Heterologous inactivated virus/mRNA vaccination response to BF.7, BQ.1.1, and XBB.1

**Background:** The emergence of highly immune-evasive Omicron subvariants of SARS-CoV-2, including BA.4.6, BF.7, BQ.1.1, XBB, and XBB.1, has raised concerns about the efficacy of existing vaccines and therapeutic monoclonal antibodies. These subvariants harbor spike protein mutations such as R346T and F486S that contribute to evasion of class III and class I/II anti-spike monoclonal antibodies, respectively. BQ.1, BQ.1.1, XBB, and XBB.1 are increasing rapidly in the United States, India, Europe, and other regions, while BF.7 is one of the dominant strains circulating in China. Humoral immune imprinting—where initial exposure to the original SARS-CoV-2 strain limits future immune responses against variants—may reduce protection from bivalent vaccine boosters and hybrid immunity. The authors previously demonstrated that an mRNA vaccine booster in individuals vaccinated with two doses of inactivated vaccine significantly increased neutralizing antibodies against Omicron BA.1. The present study investigates whether this heterologous vaccination strategy retains neutralizing activity against emerging Omicron subvariants.

**Methods:** The study used enzyme-linked immunosorbent assay and lentivirus-based pseudovirus neutralizing assay to evaluate binding and neutralizing antibody levels in 77 plasma samples collected from 67 healthy volunteers within 1–5 months after vaccination or infection. Participants were grouped into six categories based on vaccination and infection history: (1) three doses of inactivated vaccine (CoronaVac or BBIBP-CorV); (2) three doses of monovalent mRNA vaccine (BNT162b2 or mRNA-1273); (3) two doses of inactivated vaccine followed by an mRNA vaccine booster; (4) three doses of inactivated vaccine followed by one or two doses of mRNA vaccine booster; (5) infected during the G614 wave and sampled after one or two subsequent doses of mRNA vaccine; and (6) breakthrough infection during the Omicron BA.1 wave.

**Key Results:** In all six groups, levels of RBD-specific IgG and neutralizing activity were lower against all Omicron subvariants tested compared to the original G614 strain, with the lowest levels measured against XBB.1. The geometric mean NT50 of heterologous vaccination groups were 47- to 51-fold higher against G614 and 4- to 42-fold higher against Omicron subvariants compared to the three-dose homologous inactivated vaccine group. The geometric mean NT50 of each vaccinated/infected group against BF.7, BQ.1.1, and XBB.1 were 3.1- to 108.6-fold lower compared to G614. However, geometric mean NT50 against these Omicron subvariants were 20–635 in heterologous vaccination groups and the homologous mRNA vaccine group, while those in the three-dose homologous inactivated vaccine group were below the limit of detection. Among all Omicron subvariants tested, the lowest neutralization activity elicited by vaccines and breakthrough infection was against BQ.1.1 and XBB.1.

**Clinical Implications:** The results suggest that heterologous vaccination involving a parental mRNA vaccine as a booster or second booster in individuals who received two or three doses of inactivated vaccines strongly augments neutralizing activity and may remain effective against emerging Omicron subvariants. The undetectable neutralizing activity in the three-dose homologous inactivated vaccine group may account for the surge of COVID-19 cases in China, where inactivated vaccines are primarily used. Study limitations include a small cohort size (particularly for the group receiving three doses of inactivated vaccine plus mRNA vaccine booster), cross-sectional analysis, and the need for further investigation of clinical efficacy. Nevertheless, the authors recommend that heterologous boosting with mRNA-based vaccines should be considered in populations where inactivated vaccines were primarily used.