hiv中和抗体 pg9 pg16.ppt

Broad and Potent Neutralizing Antibodies from an African Donor Reveal a New HIV-1 Vaccine Target Author: Laura M. Walker Sanjay K. Phogat Dennis R. Burton Why should we find the broadly neutralizing antibodies (bNAbs)? The recent failiure of the leading T cell based HIV vaccine To develop the immunogen The patency of the bNAbs of the protection against HIV in the animal research The limintation Limited breath and protency against non-clade B virus The epitope recognized by the antibodies failed to elicit broadly neutralizing responses Protocol G PG: Protocol G, a research for broad neuturalizing antibodies of HIV Patients male or female at least 18 years of age documented HIV infection for a least three years clinically asymptomatic at the time of enrollment not currently receiving antiretroviral therapy The volunteer was identified as a broad neutralizer based on broad and potent neutralizing activity against a cross-clade pseudovirus panel Isolation of mAbs Enriching IgG-expressing memory B cells by magnetic beads gp120 and gp41 indirectly and directly ELISA micro-neutralization assay Heavy and light variable regions isolation by RT-PCR amplification VH or VL-region clones were cloned into an expression vector and transfected 293-T cells Identification: recombinant antibodies were screened by ELISA and neutralization assays Neutralization assays Neutralization activity of PG9 and PG16 against HIV-1 was measured using a TZM-BL system. Cell surface binding assays. Antibodies were added to HIV-1 Env transfected 293T cells stained with goat anti-human IgG F(ab’)2 Binding was analyzed using flow cytometry Competition assays competitor antibodies were added to the cells prior to adding of biotinylated PG9 or PG16 Antibodies were added to HIV-1 Env transfected 293T cells stained with goat anti-human IgG F(ab’)2 Binding was analyzed using flow cytometry sCD4 inhibition assays sCD4 was added to the cells p