1 Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden
2 Department of Vaccine Research, Swedish Institute for Infectious Disease Control, S-171 82 Solna, Sweden
3 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford, UK
Correspondence
Gunilla B. Karlsson
Nilla.Karlsson{at}mtc.ki.se
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ABSTRACT |
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These authors contributed equally.
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MAIN TEXT |
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An alternative strategy to increase the immunogenicity of DNA-based vaccines is to use plasmid-encoded viral replicon systems. We previously described a DNA system in which self-replicating Semliki Forest virus (SFV) vectors (replicons) were used for immunization (Berglund et al., 1998). Replicon systems have also been developed for Sindbis virus (Dubensky et al., 1996
; Hariharan et al., 1998
; Kirman et al., 2003
) and for Kunjin virus (Anraku et al., 2002
), and collectively these studies demonstrate that replicon DNA vectors provide significant dose-sparing advantages compared with conventional DNA vaccines. The increased immunogenicity of replicon DNA vaccines may result from an ability of the replicating RNA to stimulate innate antiviral signals in the transfected target cell, providing adjuvant effects that conventional DNA vaccines are unable to stimulate (Leitner et al., 2000
, 2003
). Recombinant SFV particle vaccines have been extensively evaluated in pre-clinical immunogenicity studies and protective immune responses have been demonstrated in several virus challenge models (Berglund et al., 1999
; Fleeton et al., 2001
; Morris-Downes et al., 2001b
; Mossman et al., 1996
). Antigen expression from SFV particles and from SFV replicon DNA is transient and rapid clearance of the target cells from tissues after immunization has been demonstrated (Morris-Downes et al., 2001a
). This aspect adds an important safety feature to replicon DNA vaccines, as cell death reduces the risk of chromosomal integration of the foreign DNA and prolonged antigen expression resulting in tolerance is not expected to occur. In SFV replicon DNA vectors, only the viral non-structural proteins (nsp14) and the foreign antigen of interest are encoded. The absence of the genes encoding the viral structural proteins completely excludes the risk that replication-proficient virus would be generated and it limits potential vector-directed immune responses.
In this communication, we describe an SFV replicon DNA vaccine, DREP.HIVA designed for clinical evaluation as a preventative HIV-1 vaccine. HIVA is a well-described antigen currently under evaluation in the clinic in the context of a conventional DNA plasmid, pTHr.HIVA (Hanke & McMichael, 2000; Mwau et al., 2004
). The HIVA gene encodes a scrambled HIV-1 clade A Gag protein fused to a string of HIV-1 class I epitopes recognized by human, murine and rhesus macaque cytotoxic T-lymphocytes (Hanke et al., 1998
). To generate DREP.HIVA, we made a series of modifications to the previously published pBK-SFV-E vector (Berglund et al., 1998
) to improve its immunopotency and to meet the demands of regulatory agencies and large-scale Good Manufacturing Practice (GMP) production. The modifications included the insertion of an 84 bp self-cleaving ribozyme (RZ) sequence from the hepatitis D virus (HDV) (Been et al., 1992
; Perrotta & Been, 1991
) downstream of the virus-encoded 3' sequences to obtain enhanced vector function and the removal of redundant vector sequences to obtain a vaccine product that did not carry unnecessary sequence information. Representative expression and immunogenicity results from the development of DREP.HIVA are presented in this report.
Previous studies using Sindbis virus-based replicon DNA vectors in transient transfection experiments have shown that when the RZ element is placed immediately downstream of the viral poly(A) sequence, the expression of a reporter gene is enhanced (Dubensky et al., 1996; Yamanaka & Xanthopoulos, 2004
). The HDV RZ cleaves at its own 5' end, and thus its presence downstream of the viral poly(A) sequence facilitates the generation of viral RNA molecules with authentic SFV 3' termini. This increases the chance of successful RNA replication initiation in transfected cells, yielding higher expression levels in the cell population as a whole. To investigate whether the RZ element enhanced the immunogenic profile of the SFV replicon DNA vector, we inserted the HDV RZ into pBK-SFV-E-LacZ to yield pBK-SFV-E-LacZ-RZ (Fig. 1
a) and we immunized 68-week-old BALB/c female mice intramuscularly (i.m.) with 0·1 or 1 µg of the replicon DNA plasmids in 100 µl (0·9 % saline) divided between both anterior tibialis muscles. Antibody titres in sera were measured at 10 days post-immunization using a standard ELISA and reciprocal end-point titres were calculated as the final positive dilution above the background mean+2SD (Fig. 1b
). In animals immunized with 1 µg pBK-SFV-E-LacZ and pBK-SFV-E-LacZ-RZ, antibody responses were observed in all mice in both groups, while in animals immunized with 0·1 µg replicon DNA, a greater number of animals responded in the pBK-SFV-E-LacZ-RZ-immunized group (6/7 animals) compared with the pBK-SFV-E-LacZ-immunized group (3/7 animals). Similarly enhanced immunogenicity of pBK-SFV-E-LacZ-RZ over pBK-SFV-E-LacZ was observed in mice that had been immunized twice, with 1 month between each immunization (data not shown). In this experiment, the positive contribution of the RZ was only apparent in mice immunized with low amounts of DNA. This may be because the uptake of plasmid DNA across cellular membranes is less efficient at lower doses of DNA and optimal function of the vector may consequently be more important at these doses.
