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Logo of jibtherBioMed CentralBiomed Central Web Sitesearchsubmit a manuscriptregisterthis articleJournal of Immune Based Therapies and VaccinesJournal Front Page
 
J Immune Based Ther Vaccines. 2010; 8: 7.
Published online 2010 November 19. doi:  10.1186/1476-8518-8-7
PMCID: PMC2996383

HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice

Abstract

Several approaches have been explored to eradicate HIV; however, a multigene vaccine appears to be the best option, given their proven potential to elicit broad, effective responses in animal models. The Pr55Gag protein is an excellent vaccine candidate in its own right, given that it can assemble into large, enveloped, virus-like particles (VLPs) which are highly immunogenic, and can moreover be used as a scaffold for the presentation of other large non-structural HIV antigens. In this study, we evaluated the potential of two novel chimaeric HIV-1 Pr55Gag-based VLP constructs - C-terminal fusions with reverse transcriptase and a Tat::Nef fusion protein, designated GagRT and GagTN respectively - to enhance a cellular response in mice when used as boost components in two types of heterologous prime-boost vaccine strategies. A vaccine regimen consisting of a DNA prime and chimaeric HIV-1 VLP boosts in mice induced strong, broad cellular immune responses at an optimum dose of 100 ng VLPs. The enhanced cellular responses induced by the DNA prime-VLP boost were two- to three-fold greater than two DNA vaccinations. Moreover, a mixture of GagRT and GagTN VLPs also boosted antigen-specific CD8+ and CD4+ T-cell responses, while VLP vaccinations only induced predominantly robust Gag CD4+ T-cell responses. The results demonstrate the promising potential of these chimaeric VLPs as vaccine candidates against HIV-1.

Findings

The importance of a cellular immune response against HIV-1 has been highlighted in several animal vaccine trials [1,2], with an abundance of evidence suggesting that an effective cellular immune response against HIV-1 is able to control and suppress viraemia during primary and chronic HIV infections, and to provide long-lasting protection [3-5]. Heterologous prime-boost vaccination has recently emerged as an effective means of enhancing T-cell responses [6-8], and current research suggests that HIV virus-like particles (VLPs) elicit a superior cellular immune response against HIV in animal models when used as a boost component in a prime-boost strategy [5,6]. In addition, previous studies have indicated the importance of including more than one HIV-1 proteins in a vaccine, due to the potential to induce broader and possibly more effective immune responses against HIV [9-11]. In this regard, Halsey et al. [12] showed that HIV-1 Pr55Gag-based chimaeric proteins with large C-terminal fusions both formed VLPs, and significantly enhanced T-cell responses elicited by a DNA vaccine to HIV-1 Gag and RT. The accessory proteins Tat, Nef and RT - which contain several prominent human cytotoxic T-lymphocyte (CTL) epitopes - are of particular interest in HIV vaccines: responses to Tat and Nef correlate with non-progression of HIV infections and possible protection [9], while RT-specific CTLs induce potent Th1 responses in mice, when administered in low doses [13].

This study investigates immune responses induced by chimaeric Gag VLPs incorporating RT and Tat-Nef sequences (GagRT and GagTN) as vaccine boost candidates to a DNA (pVRCgrttnC) priming vaccine expressing subtype C non-myristylated p6-deleted Gag, inactivated reverse transcriptase (RT), shuffled Tat (T) and inactivated Nef (N), as a polyprotein [14]. We further explored which combination of HIV-1 antigens in a VLP would best augment cellular immune responses induced by a complementary DNA vaccine, using DNA/VLP prime-boost vaccine regimens.

