Fig. 1

Construction of nef-, vpu-, and glycoprotein signal peptide replaced HIV-1_NL4-3 (gmHIV-1_NL3-4) and characterization of the SAV001 vaccine virus: The fragment between BsmBI and BglII (from 104 to 263 nucleotides) of HIV-1 NL4-3 nef gene was deleted and the stop codon (TAG) was inserted (a). The coding region of HIV-1 env signal peptide (30 amino acids) was replaced by the coding sequence of honeybee melittin signal peptide (21 amino acids) [24] (c). The vpu gene was deleted as the result of the Env signal peptide gene replacement due to its overlapping reading frame in the upstream of env gene. The pNL4-3 M/dNef with env signal peptide replaced plasmid was transfected into A3.01 human T-lymphocytes and recovered the genetically modified HIV-1 NL4-3 (gmHIV-1_NL4-3) virus (b). The genetic modification was confirmed by RT-PCR using honeybee melittin-specific primer with modified nef gene-specific primer (d). Electron micrograph of SAV001 vaccine showing intact virion (e). Western blot analyses of SAV001 using HIV-1 p24 antiserum (The NIH AIDS Reagent Program) and goat anti-HIV-1 gp120 polyclonal antibody (BIODESIGN), respectively (f). Aggregation of CD4⁺ AA2 [g, h (arrow)], and A3.01 [i, j (arrow)], as a result of gp120 on the SAV001 vaccine virus binding to the cell surface receptors. Induction of anti-gp120 antibody in SAV001 vaccine immunized rat sera using the gp140 trimer by ELISA (k, l)