Background: The new types of mRNA-containing lipid nanoparticle vaccines BNT162b2 and mRNA-1273 and the adenovirus-based vaccine AZD1222 were developed against SARS-CoV-2 and code for its spike (S)... Show moreBackground: The new types of mRNA-containing lipid nanoparticle vaccines BNT162b2 and mRNA-1273 and the adenovirus-based vaccine AZD1222 were developed against SARS-CoV-2 and code for its spike (S) protein. Several studies have investigated short-term antibody (Ab) responses after vaccination. Objective: However, the impact of these new vaccine formats with unclear effects on the long-term Ab response - including isotype, subclass, and their type of Fc glycosylation - is less explored. Methods: Here, we analyzed anti-S Ab responses in blood serum and the saliva of SARS-CoV-2 naive and non-hospitalized pre-infected subjects upon two vaccinations with different mRNA- and adenovirus-based vaccine combinations up to day 270. Results: We show that the initially high mRNA vaccine-induced blood and salivary anti-S IgG levels, particularly IgG1, markedly decrease over time and approach the lower levels induced with the adenovirus-based vaccine. All three vaccines induced, contrary to the short-term anti-S IgG1 response with high sialylation and galactosylation levels, a long-term anti-S IgG1 response that was characterized by low sialylation and galactosylation with the latter being even below the corresponding total IgG1 galactosylation level. Instead, the mRNA, but not the adenovirus-based vaccines induced long-term IgG4 responses - the IgG subclass with inhibitory effector functions. Furthermore, salivary anti-S IgA levels were lower and decreased faster in naive as compared to pre-infected vaccinees. Predictively, age correlated with lower long-term anti-S IgG titers for the mRNA vaccines. Furthermore, higher total IgG1 galactosylation, sialylation, and bisection levels correlated with higher long-term anti-S IgG1 sialylation, galactosylation, and bisection levels, respectively, for all vaccine combinations. Conclusion: In summary, the study suggests a comparable "adjuvant" potential of the newly developed vaccines on the anti-S IgG Fc glycosylation, as reflected in relatively low long-term anti-S IgG1 galactosylation levels generated by the long-lived plasma cell pool, whose induction might be driven by a recently described T-H1-driven B cell response for all three vaccines. Instead, repeated immunization of naive individuals with the mRNA vaccines increased the proportion of the IgG4 subclass over time which might influence the long-term Ab effector functions. Taken together, these data shed light on these novel vaccine formats and might have potential implications for their long-term efficacy. Show less
Immunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19.... Show moreImmunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19. Importantly, effector functions and resulting inflammatory responses are impacted by the structure of N-glycans linked to the Fc-tail of IgG. Studying antibody glycosylation in emerging infectious diseases such as SARS-CoV-2 allows to gain insight into specific glycan signatures at the early stages of infection, and to investigate whether these reflect how the disease would progress. For example, low fucosylation is a common glyco-phenotypic signature of IgG1 produced against the spike (S) protein of severely ill SARS-CoV-2 infected patients early on in their disease course, but has likewise been described in other disease settings, where the antigen is presented in the context of host-cell membranes (Chapter 2). In this thesis, antibody glycomics signatures of SARS-CoV-2 infection and vaccination have been explored using an established liquid chromatography – mass spectrometry-based method relying on affinity-isolation and proteolytic digestion of both total and anti-S IgG. In Chapter 3, the glycosylation of SARS-CoV-2 anti-S IgG antibodies were found to be vastly skewed relative to total IgG and to change in a highly dynamic fashion. Moreover, IgG glycosylation was shown to be an early severity marker and showed patient stratification potential, with predicting power for intensive care admission within a hospitalized patient population. Early detection of a pro-inflammatory glycosylation pattern may provide a broader intervention window and decrease the number of ICU-admissions. Furthermore, anti-S IgG1 glycosylation levels obtained with LC-MS show promise to supplement clinical parameters and biomarkers of inflammation, that have together been used for the severity score calculation of hospitalized COVID-19 patients. Similarly to SARS-CoV-2 infection, antibodies generated against the spike protein upon BNT162b2 mRNA vaccination also induced a transient afucosylated anti-S IgG1 response in antigen naïve individuals, albeit to a lower extent than in severely ill patients, exemplifying the influence of the type of immunization on antibody glycosylation (Chapter 4). Upon vaccination, the observed initial, mild afucosylated response was additionally accompanied by low fucosyltransferase (FUT8) expression in antigen-specific plasma cells. Furthermore, the observed initial anti-S IgG afucosylation signature may aided mounting a stronger immune response, as indicated by its correlation with antibody amounts following the second vaccination dose. Given the impact of glycosylation on antibody function, deciphering theunderlying regulatory mechanisms influencing IgG glycosylation will be of great importance to better understand the inflammatory potential, vaccine efficacy and protective capacity of vaccine- or pathogen-induced IgG in both body fluids and tissues in the future.In Chapter 5 and 6, the reaction steps of a previously developed linkage-specific sialic acid derivatization workflow were studied in more detail. Key players in such reactions are catalyst, of which novel types with different physico-chemical properties were introduced in Chapter 5. In Chapter 6, prior lactone formation was found to be a prerequisite for subsequent amidation of α2,3-linked sialic acids, which proceeds via direct aminolysis of the C2 lactone. Together, these new insights will be beneficial for the rational optimization of high-throughput (MALDI-)MS-based glycomics and glycoproteomics workflows relying on linkage-specific sialic acid derivatization. Show less
