Analysis of humoral and cellular immune activation up to 21 months after heterologous and homologous COVID-19 vaccination

Davide Torre¹Chiara Orlandi^2,3Ilaria Conti¹Simone Barocci⁴Eugenio Carlotti⁵Mauro Magnani^1,3Anna Casabianca^2,3*Giuseppe Stefanetti^1,2*

• ¹Department of Biomolecular Sciences, University of Urbino Carlo Bo, Campus Scientifico Enrico Mattei, Via Cà le Suore, 2/4, 61029, Urbino, PU, Italy, Urbino (PU), Italy
• ²Department of Biomolecular Sciences, Section of Biochemistry and Biotechnology, University of Urbino Carlo Bo, Via Arco d’Augusto 2, 61032 Fano, PU, Italy, Fano, Italy
• ³Laboratorio Covid, University of Urbino Carlo Bo, Via Arco d’Augusto 2, 61032 Fano, PU, Italy, Fano, PU, Italy
• ⁴Department of Clinical Pathology, Azienda Sanitaria Territoriale (AST) di Pesaro-Urbino, Viale Comandino 70, 61029 Urbino, PU, Italy, Urbino (PU), Italy
• ⁵Department of Prevention, Azienda Sanitaria Territoriale (AST) di Pesaro-Urbino, Viale Comandino 21, 61029 Urbino, PU, Italy, Urbino (PU), Italy

To address the COVID-19 pandemic, diverse vaccination strategies, including homologous and heterologous schedules, were employed to enhance immune protection. This study evaluates the longterm humoral and cellular immune responses in individuals vaccinated with homologous (ChAdOx1-S/ChAdOx1-S [ChAd/ChAd]) and heterologous (ChAdOx1-S/BNT162b2 [ChAd/BNT]) schedules, followed by a third-dose mRNA booster (BNT162b2 [BNT] or mRNA-1273). Anti-Spike IgG titers were measured at 9-, 12-, and 21-months post-primary vaccination (corresponding to 3-, 6-, and 15months post-booster), while SARS-CoV-2-specific B-and T-cell responses were assessed at 21months post-booster. Antibody titers declined by 12-months post-primary vaccination, regardless of the third dose administered, and increased significantly by 21-months, potentially due to a fourth dose (BNT or mRNA-1273) or natural SARS-CoV-2 infection. The heterologous ChAd/BNT schedule elicited a stronger and more durable immune response than the homologous ChAd/ChAd, as evidenced by higher anti-Spike IgG titers, increased IgM-/IgG+ memory B-cell activation, and enhanced cytotoxic CD8+ T-cell cytokine expression in infected individuals. SARS-CoV-2 infection further boosted humoral and cellular responses, with infected individuals showing higher anti-Spike IgG titers and greater CD8+ T-cell activation compared to uninfected individuals. These findings highlight the benefits of heterologous vaccination schedules and the role of infection-driven immune activation, providing valuable insights for optimizing vaccination strategies to improve long-term immunity against SARS-CoV-2.