Accurate evaluation of live-virus microneutralization for SARS-CoV-2 variant JN.1

Emerging SARS-CoV-2 variants require rapid assessments of pathogenicity and evasion of existing immunity to inform policy. A crucial component of these assessments is accurate estimation of serum neutralising antibody titres using cultured live virus isolates. Here, we report our updated culture methods for Omicron sub-variant JN.1 using Caco-2 cells and the subsequent evaluation of neutralising antibody titres (nAbTs) in recipients of BNT162b2-XBB.1.5 monovalent and the Ancestral/BA.5 containing bivalent vaccines. We compared culture of JN.1 in either Vero V1 cells or Caco-2 cells, finding culture in Vero V1 either resulted in low-titre stocks or induced crucial mutations at the Spike furin cleavage site. Using the sequence-clean culture stocks generated in Caco-2 cells, we assessed serum samples from 71 healthy adults eligible for a COVID-19 vaccination given as a 5 th dose booster: all participants had detectable nAbs against JN.1 prior to vaccination, with baseline/pre-existing nAbTs between both vaccine groups comparable (p = 0.240). However, nAbTs against JN.1 post-vaccination were 2.6-fold higher for recipients of the monovalent XBB1.5 vaccine than the BA.4/5 bivalent vaccine (p<0.001). Regular re-appraisal of methods involved in the evaluation of new variants is required to ensure robust data are used to underpin crucial severity assessments as variants arise and vaccine strain selection decisions.