Longitudinal Evaluation of Humoral and Cellular Immunity After BNT162b2 COVID-19 Vaccination: Influence of Booster Type, Infection and Chronic Health Conditions Chiara Orlandi, Ilaria Conti, Davide Torre, Simone Barocci, Mauro Magnani, et al. Vaccines, 2025 Background/Objectives: Understanding the durability of immunity induced by mRNA COVID-19 vaccines, especially in individuals with chronic health conditions, remains essential for guiding booster strategies. We conducted a longitudinal study to evaluate humoral and cellular immune responses up to 21 months after a primary two-dose BNT162b2 vaccination followed by a booster, either homologous (BNT162b2) or heterologous (mRNA-1273). Methods: Twenty-eight adults, mostly with chronic conditions, were assessed at approximately 9, 12 and 21 months post-primary vaccination. Serum anti-trimeric Spike IgG levels were quantified, and peripheral blood mononuclear cells were analyzed at 21 months for Spike-specific memory B-cell and T-cell responses by flow cytometry. Results: Participants were stratified by booster type, prior SARS-CoV-2 infection and health status. Anti-Spike IgG persisted in all participants but declined over time. The heterologous mRNA-1273 booster induced higher antibody titers at 9 months, while the homologous BNT162b2 booster led to more sustained antibody levels and higher frequencies of Spike-specific memory B cells at 21 months. Prior infection significantly enhanced antibody titers, particularly in homologous booster recipients. Surprisingly, individuals with chronic health conditions exhibited equal or higher antibody levels compared to healthy participants at all time points. At 21 months, robust Spike-specific class-switched memory B cells and polyfunctional CD4+ and CD8+ T-cell responses were detected. Conclusions: These findings demonstrate that BNT162b2 vaccination elicits durable, multi-layered immunity lasting nearly two years, even in individuals with chronic conditions, and support the use of both homologous and heterologous mRNA boosters to sustain protection in diverse populations.
Analysis of humoral and cellular immune activation up to 21 months after heterologous and homologous COVID-19 vaccination Davide Torre, Chiara Orlandi, Ilaria Conti, Simone Barocci, Eugenio Carlotti, et al. Frontiers in Immunology, 2025 To address the COVID-19 pandemic, diverse vaccination strategies, including homologous and heterologous schedules, were employed to enhance immune protection. This study evaluates the long-term 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 15-months post-booster), while SARS-CoV-2-specific B- and T-cell responses were assessed at 21-months 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.
Comparing Heterologous and Homologous COVID-19 Vaccination: A Longitudinal Study of Antibody Decay Chiara Orlandi, Giuseppe Stefanetti, Simone Barocci, Gloria Buffi, Aurora Diotallevi, et al. Viruses, 2023 The humoral response after vaccination was evaluated in 1248 individuals who received different COVID-19 vaccine schedules. The study compared subjects primed with adenoviral ChAdOx1-S (ChAd) and boosted with BNT162b2 (BNT) mRNA vaccines (ChAd/BNT) to homologous dosing with BNT/BNT or ChAd/ChAd vaccines. Serum samples were collected at two, four and six months after vaccination, and anti-Spike IgG responses were determined. The heterologous vaccination induced a more robust immune response than the two homologous vaccinations. ChAd/BNT induced a stronger immune response than ChAd/ChAd at all time points, whereas the differences between ChAd/BNT and BNT/BNT decreased over time and were not significant at six months. Furthermore, the kinetic parameters associated with IgG decay were estimated by applying a first-order kinetics equation. ChAd/BNT vaccination was associated with the longest time of anti-S IgG negativization and with a slow decay of the titer over time. Finally, analyzing factors influencing the immune response by ANCOVA analysis, it was found that the vaccine schedule had a significant impact on both the IgG titer and kinetic parameters, and having a Body Mass Index (BMI) above the overweight threshold was associated with an impaired immune response. Overall, the heterologous ChAd/BNT vaccination may offer longer-lasting protection against SARS-CoV-2 than homologous vaccination strategies.
Exploring the variables influencing the immune response of traditional and innovative glycoconjugate vaccines Francesca Micoli, Giuseppe Stefanetti, Calman A. MacLennan Frontiers in Molecular Biosciences, 2023 Vaccines are cost-effective tools for reducing morbidity and mortality caused by infectious diseases. The rapid evolution of pneumococcal conjugate vaccines, the introduction of tetravalent meningococcal conjugate vaccines, mass vaccination campaigns in Africa with a meningococcal A conjugate vaccine, and the recent licensure and introduction of glycoconjugates against S. Typhi underlie the continued importance of research on glycoconjugate vaccines. More innovative ways to produce carbohydrate-based vaccines have been developed over the years, including bioconjugation, Outer Membrane Vesicles (OMV) and the Multiple antigen-presenting system (MAPS). Several variables in the design of these vaccines can affect the induced immune responses. We review immunogenicity studies comparing conjugate vaccines that differ in design variables, such as saccharide chain length and conjugation chemistry, as well as carrier protein and saccharide to protein ratio. We evaluate how a better understanding of the effects of these different parameters is key to designing improved glycoconjugate vaccines.
