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 (293 FreeStyle™ cells; 293F) cells in the presence of kifunensine, which impart only high-mannose glycans; (b) insect cells (Spodoptera frugiperda, Sf9), 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; and (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-derived and wild-type mammalian-cell-derived material that is predicted to affect ligand binding sites proximal to glycans. Modeling 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 do, 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 sialylated version when administered in two different adjuvants, and induced higher titers 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 that should be considered when selecting expression systems for glycosylated antigens to be used for structure–function studies and for vaccine use.

Introduction

The only viral targets for neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) are the viral envelope glycoproteins (Env), composed of a heterotrimer of three surface subunits (gp120) noncovalently linked to three transmembrane subunits (gp41). An ideal Env-based vaccine against HIV-1 would induce long-lived NAb responses recognizing a broad range of viral strains. To move toward this goal requires considerable optimization of the antigen, adjuvant and delivery strategy. This will include an understanding of the impact of Env glycosylation with regard to its effects on antigenicity and immunogenicity. The choice of expression system might be especially relevant in the context of HIV-1 Env, since glycans contribute approximately 50% of the molecular mass of gp120,1, 2, 3, 4, 5 and enzymatic glycosylation machinery differs substantially between, for example, insect and mammalian cell expression systems. When expressed in mammalian systems, between 13 and 16 of the N-linked glycans of gp120 are of the complex-type, while the remainder (of a total of 20–26) are of the high-mannose or hybrid types.2, 6, 7, 8 High-mannose glycans occur more frequently in the conserved regions, whereas those attached to variable loops, being more exposed to the glycosylation machinery of the Golgi, are more frequently processed into complex glycans.6, 9 Conversely, in wild-type (wt) insect cell systems, glycosylation is restricted to paucimannosidic structures¹⁰ and the degree of sequon occupancy may be lower.11, 12

Insect cell expression systems have the advantage of producing large quantities of proteins cheaply and without many of the potential biohazards associated with mammalian systems. In addition, insect cells carry out many posttranslational modifications, including high-mannose-type N- and O-linked glycosylation, resulting in glycoproteins generally considered to be of similar antigenicity and functionality to those prepared in mammalian cells.¹³ When expressed in insect cell systems, Env gp160 undergoes appropriate cleavage to generate the gp41 and gp120 subunits, and the gp120 subunit retains CD4 binding.14, 15, 16 For these reasons, insect cells have been used since the early years of HIV-1 vaccine research to produce Env antigens for immunogenicity and challenge studies in animals¹⁷ and immunogenicity in humans.¹⁸ However, given the differences in glycan enzyme machinery between these different expression systems, the criteria for selection of an optimal system might go beyond yield and ease of glycoprotein production, potentially affecting parameters such as antigenicity and immunogenicity.

Few studies have compared the effect of expression system on antigenicity in a head-to-head fashion, although one recent analysis compared gp120 expressed in different human cell lines for glycan content and relative binding to polyclonal sera.⁸ By contrast, many have examined the effects of enzymatic removal of glycans or mutations deleting selected glycan sequons.19, 20, 21, 22, 23, 24 While the outcomes of individual studies vary, together they suggest that partial or complete deglycosylation generally increases the binding of antisera and monoclonal antibodies (mAbs). However, the effects can be epitope specific, with some regions being unaffected or even showing decreases in mAb binding.25, 26 The site-specific deletion of glycan sequons from Env increased recognition by important neutralizing mAbs (NmAbs) such as b12, 447-52D and 2F5, especially when the mutants were expressed in insect cells.²⁰ Moreover, deletion of three to five Env glycan sequons from wt SIVmac239 virus resulted in dramatic attenuation in rhesus macaques and the induction of substantial protection against the wt virus with higher titers of NAbs than those obtained after wt virus challenge.21, 22 Disappointingly, however, subunit antigens based on such Env glycan mutants proved to be less good than, or at best equivalent to, wt Env antigens at inducing NAb responses or controlling viremia in animal models.19, 21, 23, 24 Moreover, since deletion of glycan sequons may alter glycoprotein processing and folding, this may have unpredictable effects on the presentation of conformational and discontinuous NAb epitopes such as those present on gp120. However, one study showed that only a small number of protein-proximal glycan residues are important for folding, while the rest may function to block antibody (Ab) binding, suggesting that for immunogen design, glycans may need to be reengineered or otherwise modified rather than deleted.27, 28 Such an approach has shown promise in a recent study in which complex glycans on gp120 were replaced with trimmed oligomannose structures by expression in a cell line lacking N-acetylglucosamine transferase I, resulting in increased binding of ligands to the CD4 binding site (CD4bs) and the V3 loop.²⁷

The choice of expression system might also affect the immunogenicity of glycoproteins such as gp120 in diverse ways. Immunogenicity might be reduced in insect-cell-expressed gp120, since sialic acid residues on complex glycans, not present on insect-cell-expressed material, are important for extending glycoprotein half-life in vivo by shielding mannose from interaction with mannose receptors.²⁹ Conversely, it has recently been demonstrated that mannose receptors on professional antigen-presenting cells mediate uptake into compartments involved in cross-presentation: this function might be enhanced in the presence of insect-cell-produced glycoproteins expressing predominantly terminal mannose glycans.³⁰ The latter supposition is in agreement with the finding that gp120 expressed in insect cells was better able to induce cytotoxic T lymphocyte responses in Balb/c mice than that produced in mammalian Chinese hamster ovary (CHO) cells.³¹ The reduced response to the CHO cell material was overcome by enzymatic deglycosylation, suggesting that mammalian glycans were responsible for the poor immunogenicity.³¹ An alternative view was recently proposed—that terminal mannose groups might downmodulate Ab responses to gp120 via lectin interactions on antigen-presenting cells leading to production of the immunosuppressive cytokine IL-10.32, 33 In addition to regulating interaction with cell surface lectins including mannose receptors, the sialic acid residues of complex glycans also negatively regulate the interaction of gp120 with mannose-binding lectin in the serum,³⁴ which upon binding to an antigen can trigger the complement cascade resulting in complement opsonization and improved antigen uptake. Finally, sialic acid has been demonstrated to suppress B-cell responses via interaction with CD22, a potential mechanism to avoid self-recognition.35, 36

To investigate the effect of expression system on glycoprotein antigenicity and immunogenicity, we compared two insect systems [wt Sf9 (Sf9wt; from Spodoptera frugiperda) and Sf9 Mimic™] with a mammalian system [293 FreeStyle™ (293F)] in the presence and absence of kifunensine, in a head-to-head fashion, using gp120 from two distantly related HIV-1 strains in order to describe both general and virus-strain-specific effects. Sf9 Mimic™ cells are a recombinant Sf9 cell line that express five mammalian glycosylation enzymes and produce the majority of complex mammalian glycan modifications¹⁰ with the exception that they lack a donor for sialic acid; thus, the complex glycans they produce have terminal galactose residues.³⁷ The inclusion of this additional cell line allows the contribution to antigenicity and immunogenicity of complex glycans lacking sialic acid to be assessed without the need for enzymatic desialylation.