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To determine whether DREP.HIVA exhibited enhanced immunogenicity over the conventional DNA vaccine expressing HIVA, we performed a set of experiments using DREP.HIVA and pTH.HIVA vaccine preparations generated in the laboratory using Qiagen endotoxin-free kits. These experiments showed that DREP.HIVA stimulated detectable antigen-specific IFN- ELISPOT responses in mice after a single immunization using 10 µg DNA, while pTH.HIVA did not stimulate detectable responses using the same immunization and assay conditions (not shown). Since the HIVA plasmid used in the human clinical trials, pTHr.HIVA, is not identical to the pTH.HIVA plasmid used in these initial experiments, we next performed a set of immunogenicity experiments using clinical batches of DREP.HIVA and pTHr.HIVA. The clinical vaccine batches were produced by Cobra Biomanufacturing and provided for the current study by the International AIDS Vaccine Initiative. The pTHr.HIVA plasmid was propagated under antibiotic-free conditions (Cranenburgh et al., 2001
; Williams et al., 1998
), while pTH.HIVA encoded an antibiotic-resistance gene (Fig. 2a
) and could therefore be generated by using standard selection procedures. The clinical batch of DREP.HIVA retained the kanamycin-resistance gene as shown in Fig. 2(a)
. The availability of GMP-produced DNA batches allowed a direct comparison of the immunogenicity of two highly quality-controlled vaccine preparations. Previous experiments have shown that 50 or 100 µg pTHr.HIVA elicits detectable T-cell responses in mice (Hanke et al., 1998
, 2003
). We therefore performed a single i.m. immunization using only 10 µg DREP.HIVA or pTHr.HIVA DNA to obtain a highly sensitive measure of T-cell responses. Briefly, spleens were collected 12 days after the immunization and splenocytes were adjusted to 2x105 cells per well and added to pre-coated (anti-IFN-
; MabTech) and blocked ELISPOT plates (Millipore). Cells were stimulated with 2 µg concanavalin A ml1 (ConA; Sigma), with 2 µg HIVA-specific peptide (RGPGRAFVTI; Ana Spec Inc.) ml1 (Takahashi et al., 1988
) or were left unstimulated (medium only). After 20 h incubation in a 5 % CO2 incubator at 37 °C, a biotinylated anti-IFN-
antibody (MabTech) was added and the plates were incubated for 1 h at room temperature. After washing and incubating with an avidinperoxidase complex (ABC kit; Vector Laboratories) for another hour, the spots were developed with aminoethyl carbazole substrate and the enzymic reaction was stopped after 4 min by washing the plates in water. Spots were counted using an ELISPOT reader (Axioplan 2 Imaging; Zeiss) and expressed as spot-forming cells (SFC) per 106 cells. All mice, eight immunized with DREP.HIVA and 10 immunized with pTHr.HIVA, showed ConA responses well above 500 SFC per 106 cells confirming the viability of the splenocyte cultures (not shown). Using a criterion in the IFN-
ELIPOT assay whereby 50 SFC per 106 cells was considered a positive response, this experiment showed HIVA-specific responses in six out of eight mice immunized with DREP.HIVA, while no responders were identified amongst the pTHr.HIVA-immunized mice (Fig. 3
). The group of naïve mice included in the experiment were negative for the HIVA peptide-specific stimulation (not shown). These data confirmed the enhanced immunogenicity of replicon DNA vaccines over conventional plasmid DNA previously observed for laboratory-grade plasmids.
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The results presented here support the evaluation of DREP.HIVA in non-human primate studies and in phase I clinical trials and should stimulate further investigations into the immunogenic mechanisms of replicon vaccine systems.
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ACKNOWLEDGEMENTS |
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Received 29 July 2004;
accepted 5 November 2004.
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