Redirecting lentiviral vectors pseudotyped with Sindbis virus-derived envelope proteins to DC-SIGN by modification of N-linked glycans of envelope proteins

Abstract

Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.

FIG. 1.

(a) N-glycan structures and processing pathway. All N-glycans are first produced as the high-mannose structure in both mammalian cells and insect cells. In mammalian cells, certain N-glycans are further processed to the complex structure. In insect cells, certain N-glycans are further processed to the paucimannosidic structure. DMNJ inhibits mannosidase I, which is necessary for the formation of the complex structure; thus, all N-glycans have the high-mannose structure when generated in the presence of DMNJ. One representative structure of each N-glycan is shown. Man, mannose; GlcNAc, N-acetylglucosamine; SA, sialic acid; Gal, galactose. (b) Schematic representation of chimeric Sindbis virus envelope proteins. The Sindbis virus envelope protein is first synthesized as a polypeptide and subsequently cleaved by cellular proteases to generate the E3, E2, 6K, and E1 proteins. E1 and E2 are incorporated into the viral envelope, and E3 and 6K are leader sequences for E2 and E1, respectively. The N-linked glycosylation sites of the envelope proteins are shown. 2.2 is a modified Sindbis virus envelope protein in which the IgG-binding domain of protein A (ZZ) was inserted into the E2 region at aa 70. 2.2 1L1L has two flexible linkers (Gly-Gly-Gly-Gly-Ser) at aa 70 of the E2 protein. 2.2 ΔE2-196N does not have the N-glycan at E2 aa 196, 2.2 ΔE1-139N does not have the N-glycan at E1 aa 139, and 2.2 ΔE2-196N E1-139N does not have the N-glycans at either E2 aa 196 or E1 aa 139.

FIG. 2.

Expression of DC-SIGN on various cells, including Jurkat (human T-cell line), DC-SIGN Jurkat (Jurkat cells transduced with a DC-SIGN expression vector), 293T (human embryonic kidney cell line), CD20 293T (293T cells transduced with a CD20 expression vector), and DC-SIGN 293T (293T cells transduced with a DC-SIGN expression vector) cells. Each cell type was stained with either PE-conjugated isotype control antibody (blue line) or PE-conjugated anti-DC-SIGN antibody (red line).

FIG. 3.

Transduction of cells with lentiviral vectors pseudotyped with various types of envelope proteins. Jurkat, DC-SIGN Jurkat, 293T, DC-SIGN 293T, and CD20 293T cells were transduced with HIV vectors pseudotyped with (A) Sindbis virus, (B) 2.2 1L1L, (C) 2.2, (D) Sindbis virus DMNJ (Sindbis virus pseudotype produced in the presence of DMNJ), (E) 2.2 1L1L DMNJ (2.2 1L1L pseudotype produced in the presence of DMNJ), or (F) 2.2 DMNJ (2.2 pseudotype produced in the presence of DMNJ). EGFP expression was analyzed by flow cytometry 3 days posttransduction. The percentage of EGFP-positive cells is shown as an average of triplicate experiments. The y axis is forward scatter, and the x axis is EGFP expression. The virus amounts used for transduction are shown in Table 1.

FIG. 4.

Blocking of transduction by mannan and anti-DC-SIGN antibody. DC-SIGN Jurkat (a) and DC-SIGN 293T (b) cells were each incubated with mannan (polymer of mannose sugar), isotype control antibody, or anti-DC-SIGN antibody before and during transduction with the HIV vector pseudotyped with 2.2 1L1L DMNJ (1 ng of p24 for DC-SIGN Jurkat and 200 pg of p24 for DC-SIGN 293T cells). EGFP expression was analyzed 3 days posttransduction. The percentage of EGFP-positive cells is shown as an average of triplicate experiments with standard deviations.

FIG. 5.

Identification of N-glycans that bind DC-SIGN. HIV vectors pseudotyped with 2.2, 2.2 ΔE2-196N, 2.2 ΔE1-139N, or 2.2 ΔE2-196N E1-139N were produced in the presence of DMNJ and designated 2.2 DMNJ, 2.2 ΔE2-196N DMNJ, 2.2 ΔE1-139N DMNJ, and 2.2 ΔE2-196N E1-139N DMNJ, respectively. The viruses were titrated on Jurkat cells by conjugating them with the antibody against HLA class I, which is abundantly expressed on Jurkat cells. The same titer of each virus was used to transduce DC-SIGN Jurkat cells without conjugation of an antibody. The p24 values of the viruses used for transduction are 2, 2, 8, and 8 ng for 2.2 DMNJ, 2.2 ΔE2-196N DMNJ, 2.2 ΔE1-139N DMNJ, and 2.2 ΔE2-196N E1-139N DMNJ pseudotype, respectively. EGFP expression was analyzed by flow cytometry 3 days posttransduction. The percentage of EGFP-positive cells is shown as an average of triplicate experiments with standard deviations. The y axis is forward scatter, and the x axis is EGFP expression.

FIG. 6.

(a) Expression of surface markers on human dendritic cells. Dendritic cells were prepared by culturing the adherent fraction of peripheral blood mononuclear cells in GM-SCF and IL-4 and then depleting T, B, and NK cells using anti-CD3, -CD19, and -CD56 antibodies and anti-mouse IgG-conjugated immunomagnetic beads. The cells were costained with the anti-HLA DR antibody conjugated with FITC, the anti-CD86 antibody conjugated with PE, and the anti-CD11c antibody conjugated with APC. The cells were also stained with the anti-DC-SIGN antibody conjugated with APC. All staining was performed in the presence of human AB serum to block nonspecific staining of Fc receptors, and isotype control antibodies conjugated with FITC, PE, or APC were used as negative controls for staining. (b) Dendritic cells (2 × 10⁵) were incubated for 1 h with mannan (200 μg/ml), control antibody (20 μg/ml), anti-DC-SIGN antibody (20 μg/ml), or nevirapine (5 μM). The cells were then infected with the VSV-G, 2.2 1L1L, or 2.2 1L1L DMNJ pseudotype (2.5 μg p24) for 6 h in the presence of blocking reagents. EGFP expression was assayed by flow cytometry 4 days posttransduction. Experiments necessary to show statistically significant differences were performed in triplicate, and averages of the triplicate experiments are shown with standard deviations.

FIG. 7.

Two lots of human AB serum were used. Heat-inactivated serum was made by incubation at 56°C for 40 min. Wild-type Sindbis virus envelope protein, 2.2 1L1L DMNJ pseudotypes, and 2.2 pseudotype conjugated with anti-DC-SIGN antibody (Ab; 50 μg/ml) (virus amount, 100 μl; 5 μg HIV p24/ml) were incubated with 100 μl of serum or heat-inactivated serum at 37°C for 1 h. Each virus was diluted 50-fold with PBS (+) and then incubated with DC-SIGN 293T. EGFP expression was assayed by flow cytometry 3 days posttransduction. All experiments were performed in triplicate. The transduction efficiency of the virus treated with heat-inactivated serum was considered 100%. The relative transduction efficiency was calculated by the formula presented in Materials and Methods.