Impact of the Host Microbiome on Vaccine Responsiveness: Lessons Learned and Future Perspective Giuseppe Stefanetti, Dennis L. Kasper Biochemistry, 2022 Vaccination shows high variability in the elicited immune responses among individuals and populations for reasons still poorly understood. An increasing number of studies is supporting the evidence that gut microbiota, along with other interplaying variables, is able to modulate both humoral and cellular responses to infection and vaccination. Importantly, vaccine immunogenicity is often suboptimal at the extremes of age and also in low- and middle-income countries (LMICs), where the microbiota is believed to have an important role on immune responses. Still, contrasting findings and lack of causal evidence are calling for sophisticated methodologies to be able to overcome scientific and technical challenges to better decipher the immunomodulatory role of microbiota. In this perspective, we briefly review the status of the vaccine field in relation to the microbiome and offer possible scientific approaches to better understand the impact of the host microbiome on vaccine responsiveness.
Impact and Control of Sugar Size in Glycoconjugate Vaccines Giuseppe Stefanetti, Calman Alexander MacLennan, Francesca Micoli Molecules, 2022 Glycoconjugate vaccines have contributed enormously to reducing and controlling encapsulated bacterial infections for over thirty years. Glycoconjugate vaccines are based on a carbohydrate antigen that is covalently linked to a carrier protein; this is necessary to cause T cell responses for optimal immunogenicity, and to protect young children. Many interdependent parameters affect the immunogenicity of glycoconjugate vaccines, including the size of the saccharide antigen. Here, we examine and discuss the impact of glycan chain length on the efficacy of glycoconjugate vaccines and report the methods employed to size polysaccharide antigens, while highlighting the underlying reaction mechanisms. A better understanding of the impact of key parameters on the immunogenicity of glycoconjugates is critical to developing a new generation of highly effective vaccines.
Immunobiology of Carbohydrates: Implications for Novel Vaccine and Adjuvant Design Against Infectious Diseases Giuseppe Stefanetti, Francesco Borriello, Barbara Richichi, Ivan Zanoni, Luigi Lay Frontiers in Cellular and Infection Microbiology, 2022 Carbohydrates are ubiquitous molecules expressed on the surface of nearly all living cells, and their interaction with carbohydrate-binding proteins is critical to many immunobiological processes. Carbohydrates are utilized as antigens in many licensed vaccines against bacterial pathogens. More recently, they have also been considered as adjuvants. Interestingly, unlike other types of vaccines, adjuvants have improved immune response to carbohydrate-based vaccine in humans only in a few cases. Furthermore, despite the discovery of many new adjuvants in the last years, aluminum salts, when needed, remain the only authorized adjuvant for carbohydrate-based vaccines. In this review, we highlight historical and recent advances on the use of glycans either as vaccine antigens or adjuvants, and we review the use of currently available adjuvants to improve the efficacy of carbohydrate-based vaccines. A better understanding of the mechanism of carbohydrate interaction with innate and adaptive immune cells will benefit the design of a new generation of glycan-based vaccines and of immunomodulators to fight both longstanding and emerging diseases.
