Immunogenicity Evaluation of N-Glycans Recognized by HIV Broadly Neutralizing Antibodies.

While the improved treatment of human immunodeficiency virus type 1 (HIV-1) infection is available, the development of an effective and safe prophylactic vaccine against HIV-1 is still an unrealized goal. Encouragingly, the discovery of broadly neutralizing antibodies (bNAbs) from HIV-1 positive patients that are capable of neutralizing a broad spectrum of HIV-1 isolates of various clades has accelerated the progress of vaccine development in the past few years. Some of these bNAbs recognize the N-glycans on the viral surface gp120 glycoprotein. We have been interested in using the glycan epitopes recognized by bNAbs for the development of vaccines to elicit bNAb-like antibodies with broadly neutralizing activities. Toward this goal, we have identified novel hybrid-type structures with subnanomolar avidity toward several bNAbs including PG16, PGT121, PGT128-3C, 2G12, VRC13, VRC-PG05, VRC26.25, VRC26.09, PGDM1400, 35O22, and 10-1074. Here, we report the immunogenicity evaluation of a novel hybrid glycan conjugated to carrier DTCRM197, a nontoxic mutant of the diphtheria toxin, for immunization in mice. Our results indicated that the IgG response was mainly against the chitobiose motif with nonspecific binding to a panel of N-glycans with reducing end GlcNAc-GlcNAc (chitobiose) printed on the glass slides. However, the IgM response was mainly toward the reducing end GlcNAc moiety. We further used the glycoconjugates of Man3GlcNAc2, Man5GlcNAc2, and Man9GlcNAc2 glycans for immunization, and a similar specificity pattern was observed. These findings suggest that the immunogenicity of chitobiose may interfere with the outcome of N-glycan-based vaccines, and modification may be necessary to increase the immunogenicity of the entire N-glycan epitope.

Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies.

The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.

Influenza virus neuraminidase regulates host CD8+ T-cell response in mice.

Influenza A virus (IAV)-specific CD8+ T-cell response was shown to provide protection against pandemic and seasonal influenza infections. However, the response was often relatively weak and the mechanism was unclear. Here, we show that the composition of IAV released from infected cells is regulated by the neuraminidase (NA) activity and the cells infected by NA-defective virus cause intracellular viral protein accumulation and cell death. In addition, after uptake of NA-defective viruses by dendritic cells (DCs), an expression of the major histocompatibility complex class I is induced to activate IAV-specific CD8+ T-cell response. When mice were infected by NA-defective IAV, a CD8+ T-cell response to the highly conserved viral antigens including PB1, NP, HA, M1, M2 and NS1 was observed along with the increasing expression of IL10, IL12 and IL27. Vaccination of mice with NA-defective H1N1 A/WSN/33 induced a strong IAV-specific CD8+ T cell response against H1N1, H3N2 and H5N1. This study reveals the role of NA in the IAV-specific CD8+ T-cell response and virion assembly process, and provides an alternative direction toward the development of universal influenza vaccines.

Influenza A surface glycosylation and vaccine design.

We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAV-specific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.

A common glycan structure on immunoglobulin G for enhancement of effector functions.

Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.

Efficient convergent synthesis of bi-, tri-, and tetra-antennary complex type N-glycans and their HIV-1 antigenicity.

The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.

Synthesis and vaccine evaluation of the tumor-associated carbohydrate antigen RM2 from prostate cancer.

We have successfully developed a [1+2+3] one-pot strategy to synthesize the RM2 antigen hexasaccharide that was proposed to be a prostate tumor antigen. The structure of the synthetic product was verified by NMR analysis and antibody binding assay using a glycan microarray. In addition, the synthetic antigen was conjugated to a mutated diphtheria toxin (DT, CRM197) with different copy numbers and adjuvant combinations to form the vaccine candidates. After vaccination in mice, we used glycan microarrays to monitor their immune response, and the results indicated that, when one molecule of DT was incorporated with 4.7 molecules of RM2 on average (DT-RM4.7) and adjuvanted with the glycolipid C34, the combination exhibited the strongest anti-RM2 IgG titer. Moreover, the induced mouse antibodies mediated effective complement-dependent cytotoxicity (CDC) against the prostate cancer cell line LNCap.