The pVRCgrttnC DNA vaccine (1 mg/ml in PBS, manufactured by Aldevron, Fargo, ND, USA) is based on the pTHgrttnC vaccine described previously [14], but has the pVRC backbone (provided by the Vaccine Research Centre of the National Institutes of Health, Bethesda, Maryland, USA) in place of the pTH vector [15]. GagRT VLPs were expressed from the HIV-1 subtype C Gag precursor Pr55Gag gene fused to the RT-encoding sequence from grttnC, and GagTN VLPs from a similar GagTatNef fusion [12]. Production of recombinant baculovirus-expressed GagRT and GagTN VLPs was optimized as described in Pillay et al. [16]. VLPs were purified from 2.5 L of Sf9 cell culture supernatants after 96 h incubation at 27°C. They were filtered through a 0.45 μm CFP-4-E-4MA polysulfone membrane capsule filter, and subsequently through a UFP-300-C-4MA polyethersulfone membrane (MWCO = 300 kDa (both Amersham)). Both filtration steps were necessary to remove cell debris and baculovirus contaminants from VLP samples. VLPs were pelleted by ultracentrifugation at 12 000 g for 60 min and re-suspended in PBS. Purity of the resulting VLPs was assessed using SDS-PAGE (Figure (Figure1a1a and and1b).1b). The presence of only the appropriate-sized protein bands indicated that no detectable contaminating material was present in the VLP samples. Endotoxin levels were < 0.125 endotoxin units per ml. Transmission electron microscopy (TEM) using a Zeiss S1109 electron microscope showed characteristic VLPs, albeit with a distribution of sizes [12]. Western blots probed with a 1:10 000 dilution of HIV-1 Gag p24 antibody (ARP432, NIBSC Centralised Facility for AIDS reagents, MRC, UK) and developed with goat anti-rabbit alkaline phosphatase conjugate (1:5 000; Sigma) and 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium phosphatase substrate (BCIP/NBT; KPL) were used to quantify the Gag content of the VLP samples. The intensity of the Gag band in the VLP samples was compared to that of p17/p24-C standards (ARP695.2. FIT Biotech) using densitometry (Figure (Figure1c1c and and1d1d).

Figure 1
Purity and quantification of Gag in VLP preparations. Coomassie-stained SDS-PAGE gels of (a) GagTN VLP samples and (b) GagRT VLP samples indicating the purity of the samples. Western blots of (c) GagTN VLP samples and (d) GagRT VLP samples probed with ...

Immunization of female BALB/c mice (8-10 weeks old, five per group; approved by the UCT Animal Research Ethics Committee, AEC No. 006-007) was by injection of 50 μl of the DNA dose (100 μg DNA/100 μl PBS) or 50 μl of the VLP dose (Gag protein dose in 100 μl PBS) into each hind leg muscle. Mice were primed with DNA on day 0 and boosted on day 28 with DNA or GagRT or GagTN or a mix of GagRT and GagTN. Initially, three doses of the chimaeric VLP boosts were evaluated to determine the optimal dose necessary for cellular immune response enhancement. These doses were 50 ng, 100 ng or 200 ng of Gag per 100 μl PBS, selected based on a previous DNA prime-VLP boost study [17]. Once determined, the optimal dose was used for the comparative mouse experiments. To test the immunogenicity of GagRT or GagTN alone, mice were left unprimed then vaccinated on day 28. Immune responses were detected on day 40.

Harvested spleens were pooled from a group of mice and isolated splenocytes were resuspended after erythrocyte lysis at 2 × 107 cells/ml R10 culture medium (RPMI with 10% heat inactivated FCS, Gibco, containing 15 mM β-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin). Splenocytes were cultured in triplicate reactions with peptides (4 μg/ml) restricted to BALB/c mouse epitopes in Gag (CD8 peptide AMQMLKDTI; CD4(13) peptide NPPIPVGRIYKRWIILGLNK and CD4(17) peptide FRDYVDRFFKTLRAEQATQE), and RT (CD8 peptide VYYDPSKDLIA and CD4 peptide PKVKQWPLTEVKIKALTAI) in IFN-γ and IL-2 ELISPOT assays in triplicate (BD™Biosciences) [18]. Responses after subtraction of background in the absence of peptides are reported as mean spot forming units (sfu) ± standard deviation (SD) of the mean/106 splenocytes.

Of the three doses assessed, the most prominent cellular immune responses were observed using a 100 ng VLP dose for the boost, with a two-fold differential in IFN-γ RT CD4 and CD8 responses for GagRT (Figure (Figure2a),2a), and a 10-fold increase in the IFN-γ Gag CD8 response for GagTN (Figure (Figure2b).2b). The overall stronger responses induced by the 100 ng dose indicated that this was the optimum dose for both GagRT and GagTN VLP boost vaccinations, and was used in subsequent experiments.

Figure 2
Dose assessment for chimaeric VLP boost. Three groups of five BALB/c mice were primed on day 0 with the DNA vaccine pVRCgrttnC and boosted with (a) GagRT or (b) GagTN on day 28. For all mouse groups spleens were harvested on day 40 and the splenocytes ...

After VLP-only vaccination with GagRT or GagTN, Gag- and RT- specific CD4+ cells contributed 84% to the cumulative IFN-γ ELISPOT response for GagRT, and 100% for GagTN. These VLPs also induced IL-2 Gag- and RT- specific responses (Figures (Figures33 and and4).4). These responses were considerably enhanced using the heterologous prime-boost regimen.

