Our study focused on demonstrating the effect of vaccine boosters in infection- naïve individuals, unconfounded by prior infection or vaccine breakthrough infection and thus demonstrates the intrinsic virus neutralizing antibody responses elicited by the vaccine alone. Other studies demonstrated that the BNT162b2(WT + BA.4/5) bivalent vaccine including recent omicron subvariant BA.5 immunogen was superior to monovalent vaccine against omicron subvariants BA.1, BA.2.75.2, BQ.1.1 and XBB.1.5, but these did not exclude individuals with past infection [11, 12]. An approach to understand immune evasion in immune naïve individuals has been to use mono-specific hamster immune sera [7], but hamsters may not fully recapitulate human immune responses. There are fewer reports on immune evasion of JN.1 [13, 14], none of them including infection-naïve individuals. Participants in these studies had past SARS-CoV-2 infection or only excluded subjects with natural infection within 3 months before assessment. We have demonstrated that BBB + BIV booster elicited significantly higher neutralizing antibody to Omicron BA.5 compared to BBBB as expected, but the BBB + BIV also elicited significantly higher PRNT50 titres to all the more recent Omicron subvariants tested, including JN.1.

Spike proteins of BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1 and HK.3 have over 30 amino acids (a.a.) substitutions relative to WT, while BA.2.86 has over 60 a.a. substitutions relative to WT. Compared to BA.5, the RBD region of XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3 and BA.2.86 have additional V445P, G446S, N460K a.a. substitutions which may explain the greater evasion from neutralizing antibody. R346T, L368I and F490S were additional mutations in XBB.1 descendent lineage (XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3), but these were not observed in BA.5 or BA.2.86, which may explain the relatively higher neutralizing titres observed with BA.2.86. In our BBB + BIV vaccinated group, the lowest GMT were found with EG.5.1, HK.3 and JN.1 viruses, possibly a result of the F456L mutation. This was also noted in another study which compared the immune evasion of EG.5.1 and XBB.2.3.2 [15]. GMTs to EG.5.1 and HK.3 differed significantly in the BBBB vaccine group which may be due to the L455F mutation in HK.3, a mutation shown by computational analysis to change the protein structure [16]. The greater immune evasion in JN.1 compared to BA.2.86 could be explained by a key mutation L455S in spike protein. This was also demonstrated in other studies by pseudovirus neutralization [13]. However, the difference in neutralizing titres to BA.2.86 and JN.1 was not seen in those recently infected with XBB.1.5 [14].

Our study had a number of limitations including a small sample size, but this was the result of excluding those with prior natural immunity, to allow us to exclusively investigate the intrinsic immunogenicity of the vaccines, unconfounded by prior infection immunity. We used a correlate of protection as described previously [8,9,10], but we recognize that neutralizing antibody may not be a sole correlate of protection for all vaccines, including inactivated vaccines. Populations in many parts of the world now have complex “hybrid” immune landscapes elicited by vaccination as well as natural infection, making it difficult to extrapolate our data to populations in general but our data are relevant to understanding the intrinsic antibody responses of vaccines. A new monovalent XBB.1.5 vaccine is now being rolled out and it would be important to conduct studies to understand neutralizing antibody responses to this vaccine.

In summary, we assessed the neutralizing antibody to a panel of SARS-CoV-2 subvariants, BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3, BA.2.86 and JN.1 in comparison to WT, in infection-naïve vaccinees who received 4 doses of BNT or 3 doses of BNT162b2 boosted by a dose of BNT162b2(WT + BA.4/5). The bivalent vaccine elicited significantly higher neutralizing antibody levels to new omicron subvariants compared to boosting with the monovalent BNT162b2 vaccine. We found that BA.2.86 and JN.1 differed in neutralizing antibody evasion in infection-naïve BBB + BIV vaccinated sera. The continued monitoring for new variants that evade neutralizing antibody responses remains a priority.