Secreted antiviral entry inhibitory (SAVE) peptides for gene therapy of HIV infection

Abstract

Gene therapeutic strategies for human immunodeficiency virus type 1 (HIV-1) infection could potentially overcome the limitations of standard antiretroviral drug therapy (ART). However, in none of the clinical gene therapy trials published to date, therapeutic levels of genetic protection have been achieved in the target cell population for HIV-1. To improve systemic antiviral efficacy, C peptides, which are efficient inhibitors of HIV-1 entry, were engineered for high-level secretion by genetically modified cells. The size restrictions for efficient peptide export through the secretory pathway were overcome by expressing the C peptides as concatemers, which were processed into monomers by furin protease cleavage. These secreted antiviral entry inhibitory (SAVE) peptides mediated a substantial protective bystander effect on neighboring nonmodified cells, thus suppressing virus replication even if only a small fraction of cells was genetically modified. Accordingly, these SAVE peptides may provide a strong benefit to AIDS patients in future, and, if applied by direct in vivo gene delivery, could present an effective alternative to antiretroviral drug regimen.

Figure 1

Multimerization and addition of scaffold enhances C peptide secretion. (a) Schematic overview of retroviral vector constructs encoding elongated C peptides. The amino acid sequences of the depicted domains are given in the Supplementary Materials and Methods section. (b) C46 fusion peptides were analyzed by western blot using a human monoclonal antibody to glycoprotein subunit 41 (gp41) (C heptad repeat, mab2F5). Deglycosylated supernatants and cell lysates of 293T cells transfected with the indicated retroviral vector constructs are shown. *IgC supernatant was diluted 1:5 before loading. Defined amounts of synthetic (syn) C46 peptide were analyzed in parallel (10, 20, and 50 nmol/l, respectively). (c) Single-round infection assay. PM1 cells were infected with lentiviral vector particles packaged with HIV-1_JRFL Env and encoding enhanced green fluorescent protein (eGFP) in the presence of increasing concentrations of peptide-containing supernatants from 293T cells transfected with the indicated constructs. Peptide concentrations of 293T supernatants were estimated as shown in b. eGFP-positive cells were determined by fluorescence-activated cell sorting analysis. 100% relative infection corresponds to 6.5% eGFP-positive cells. Data are means from duplicates; error bars show SEM. aa, amino acid; GAr, glycine–alanine repeat; H, hinge (HIV gp41 membrane proximal region); IgC, linker from human IgG2 containing four cysteine residues; IgS, linker from human IgG2 with cysteine residues mutated to serines; L, loop derived from gp41; M, c-myc tag; S, signal peptide from human tissue type plasminogen activator; SPase, cleavage site for signal peptidase.

Figure 2

Secreted peptide concatemers with cleavable linkers efficiently block HIV-1 entry. (a) Schematic overview of the concatemeric C peptides encoded by retroviral vectors. Two C46 peptides were connected by the indicated linker sequences. (b) To test the antiviral activity of the secreted C46, PM1 cells were challenged with lentiviral vector particles packaged with HIV-1_JRFL Env and encoding enhanced green fluorescent protein (eGFP) in the presence of increasing concentrations of supernatant from 293T cells previously transfected with the indicated constructs. The concentration of peptide in the cell culture supernatants was determined in a fluorescence-activated cell sorting (FACS)-based assay as described in Materials and Methods section. eGFP-positive cells were determined by FACS analysis. 100% relative infection corresponds to 11% eGFP-positive cells. Data are means from duplicates; error bars show SEM. (c,d) Western blots of deglycosylated cell culture supernatants and cell lysates of 293T cells transfected with the indicated constructs are shown. A furin expression plasmid was cotransfected were indicated in d. D, noncleaved dimeric concatemers; M, cleaved monomeric peptides; S, signal peptide from human tissue type plasminogen activator; SPase, cleavage site for signal peptidase.

Figure 3

Efficient cleavage of the C peptide precursor with two GA repeats flanking the furin cleavage site. (a) 293T cells were transiently transfected with plasmid DNA coding for Fur_o sequence on both sides with GA repeats (GAFur_oGA) (see Figure 2a). Twenty-four hours post-transfection cells were pulse-labeled with ³⁵S-L-methionine for 30 minutes and chased with an excess of unlabeled methionine (Dulbecco's modified Eagle's medium) for the indicated period of time. C46 peptides in the cell lysates and cell culture supernatants were immunoprecipitated with an antibody specific for the HIV-1 glycoprotein subunit 41 (gp41) C heptad repeat (mab2F5) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by phosphorimaging. Western blot for actin was performed as a loading control. Immunoprecipitated secreted peptides were digested with PNGase F to discriminate between monomeric C46 and the concatemeric precursor. (b) The signals were quantified (ImageQuant) and a graphic representation is shown for duplicate experiments; error bars show SEM. Values are given relative to the initial amount of labeled C46 precursor. (c) The amount of C46 monomers in the cell culture supernatant relative to the total amount of secreted Fur_oGA (in 293T cells, n = 9) and GAFur_oGA (in 293T cells, n = 16; and peripheral blood lymphocytes (PBL), n = 5) was calculated. Error bars indicate SEM. D, noncleaved dimeric concatemers; GAFur_oGA, Fur_o which contains two GA repeats flanking the cleavage site; M, cleaved monomeric peptides.

Figure 4

Monomeric secreted peptides show broad and specific neutralization. (a) In a single-round infection assay, PM1 cells were challenged with lentiviral vector particles packaged with vesicular stomatitis virus (VSV)-G as control or Envs from the HIV-1 strains JRFL, HxB2, or HxB2-SIM. The latter contains a mutation in the GIV motif that confers resistance to T-20. The vectors encode enhanced green fluorescent protein (eGFP). Infection was performed in the presence of supernatants from 293T cells expressing GAFur_oGA or different concentrations of synthetic T-20. The concentration of secreted antiviral entry inhibitory peptide in the supernatants was determined in a fluorescence-activated cell sorting (FACS)-based assay as described in Materials and Methods. eGFP-positive cells were measured by FACS analysis. 100% relative infection corresponds to ~20% and 10% eGFP-positive cells for HIV-1 Envs and VSV-G, respectively. Data are means from duplicates; error bars show SEM. (b) In a single-round-infection assay Jurkat T cells transduced to 3% with GAFur_oGA or mock-transduced cells were infected with replication incompetent eGFP-encoding lentiviral vectors packaged with HIV-1_HxB2 Env. eGFP-positive cells were determined by FACS. (c) Jurkat T cells transduced to 3% with GAFur_oGA or mock-transduced cells were infected with replicating CXCR4-tropic HIV-1_NL4–3 and p24 antigen was measured by enzyme-linked immunosorbent assay (ELISA). The dotted line shows the detection limit. n = 3; error bars show SEM. (d) Primary human CD4⁺ T cells transduced to 37% with GAFur_oGA or mock-transduced were infected with replicating CCR5-tropic HIV-1_JR-CSF and p24 antigen was measured by ELISA. The dotted line shows the detection limit. n = 3; error bars show SEM.