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2022;7(1):241. and heterogenous antibody responses in a mouse model. The results indicated that monovalent vaccine strategy with individual spike trimer could only induce binding and neutralizing antibodies against homologous viruses. However, sequential and bivalent immunization with Delta and Omicron spike trimers could induce significantly broader neutralizing antibody responses against heterogenous SARS\CoV\2. Interestingly, the spike trimer from Omicron variant showed Eltrombopag Olamine superior immunogenicity in inducing antibody response against recently emerging XE variant. Taken together, our data supported the development of novel vaccination strategies or multivalent vaccine against emerging variants. Keywords: immunogenicity, SARS\CoV\2, spike, trimer, variant 1.?INTRODUCTION Coronavirus disease 2019 (COVID\19) caused by severe acute respiratory syndrome coronavirus 2 (SARS\CoV\2) has caused a devastating impact on the global pubic health security. The COVID\19 vaccine inducing neutralizing Eltrombopag Olamine antibodies against SARS\COV\2 has showed significant effect on the control of the pandemic. However, the emergence of SARS\CoV\2 variants of concern (VOCs) bringing new waves of the infection even several approved COVID\19 vaccines are widely available and used. At present, five Plxna1 SARS\COV\2 VOCs have been identified: for example, Alpha (B.1.1.7), 1 Beta (B.1.351), 2 Gamma (P.1), 3 Delta (B.1.617.2), 4 and Omicron (B.1.1.529) 5 were associated with increased transmissibility. 6 Since emerging in November 2021, Omicron variant has evolved into many subtypes and recombinant strains, including early BA.1, BA.2, BA.3, and recently emerged BA.4, BA.5, and BA.2.12.1, as well as XD variant (Delta AY.4 and BA.1 recombinant), XE variant (BA.1 and BA.2 recombinant), and XF variant (Delta and the BA.1 recombinant). 7 XE recombinant variant appears to be roughly 10% more transmissible than its parent variant BA.2, implying that the XE may have the potential for greater range prevalence in the near future. 8 VOCs showed increased transmissibility and might have the potential for increasing disease severity when compared with the wild\type virus. 9 The diverse spikes of SASR\CoV\2 not only affect the replication and infection ability of the virus, but also have an influence on host immune response. Natural spike trimer is the dominated immunogen that induces humoral immune response, thus becomes the main target of neutralizing antibodies and currently approved COVID\19 vaccines. Eltrombopag Olamine 10 , 11 , 12 Generally, antigenic drift could occur in the glycoprotein of emerging SARS\CoV\2 variants; 13 thus, antibodies induced by parent strain might not afford sufficient cross\neutralizing effect against these variants. The immune escape of VOCs to the current vaccines based on wild\type SARS\CoV\2 has become the major obstacle to end the pandemic. 14 Therefore, it is necessary to fully explore the difference of immunogenicity of spike proteins from different emerging SARS\CoV\2 variants. In addition, vaccines and vaccination strategies inducing potent and broad neutralizing antibody responses against various variants, for example, Delta and Omicron, are urgently needed. In this study, we applied three immunization strategies including monovalent, sequential, and divalent vaccination to explore the difference of resultant humoral immune responses, aiming to provide theoretical basis for development of efficient COVID\19 vaccine targeting the viral diversity. Specifically, the binding antibody titers and neutralizing antibody titers of three vaccination strategies, including individual, sequential, and bivalent regimens based on wild type, Delta, and Omicron spike trimer were tested in mouse model. We observed that even though all of the three strategies could induce cross\binding antibody, only sequential and bivalent immunization with Delta and Omicron spike trimers induced broader neutralizing antibody that even neutralized the newly emerging variants, XE recombinant strain. 2.?METHODS AND MATERIALS 2.1. Construction of plasmids and cell lines SARS\CoV\2 surface glycoprotein gene (GenBank: MN_908947) with C\terminal 19 amino acids deletion was codon\optimized for and cloned into eukaryotic expression plasmid pcDNA3.1(+) between HindIII and BamHI sites to generate the spike expression plasmids pcDNA3.1(+)\OPS. The lentiviral packaging plasmid pNL4\3 Luc+R\E\ carrying an Env\defective, luciferase\expressed HIV\1 genome was generously gifted by Binlian Sun, Jianghan University. Wild\type SARS\CoV\2 pseudovirus was produced by cotransfection of HEK293T cells with pNL4\3 Luc+R\E\ and pcDNA3.1(+)\OPS. To produce various spike pseudoviruses, pcDNA3.1(+)\OPS plasmid was subsequently used as a template to generate plasmids encoding various spike mutants, including D614G, emerging Delta, Omicron, and XE variants. The amino acid mutations of each variant in this study were.