein was identified by Western blot. Its in vitro binding activity to human angiotensin converting enzyme 2 (hACE2) was analyzed by ELISA. The immunogenicity of RBD-sc-trimer as well as RBD proteins of other forms including RBD dimer (RBD-Fc), RBD monomer (RBD) and S protein trimer (S trimer) as a booster dose was evaluated in BALB/c mice. Results:In terms of both binding and neutralizing antibodies against SARS-CoV-2, RBD-sc-trimer showed an immunogenicity that was superior to that of RBD-Fc and RBD and close to the level of S trimer. The antibody response induced by RBD-sc-trimer was characterized as Th1-biased. Moreover, it displayed a stronger cross-neutralization activity against SARS-CoV-2 Beta, Delta and Omicron variants. The titer of neutralizing antibody against Omicron induced by RBD-sc-trimer only decreased by 9.1 folds relative to the prototype strain, while the antibody response induced by RBD-Fc and S trimer decreased by 68.4 and 70.8 folds, respectively.Conclusions:The recombinant protein, RBD-sc-trimer, which was capable of eliciting stronger humoral response in mice as a booster dose and showed the superiority in raising cross-reactive antibodies against SARS-CoV-2 variants over non-trimeric RBD forms, should be considered as an optimal immunogen for the development of more effective SARS-CoV-2 vaccines.

To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.

The ongoing SARS-CoV-2 pandemic poses a severe global threat to public health, as do influenza viruses and other coronaviruses. Here, we present chimpanzee adenovirus 68 (AdC68)-based vaccines designed to universally target coronaviruses and influenza. Our design is centered on an immunogen generated by fusing the SARS-CoV-2 receptor-binding domain (RBD) to the conserved stalk of H7N9 hemagglutinin (HA). Remarkably, the constructed vaccine effectively induced both SARS-CoV-2-targeting antibodies and anti-influenza antibodies in mice, consequently affording protection from lethal SARS-CoV-2 and H7N9 challenges as well as effective H3N2 control. We propose our AdC68-vectored coronavirus-influenza vaccine as a universal approach toward curbing respiratory virus-causing pandemics. IMPORTANCE The COVID-19 pandemic exemplifies the severe public health threats of respiratory virus infection and influenza A viruses. The currently envisioned strategy for the prevention of respiratory virus-causing diseases requires the comprehensive administration of vaccines tailored for individual viruses. Here, we present an alternative strategy by designing chimpanzee adenovirus 68-based vaccines which target both the SARS-CoV-2 receptor-binding-domain and the conserved stalk of influenza hemagglutinin. When tested in mice, this strategy attained potent neutralizing antibodies against wild-type SARS-CoV-2 and its emerging variants, enabling an effective protection against lethal SARS-CoV-2 challenge. Notably, it also provided complete protection from lethal H7N9 challenge and efficient control of H3N2-induced morbidity. Our study opens a new avenue to universally curb respiratory virus infection by vaccination.