Symbionts exploit complex signaling to educate the immune system Deniz Erturk-Hasdemir, Sungwhan F. Oh, Nihal A. Okan, Giuseppe Stefanetti, Francesca S. Gazzaniga, et al. Proceedings of the National Academy of Sciences of the United States of America, 2019
Profiling immune responses to novel Salmonella vaccines: a path to clinical trials G Stefanetti, MM Gibani, F Fiorino Frontiers in Immunology 17, 1817651 , 2026 2026
Longitudinal Evaluation of Humoral and Cellular Immunity After BNT162b2 COVID-19 Vaccination: Influence of Booster Type, Infection and Chronic Health Conditions C Orlandi, I Conti, D Torre, S Barocci, M Magnani, G Stefanetti, ... Vaccines 13 (10), 1031 , 2025 2025 Citations: 1
Analysis of humoral and cellular immune activation up to 21 months after heterologous and homologous COVID-19 vaccination D Torre, C Orlandi, I Conti, S Barocci, E Carlotti, M Magnani, ... Frontiers in Immunology 16, 1579163 , 2025 2025 Citations: 3
Glycoconjugate antigen processing and immune response JF Cipollo, G Stefanetti Frontiers in Molecular Biosciences 10, 1350141 , 2023 2023
Exploring the variables influencing the immune response of traditional and innovative glycoconjugate vaccines F Micoli, G Stefanetti, CA MacLennan Frontiers in molecular biosciences 10, 1201693 , 2023 2023 Citations: 30
Comparing heterologous and homologous COVID-19 vaccination: A longitudinal study of antibody decay C Orlandi, G Stefanetti, S Barocci, G Buffi, A Diotallevi, E Rocchi, ... Viruses 15 (5), 1162 , 2023 2023 Citations: 28
Impact and control of sugar size in glycoconjugate vaccines G Stefanetti, CA MacLennan, F Micoli Molecules 27 (19), 6432 , 2022 2022 Citations: 21
Impact of the host microbiome on vaccine responsiveness: lessons learned and future perspective G Stefanetti, DL Kasper Biochemistry 61 (24), 2849-2855 , 2022 2022 Citations: 11
Immunobiology of carbohydrates: implications for novel vaccine and adjuvant design against infectious diseases G Stefanetti, F Borriello, B Richichi, I Zanoni, L Lay Frontiers in cellular and infection microbiology 11, 808005 , 2022 2022 Citations: 66
Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella Typhimurium GMMA Is Critical to Induce an Effective Immune Response against … F Necchi, G Stefanetti, R Alfini, E Palmieri, M Carducci, R Di Benedetto, ... Pathogens 10 (6), 726 , 2021 2021 Citations: 9
Conjugation Techniques and Linker Strategies for Carbohydrate-Based Vaccines B Richichi, G Biagiotti, L Lay, G Stefanetti Conjugation Techniques and Linker Strategies for Carbohydrate-Based Vaccines … , 2021 2021 Citations: 3
Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM 197 as carrier protein G Stefanetti, M Allan, A Usera, F Micoli Glycoconjugate Journal 37 (5), 611-622 , 2020 2020 Citations: 21
Harnessing the immunogenicity of peptide-based glycoconjugate vaccines G Stefanetti, N Okan, DL Kasper The Journal of Immunology 204 (1_Supplement), 168.18-168.18 , 2020 2020 Citations: 1
Microbiota-targeted maternal antibodies protect neonates from enteric infection W Zheng, W Zhao, M Wu, X Song, F Caro, X Sun, F Gazzaniga, ... Nature 577 (7791), 543-548 , 2020 2020 Citations: 169
Symbionts exploit complex signaling to educate the immune system D Erturk-Hasdemir, SF Oh, NA Okan, G Stefanetti, FS Gazzaniga, ... Proceedings of the National Academy of Sciences 116 (52), 26157-26166 , 2019 2019 Citations: 153
Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis G Stefanetti, N Okan, A Fink, E Gardner, DL Kasper Proceedings of the National Academy of Sciences 116 (14), 7062-7070 , 2019 2019 Citations: 45
Polysaccharide structure dictates mechanism of adaptive immune response to glycoconjugate vaccines X Sun, G Stefanetti, F Berti, DL Kasper Proceedings of the National Academy of Sciences 116 (1), 193-198 , 2019 2019 Citations: 133
Click Chemistry Applied to the Synthesis of Salmonella Typhimurium O-Antigen Glycoconjugate Vaccine on Solid Phase with Sugar Recycling G Stefanetti, A Saul, CA MacLennan, F Micoli Bioconjugate Chemistry 26 (12), 2507-2513 , 2015 2015 Citations: 26
Frontispiz: Sugar–Protein Connectivity Impacts on the Immunogenicity of Site‐Selective Salmonella O‐Antigen Glycoconjugate Vaccines G Stefanetti, QY Hu, A Usera, Z Robinson, M Allan, A Singh, H Imase, ... Angewandte Chemie 127 (45) , 2015 2015
Sugar–Protein Connectivity Impacts on the Immunogenicity of Site‐Selective Salmonella O‐Antigen Glycoconjugate Vaccines G Stefanetti, QY Hu, A Usera, Z Robinson, M Allan, A Singh, H Imase, ... Angewandte Chemie 127 (45), 13396-13401 , 2015 2015 Citations: 97
