Sustained phenotypic characterization and molecular epidemiological monitoring are crucial for the ongoing surveillance of newly emerging SARS-CoV-2 lineages. In this work, we implemented practical approaches to monitor the appearance, dissemination, and biological properties of SARS-CoV-2 variants across Australia at a time when diagnostic PCR testing had substantially declined, and research relied more heavily on targeted cohort studies. These activities were integrated with long-term investigations spanning four years of the COVID-19 pandemic. Throughout 2023, we collaborated with diagnostic pathology services and genomics laboratories to detect circulating and newly arising variants in the New South Wales (NSW) population. We evaluated these variants using viral isolation in culture, assessment of replication dynamics, neutralization assays, and evaluation of entry mechanisms dependent on ACE2 and TMPRSS2 receptors. To place these observations in the wider pandemic context, we performed ongoing longitudinal monitoring of neutralizing antibody activity at the population level using pooled intravenous immunoglobulins (IVIG) obtained from over 700,000 donations. Antibodies in both recent individual plasma samples and large IVIG pools derived from thousands of donations retained neutralizing capability against historical and contemporary SARS-CoV-2 variants. The variants EG.5.1, HV.1, XCT, and JN.1 displayed the strongest capacity to evade neutralization. Specific modifications at Spike positions 452, 455, and 456 in the type I antibody epitope region correlated with reduced neutralization titers in XBB lineages. Over three years of population-level immunity tracking, neutralization breadth against all tested SARS-CoV-2 variants continued to expand. Although early responses showed strong imprinting biased toward the Ancestral strain and pre-Omicron variants, this bias gradually diminished as cross-reactive neutralization broadened. We forecasted that JN.1 would confer a significant transmission benefit by late 2023, which was later confirmed by its worldwide predominance beginning in early 2024. This growth advantage was not primarily attributable to neutralization escape; rather, we suggest it resulted from enhanced utilization of ACE2 receptor populations that do not interact with TMPRSS2 via its Collectrin-Like Domain (CLD). Multiple SARS-CoV-2 lineages that arose toward the close of 2023 initially showed partial resistance to neutralization. Over time, this limitation was increasingly overcome by the progressive development of broader cross-reactive neutralizing responses. The eventual dominance of the highly divergent JN.1 lineage cannot be explained by neutralization resistance alone. Our results indicate that its success arose from the interplay between moderate immune evasion and altered preferences for ACE2/TMPRSS2-mediated cellular entry.
