Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

Simple item page
dc.contributor.authorValley-Omar, Ziyaaden_ZA
dc.contributor.authorMeyers, Annen_ZA
dc.contributor.authorShephard, Eniden_ZA
dc.contributor.authorWilliamson, Anna-Liseen_ZA
dc.contributor.authorRybicki, Edwarden_ZA
dc.date.accessioned2015-11-18T03:59:24Z
dc.date.available2015-11-18T03:59:24Z
dc.date.issued2011en_ZA
dc.description.abstractBACKGROUND: 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/mug 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.en_ZA
dc.identifier.apacitationValley-Omar, Z., Meyers, A., Shephard, E., Williamson, A., & Rybicki, E. (2011). Abrogation of contaminating RNA activity in HIV-1 Gag VLPs. <i>Virology Journal</i>, http://hdl.handle.net/11427/15090en_ZA
dc.identifier.chicagocitationValley-Omar, Ziyaad, Ann Meyers, Enid Shephard, Anna-Lise Williamson, and Edward Rybicki "Abrogation of contaminating RNA activity in HIV-1 Gag VLPs." <i>Virology Journal</i> (2011) http://hdl.handle.net/11427/15090en_ZA
dc.identifier.citationValley-Omar, Z., Meyers, A. E., Shephard, E. G., Williamson, A. L., & Rybicki, E. P. (2011). Abrogation of contaminating RNA activity in HIV-1 Gag VLPs. Virol J, 8, 462.en_ZA
dc.identifier.ris TY - Journal Article AU - Valley-Omar, Ziyaad AU - Meyers, Ann AU - Shephard, Enid AU - Williamson, Anna-Lise AU - Rybicki, Edward AB - 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/mug 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. DA - 2011 DB - OpenUCT DO - 10.1186/1743-422X-8-462 DP - University of Cape Town J1 - Virology Journal LK - https://open.uct.ac.za PB - University of Cape Town PY - 2011 T1 - Abrogation of contaminating RNA activity in HIV-1 Gag VLPs TI - Abrogation of contaminating RNA activity in HIV-1 Gag VLPs UR - http://hdl.handle.net/11427/15090 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/15090
dc.identifier.urihttp://dx.doi.org/10.1186/1743-422X-8-462
dc.identifier.vancouvercitationValley-Omar Z, Meyers A, Shephard E, Williamson A, Rybicki E. Abrogation of contaminating RNA activity in HIV-1 Gag VLPs. Virology Journal. 2011; http://hdl.handle.net/11427/15090.en_ZA
dc.language.isoengen_ZA
dc.publisherBioMed Central Ltden_ZA
dc.publisher.departmentDepartment of Molecular and Cell Biologyen_ZA
dc.publisher.facultyFaculty of Scienceen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an Open Access article distributed under the terms of the Creative Commons Attribution Licenseen_ZA
dc.rights.holder2011 Valley-Omar et al; licensee BioMed Central Ltd.en_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/2.0en_ZA
dc.sourceVirology Journalen_ZA
dc.source.urihttp://virologyj.biomedcentral.com/en_ZA
dc.subject.otherHIV-1 Gag virusen_ZA
dc.subject.otherchloramphenicol acetyl transferase (CAT) RNAen_ZA
dc.subject.otherCD4 T cellen_ZA
dc.titleAbrogation of contaminating RNA activity in HIV-1 Gag VLPsen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Valley_Omar_Abrogation_contaminating_RNA_2011.pdf
Size:
610 KB
Format:
Adobe Portable Document Format
Description:
Download
Collections
Journal Articles