The SARS-CoV-2 JN.1 variant, derived from BA.2.86.1 with the S:L455S substitution, showed increased fitness and outcompeted XBB lineages by early 2024. Subsequently, JN.1 subvariants including KP.2, KP.3, LB.1, and KP.2.3 emerged, with KP.2.3 acquiring the S:S31del deletion in addition to other substitutions. The study investigated the virological properties of KP.3, LB.1, and KP.2.3. Using a Bayesian multinomial logistic model, the relative effective reproduction number (Re) of these variants was estimated based on genome surveillance data from Canada, the UK, and the USA. KP.3 had a Re more than 1.2-fold higher than JN.1 and comparable to KP.2. LB.1 and KP.2.3 had even higher Re values than KP.2 and KP.3, suggesting they may become major circulating variants. Pseudovirus infectivity of KP.2 and KP.3 was significantly lower than JN.1, while LB.1 and KP.2.3 had infectivity comparable to JN.1. Neutralization assays showed that LB.1 and KP.2.3 were more resistant to neutralization by breakthrough infection sera than JN.1. KP.3 also showed resistance, but no significant difference from KP.2. In vaccinated individuals, JN.1 subvariants had low neutralization titers, while KP.3, LB.1, and KP.2.3 had significantly lower titers than JN.1. Overall, the S substitutions in JN.1 subvariants contributed to immune evasion and increased Re compared to JN.1. LB.1 and KP.2.3 showed higher infectivity and stronger immune resistance than KP.2. These findings suggest that S:S31del is critical for increased infectivity and immune evasion, potentially contributing to increased Re.The SARS-CoV-2 JN.1 variant, derived from BA.2.86.1 with the S:L455S substitution, showed increased fitness and outcompeted XBB lineages by early 2024. Subsequently, JN.1 subvariants including KP.2, KP.3, LB.1, and KP.2.3 emerged, with KP.2.3 acquiring the S:S31del deletion in addition to other substitutions. The study investigated the virological properties of KP.3, LB.1, and KP.2.3. Using a Bayesian multinomial logistic model, the relative effective reproduction number (Re) of these variants was estimated based on genome surveillance data from Canada, the UK, and the USA. KP.3 had a Re more than 1.2-fold higher than JN.1 and comparable to KP.2. LB.1 and KP.2.3 had even higher Re values than KP.2 and KP.3, suggesting they may become major circulating variants. Pseudovirus infectivity of KP.2 and KP.3 was significantly lower than JN.1, while LB.1 and KP.2.3 had infectivity comparable to JN.1. Neutralization assays showed that LB.1 and KP.2.3 were more resistant to neutralization by breakthrough infection sera than JN.1. KP.3 also showed resistance, but no significant difference from KP.2. In vaccinated individuals, JN.1 subvariants had low neutralization titers, while KP.3, LB.1, and KP.2.3 had significantly lower titers than JN.1. Overall, the S substitutions in JN.1 subvariants contributed to immune evasion and increased Re compared to JN.1. LB.1 and KP.2.3 showed higher infectivity and stronger immune resistance than KP.2. These findings suggest that S:S31del is critical for increased infectivity and immune evasion, potentially contributing to increased Re.