OBJECTIVES: We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy. METHODS: Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay. RESULTS: At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity. CONCLUSIONS: The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.
OBJECTIVES: We previously constructed a trimeric coiled coil, N28Fd, based on the N-heptad repeat (NHR) sequence of HIV-1 gp41, as a promising HIV-1 entry inhibitor. Here, we attempted to engineer a stabilized trimeric coiled coil, ccN28Fd, by adding interchain disulphide bonds at the N terminus of N28Fd to improve its biophysical properties and anti-HIV-1 efficacy. METHODS: Molecular biology techniques were applied to engineer ccN28Fd. Circular dichroism and sedimentation velocity analysis were used to determine its secondary structure and thermostability and polymeric states, respectively. The anti-HIV-1 activity was assessed by p24 or luciferase expression. Its cytotoxicity was evaluated by XTT assay. RESULTS: At low pH, ccN28Fd and N28Fd were in trimeric and monomeric conformation, respectively. ccN28Fd showed higher thermostability and much stronger antiviral activity against HIV-1 IIIB (X4) and Bal (R5) strains than N28Fd. ccN28Fd was effective in inhibiting infection by a broad spectrum of primary HIV-1 isolates and enfuvirtide-resistant strains and blocking cell-to-cell HIV-1 transmission. A combination of ccN28Fd with tenofovir, a nucleoside reverse transcriptase inhibitor-based microbicide, exhibited potent synergistic anti-HIV-1 activity. ccN28Fd was highly resistant to digestion by proteinase K at pH 7.2 and pepsin at pH 1.5, and its anti-HIV-1 activity was not significantly affected by the presence of hydroxyethyl cellulose gel, seminal fluid or vaginal fluid simulant. It possessed no significant in vitro cytotoxicity. CONCLUSIONS: The engineered ccN28Fd maintains high stability in a low pH environment and exhibits potent and broad anti-HIV-1 activity, suggesting good potential for development as an effective and safe vaginal microbicide to prevent HIV sexual transmission.
Authors: Sara Crespillo; Ana Cámara-Artigas; Salvador Casares; Bertrand Morel; Eva S Cobos; Pedro L Mateo; Nicolas Mouz; Christophe E Martin; Marie G Roger; Raphaelle El Habib; Bin Su; Christiane Moog; Francisco Conejero-Lara Journal: Proc Natl Acad Sci U S A Date: 2014-12-08 Impact factor: 11.205
Authors: Yue Tan; Pei Tong; Junyi Wang; Lei Zhao; Jing Li; Yang Yu; Ying-Hua Chen; Ji Wang Journal: Front Immunol Date: 2017-04-24 Impact factor: 7.561