Reduced Spike specific T-cell responses in COVID-19 vaccinated subjects undergoing SARS-CoV-2 breakthrough infection

Stefania Varchetta^1*Federica Sole Golfetto¹Patrizia Bono²Annapaola Callegaro²Tanya Fabbris³ANDREA FAVALLI³MARIACRISTINA CROSTI³Tullia Maria De Feo⁴Nathalie Iannotti¹Giorgio Bozzi¹Valeria Castelli¹Bianca Mariani¹Antonio Muscatello¹Sergio Abrignani^3,5Renata Maria Grifantini³Alessandra Bandera^1,6ANDREA LOMBARDI^1,6

• ¹Infectious Disease Unit, Department of Internal Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico., Milan, Italy
• ²Microbiology and Virology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
• ³INGM, Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy
• ⁴North Italy Transplant program (NITp). Transplant Coordination Unit, Fondazione IRCSS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
• ⁵Department of Clinical Sciences and Community Health, Università degli Studi di Milano., Milan, Italy
• ⁶Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy

Abstract Introduction: T-cell responses to SARS-CoV-2 remain largely preserved across variants despite waning neutralizing antibodies. However, T-cell immunity may vary with the host's immune status, and data on T-cell responses in post-vaccine infections (PVI) are limited. Methods: We assessed Spike-specific T-cell responses in 32 vaccinated individuals, 16 of whom experienced PVI. Immune responses were evaluated at three time points: 1 month after the second vaccine dose (T1), 1 month after the booster dose (T2), and, in the PVI group, 1-3 months after the first positive nasal swab (T3). Additionally, we evaluated anti-spike antibody levels, T-cell exhaustion markers, and natural killer cell subsets, focusing on memory-like CD57+ NKG2C⁺ cells. Results: Subjects who developed PVI exhibited significantly reduced Spike-specific CD4 T-cell responses following the booster dose compared to vaccinated individuals who remained uninfected. This was accompanied by increased frequencies of LAG-3⁺ CD4⁺ and CD8⁺ T-cells. A positive correlation was observed between AIM⁺ CD4⁺ T-cells and NKG2C⁺ NK cells at T2 in PVI subjects. Following natural infection, T-cell responses were enhanced and associated with an expansion of NKG2C+ NK cells. Conclusions: Individuals experiencing PVI displayed impaired booster-induced CD4⁺ T-cell responses and increased expression of the immune checkpoint LAG-3. Natural infection restored and enhanced cellular immunity, particularly through the expansion of Spike-specific T-cells and memory NK cell populations. This study identifies an immune profile characterized by low spike-specific responses, which are associated with an increased susceptibility to breakthrough infections.