1 Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town, Rondebosch 7701, South Africa
2 MRC Liver Research Centre, Department of Medicine, University of Cape Town, Observatory 7925, South Africa
3 Division of Virology, University of Cape Town, Observatory 7925, South Africa
4 Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Observatory 7925, South Africa
5 National Health Laboratory Service, University of Cape Town, Observatory 7925, South Africa
Correspondence
Edward P. Rybicki
ed{at}science.uct.ac.za
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ABSTRACT |
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MAIN TEXT |
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For some years there has been great interest in using HIV-1 Gag as a candidate prophylactic vaccine. The HIV-1 Gag polyprotein (Pr55gag) is the major structural protein which forms a submembrane shell in immature virus particles (Freed, 1998). Although in natural infections it associates with the env-encoded transmembrane glycoprotein (gp160) and viral RNA to mature into infectious virions, Pr55gag expressed in isolation in a number of recombinant systems may also associate into non-infectious virus-like particles (VLPs) which are morphologically similar to immature HIV virions (Gheysen et al., 1989
; Jowett et al., 1992
; Mergener et al., 1992
; Nermut et al., 1994
, 2003
; Overton et al., 1989
; Royer et al., 1991
, 1992
; Shioda & Shibuta, 1990
; Vernon et al., 1991
; Wills & Craven, 1991
).
Gag is relatively well conserved across diverse HIV-1 subtypes (van Baalen et al., 1996; Gaschen et al., 2002
), and a potent cellular response is elicited to this protein in humans following infection with HIV-1 (Novitsky et al., 2002
; Addo et al., 2003
). HIV-1 subtype B Gag VLPs or pseudoparticles also stimulate strong CTL responses in mice and other animals: this is a reason for selecting Gag as a vaccine candidate for humans (Huang et al., 2001
; Shiver et al., 2002
; Vajdy et al., 2001
; Wee et al., 2002
).
It appears that to achieve a broad cellular and humoral response to a vaccine that is also prolonged and protective, a combination of different approaches will be required (Bojak et al., 2002; Nabel, 2002
). Several HIV vaccine studies have shown that a heterologous prime/boost vaccination strategy can elicit the necessary immune response for protection against disease (Amara et al., 2001
; Haglund et al., 2002
). Robinson et al. (1999)
have shown that a gagpol DNA prime immunization followed by a recombinant fowlpox virus protein booster immunization afforded protection against a simian immunodeficiency virus (SIV) challenge in rhesus macaques. Moreover, it appeared that this immunity was cell-mediated. Barouch et al. (2000)
showed that rhesus macaques immunized with an HIV-1 gag DNA vaccine and boosted with IL-2/Ig protein (a fusion protein having IL-2 functional activity) augmented the DNA vaccine-elicited cell-mediated immune response significantly.
Our group has shown in previous studies that an HIV-1 subtype C DNA gag vaccine induces a significant CTL response in mice (van Harmelen et al., 2003). In this study, we have investigated first whether the Pr55gag polyprotein of a typical southern African HIV-1 subtype C would form VLPs when expressed via recombinant baculovirus, and second, whether immunization with these Gag VLPs enhances the cellular immune response to our gag subtype C DNA vaccine candidate in mice.
The HIV-1 subtype C gag gene from the South African HIV isolate DU422 (Williamson et al., 2003) was cloned into the multiple cloning site pFastBac1, and transposed into competent E. coli DH10Bac cells which were then screened for successful transposition into the baculovirus shuttle vector (bacmid). Gag VLPs were produced in Spodoptera frugiperda (Sf21) cells via recombinant baculovirus expressing the full-length myristylated Pr55gag precursor protein, according to the manufacturer's protocols (Gibco Life Sciences). The cells were incubated in TC100 medium (Gibco Life Sciences) supplemented with fetal calf serum at 28 °C for 84 h. Transfected Sf21 cells were separated from VLPs which had budded into the culture medium by centrifugation at 3000 g. Putative Pr55gag VLPs were purified from the culture fluid on sucrose gradients as described by Nermut et al. (1994)
. Purified VLPs were dialysed for 16 h in 1x PBS at 4 °C, and Gag content and integrity were evaluated by Western blotting using antiserum to HIV-1 p17 (ARP431, NIBSC) diluted 1 in 1000 in 1x PBS (pH 7·4) after SDS-PAGE on 10 % gels.
The process of VLP production by Sf21 cells was visualized by transmission electron microscopy (TEM). Recombinant virus-infected cells were prepared for ultrathin sectioning by fixing cells sequentially in 2·5 % glutaraldehyde and 1 % osmium tetroxide in 1x PBS (pH 7·4). Fixed cells were washed in 1x PBS and water, and then dehydrated in graded ethanol solutions and 100 % acetone, after which they were embedded in Spurr's resin and sectioned. Sections were stained with both 2 % uranyl acetate and Reynolds' lead citrate and viewed using a Zeiss S1109 electron microscope at magnifications of 12 000x to 100 000x using an accelerating voltage of 80 kV.
Gag VLPs harvested from the extracellular medium were prepared for TEM by adsorption onto carbon-coated copper grids and staining with 2 % uranyl acetate or 2 % methylamine tungstate.
The preparation of the HIV-1 subtype C gag DNA candidate vaccine pTHgagC has been reported previously (van Harmelen et al., 2003). Briefly, the gag gene from the subtype C isolate DU422 (European Collection of Cell Cultures provisional accession no. 01032114) was resynthesized for human codon usage, and inserted into the pTH DNA vaccine vector (T. Hanke, Oxford, UK) to result in the vaccine construct pTHgagC. The DNA vaccine was manufactured for animal immunizations by Aldevron (Fargo, USA) and resuspended at 1 mg ml-1 in sterile PBS (Sigma).
To test the immune response in mice to the gag DNA prime/Gag VLP boost vaccination strategy, BALB/c mice (five per group) were inoculated and subsequently boosted 4 weeks later, as indicated in Table 1. DNA inoculations were intramuscular with 100 µg of plasmid, and VLP inoculations were intraperitoneal with 2 ng of protein. This strategy allowed the following prime/boost combinations: gag prime/empty vector boost, gag prime/gag boost, gag prime/VLP boost and vector prime/VLP boost. Ten days after the boost inoculation, mice were killed and splenocytes isolated from harvested spleens. Effector cells were generated by stimulating splenocytes in a bulk culture for 5 days with the Gag-specific MHC class I-restricted peptide AMQMLKDTI, followed by a restimulation with or without Gag peptide for 4 h. Functional cytotoxicity of these effector cells was then detected in a standard 4 h 51Cr release assay using P815 cells as antigen presenting cells. In addition, Gag peptide-specific IFN-
+/CD8+ cells were detected by flow cytometry after the 4 h restimulation of these effector cells with or without the Gag peptide and P815 cells as antigen presenting cells at a 1 : 1 ratio in the presence of 2 µg Brefeldin A. A 4 h restimulation with an irrelevant peptide was included as an additional control. Cells were stained with anti-CD8 (PharMingen), and then permeabilized using the cytofix/cytoperm kit before staining with anti-IFN-
according to the manufacturer's instructions (PharMingen). Labelled cells were acquired on a FACSCalibur flow cytometer (500 000 gated events acquired per sample) and analysed using Cellquest software (Becton Dickinson).
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ACKNOWLEDGEMENTS |
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Received 4 June 2003;
accepted 7 November 2003.