Figure 3
Immunogenicity of GagRT VLPs. BALB/c mice were primed on day 0 with the DNA vaccine pVRCgrttnC then boosted on day 28 with the DNA vaccine (DNA+DNA group) or GagRT VLPs (DNA+GagRT group). A further group was left unprimed then vaccinated on day 28 with ...
Figure 4
BALB/c mice were primed on day 0 with the DNA vaccine pVRCgrttnC then boosted on day 28 with DNA (DNA+DNA group) or GagTN VLPs (DNA+GagTN group). A further group was left unprimed then vaccinated on day 28 with GagTN VLPs. For all mouse groups spleens ...

The cumulative IFN-γ ELISPOT responses to a prime with DNA and boost with either GagRT or GagTN were greater by 2.4- and 16-fold respectively than two DNA vaccinations. The elicited responses after VLP boosts were predominantly from CD4+ cells (Figures (Figures33 and and4).4). DNA prime/GagRT boost induced Gag and RT-specific IL-2 producing CD8 and CD4 cells while DNA prime/GagTN boost induced IL-2-producing Gag CD4 cells only. These IL-2 responses were 3.4- and 4.6-fold above that for two DNA vaccinations (Figures (Figures33 and and44).

The DNA prime/mixed VLP boost vaccination increased the cumulative Gag- and RT-specific IFN-γ response relative to two DNA vaccinations 4-fold, and Gag- and RT-specific IL-2 response 2.8 fold. CD4+ cells contributed 41% to the cumulative IFN-γ and 31% to IL-2 ELISPOT responses, respectively (Figure (Figure55).

Figure 5
Use of a mixture of GagRT and GagTN VLPs as vaccine boosts. BALB/c mice were vaccinated with the DNA vaccine pVRCgrttnC on day 0 and day 28 (DNA+DNA group) or primed on day 0 with the DNA vaccine and boosted on day 28 with a mix of GagRT and GagTN (DNA+VLPmix ...

In conclusion, this study extends and confirms results of our previous work in clearly showing that enhanced cellular immune responses against Gag and RT result when the chimaeric VLPs GagRT and GagTN are used in mice in a DNA vaccine prime/VLP boost vaccination regimen [12]. Novel results presented here include the induction of predominantly specific CD4+ cells producing IFN-γ and IL-2 by GagRT and GagTN vaccination alone. The lack of response to these VLPs in our previous study is probably due to the VLP dose being 1/5 of that used in this study [12]. Surprisingly, this 5-fold increase in GagRT dose did not increase the magnitude of the reported DNA prime/GagRT boost response, suggesting that only a low VLP dose is required for induction of recall cellular responses by primary vaccine-induced memory cells.

In general, the magnitude of responses induced were better using a single VLP boost, than with the mixed VLP boost. This is possibly related to an antigen dose effect, where above the optimum dose, antigen-specific cellular immune responses decline [19]. The results of the boost dose assessment (Figure (Figure2)2) support this hypothesis, as the 200 ng VLP boost (which is equivalent to the mixed VLP dose administered) induced much lower responses than the 100 ng dose. That being said, the mixed VLP boost was able to elicit more proportionate, cumulative CD4+ and CD8+ specific T-cell responses. Expansion of specific CD4+ cells in response to vaccination is important in that these cells play a key role in activation of both B cells and CD8+ cells, and in controlling CTL responses [20], and are linked to the control of HIV-1 infection and replication [21,22]. However, it is the CD8+ cell response that is mainly responsible for controlling the viral infection and suppressing viraemia in HIV-infected individuals [23]. Hence, the CD4+ and CD8+ cell boost observed with vaccination of a VLP mix, and the finding that HIV-specific CD4+ and CD8+ cells produce IFN-γ and IL-2, are of strong significance. Collectively, the data shown here demonstrates that chimaeric VLP boosts such as these are a promising option for future HIV-1 vaccine studies.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SP produced and purified the chimaeric VLPs, participated in performing the ELISPOT assays, analysed the immunology data with ES, and drafted the manuscript. ES and her group were responsible for carrying out the mouse experiments and performing the ELISPOT assays. AM and EPR were involved in supervision of the work, critically revising the manuscript for important intellectual content, and together with A-LW, conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

Acknowledgements

This research was supported by The South African AIDS Vaccine Initiative (SAAVI). We thank Dr Carolyn Williamson for providing gag, RT, tat and nef from isolate Du422, Allison Lynch for technical expertise, Richard Halsey for the VLP constructs, and Shireen Galant, Desiree Bowers, Zaahier Isaacs and Anke Binder for their contributions to the immunology studies. The polyclonal antiserum ARP432 (donated by G. Reid) and HIV-1 protein standard were provided by the EU Programme EVA Centralised Facility for AIDS Reagents, NIBSC, UK (AVIP Contract Number LSHP-CT-2004-503487).

