A comprehensive longitudinal analysis of the cellular immune response specific to the spike protein in healthcare workers vaccinated against SARS-CoV-2– ORCHESTRA Project

Stefano Ugel¹Akshita Gupta²Gianluca Spiteri^3*Pierpaolo Marchetti⁴Francesco De Sanctis¹Shari Wouters²Angelina Konnova²Maria Grazia Lourdes Monaco^1,4Angela Carta^1,4Maria Diletta Pezzani⁵Filippo Liviero^6,7Sofia Pavanello^6,7,8Marco dell'Omo⁹Eleonóra Fabiánová¹⁰Jana Bérešová¹¹Francesca Larese Filon¹²Marcella Mauro¹²Giuseppe Verlato⁴Vincenzo Bronte¹Samir Kumar-Singh²Stefano Porru^4,5

• ¹Universita degli Studi di Verona Dipartimento di Medicina, Verona, Italy
• ²Universiteit Antwerpen Vaccin en Infectieziekten Instituut, Antwerp, Belgium
• ³Other
• ⁴Universita degli Studi di Verona Dipartimento di Diagnostica e Sanita Pubblica, Verona, Italy
• ⁵Azienda Ospedaliera Universitaria Integrata Verona, Verona, Italy
• ⁶Universita degli Studi di Padova Dipartimento di Scienze Cardio-Toraco-Vascolari e Sanita pubblica, Padua, Italy
• ⁷Azienda Ospedale Universita Padova, Padua, Italy
• ⁸Universita degli Studi di Padova Centro di Ateneo di Studi e Attivita Spaziali 'Giuseppe Colombo', Padua, Italy
• ⁹Universita degli Studi di Perugia Dipartimento di Medicina e Chirurgia, Perugia, Italy
• ¹⁰Occupational Health Department., Regional Authority of Public Health (RAPH), Banská Bystrica, Slovakia
• ¹¹Occupational Health Department, Regional Authority of Public Health (RAPH), Banská Bystrica, Slovakia
• ¹²Universita degli Studi di Trieste Dipartimento Universitario Clinico di Scienze Mediche e Chirurgiche e della Salute, Trieste, Italy

The long-term dynamics of T-cell immunity following SARS-CoV-2 vaccination, essential for durable protection, remain incompletely understood. This study, therefore, aimed to investigate the kinetics and persistence of spike-specific T-cell responses in vaccinated healthcare workers. Within the framework of the ORCHESTRA Project, we conducted a longitudinal study on the kinetics and persistence of CD4+ and CD8+ T cell immunity in healthcare workers (n=305) from four hospitals and public health centers across two European countries who received either 2 or 3 doses of an mRNA vaccine, with or without prior SARS-CoV-2 infection. Specifically, the anti-spike adaptive immune cellular response was evaluated, focusing on its crosstalk with the B cell response as measured by serology. Circulating cellular adaptive immune cells were extensively analyzed using flow cytometry to assess pro-inflammatory cytokine production (TNF-α, IFN-γ, IL-2), functional activation (CD154), and memory differentiation (CD45RO). Our findings show that anti-spike T cell reactivity is not influenced by age, with the only exception of a weak positive correlation with spike-specific CD8+CD45RO+ T lymphocytes (Spearman's rho = 0.34, p<0.001), and an equally weak negative correlation with CD8+TNF+ (Spearman's rho = -0.23, p<0.01). Other variables, such as gender and job category, did not significantly impact the vaccine-induced, anti-spike T cell immune response. No distinct relationship between CD4+ and CD8+ T cell subsets was observed post-vaccination. However, specific dynamic changes in vaccine-induced T cells were identified showing clear dose-and time-dependence. Finally, the median level of CD8+CD154+ lymphocytes, indicative of activated T cells, was significantly associated with infection incidence and may represent a reliable predictive biomarker. . This study provides evidence that the vaccine-induced anti-spike cellular immune response should be considered when making vaccination decisions, as it has predictive value for infection risk.