Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity

Abstract

The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications.

Importance: The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced immune responses possibly due to differences in their interaction with immune cells. We have generated virus-like particles (VLPs) composed of hepatitis B virus envelope proteins (HBsAgS) with additional N-glycosylation sites. Hyperglycosylated VLPs were synthesized and characterized, and the results demonstrated that they carry the same types of glycans as wild-type VLPs. Comparative immunization studies demonstrated that the VLPs with the highest N-glycan density induce earlier and longer-lasting antibody immune responses than do wild-type or hypoglycosylated VLPs, possibly allowing reduced numbers of vaccine injections. The ability to modulate the immunogenicity of an immunogen will provide opportunities to develop optimized vaccines and VLP delivery platforms for foreign antigenic sequences, possibly in synergy with the use of suitable adjuvanting compounds.

FIG 1

Schematic diagram of HBsAgS. Wild-type (WT) HBsAgS has glycosylation site at amino acid (aa) 146. To introduce new potential glycosylation sites, site-directed mutagenesis was performed at positions T116 and G130 to introduce an asparagine (N) in a NXT/S sequon. Cysteine residues are indicated by orange circles, and disulfide bonds are indicated by “-S-S-.” Alpha-helix transmembrane regions are shown as cylinders.

FIG 2

To identify the synthesis of differentially glycosylated virus-like particles (VLPs), HEK293T cells were transfected with plasmids expressing the VLP subunits, HBsAgS. The culture supernatant (A) and the cell lysate (B) were harvested, [³⁵S]methionine-cysteine-labeled VLPs were immunoprecipitated with anti-HBsAgS antibodies, and the HBsAgS subunits were separated by gel electrophoresis under reducing conditions. To verify the presence of N-glycans, [³⁵S]methionine-cysteine-labeled VLPs from the cell culture supernatant were immunoprecipiated (C) and digested with PNGase F (D) and then separated by gel electrophoresis and visualized. Lane 1, VLPs composed of wild-type HBsAgS subunits with the N146 glycosylation site present. Lane 2, N146 glycosylation site (N146Q) of HBsAgS removed. Lanes 3 and 4: VLPs composed of HBsAgS subunits with introduced glycosylation sites at positions T116N and G130N, respectively. In addition to the newly introduced sites, the N146 glycosylation site is retained. Lane 5, samples derived from cells transfected with the empty pCI vector. N, M, and D, nonglycosylated (24-kDa), monoglycosylated (27-kDa), and diglycosylated (30/31-kDa) HBsAgS proteins, respectively.

FIG 3

Electron micrograph of VLP samples. VLPs were derived from the cell culture medium and purified by sucrose density gradient centrifugation, dialyzed against phosphate-buffered saline (PBS), and visualized by negative staining with phosphotungstic acid (PTA). (A) Wild-type (WT) HBsAgS VLPs; (B) N146Q-HBsAgS VLPs; (C) T116N-HBsAgS VLPs; (D) G130N-HBsAgS VLPs.

FIG 4

(A) N-Glycan abundance of HBsAgS variants as measured by the protein N-glycan mass spectrometry (MS) intensity of HBsAgS variants normalized to the wild-type (WT) HBsAgS level (red broken line). (B) Structures of the 10 most abundant glycoforms of HBsAgS. Symbols and nomenclature are according to the Consortium for Functional Glycomics (CFG) convention. Means ± SDs are represented in panels A and C *, P < 0.05; **, P < 0.01. (C) Relative N-glycan structure distribution on WT HBsAgS versus T116N HBsAgS (left) and G130N (right). Only N-glycans which were present at more than 0.5% of the total N-glycome in at least one of the HBsAgS variants were included in the quantitative distribution (see Table S1 in the supplemental material for all glycan structures). High correlation coefficients (R²) indicate similar N-glycomes. Symbols for the monosaccharides are as follows: for galactose, yellow circles; for mannose, green circles; for N-acetylglucosamine, blue squares; for fucose, red triangles; and for N-acetylneuraminic acid, purple diamonds. The linkages are indicated.

FIG 5

Mapping of the antigenic profile of the HBsAgS VLPs using the Bio-Plex bead-based flow cytometric platform (Bio-Rad, Hercules, CA). The HBsAgS multiplex antibody panel consists of 19 monoclonal antibodies (MAbs) directed against epitopes located between residues 99 and 226 of HBsAgS. The x axis defines the HBsAgS region of antibody binding, Nterm, N-terminal region preceding loop 1 domain; loop 1, amino acids 107 to 138; loop 2, amino acids 139 to 149; Combo, semiconformational loop 1/2 epitope; Cterm, C-terminal region amino acids 160 to 226; Conform, conformational epitope. The number after the hyphen stands for the antibody. The y axis shows the ratio of antibody binding to wild-type (WT) VLPs versus N146Q VLPs, T116N VLPs, or G130N VLPs. A gain-of-epitope recognition corresponds to positive fold change (>0.5-fold), and loss or reduction of epitope binding corresponds to negative fold changes (>0.5-fold), a range larger or smaller than the variation of epitope recognition of WT VLPs.

FIG 6

Groups of eight BALB/c mice were immunized with 2 μg of VLPs in weeks 1, 3, 5, and 7 in the absence of adjuvant. Serum samples were taken in week 4 (A), week 6 (B), and week 14 (C) and at the last bleed in week 20 (D). HBsAgS-specific antibody responses were measured by an enzyme-linked immunosorbent assay (ELISA) against yeast-derived HBsAg VLPs (serotype ayw). Endpoint titer was determined to be the highest serum dilution that gave an optical density OD value of at least two times above background. Standard deviations were calculated and are indicated as error bars. The red asterisk indicates that the difference between the T116N VLPs and WT VLPs is statistically significant (P < 0.01).