References

  • Buonaguro L, Tornesello ML, Tagliamonte M, Gallo RC, Wang LX, Kamin-Lewis R, Abdelwahab S, Lewis GK, Buonaguro FM. Baculovirus-derived human immunodeficiency virus type 1 virus-like particles activate dendritic cells and induce ex vivo T-cell responses. J Virol. 2006;80(18):9134–9143. doi: 10.1128/JVI.00050-06. [PMC free article] [PubMed] [Cross Ref]
  • Chege GK, Thomas R, Shephard EG, Meyers A, Bourn W, Williamson C, Maclean J, Gray CM, Rybicki EP, Williamson AL. A prime-boost immunisation regimen using recombinant BCG and Pr55(gag) virus-like particle vaccines based on HIV type 1 subtype C successfully elicits Gag-specific responses in baboons. Vaccine. 2009;27(35):4857–4866. doi: 10.1016/j.vaccine.2009.05.064. [PubMed] [Cross Ref]
  • Amara RR, Sharma S, Patel M, Smith JM, Chennareddi L, Herndon JG, Robinson HL. Studies on the cross-clade and cross-species conservation of HIV-1 Gag-specific CD8 and CD4 T cell responses elicited by a clade B DNA/MVA vaccine in macaques. Virology. 2005;334(1):124–133. doi: 10.1016/j.virol.2005.01.026. [PubMed] [Cross Ref]
  • Deml L, Speth C, Dierich MP, Wolf H, Wagner R. Recombinant HIV-1 Pr55gag virus-like particles: potent stimulators of innate and acquired immune responses. Mol Immunol. 2005;42(2):259–277. doi: 10.1016/j.molimm.2004.06.028. [PubMed] [Cross Ref]
  • Jaffray A, Shephard E, van Harmelen J, Williamson C, Williamson AL, Rybicki EP. Human immunodeficiency virus type 1 subtype C Gag virus-like particle boost substantially improves the immune response to a subtype C gag DNA vaccine in mice. J Gen Virol. 2004;85(Pt 2):409–413. doi: 10.1099/vir.0.19396-0. [PubMed] [Cross Ref]
  • Ye L, Wen Z, Dong K, Pan L, Bu Z, Compans RW, Zhang H, Yang C. Immunization with a Mixture of HIV Env DNA and VLP Vaccines Augments Induction of CD8 T Cell Responses. J Biomed Biotechnol. 2010. p. 497219. [PMC free article] [PubMed]
  • Zanotto C, Elli V, Basavecchia V, Brivio A, Paganini M, Pinna D, Vicenzi E, De Giuli Morghen C, Radaelli A. Evaluation in rabbits of different anti-SHIV vaccine strategies based on DNA/fowlpox priming and virus-like particle boosting. FEMS Immunol Med Microbiol. 2003;35(1):59–65. doi: 10.1111/j.1574-695X.2003.tb00649.x. [PubMed] [Cross Ref]
  • Woodland DL. Jump-starting the immune system: prime-boosting comes of age. Trends Immunol. 2004;25(2):98–104. doi: 10.1016/j.it.2003.11.009. [PubMed] [Cross Ref]
  • Scriba TJ, zur Megede J, Glashoff RH, Treurnicht FK, Barnett SW, van Rensburg EJ. Functionally-inactive and immunogenic Tat, Rev and Nef DNA vaccines derived from sub-Saharan subtype C human immunodeficiency virus type 1 consensus sequences. Vaccine. 2005;23(9):1158–1169. doi: 10.1016/j.vaccine.2004.08.026. [PubMed] [Cross Ref]
  • Nkolola JP, Wee EG, Im EJ, Jewell CP, Chen N, Xu XN, McMichael AJ, Hanke T. Engineering RENTA, a DNA prime-MVA boost HIV vaccine tailored for Eastern and Central Africa. Gene Ther. 2004;11(13):1068–1080. doi: 10.1038/sj.gt.3302241. [PubMed] [Cross Ref]
  • Ellenberger D, Wyatt L, Li B, Buge S, Lanier N, Rodriguez IV, Sariol CA, Martinez M, Monsour M, Vogt J. et al. Comparative immunogenicity in rhesus monkeys of multi-protein HIV-1 (CRF02_AG) DNA/MVA vaccines expressing mature and immature VLPs. Virology. 2005;340(1):21–32. doi: 10.1016/j.virol.2005.06.014. [PubMed] [Cross Ref]
  • Halsey RJ, Tanzer FL, Meyers A, Pillay S, Lynch A, Shephard E, Williamson AL, Rybicki EP. Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles. Virus Res. 2008;133(2):259–268. doi: 10.1016/j.virusres.2008.01.012. [PubMed] [Cross Ref]
