Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants

Abstract

Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and β1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants.

Keywords: chaperones; glyco-engineering; glycosylation; immunogenicity; neutralization; occupancy; vaccine.

Figure 1

Production of HIV envelope (Env) gp140 in plants with improved glycosylation. (a) Overlayed normalized size exclusion chromatography elution profiles of gp140 when produced in human embryonic kidney 293 (HEK293) cells or in plants with modified (glycan‐enhanced [GE]) or unmodified glycosylation (wild type [WT]). The peaks corresponding to aggregates and putative trimers are indicated by the cartoon image above the figure. (b) Coomassie‐stained blue native polyacrylamide gel electrophoresis (BN‐PAGE) gel comprising of size exclusion chromatography (SEC)‐fractionated material from all three protein variants. The number above each lane refer to a corresponding fraction from size exclusion chromatography. Top: WT; middle: GE; bottom: HEK293. (c) Western blot analysis of the pooled fractions following SEC. HIV Env was detected using polyclonal goat anti‐gp120 antibody.

Figure 2

Site‐specific glycosylation of CAP256 gp140 produced in mammalian cells and in plants with either unmodified or modified glycosylation. (a) Heat map depicting the abundance of oligomannose‐type glycans when HIV envelope (Env) gp140 is produced in human embryonic kidney 293 (HEK293) mammalian cells. The model was constructed using SWISS‐MODEL using the structure of BG505 NFL as a template (6B0N). A representative glycan corresponding to the most abundant glycoform present at each site was modeled and sites are colored according to the abundance of oligomannose‐type glycans detected at each site. All glycans are labeled according to the asparagine they are attached to, following alignment with HxB2. Where an N‐linked glycan site was occupied by N‐linked glycans on <50% of sites, the glycan modeled was colored blue. (b) The model generated for HEK293 produced CAP256 Env was recolored according to the site‐specific glycan data for WT material. (c) The model generated for HEK293 produced CAP256 Env was recolored according to the site‐specific glycan data for “glycan‐enhanced” Env.

Figure 3

Comparison of the glycan occupancy of recombinant HIV envelope (Env) gp140 when produced in N. benthamiana using integrated host and transient glyco‐engineering (“glycan‐enhanced”) or no glyco‐engineering expression approaches (wild type [WT]). (a) The percentage point changes in glycan occupancy are labeled onto the model generated in Figure 2. A positive change represents a glycan site that is more occupied on the “glycan‐enhanced” material compared with the WT. (b) Site‐specific glycan occupancy of recombinant HIV Env gp140 produced in WT and “glycan‐enhanced” N. benthamiana. The change in glycan occupancy is colored, with red representing a decrease and blue an increase.

Figure 4

Comparison of the glycan occupancy of recombinant HIV envelope (Env) gp140 when produced in N. benthamiana using integrated host and transient glyco‐engineering (“glycan‐enhanced”) or mammalian cells (HEK293). (a) The percentage point changes in glycan occupancy are labeled onto the model generated in Figure 2. A positive change represents a glycan site that is more occupied on the “glycan‐enhanced” material compared with the HEK293‐derived material. (b) Site‐specific glycan occupancy of recombinant HIV Env gp140 produced in WT and “glycan‐enhanced” N. benthamiana. The change in glycan occupancy is colored, with red representing a decrease and blue an increase.

Figure 5

The average change in glycosylation across all detected sites on recombinant HIV envelope (Env) gp140 when produced in plants and mammalian cells. (a) Comparison of the glycosylation of “glycan‐enhanced” gp140 compared with the nonoptimized protein, both proteins are produced in N. Benthamiana. (b) Comparison of the glycosylation of the “glycan enhanced” gp140 compared with the mammalian cell‐derived protein. In both a and b, the glycosylation of the “glycan‐enhanced” protein is presented relative to the comparator protein and, therefore, positive and negative values indicate an increase or decrease in the abundance of a particular glycan species in the “glycan‐enhanced” antigen. Schematics above each graph display the HxB2 aligned CAP256 NFL N‐linked glycan sites, with sites labeled in black representing sites that were resolved by liquid chromatography‐mass spectrometry (LC‐MS) and gray the ones that were not.

Figure 6

Estimating the proportion of correctly assembled trimeric gp140. (a) Sample of two‐dimensional (2D) classes of negatively stained gp140 expressed in HEK293 cells. The second most populous class (*) contains 5% of the particles (355/7158) and shows approximately the correct size and 3‐fold symmetry to be identified as trimeric gp140. (b) Sample of 2D classes of gp140 expressed in plants with modified glycosylation (“glycan‐enhanced”). Two classes (*) 6% of the images (1263/30444) show ~3‐fold symmetry and could represent correctly assembled gp140.

Figure 7

Immunogenicity comparison of “Glycan‐enhanced” and human embryonic kidney 293 (HEK293) cell‐produced HIV envelope (Env) gp140 in rabbits. (a) Schedule of immunizations and bleeds. Animals (n = 5) in Group 1 and 2 were immunized four times with purified “glycan‐enhanced” gp140 or gp140 from HEK293 cells. Animals in Groups 3 and 4 were primed with two DNA and two modified vaccinia ankara (MVA) vaccinations encoding mosaic Gag and CAP256 SU gp150, before boosting with purified protein. Animals in Group 3 were injected with the plant‐produced “glycan‐enhanced” antigen as a boost, whereas animals in Group 4 received the HEK293 cell‐derived protein as a boost. D,  DNA; M, MVA; ×, bleed, Ɨ, sacrifice; P, protein; P (green), glycan‐enhanced; P (blue), HEK293 cell‐derived). (b) Serum‐binding antibodies induced by the PPPP vaccination regimen. (c) Serum‐binding antibodies induced by the DDMMPP vaccination regimen.