Comparative analysis of spike-specific IgG Fc glycoprofiles elicited by adenoviral, mRNA, and protein-based SARS-CoV-2 vaccines.

IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps.

Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages.

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences.

mRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine.

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) 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 naïve 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 naïve 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 TH1-driven B cell response for all three vaccines. Instead, repeated immunization of naïve 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.

High titers and low fucosylation of early human anti-SARS-CoV-2 IgG promote inflammation by alveolar macrophages.

Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fcγ receptor (FcγR) IIa and FcγRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase.

Profiling the proteoforms of urinary prostate-specific antigen by capillary electrophoresis - mass spectrometry.

Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients' urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA. SIGNIFICANCE: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and non-cleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

High-throughput glycopeptide profiling of prostate-specific antigen from seminal plasma by MALDI-MS.

An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts.

Semiautomated glycoproteomics data analysis workflow for maximized glycopeptide identification and reliable quantification.

Glycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly evolving in many research areas, leading to a demand in reliable data analysis tools. In recent years, several bioinformatic tools were developed to facilitate and improve both the identification and quantification of glycopeptides. Here, a selection of these tools was combined and evaluated with the aim of establishing a robust glycopeptide detection and quantification workflow targeting enriched glycoproteins. For this purpose, a tryptic digest from affinity-purified immunoglobulins G and A was analyzed on a nano-reversed-phase liquid chromatography-tandem mass spectrometry platform with a high-resolution mass analyzer and higher-energy collisional dissociation fragmentation. Initial glycopeptide identification based on MS/MS data was aided by the Byonic software. Additional MS1-based glycopeptide identification relying on accurate mass and retention time differences using GlycopeptideGraphMS considerably expanded the set of confidently annotated glycopeptides. For glycopeptide quantification, the performance of LaCyTools was compared to Skyline, and GlycopeptideGraphMS. All quantification packages resulted in comparable glycosylation profiles but featured differences in terms of robustness and data quality control. Partial cysteine oxidation was identified as an unexpectedly abundant peptide modification and impaired the automated processing of several IgA glycopeptides. Finally, this study presents a semiautomated workflow for reliable glycoproteomic data analysis by the combination of software packages for MS/MS- and MS1-based glycopeptide identification as well as the integration of analyte quality control and quantification.

Serum N-Glycome analysis reveals pancreatic cancer disease signatures.

BACKGROUND &AIMS: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer type with loco-regional spread that makes the tumor surgically unresectable. Novel diagnostic tools are needed to improve detection of PDAC and increase patient survival. In this study we explore serum protein N-glycan profiles from PDAC patients with regard to their applicability to serve as a disease biomarker panel. METHODS: Total serum N-glycome analysis was applied to a discovery set (86 PDAC cases/84 controls) followed by independent validation (26 cases/26 controls) using in-house collected serum specimens. Protein N-glycan profiles were obtained using ultrahigh resolution mass spectrometry and included linkage-specific sialic acid information. N-glycans were relatively quantified and case-control classification performance was evaluated based on glycosylation traits such as branching, fucosylation, and sialylation. RESULTS: In PDAC patients a higher level of branching (OR 6.19, P-value 9.21 × 10-11 ) and (antenna)fucosylation (OR 13.27, P-value 2.31 × 10-9 ) of N-glycans was found. Furthermore, the ratio of α2,6- vs α2,3-linked sialylation was higher in patients compared to healthy controls. A classification model built with three glycosylation traits was used for discovery (AUC 0.88) and independent validation (AUC 0.81), with sensitivity and specificity values of 0.85 and 0.71 for the discovery set and 0.75 and 0.72 for the validation set. CONCLUSION: Serum N-glycome analysis revealed glycosylation differences that allow classification of PDAC patients from healthy controls. It was demonstrated that glycosylation traits rather than single N-glycan structures obtained in this clinical glycomics study can serve as a basis for further development of a blood-based diagnostic test.

A Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Assay for the Relative Quantitation of Antennary Fucosylated N-Glycans in Human Plasma.

Changes in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N-linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of α2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer.

An In-Depth Glycosylation Assay for Urinary Prostate-Specific Antigen.