  • Pacheco SE, Gibbs RA, Ansari-Lari A, Rogers P. Intranasal immunization with HIV reverse transcriptase: effect of dose in the induction of helper T cell type 1 and 2 immunity. AIDS Res Hum Retroviruses. 2000;16(18):2009–2017. doi: 10.1089/088922200750054747. [PubMed] [Cross Ref]
  • Burgers WA, van Harmelen JH, Shephard E, Adams C, Mgwebi T, Bourn W, Hanke T, Williamson AL, Williamson C. Design and preclinical evaluation of a multigene human immunodeficiency virus type 1 subtype C DNA vaccine for clinical trial. J Gen Virol. 2006;87(Pt 2):399–410. doi: 10.1099/vir.0.81379-0. [PubMed] [Cross Ref]
  • Barouch DH, Yang ZY, Kong WP, Korioth-Schmitz B, Sumida SM, Truitt DM, Kishko MG, Arthur JC, Miura A, Mascola JR. et al. A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates. J Virol. 2005;79(14):8828–8834. doi: 10.1128/JVI.79.14.8828-8834.2005. [PMC free article] [PubMed] [Cross Ref]
  • Pillay S, Meyers A, Williamson AL, Rybicki EP. Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system. Biotechnol Prog. 2009;25(4):1153–1160. doi: 10.1002/btpr.187. [PubMed] [Cross Ref]
  • Radaelli A, Bonduelle O, Beggio P, Mahe B, Pozzi E, Elli V, Paganini M, Zanotto C, De Giuli Morghen C, Combadiere B. Prime-boost immunization with DNA, recombinant fowlpox virus and VLP(SHIV) elicit both neutralizing antibodies and IFNgamma-producing T cells against the HIV-envelope protein in mice that control env-bearing tumour cells. Vaccine. 2007;25(11):2128–2138. doi: 10.1016/j.vaccine.2006.11.009. [PubMed] [Cross Ref]
  • Shephard E, Burgers WA, van Harmelen JH, Monroe JE, Greenhalgh T, Williamson C, Williamson AL. A Multigene HIV Type 1 Subtype C Modified Vaccinia Ankara (MVA) Vaccine Efficiently Boosts Immune Responses to a DNA Vaccine in Mice. AIDS Res Hum Retroviruses. 2008;24(2):207–17. doi: 10.1089/aid.2007.0206. [PubMed] [Cross Ref]
  • Janeway C, Travers P, Walport M, Shlomchik M. Immunobiology 5: the immune system in health and disease. 5. New York: Garland Pub; 2001. Immunology tool box: Appendix I.
  • Sadagopal S, Amara RR, Montefiori DC, Wyatt LS, Staprans SI, Kozyr NL, McClure HM, Moss B, Robinson HL. Signature for long-term vaccine-mediated control of a Simian and human immunodeficiency virus 89.6P challenge: stable low-breadth and low-frequency T-cell response capable of coproducing gamma interferon and interleukin-2. J Virol. 2005;79(6):3243–3253. doi: 10.1128/JVI.79.6.3243-3253.2005. [PMC free article] [PubMed] [Cross Ref]
  • Jansen CA, De Cuyper IM, Hooibrink B, van der Bij AK, van Baarle D, Miedema F. Prognostic value of HIV-1 Gag-specific CD4+ T-cell responses for progression to AIDS analyzed in a prospective cohort study. Blood. 2006;107(4):1427–1433. doi: 10.1182/blood-2005-07-2907. [PubMed] [Cross Ref]
  • Kalams SA, Buchbinder SP, Rosenberg ES, Billingsley JM, Colbert DS, Jones NG, Shea AK, Trocha AK, Walker BD. Association between virus-specific cytotoxic T-lymphocyte and helper responses in human immunodeficiency virus type 1 infection. J Virol. 1999;73(8):6715–6720. [PMC free article] [PubMed]
  • Paliard X, Liu Y, Wagner R, Wolf H, Baenziger J, Walker CM. Priming of strong, broad, and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T cells after administration of a virus-like particle vaccine in rhesus macaques. AIDS Res Hum Retroviruses. 2000;16(3):273–282. doi: 10.1089/088922200309368. [PubMed] [Cross Ref]

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