HIV-1 sub-type C chimaeric VLPs boost cellular immune responses in mice
© Pillay et al; licensee BioMed Central Ltd. 2010
Received: 16 July 2010
Accepted: 19 November 2010
Published: 19 November 2010
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.
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.  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 , while RT-specific CTLs induce potent Th1 responses in mice, when administered in low doses .
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 . 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.
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 . 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) . 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.
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 3 and 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 3 and 4).
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 . 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 . 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 . The results of the boost dose assessment (Figure 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 , 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 . 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.
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).
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