Background: Vaccines against COVID-19 target the spike protein. There is minimal information on longitudinal COVID-19 immune profiling in recovered versus naïve and vaccinated versus non-vaccinated healthcare workers (HCWs). Methods: This is a prospective longitudinal observational cohort of pediatric HCWs (pHCWs) conducted during 2020–2022 at an academic center, exploring the impact of COVID-19 vaccination on immunoglobulin G (IgG) antibody titers over time and cross-reactivity with other coronaviruses, including SARS-CoV-1, MERS-CoV, and seasonal coronaviruses (HCoV-HKU1 and HCoV-OC43). Results: A total of 642 pHCWs initially enrolled, and 337 participants had repeat IgG titers measured post-vaccine and post-booster. Most participants were female, median age range of 31–40 years. Anti-spike was higher in all vaccinated individuals versus non-vaccinated (p < 0.0001) and naïve versus infected (p < 0.0001). A single dose of vaccine was sufficient to attain maximum titers in recovered participants versus naïve who received both doses of vaccine. Anti-spike titers dropped significantly at 9 months after the primary series, whereas sustained anti-spike titers were observed at 9 months post-booster. Conclusions: All vaccinated pHCWs developed antibodies to spike. COVID-19 infection and/or vaccination yielded antibodies that cross-reacted to SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43. Anti-spike titers were more durable post-booster compared to the primary series. Longitudinal immune profiling of COVID-19 responses provides vital data to shape public health policies, optimize vaccine strategies, and strengthen pandemic preparedness.