title: Derivation of non-infectious envelope proteins from virions isolated from plasma negative for HIV antibodies.
abstract: Natural membrane-bound HIV-1 envelope proteins (mHIVenv) could be used to produce an effective subunit vaccine against HIV infection, akin to effective vaccination against HBV infection using the hepatitis B surface antigen. The quaternary structure of mHIVenv is postulated to elicit broadly neutralizing antibodies protective against HIV-1 transmission. The founder virus transmitted to infected individuals during acute HIV-1 infection is genetically homogeneous and restricted to CCR5-tropic phenotype. Therefore, isolates of plasma-derived HIV-1 (PHIV) from infected blood donors while negative for antibodies to HIV proteins were selected for expansion in primary lymphocytes as an optimized cell substrate (OCS). Virions in the culture supernatants were purified by removing contaminating microvesicles using immunomagnetic beads coated with anti-CD45. Membrane cholesterol was extracted from purified virions with beta-cyclodextrin to permeabilize them and expel p24, RT and viral RNA, and permit protease-free Benzonase to hydrolyze the residual viral/host DNA/RNA without loss of gp120. The resultant mHIVenv, containing gp120 bound to native gp41 in immunoreactive form, was free from infectivity in vitro in co-cultures with OCS and in vivo after inoculating SCID-hu Thy/Liv mice. These data should help development of mHIVenv as a virally safe immunogen and enable preparation of polyclonal hyper-immune globulins for immunoprophylaxis against HIV-1 infection.
authors.affiliation: Department of Laboratory Medicine, University of California, School of Medicine, UCSF Box 0134, 185 Berry Street, Suite 2010-07, San Francisco, CA 94143, USA. girish.vyas@ucsf.edu
authors.forename: Girish N
authors.initials: GN
authors.lastname: Vyas
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Stoddart
authors.forename: M Scott
authors.initials: MS
authors.lastname: Killian
authors.forename: Todd V
authors.initials: TV
authors.lastname: Brennan
authors.forename: Tiffany
authors.initials: T
authors.lastname: Goldberg
authors.forename: Alyssa
authors.initials: A
authors.lastname: Ziman
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Bryson
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D015699:HIV Envelope Protein gp120 / Q000097:blood / Q000276:immunology* / Q000494:pharmacology; D015700:HIV Envelope Protein gp41 / Q000097:blood / Q000276:immunology* / Q000494:pharmacology; D015658:HIV Infections / Q000097:blood / Q000276:immunology*; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D014771:Virion / Q000276:immunology* / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; C514793:gp120 protein, Human immunodeficiency virus 1; C514787:gp41 protein, Human immunodeficiency virus 1