The study investigates the virological characteristics of the SARS-CoV-2 variants KP.3, LB.1, and KP.2.3, which emerged as subvariants of the JN.1 variant. These variants, characterized by specific substitutions in the spike (S) protein, have shown increased fitness and are spreading globally. The relative effective reproduction number (Rₑ) of KP.3 is more than 1.2-fold higher than that of JN.1, while LB.1 and KP.2.3 have even higher Rₑ values. Pseudovirus infectivity assays revealed that KP.2 and KP.3 are less infectious than JN.1, whereas LB.1 and KP.2.3 are comparable to JN.1. Neutralization assays using breakthrough infection (BTI) sera and XBB.1.5 vaccine sera showed that LB.1 and KP.2.3 have significantly lower neutralization titers compared to JN.1 and KP.2. KP.3 exhibits higher neutralization resistance than JN.1. The study concludes that the S substitutions in these variants contribute to immune evasion and increased Rₑ, with S:S31del being critical for enhanced infectivity and immune evasion.The study investigates the virological characteristics of the SARS-CoV-2 variants KP.3, LB.1, and KP.2.3, which emerged as subvariants of the JN.1 variant. These variants, characterized by specific substitutions in the spike (S) protein, have shown increased fitness and are spreading globally. The relative effective reproduction number (Rₑ) of KP.3 is more than 1.2-fold higher than that of JN.1, while LB.1 and KP.2.3 have even higher Rₑ values. Pseudovirus infectivity assays revealed that KP.2 and KP.3 are less infectious than JN.1, whereas LB.1 and KP.2.3 are comparable to JN.1. Neutralization assays using breakthrough infection (BTI) sera and XBB.1.5 vaccine sera showed that LB.1 and KP.2.3 have significantly lower neutralization titers compared to JN.1 and KP.2. KP.3 exhibits higher neutralization resistance than JN.1. The study concludes that the S substitutions in these variants contribute to immune evasion and increased Rₑ, with S:S31del being critical for enhanced infectivity and immune evasion.