A booster of Delta-Omicron RBD-dimer protein subunit vaccine augments sera neutralization of Omicron sub-variants BA.1/BA.2/BA.2.12.1/BA.4/BA.5

ABSTRACT The SARS-CoV-2 Omicron variants of concern (VOCs) showed severe resistance to the early-approved COVID-19 vaccines-induced immune responses. The breakthrough infections by the Omicron VOCs are currently the major challenge for pandemic control. Therefore, booster vaccination is crucial to enhance immune responses and protective efficacy. Previously, we developed a protein subunit COVID-19 vaccine ZF2001, based on the immunogen of receptor-binding domain (RBD) homodimer, which was approved in China and other countries. To adapt SARS-CoV-2 variants, we further developed chimeric Delta-Omicron BA.1 RBD-dimer immunogen which induced broad immune responses against SARS-CoV-2 variants. In this study, we tested the boosting effect of this chimeric RBD-dimer vaccine in mice after priming with two doses of inactivated vaccines, compared with a booster of inactivated vaccine or ZF2001. The results demonstrated that boosting with bivalent Delta-Omicron BA.1 vaccine greatly promoted the neutralizing activity of the sera to all tested SARS-CoV-2 variants. Therefore, the Delta-Omicron chimeric RBD-dimer vaccine is a feasible booster for those with prior vaccination of COVID-19 inactivated vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern (VOC) is continuously dominating the current pandemic. The sub-variants of Omicron VOC contain far more mutations in spike (S) protein compared with their preceding VOCs ( Figure S1) and showed largely reduced sensitivity to the currently-available vaccines [1,2]. In China, more than a billion people have received two or three doses of coronavirus disease 2019 (COVID-19) vaccines, and most of them took inactivated vaccines. We previously developed a protein subunit COVID-19 vaccine ZF2001, using the immunogen of tandem-repeat dimeric receptor-binding domain (RBD), which was approved in China and other countries [3][4][5]. ZF2001 was also approved as a booster for those who had received two doses of inactivated vaccine (e.g. BBIBP-CorV). Both these inactivated and protein subunit vaccines are based on the early SARS-CoV-2 strain identified in Wuhan (prototype) and elicited sera with greatly reduced neutralization to Omicron [1].
To adapt SARS-CoV-2 variants, we further developed a chimeric Delta-Omicron BA.1 RBD-dimer immunogen which elicited broader sera neutralization of SARS-CoV-2 variants and conferred better protection in mice in comparison to the prototypic homodimer immunogen being used in ZF2001 [6]. COVID-19 protein subunit vaccine ZF2202, based on the chimeric Delta-Omicron BA.1 RBD-dimer antigen, has been approved for clinical trial studies in November 2022 (NCT05616754). Here, we tested the boosting effect of this chimeric RBD-dimer vaccine in mice after receipt of two doses of inactivated vaccines, compared with either the homologous boosting with the inactivated vaccine or the heterologous boosting with ZF2001 (prototype RBD-dimer).
Thirty BALB/c mice were injected with two doses of inactivated vaccine BBIBP-CorV, 21 days apart ( Figure S2). Vaccine-elicited RBD-binding antibodies were measured 14 days after dose 2. These 30 mice were evenly distributed into three groups (n = 10) according to the mean titer of RBD-binding IgG for synchronization ( Figure S3). Next, each group of mice was given a homologous booster of BBIBP-CorV, a heterologous booster of ZF2001, or a heterologous booster of Delta-Omicron BA.1, at 21 days after the dose two ( Figure S2). A group of mice (n = 10) received three doses of sham as control. Serum samples were collected 14 days later to measure the antibody responses. As expected, both homologous and heterologous boosters induced substantial binding IgG to RBD of prototype, Delta, or Omicron. The heterologous boosters were superior to the homologous booster (Figure 1(A)).
Splenic cellular immune responses after sequential vaccination were also measured 14 days after boosting. Peptide pools spanning either the prototype or Omicron antigen elicited substantial secretion of Th1 cytokines (IFN-γ and IL-2) in all booster groups ( Figure  S5). The levels of cellular immunity against Omicron BA.1 induced by a booster of homologous inactivated vaccine or heterologous Delta-Omicron RBD-dimer vaccine were similar without significant difference ( Figure S5). Recently, a similar finding was also reported from the clinical study of BA.5 bivalent mRNA vaccine (Pfizer/BioNTech and Moderna) boosters [11]. In addition, inactivated vaccine and the RBD-dimer protein subunit vaccines are all adjuvanted by aluminum hydroxide. The cellular immune responses elicited by them were not at robust levels, though substantial responses were detected.
Thanks to the rapid vaccine development and mass vaccination campaign worldwide, most people have acquired specific immunity against SARS-CoV-2. However, the breakthrough infections by the Omicron sub-variants are currently the major challenge for pandemic control. Recent studies in macaques indicated that boost vaccination with an Omicron-matched vaccine did not augment sera neutralization against the Omicron variant, compared with boosting with prototype vaccine [12]. Therefore, it is yet unclear what formulations should be used for future COVID-19 vaccines: a booster using the available prototype vaccine, Omicron-matched monovalent vaccine or the multivalent vaccine?
Our results demonstrated that boosting with prototype vaccines had limited augment of sera neutralization of Omicron VOCs, in particular, the sub-variants BA.2.12.1 and BA.4/BA5 in the animal model (Figure 1(H, I)). By contrast, boosting with multivalent Delta-Omicron BA.1 vaccine greatly promoted the neutralizing activity of the sera to all tested Omicron sub-variants, yet largely preserved the activity to all early VOCs.
The Omicron-containing bivalent COVID-19 vaccines, developed by Pfizer/BioNTech and Moderna, have been approved for use in the United States and other countries. The published data showed that a booster of bivalent (prototype + Omicron BA.1) mRNA vaccines induced broad antibody responses against SARS-CoV-2 variants, which were superior to prototype sequence-based vaccines (BNT162b2 and mRNA-1273) [13,14]. In addition, another study analyzed the effect of boosting immunization of bivalent protein vaccines (produced by Livzon, China) after three doses of inactivated vaccines and found that bivalent BV-01-B5 vaccine induced stronger neutralizing responses against multiple Omicron sublineages than monovalent V-01 vaccine (prototype) and homologous inactivated vaccine in human participants [15]. These conclusions were similar to ours from the murine model study. However, the bivalent mRNA vaccines from Pfizer/BioNTech and Moderna and protein vaccine BV-01-B5 from Livzon were produced with a 1:1 mixture formulation. The chimeric RBD-heterodimer design strategy allows the single antigen production as the bivalent vaccine. In the context of uncertain future evolutionary steps for SARS-CoV-2 variants, our data support the advice by World Health Organization (WHO) for developing multivalent variant-adapted vaccines to elicit broader immune responses against both co-circulating and emerging SARS-CoV-2 variants [16]. The Delta-Omicron chimeric RBD-dimer vaccine is a feasible booster for those with prior vaccination of COVID-19 inactivated vaccines.
Given that the sequences of influenza vaccines were updated annually with the data from global surveillance and prediction, the strategy of future updated COVID-19 vaccines will possibly be similar to those from influenza vaccines. Currently, the Omicron sub-variants are co-circulating in the world, including XBB, BQ.1.1 and BF.7 with strong immune evasion. Next-generation and updated vaccines were urgently needed. Our study provided a bivalent booster vaccine candidate with high and broad-spectrum immunogenicity.