Search

Respiratory syncytial virus (RSV) infection is a major cause of severe lower respiratory tract infection and death in young infants and the elderly. With no effective prophylactic treatment available, current vaccine candidates aim to elicit neutralizing antibodies. However, binding and neutralization have poorly predicted protection in the past, and accumulating data across epidemiologic cohorts and animal models collectively point to a role for additional antibody Fc-effector functions. To begin to define the humoral correlates of immunity against RSV, here we profiled an adenovirus 26 RSV-preF vaccine-induced humoral immune response in a group of healthy adults that were ultimately challenged with RSV. Protection from infection was linked to opsonophagocytic functions, driven by IgA and differentially glycosylated RSV-specific IgG profiles, marking a functional humoral immune signature of protection against RSV. Furthermore, Fc-modified monoclonal antibodies able to selectively recruit effector functions demonstrated significant antiviral control in a murine model of RSV.

Neutrophils, the most abundant white blood cell, play a critical role in anti-pathogen immunity via phagocytic clearance, secretion of enzymes and immunomodulators, and the release of extracellular traps. Neutrophils non-specifically sense infection through an array of innate immune receptors and inflammatory sensors, but are also able to respond in a pathogen/antigen-specific manner when leveraged by antibodies via Fc-receptors. Among neutrophil functions, antibody-dependent neutrophil phagocytosis (ADNP) results in antibody-mediated opsonization, enabling neutrophils to sense and respond to infection in a pathogen-appropriate manner. Here, we describe a high-throughput flow cytometric approach to effectively visualize and quantify ADNP and its downstream consequences. The assay is easily adaptable, supporting both the use of purified neutrophils or white blood cells, the use of purified Ig or serum, and the broad utility of any target antigen. Thus, this ADNP assay represents a high-throughput platform for the in-depth characterization of neutrophil function., Graphical abstract Unlabelled Image, Highlights • A high-throughput antibody-dependent neutrophil phagocytosis (ADNP) assay was developed. • This flow cytometry assay is flexible and can be easily adapted to any pathogen. • Analysis of sample sets by ADNP assay is fast, robust and cost-effective. • Additional neutrophil functions can be profiled in secondary analyses.

Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N-glycosylation on the CH2 domain, and the remodeling of the N-linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N-glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N-glycosylation. Although IgGs usually have a single N-glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N-glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N-glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N-linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N-glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N-glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N-glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.

Respiratory syncytial virus (RSV) infection is a major cause of severe respiratory illness in both young infants and the elderly. While antibodies represent key correlates of protection, the antibody mechanisms remain poorly understood. Emerging data point to additional humoral Fc-mediated mechanisms of action beyond neutralization. Therefore, to map the humoral correlates of immunity against RSV, antibody responses were profiled in a highly controlled non-human primate challenge model. Six different vaccine platforms were tested in African Green Monkeys, which post-challenge, yielded distinct antibody profiles and viral loads in both upper and lower respiratory tracts. Machine learning was then used to determine antibody features associated with protection. Upper respiratory control involved virus specific IgA levels, neutralization, and complement whereas lower respiratory control involved Fc-mediated mechanisms. These data highlight a collaborative Fab and Fc protective signature, induced selectively by distinct vaccine platforms, and provides mechanistic insights for the rational development of vaccines.

Antibodies are the primary correlate of protection for most licensed vaccines; however, their mechanisms of protection may vary, ranging from physical blockade to clearance via the recruitment of innate immunity. Here, we uncover striking functional diversity in vaccine-induced antibodies that is driven by immunization site and is associated with reduced risk of SIV infection in nonhuman primates. While equivalent levels of protection were observed following intramuscular (IM) and aerosol (AE) immunization with an otherwise identical DNA prime-Ad5 boost regimen, reduced risk of infection was associated with IgG-driven antibody-dependent monocyte-mediated phagocytosis in the IM vaccinees, but with vaccine-elicited IgA-driven neutrophil-mediated phagocytosis in AE-immunized animals. Thus, although route-independent correlates indicate a critical role for phagocytic Fc-effector activity in protection from SIV, the site of immunization may drive this Fc activity via distinct innate effector cells and antibody isotypes. Moreover, the same correlates predicted protection from SHIV infection in a second nonhuman primate vaccine trial using a disparate IM canarypox prime-protein boost strategy, analogous to that used in the first moderately protective human HIV vaccine trial. These data identify orthogonal functional humoral mechanisms, initiated by distinct vaccination routes and immunization strategies, pointing to multiple, potentially complementary correlates of immunity that may support the rational design of a protective vaccine against HIV.

Respiratory syncytial virus (RSV) infection is a major cause of respiratory illness in infants and the elderly. Although several vaccines have been developed, none have succeeded in part due to our incomplete understanding of the correlates of immune protection. While both T cells and antibodies play a role, emerging data suggest that antibody-mediated mechanisms alone may be sufficient to provide protection. Therefore, to map the humoral correlates of immunity against RSV, antibody responses across six different vaccines were profiled in a highly controlled nonhuman primate-challenge model. Viral loads were monitored in both the upper and lower respiratory tracts, and machine learning was used to determine the vaccine platform-agnostic antibody features associated with protection. Upper respiratory control was associated with virus-specific IgA levels, neutralization, and complement activity, whereas lower respiratory control was associated with Fc-mediated effector mechanisms. These findings provide critical compartment-specific insights toward the rational development of future vaccines.

HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations., Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components. IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Acid alpha-glucosidase (GAA) is a lysosomal glycogen-catabolizing enzyme, the deficiency of which leads to Pompe disease. Pompe disease can be treated with systemic recombinant human GAA (rhGAA) enzyme replacement therapy (ERT), but the current standard of care exhibits poor uptake in skeletal muscles, limiting its clinical efficacy. Furthermore, it is unclear how the specific cellular processing steps of GAA after delivery to lysosomes impact its efficacy. GAA undergoes both proteolytic cleavage and glycan trimming within the endolysosomal pathway, yielding an enzyme that is more efficient in hydrolyzing its natural substrate, glycogen. Here, we developed a tool kit of modified rhGAAs that allowed us to dissect the individual contributions of glycan trimming and proteolysis on maturation-associated increases in glycogen hydrolysis using in vitro and in cellulo enzyme processing, glycopeptide analysis by MS, and high-pH anion-exchange chromatography with pulsed amperometric detection for enzyme kinetics. Chemical modifications of terminal sialic acids on N-glycans blocked sialidase activity in vitro and in cellulo, thereby preventing downstream glycan trimming without affecting proteolysis. This sialidase-resistant rhGAA displayed only partial activation after endolysosomal processing, as evidenced by reduced catalytic efficiency. We also generated enzymatically deglycosylated rhGAA that was shown to be partially activated despite not undergoing proteolytic processing. Taken together, these data suggest that an optimal rhGAA ERT would require both N-glycan and proteolytic processing to attain the most efficient enzyme for glycogen hydrolysis and treatment of Pompe disease. Future studies should examine the amenability of next-generation ERTs to both types of cellular processing.

Antibodies are critical glycoproteins that bridge the innate and adaptive immune systems to provide protection against infection. The isotype/subclass of the antibody, the co-translational N-glycosylation on the CH2 domain, and the remodeling of the N-linked glycans during passage through the ER and Golgi are the known variables within the Fc domain that program antibody effector function. Through investigations of monoclonal therapeutics, it has been observed that addition or removal of specific monosaccharide residues from antibody N-glycans can influence the potency of antibodies, highlighting the importance of thoroughly characterizing antibody N-glycosylation. Although IgGs usually have a single N-glycosylation site and are well studied, other antibody isotypes, e.g. IgA and IgM, that are the first responders in certain diseases, have two to five sites/monomer of antibody, and little is known about their N-glycosylation. Here we employ a nLC-MS/MS method using stepped-energy higher energy collisional dissociation to characterize the N-glycan repertoire and site occupancy of circulating serum antibodies. We simultaneously determined the site-specific N-linked glycan repertoire for IgG1, IgG4, IgA1, IgA2, and IgM in individual healthy donors. Compared with IgG1, IgG4 displayed a higher relative abundance of G1S1F and a lower relative abundance of G1FB. IgA1 and IgA2 displayed mostly biantennary N-glycans. IgA2 variants with the either serine (S93) or proline (P93) were detected. In digests of the sera from a subset of donors, we detected an unmodified peptide containing a proline residue at position 93; this substitution would strongly disfavor N-glycosylation at N92. IgM sites N46, N209, and N272 displayed mostly complex glycans, whereas sites N279 and N439 displayed higher relative abundances of high-mannose glycoforms. This multi-isotype approach is a crucial step toward developing a platform to define disease-specific N-glycan signatures for different isotypes to help tune antibodies to induce protection. Data are available via ProteomeXchange with identifier PXD010911.

The congenital disorders of glycosylation (CDG), a group of inherited diseases characterized by aberrant glycosylation, encompass a wide range of defects, including glycosyltransferases, glycosidases, nucleotide-sugar transporters as well as proteins involved in maintaining Golgi architecture, pH and vesicular trafficking. Mutations in a previously undescribed protein, TMEM165, were recently shown to cause a new form of CDG, termed TMEM165-CDG. TMEM165-CDG patients exhibit cartilage and bone dysplasia and altered glycosylation of serum glycoproteins. We utilized a morpholino knockdown strategy in zebrafish to investigate the physiologic and pathogenic functions of TMEM165. Inhibition of tmem165 expression in developing zebrafish embryos caused craniofacial abnormalities, largely attributable to fewer chondrocytes. Decreased expression of several markers of cartilage and bone development suggests that Tmem165 deficiency alters both chondrocyte and osteoblast differentiation. Glycomic analysis of tmem165 morphants also revealed altered initiation, processing and extension of N-glycans, paralleling some of the glycosylation changes noted in human patients. Collectively, these findings highlight the utility of zebrafish to elucidate pathogenic mechanisms associated with glycosylation disorders and suggest that the cartilage and bone dysplasia manifested in TMEM165-CDG patients may stem from abnormal development of chondrocytes and osteoblasts.