Expression-System-Dependent Modulation of HIV-1 Envelope Glycoprotein Antigenicity and Immunogenicity

Abstract

Recombinant expression systems differ in the type of glycosylation they impart on expressed antigens such as the Human Immunodeficiency Virus Type-1 (HIV-1) envelope glycoproteins, potentially affecting their biological properties. We performed head-to-head antigenic, immunogenic and molecular profiling of two distantly-related Env surface (gp120) antigens produced in different systems: a) mammalian (293F) cells in the presence of kifunensine which impart only high mannose glycans; b) insect (Spodoptera frugiperda, Sf9) cells, which confer mainly paucimannosidic glycans; c) Sf9 cells recombinant for mammalian glycosylation enzymes (Sf9 Mimic™), which impart high mannose, hybrid and complex glycans without sialic acid; d) 293F cells, which impart high mannose, hybrid and complex glycans with sialic acid. Molecular models revealed a significant difference in gp120 glycan coverage between the Sf9- and wild-type mammalian cell-derived material that is predicted to impact upon ligand binding sites proximal to glycans. Modelling of solvent-exposed surface electrostatic potentials showed that sialic acid imparts a significant negative surface charge that may influence gp120 antigenicity and immunogenicity. Gp120 expressed in systems that do not incorporate sialic acid displayed increased ligand binding to the CD4-binding and CD4–induced sites compared to those expressed in the system that does, and imparted other more subtle differences in antigenicity in a gp120 subtype-specific manner. Non-sialic acid-containing gp120 was significantly more immunogenic than the sialyated version when administered in two different adjuvants, and induced higher titres of antibodies competing for CD4 binding site ligand-gp120 interaction. These findings suggest that non-sialic acid imparting systems yield gp120 immunogens with modified antigenic and immunogenic properties, considerations which should be considered when selecting expression systems for glycosylated antigens to be used for structure/function studies and for vaccine use.

High-Mannose Glycan-Dependent Epitopes Are Frequently Targeted in Broad Neutralizing Antibody Responses during Human Immunodeficiency Virus Type 1 Infection

“…Glycan-specific antibody responses can be an attractive line of vaccine development, since some glycan-specific MAbs demonstrate great neutralizing breadth and potency (51,54). It has also been shown that glycan-binding antibodies can be induced by synthetic glycans (2,3,17,19,26,57) or HM-glycan on non-HIV-1 proteins (1, 25, 34, 35). Unfortunately, a glycan-binding antibody capable of neutralizing diverse strains of primary HIV-1 has yet to be achieved.…”

Enhancing the Quality of Antibodies to HIV-1 Envelope by GagPol-Specific Th Cells

“…DNA electroporation. Genetic vaccines avoid a bias in the immune response imparted by the Ag production system (35), and DNA electroporation has been shown to favor the induction of immune responses with a Th1 phenotype (36). Nevertheless, the Env-specific humoral immune response induced by our vaccine was highly dominated by the Th2-associated IgG1 subclass (Fig.…”

Characterization of Glycosylation Profiles of HIV-1 Transmitted/Founder Envelopes by Mass Spectrometry

“…Glycan modifications on proteins undergo a series of glycan processing steps from the endoplasmic reticulum (ER) to the Golgi apparatus with a diverse array of glycan processing enzymes that compete for available substrate, resulting in multiple glycosylation patterns for each glycosylation site for a given protein and variation in glycosylation site occupancy (35,52,53). Moreover, protein glycosylation varies significantly across different cell types, cell states, tissues, and organisms (3,12,16,34,35,53,56,62). Despite these challenges, recent advances in proteomics have accelerated the pace of the development of efficient methods and technologies that can be tailored for the analysis of protein glycosylation (6,51,66).…”

“…The types of glycans on recombinant viral Env are beginning to be characterized (5,34), and the carbohydrates on recombinantly expressed envelopes may not be fully representative of those glycans on virion Env spikes (15). Nonetheless, comparison of recombinant transmitted/founder and chronic HIV-1 Env glycosylation with new carbohydrate analysis technology is an important initial analysis, primarily because of the anticipated use of recombinant transmitted/founder Envs in human immunogenicity trials.…”