The concentration of prostate-specific antigen (PSA) in serum is used as an early detection method of prostate cancer (PCa); however, it shows low sensitivity, specificity, and a poor predictive value. Initial studies suggested the glycosylation of PSA to be a promising marker for a more specific yet noninvasive PCa diagnosis. Recent studies on the molecular features of PSA glycosylation (such as antenna modification and core fucosylation) were not successful in demonstrating its potential for an improved PCa diagnosis, probably due to the lack of analytical sensitivity and specificity of the applied assays. In this study, we established for the first time a high-performance PSA Glycomics Assay (PGA), allowing differentiation of α2,6- and α2,3-sialylated isomers, the latter one being suggested to be a hallmark of aggressive types of cancer. After affinity purification from urine and tryptic digestion, PSA samples were analyzed by CE-ESI-MS (capillary electrophoresis-electrospray ionization coupled to mass spectrometry). Based on positive controls, an average interday relative standard deviation of 14% for 41 N-glycopeptides was found. The assay was further verified by analyzing PSA captured from patients' urine samples. A total of 67 N-glycopeptides were identified from the PSA pooled from the patients. In summary, the first PGA successfully established in this study allows an in-depth relative quantitation of PSA glycoforms from urine. The PGA is a promising tool for the determination of potential glycomic biomarkers for the differentiation between aggressive PCa, indolent PCa, and benign prostate hyperplasia in larger cohort studies.

Detection and functional analysis of CD8+ T cells specific for PRAME: a target for T-cell therapy.

PURPOSE: Preferentially expressed antigen on melanomas (PRAME) is an interesting antigen for T-cell therapy because it is frequently expressed in melanomas (95%) and other tumor types. Moreover, due to its role in oncogenic transformation, PRAME-negative tumor cells are not expected to easily arise and escape from T-cell immunity. The purpose of this study is to investigate the usefulness of PRAME as target for anticancer T-cell therapies. EXPERIMENTAL DESIGN: HLA-A*0201-subtyped healthy individuals and advanced melanoma patients were screened for CD8+ T cells directed against previously identified HLA-A*0201-binding PRAME peptides by IFN-gamma enzyme-linked immunosorbent spot assays and tetramer staining. PRAME-specific T-cell clones were isolated and tested for recognition of melanoma and acute lymphoid leukemia (ALL) cell lines. PRAME mRNA expression was determined by quantitative real-time reverse transcription-PCR. RESULTS: In 30% to 40% of healthy individuals and patients, PRA(100-108)-specific CD8+ T cells were detected both after in vitro stimulation and directly ex vivo after isolation by magnetic microbeads. Although CD45RA- memory PRA(100-108)-specific T cells were found in some individuals, the majority of PRA(100-108)-tetramer+ T cells expressed CD45RA, suggesting a naive phenotype. PRA(100-108)-tetramer+ T-cell clones were shown to recognize and lyse HLA-A*0201+ and PRAME+ melanoma but not ALL cell lines. Quantitative real-time reverse transcription-PCR showed significantly lower PRAME mRNA levels in ALL than in melanoma cell lines, suggesting that PRAME expression in ALL is below the recognition threshold of our PRA(100-108)-tetramer+ T cells. CONCLUSION: These data support the usefulness of PRAME and in particular the PRA(100-108) epitope as target for T-cell therapy of PRAME-overexpressing cancers.

Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine.

Dendritic cells (DCs) play an important role in the induction of T cell responses. Fc gammaRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual Fc gammaRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that Fc gammaRI and Fc gammaRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating Fc gammaRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating Fc gammaRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication.

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.

Chemically synthesized protein as tumour-specific vaccine: immunogenicity and efficacy of synthetic HPV16 E7 in the TC-1 mouse tumour model.

Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8+ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNgamma production and the ability to protect mice against a challenge with HPV16-E7+ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.

Established human papillomavirus type 16-expressing tumors are effectively eradicated following vaccination with long peptides.

Peptide-based vaccines aimed at the induction of effective T cell responses against established cancers have so far only met with limited clinical success and clearly need to be improved. In a preclinical model of human papillomavirus (HPV)16-induced cervical cancer we show that prime-boost vaccinations with the HPV16-derived 35 amino-acid long peptide E7(43-77), containing both a CTL epitope and a Th epitope, resulted in the induction of far more robust E7-specific CD8(+) T cell responses than vaccinations with the minimal CTL epitope only. We demonstrate that two distinct mechanisms are responsible for this effect. First, vaccinations with the long peptide lead to the generation of E7-specific CD4(+) Th cells. The level of the induced E7-specific CD8(+) T cell response proved to be dependent on the interactions of these Th cells with professional APC. Second, we demonstrate that vaccination with the long peptide and dendritic cell-activating agents resulted in a superior induction of E7-specific CD8(+) T cells, even when T cell help was excluded. This suggests that, due to its size, the long peptide was preferably endocytosed, processed, and presented by professional APCs. Moreover, the efficacy of this superior HPV-specific T cell induction was demonstrated in therapeutic prime-boost vaccinations in which the long peptide admixed with the dendritic cell-activating adjuvant oligodeoxynucleotide-CpG resulted in the eradication of large, established HPV16-expressing tumors. Because the vaccine types used in this study are easy to prepare under good manufacturing practice conditions and are safe to administer to humans, these data provide important information for future clinical trials.