Adrian Sutta ¹ Rafael Bayarri-Olmos ^1* Anne Rosbjerg ¹ Mie Mandal Mortensen ² Charlotte Helgstrand ² Per Franklin Nielsen ² Laura Pérez-Alós ¹ Beatriz González García ¹ Laust Johnsen ² Finn Matthiesen ² Thomas Egebjerg ² Cecilie Bo Hansen ¹ Alessandro Sette ^3,4 Alba Grifoni ³ Ricardo Da Silva Antunes ³ Peter Garred ¹

• ¹ Rigshospitalet, University of Copenhagen, Copenhagen, Capital Region of Denmark, Denmark
• ² Novo Nordisk (Denmark), Copenhagen, Denmark
• ³ Center for Vaccine Innovation, La Jolla Institute for Immunology (LJI), San Diego, California, United States
• ⁴ Division of Infectious Diseases and Global Public Health, School of Medicine, University of California, San Diego, La Jolla, California, United States

Throughout the COVID-19 pandemic, the emergence of new viral variants has challenged public health efforts, often evading antibody responses generated by infections and vaccinations. This immune escape has led to waves of breakthrough infections, raising questions about the efficacy and durability of immune protection. Here we focus on the impact of SARS-CoV-2 Delta and Omicron spike mutations on ACE-2 receptor binding, protein stability, and immune response evasion. Delta and Omicron variants had 3–5 times higher binding affinities to ACE-2 than the ancestral strain (KDwt = 23.4 nM, KDDelta = 8.08 nM, KDBA.1 = 4.77 nM, KDBA.2 = 4.47 nM). The pattern recognition molecule mannose-binding lectin (MBL) has been shown to recognize the spike protein. Here we found that MBL binding remained largely unchanged across the variants, even after introducing mutations at single glycan sites. Although MBL binding decreased post-vaccination, it increased by 2.6-fold upon IgG depletion, suggesting a compensatory or redundant role in immune recognition. Notably, we identified two glycan sites (N717 and N801) as potentially essential for the structural integrity of the spike protein. We also evaluated the antibody and T cell responses. Neutralization by serum immunoglobulins was predominantly mediated by IgG rather than IgA and was markedly impaired against the Delta (5.8-fold decrease) and Omicron variants BA.1 (17.4-fold) and BA.2 (14.2-fold). T cell responses, initially conserved, waned rapidly within 3 months post-Omicron infection. Our data suggests that immune imprinting may have hindered antibody and T cell responses toward the variants. Overall, despite decreased antibody neutralization, MBL recognition and T cell responses were generally unaffected by the variants. These findings contribute to our understanding of the complex interplay between viral adaptation and immune response, underscoring the importance of considering MBL interactions, immune imprinting, and viral evolution dynamics in developing future vaccines and therapeutic strategies.