MOST CITED SCHOLAR PUBLICATIONS
Microbiota-targeted maternal antibodies protect neonates from enteric infection W Zheng, W Zhao, M Wu, X Song, F Caro, X Sun, F Gazzaniga, ... Nature 577 (7791), 543-548 , 2020 2020 Citations: 169
Symbionts exploit complex signaling to educate the immune system D Erturk-Hasdemir, SF Oh, NA Okan, G Stefanetti, FS Gazzaniga, ... Proceedings of the National Academy of Sciences 116 (52), 26157-26166 , 2019 2019 Citations: 153
Polysaccharide structure dictates mechanism of adaptive immune response to glycoconjugate vaccines X Sun, G Stefanetti, F Berti, DL Kasper Proceedings of the National Academy of Sciences 116 (1), 193-198 , 2019 2019 Citations: 133
Sugar–Protein Connectivity Impacts on the Immunogenicity of Site‐Selective Salmonella O‐Antigen Glycoconjugate Vaccines G Stefanetti, QY Hu, A Usera, Z Robinson, M Allan, A Singh, H Imase, ... Angewandte Chemie 127 (45), 13396-13401 , 2015 2015 Citations: 97
Structural analysis of O-polysaccharide chains extracted from different Salmonella Typhimurium strains F Micoli, N Ravenscroft, P Cescutti, G Stefanetti, S Londero, S Rondini, ... Carbohydrate research 385, 1-8 , 2014 2014 Citations: 80
Immunobiology of carbohydrates: implications for novel vaccine and adjuvant design against infectious diseases G Stefanetti, F Borriello, B Richichi, I Zanoni, L Lay Frontiers in cellular and infection microbiology 11, 808005 , 2022 2022 Citations: 66
Impact of conjugation chemistry on the immunogenicity of S. Typhimurium conjugate vaccines G Stefanetti, S Rondini, L Lanzilao, A Saul, CA MacLennan, F Micoli Vaccine 32 (46), 6122-6129 , 2014 2014 Citations: 53
Structural analysis of the O-acetylated O-polysaccharide isolated from Salmonella paratyphi A and used for vaccine preparation N Ravenscroft, P Cescutti, M Gavini, G Stefanetti, CA MacLennan, ... Carbohydrate Research 404, 108-116 , 2015 2015 Citations: 52
Strain selection for generation of O-antigen-based glycoconjugate vaccines against invasive nontyphoidal Salmonella disease L Lanzilao, G Stefanetti, A Saul, CA MacLennan, F Micoli, S Rondini PLoS One 10 (10), e0139847 , 2015 2015 Citations: 48
Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis G Stefanetti, N Okan, A Fink, E Gardner, DL Kasper Proceedings of the National Academy of Sciences 116 (14), 7062-7070 , 2019 2019 Citations: 45
Exploring the variables influencing the immune response of traditional and innovative glycoconjugate vaccines F Micoli, G Stefanetti, CA MacLennan Frontiers in molecular biosciences 10, 1201693 , 2023 2023 Citations: 30
Comparing heterologous and homologous COVID-19 vaccination: A longitudinal study of antibody decay C Orlandi, G Stefanetti, S Barocci, G Buffi, A Diotallevi, E Rocchi, ... Viruses 15 (5), 1162 , 2023 2023 Citations: 28
Click Chemistry Applied to the Synthesis of Salmonella Typhimurium O-Antigen Glycoconjugate Vaccine on Solid Phase with Sugar Recycling G Stefanetti, A Saul, CA MacLennan, F Micoli Bioconjugate Chemistry 26 (12), 2507-2513 , 2015 2015 Citations: 26
Impact and control of sugar size in glycoconjugate vaccines G Stefanetti, CA MacLennan, F Micoli Molecules 27 (19), 6432 , 2022 2022 Citations: 21
Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM 197 as carrier protein G Stefanetti, M Allan, A Usera, F Micoli Glycoconjugate Journal 37 (5), 611-622 , 2020 2020 Citations: 21
Impact of the host microbiome on vaccine responsiveness: lessons learned and future perspective G Stefanetti, DL Kasper Biochemistry 61 (24), 2849-2855 , 2022 2022 Citations: 11
Neisseria meningitidis Factor H Binding Protein Surface Exposure on Salmonella Typhimurium GMMA Is Critical to Induce an Effective Immune Response against … F Necchi, G Stefanetti, R Alfini, E Palmieri, M Carducci, R Di Benedetto, ... Pathogens 10 (6), 726 , 2021 2021 Citations: 9
Analysis of humoral and cellular immune activation up to 21 months after heterologous and homologous COVID-19 vaccination D Torre, C Orlandi, I Conti, S Barocci, E Carlotti, M Magnani, ... Frontiers in Immunology 16, 1579163 , 2025 2025 Citations: 3
Conjugation Techniques and Linker Strategies for Carbohydrate-Based Vaccines B Richichi, G Biagiotti, L Lay, G Stefanetti Conjugation Techniques and Linker Strategies for Carbohydrate-Based Vaccines … , 2021 2021 Citations: 3
Longitudinal Evaluation of Humoral and Cellular Immunity After BNT162b2 COVID-19 Vaccination: Influence of Booster Type, Infection and Chronic Health Conditions C Orlandi, I Conti, D Torre, S Barocci, M Magnani, G Stefanetti, ... Vaccines 13 (10), 1031 , 2025 2025 Citations: 1
