VLPs Carring HIV-1 Env with Modulated Glycan Composition

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Previously obtained highly immunogenic Env-VLPs ensure overcoming the natural resistance of HIV-1 surface proteins associated with their low level of incorporation and inaccessibility of conserved epitopes to induce neutralizing antibodies. We also adopted this technology to modify Env trimers of ZM53(T/F) strain to produce Env-VLPs by recombinant vaccinia viruses (rVVs). These rVVs expressing Env, Gag-Pol (HIV-1/SIV), as well as the cowpox virus hr gene allowing to avoid the restriction of vaccinia virus replication in CHO cells were used for VLP production. The CHO Lec1 engineered cell line lacking GlcNAc-TI was used for generating VLPs with Env proteins containing a cytoplasmic domain (CT) affecting on surface subunit (SU) conformation. This has created the opportunity to modulate the glycan composition, and refine the conditions for their production, and optimize approaches to overcoming HIV-1 resistance associated with abundant glycosylation.

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作者简介

G. Kaevitser

Gamaleya Federal Research Center of Epidemiology and Microbiology

编辑信件的主要联系方式.
Email: anvzorov@mail.ru
俄罗斯联邦, Moscow, 123098

E. Samokhvalov

Gamaleya Federal Research Center of Epidemiology and Microbiology

Email: anvzorov@mail.ru
俄罗斯联邦, Moscow, 123098

D. Scheblyakov

Gamaleya Federal Research Center of Epidemiology and Microbiology

Email: anvzorov@mail.ru
俄罗斯联邦, Moscow, 123098

A. Gintsburg

Gamaleya Federal Research Center of Epidemiology and Microbiology; Sechenov First Moscow State Medical University, Ministry of Health of the Russian Federation

Email: anvzorov@mail.ru
俄罗斯联邦, Moscow, 123098; Moscow, 123098

A. Vzorov

Gamaleya Federal Research Center of Epidemiology and Microbiology; Department of Immunology, Faculty of Biology, Lomonosov Moscow State University

Email: anvzorov@mail.ru
俄罗斯联邦, Moscow, 123098; Moscow, 119234

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2. Fig. 1. Effect of vaccinia virus hr gene expression on HIV-1 Env and Gag protein synthesis in CHO and Hep2 cells. Hep2 (lanes 1, 3, 5) or CHO (lanes 2, 4, 6) cells were coinfected with VVEnvIIIB (lanes 1–6), VVGag-Pol (lanes 1, 2), VVCP (lanes 1–4), or VVSC11 (control virus lacking VKO and HIV-1 hr genes) (lanes 5, 6). Numbers on the right indicate the locations of protein molecular mass standards (kDa).

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3. Fig. 2. Translation of the HIV-1 Env protein is accompanied by glycosylation of the synthesized polypeptide chain and oligomerization into trimers in the endoplasmic reticulum (ER) of the cell. Oligomerization promotes the movement of trimers to the Golgi apparatus (G), where the polypeptide chain is cut into SU and a transmembrane subunit and further maturation of the carbohydrate chain occurs. In the Env trimer, in the presence of an α-helix in the CT domain, some substrates may be inaccessible to enzymes. (1) Env precursor; (2) oligomannose chains (beige) attached at an asparagine residue in the site of potential N-glycosylation (Asn-X-Thr/Ser, where X ≠ Pro) on the Env monomer (gp160); (3) oligomerization and movement of Env trimers; (4) proteolytic cleavage of gp160 by cellular furin to form gp120 and gp41; (5) processing of carbohydrate chains and formation of complex glycans (green). For a description of the composition of the Env-22 and Env-22hb proteins, see Table 1.

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4. Fig. 3. Trimer formation by HIV-1 Env protein expressed by VVEnv-22. Hep2 cells were infected with the virus at different multiplicities of infection (MOI): 0.1 (lane 1), 0.2 (lane 2), 0.5 (lanes 3, 5) PFU/cell. Negative control – uninfected Hep2 cells (lane 4). After 48 h, the cells were removed and the plasma membrane fraction containing HIV-1 proteins was purified. Samples were dissolved in sample loading buffer without reducing agents at room temperature (lanes 1‒3) or with reducing agents (200 mM dithiothreitol, 200 mM β-mercaptoethanol, 8 M urea) for 5 min at 96°C (lane 5). Proteins were separated in 4–15% SDS-PAAG and analyzed by immunoblotting.

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5. Fig. 4. Kinetics of HIV-1 VLP production. a – Analysis of VLP production by immunoblotting. CHO cells were coinfected with three viruses: VVCP : VVEnv-22 : VVGag-Pol in a ratio of 1 : 1 : 1 – with an MOI of 0.5 PFU/cell; the culture medium containing VLPs was collected at 0 (lane 6), 48 (1), 60 (2), 72 (3), 84 (4) and 96 (5) h after the start of infection. b – Densitometric analysis was performed using ImageJ software. Data are presented based on two independent experiments.

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6. Fig. 5. Immunoblot analysis of the molecular weight of HIV-1 Env proteins on the surface of CHO cells coinfected with the rVV mixture. The content of VVCP + VVEnv-22 + VVGag-Pol viruses in the inoculum was 0.5 : 0.5 : 0.5 (lane 2), 1 : 1 : 1 (lane 3), 0.5 : 1.5 : 0.5 (lane 4), 1 : 1.5 : 0.5 (lane 5) PFU/cell. Negative control – CHO cells coinfected with VVCP and VVGag-Pol in a ratio of 1 : 1 PFU/cell (lane 1). Proteins were separated in 8% SDS-PAAG and analyzed by immunoblotting as described in the Experimental section.

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7. Fig. 6. Immunoblotting of HIV-1 Env proteins expressed by CHO (1–3) and CHO Lec1 (4–6) cells infected with VVEnvIIIB. The plasma membrane fraction was treated with Endo H (lanes 1, 4), PNGase F (lanes 2, 5), or left untreated (lanes 3, 6). Proteins were separated on 8% SDS-PAAG and analyzed by immunoblotting as described in the Experimental Section.

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8. Fig. 7. BN-PAGE analysis of VLPs treated with glycosidases or proteases. VLPs obtained in Lec1 cells infected with the same multiplicity of VVGag-Pol and VVEnv-22 (a), with an excess of VVEnv-22 or VVEnv-22hb relative to VVGag-Pol (b). Env-22 VLPs (a, 1–3; b, 1–3); Env-22hb VLPs (b, 4–6). Untreated VLPs (a, 1; b, 3 and 6), Endo H-treated VLPs (a, 2; b, 2 and 5), and a mixture of chymotrypsin, trypsin, subtilisin/proteinase K enzymes (a, 3; b, 1 and 4).

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