title: Abrogation of contaminating RNA activity in HIV-1 Gag VLPs.
abstract: BACKGROUND
  HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine.
  
  
  METHODS
  HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen.
  
  
  RESULTS
  HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold.
  
  
  CONCLUSIONS
  Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice.
authors.affiliation: Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town, University Ave, Rondebosch 7701, South Africa.
authors.forename: Ziyaad
authors.initials: Z
authors.lastname: Valley-Omar
authors.forename: Ann E
authors.initials: AE
authors.lastname: Meyers
authors.forename: Enid G
authors.initials: EG
authors.lastname: Shephard
authors.forename: Anna-Lise
authors.initials: AL
authors.lastname: Williamson
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
mesh_terms: D016915:AIDS Vaccines / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D017930:Genes, Reporter; D057809:HEK293 Cells; D015658:HIV Infections / Q000517:prevention & control* / Q000821:virology; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D058425:Vaccines, Virus-Like Particle / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics*; D014771:Virion / Q000235:genetics / Q000276:immunology
chemical_list: D016915:AIDS Vaccines; D015683:Gene Products, gag; D019444:Vaccines, DNA; D058425:Vaccines, Virus-Like Particle; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase