1 Laboratory of Viral Pathogenesis, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
2 Laboratory of Viral Control, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
3 Laboratory Animal Research Center, Toyama Medical and Pharmaceutical University, Toyama 930-0152, Japan
4 College of Medical Technology, Kyoto University, Kyoto 606-8507, Japan
5 Department of Microbiology and Immunology, Nippon Medical School, Tokyo 113-8602, Japan
Correspondence
Eiji Ido
eido{at}virus.kyoto-u.ac.jp
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ABSTRACT |
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Published ahead of print on 27 May 2003 as DOI 10.1099/vir.0.19082-0
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INTRODUCTION |
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Another candidate for an AIDS vaccine is DNA, which can elicit both humoral and cell-mediated immunity. This method consists of injection of DNA plasmids that encode antigenic proteins under a strong enhancer/promoter, such as the human cytomegalovirus immediate/early promoter. DNA vaccines are considered to be much safer than live attenuated vaccines and initial results have been promising. Recent reports employed DNA vaccination in combination with boosting of antigenic proteins (Boyer et al., 1997; Letvin et al., 1997
), live virus vectors (Robinson et al., 1999
) or cytokine augmentation (Barouch et al., 2000
). None of the vaccination regimes using DNA alone have achieved complete protection, which is in contrast to the rather satisfactory results obtained with live attenuated vaccines.
Previously, we reported a DNA vaccination regime using a full-genome plasmid producing non-infectious whole particles of human immunodeficiency virus type 1 strain NL432 (HIV-1NL432), transcribed under its original promoter, HIV-1 LTR (Akahata et al., 2000). The plasmid has mutations (Cys...Cys...His...Cys
Ser...Ser...His...Cys) in an N-terminal zinc-finger motif of the nucleocapsid protein (NC), located in the gag region of HIV-1. Zinc-finger motifs are believed to play an important role in packaging the viral genomic RNA. The mutant plasmid expresses whole virus component proteins to produce virus particles that do not possess genomic RNA (Mizuno et al., 1996
). All monkeys that had been injected intramuscularly with the plasmid 14 times showed immunological responses against HIV-1: stable anti-HIV-1 Env antibodies were raised in two monkeys and CTL activity against HIV-1 was induced in the other monkeys. All monkeys showed partial protection against challenge with 100 TCID50 of a simian immunodeficiency virus/HIV chimeric virus (SHIV), termed SHIV-NM-3rN (Kuwata et al., 1995
). Namely, peak plasma virus loads of the challenge virus were two to three orders of magnitude lower than they were in naive controls. However, we repeated the DNA injections a total of 14 times because of the inefficient expression of the proteins encoded by the plasmid.
To improve the DNA vaccination regime described previously, we vaccinated animals with a novel SHIV plasmid, termed pSHIV-NM-3rn ZF1*. The mutant plasmid pSHIV-NM-3rn ZF1* produces non-infectious SHIV whole virus particles similar to those produced by pNL432 ZF1* due to the mutation in the NC proteins. We expected that the expression of pSHIV-NM-3rn ZF1*, which is driven by the SIV LTR promoter, might be better in monkeys than the expression of pNL432 ZF1*, which is driven by the HIV-1 LTR promoter.
An advantage of a full-genome plasmid is that it is capable of expressing all of the viral antigenic components, including the Gag, Pol and Env proteins. Recently, some reports suggested that a single viral epitope-specific CTL response may not be sufficient to block infection with pathogenic SIV (Hanke et al., 1999; Yasutomi et al., 1995
). Our DNA vaccine included all viral epitopes. In addition, this regime more closely resembles the use of a live attenuated vaccine than the foregoing DNA vaccination regimes. Live attenuated vaccines replicate in infected cells and viral antigenic components are presented by way of MHC molecules on the infected cell surface, thus eliciting a strong immunity against the virus in the host. Viral genes contained in the full-genome plasmid are expected to be expressed in a similar manner, leading to the assembly of virus components and budding of virus particles in the cells that took up the plasmid. This series of events is believed to be effective for acquisition of cellular and humoral immunity in the host.
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METHODS |
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Cell culture and transfections.
COS-1 cells were cultured in DMEM containing 10 % heat-inactivated FBS (Gibco-BRL) and 20 mM L-glutamine. M8166 cells (a human CD4+ lymphoid cell line) were cultured in RPMI 1640 medium containing 10 % FBS and 20 mM L-glutamine. Cells were transfected using the FuGENE-6 Transfection Reagent kit (Roche) with 20 µg of the viral plasmid DNAs, following the manufacturer's recommendations.
Reverse transcriptase (RT) assay and ELISA.
The RT assay was performed as described (Mizuno et al., 1996). The amount of p27CA in the culture supernatant was measured using the SIV Core Antigen ELISA kit (Coulter).
Quantitative RT-PCR.
To measure the genomic RNA in virus particles, we performed quantitative RT-PCR as follows. The transfected culture supernatant was filtered through a 0·45 µm pore-size filter and viral RNA was extracted using the QIAamp Viral RNA Mini kit (Qiagen). To remove contaminating plasmid DNA, the precipitated RNA was digested with DNase I (Gibco-BRL) for 15 min at 37 °C, followed by heat treatment (15 min at 70 °C) to inactivate the DNase I. RT reactions and subsequent PCR conditions were performed using the Taqman RT-PCR kit (Perkin Elmer), according to the manufacture's recommendations. SIVII-696F (5'-GGAAATTACCCAGTACAACAAATAGG-3') and SIVII-784R (5'-TCTATCAATTTTACCCAGGACTTTA-3') were used as primers, and a labelling probe, SIVII-731 (5'-Fam-TGTCCACCTGCCATTAAGCCCG-Tamra-3') (Perkin Elmer), was also used.
Immunoblot analysis.
Immunoblot analysis was performed as described (Mizuno et al., 1996). In brief, for an immunoblot analysis of transfected cells, the cells were washed three times and, at 3 days after transfection, were lysed in lysis buffer and subjected to analysis. Plasma from a SHIV-NM-3rN-infected monkey (Kuwata et al., 1995
) was used to provide the first antibody. To analyse for virus particles, the transfected culture supernatant was filtered through a 0·45 µm pore-size filter. Each sample was adjusted to contain an equal amount of RT activity in the supernatant. The virus particles released were pelleted by centrifugation at 14 000 r.p.m. for 2 h at 4 °C. The pelleted virions were then lysed in lysis buffer and subjected to immunoblot analysis.
Electron microscopy.
The morphology of the virus particles produced by full-genome plasmids was examined under electron microscopy. Transfected COS-1 cells were prefixed for 2 h in 2 % glutaraldehyde in PBS and washed thoroughly with PBS. The cells were then fixed with 1 % osmium tetroxide in PBS, dehydrated and embedded in epoxy resin. Ultrathin sections of the cells were stained with uranyl acetate and lead citrate and then observed under a Hitachi H-7100 electron microscope, as described (Mizuno et al., 1996).
Vaccination protocol.
Plasmids were amplified in Escherichia coli on a large scale and extracted from the cells by conventional alkaline lysis methodology followed by purification with ethanol and polyethylene glycol precipitation. Four mature male rhesus macaques (Macaca mulatta) (MM212, MM214, MM231 and MM234) were injected intramuscularly at two points on the right quadriceps and at two points on the calves with a solution containing 500 µg pSHIV-NM-3rn ZF1* in 450 µl PBS and 450 µl bupivacaine (Fujisawa Pharmaceutical) per injection for a total of eight injections. We injected the plasmid in two sets of DNA immunization series. Each set comprised one injection per week for four consecutive weeks. We injected at 03 weeks post initial vaccination (p.v.) and, after an interval of 5 weeks, we gave the second set of DNA immunizations, at 1013 p.v.
Animals.
All macaques used in this study were serologically negative for SIV and simian T cell lymphotropic virus type 1 before vaccination. They were housed throughout the experimental period and autopsies were carried out in accordance with regulations approved by the Institutional Animal Care and Use Committee of the Institute for Virus Research, Kyoto University, Japan.
Assays for antibodies.
The titres of anti-Env antibodies in the plasma of the monkeys vaccinated were determined using the Genscreen HIV-1/2 kit (Fujirebio), following the manufacturer's recommendations. The kit is based on a sandwich ELISA and the wells of microtitre plates were coated with the recombinant proteins HIV-1 gp160 p25, and synthetic peptides HIV-1 gp41 and HIV-2 gp36.
Particle agglutination was measured using the Serodia HIV-1/2 kit (Serodia HIV, Fujirebio) and immunoblotting was measured using the LAV BLOT 1 and LAV BLOT 2 kits (LAV BLOT, Fujirebio), following the manufacturer's recommendations. The LAV BLOT 1 kit was used for detection of HIV-1 Env antibodies and the LAV BLOT 2 kit was used for detection of SIV Gag antibodies.
Assay for HIV-1 Env- and SIV Gag-specific killer cell activities.
Specific killer cell activities for HIV-1 Env and SIV Gag were measured as described previously (Akahata et al., 2000; Yamamoto et al., 1990
). Herpesvirus papio-transformed B lymphocyte cell lines (B-LCL) established from the respective monkey PBMCs were infected with an HIV-1 Env (HIV-1IIIb) or SIV Gag (SIVmac239)-expressing recombinant vaccinia virus and used as target cells. The parental vaccinia virus-infected and non-infected cells were used as control targets. HIV-1 Env- and SIV Gag-specific killer cell activities were expressed as the percentage of specific lysis: (% specific lysis)=(% lysis of the HIV-1 Env- or SIV Gag-expressing B-LCL)-(% lysis of the parental vaccinia virus-infected B-LCL), when the effector : target cell ratio was 50 : 1. (In some cases, slightly lower ratios were adopted due to a limited number of the available cells.) The respective monkey B-LCLs infected with HIV-1 Env- or SIV Gag-expressing vaccinia virus were killed by treatment with glutaraldehyde and were used as antigen-stimulator cells. The respective monkey PBMCs were stimulated by the HIV-1 Env- or SIV Gag-expressing B-LCLs and incubated for 5 days. Stimulated PBMCs were used as the effector cells. The values of per cent specific lysis were measured in triplicate and mean values are shown. A per cent specific lysis value of above 7·0 was considered as specific killer activity. HIV-1 Env- and SIV Gag-specific killer cell activities were measured in MM212 and MM214 at 16 and 24 weeks p.v. and in MM231 and MM234 at 14 and 19 weeks p.v., respectively.
Lymphocyte proliferation assay.
PBMCs were obtained from the vaccinated monkeys and cultured in a 96-well plate (2x105 cells per well) in 200 µl RPMI in the presence of 2·5 µg HIV-1 rgp160 ml-1 (HIV-1IIIB gp160 recombinant viral protein) (Advanced Biotechnologies) for 72 h. Then lymphocyte proliferation was measured using a cell proliferation ELISA kit (Roche Diagnostics), following the manufacturer's recommendations. The ratio of incorporated BrdU by PBMCs for 24 h in the presence of antigen to that in media alone was expressed as the stimulation index (SI). An SI value above 2·5 was considered as antigen-specific stimulation. As a positive control, PBMCs were cultured in the presence of 1 µg concanavalin A (ConA) as a polyclonal stimulator. ConA-stimulated proliferation was found and the SI values were above 5·0 in all assays.
Challenge virus.
SHIV-NM-3rN was used as a challenge virus, in which the env, rev, tat, vpu and vpr genes are derived from HIV-1NL432 and the rest of the genome is derived from SIVmac239 (Kuwata et al., 1995). NM-3rN possesses identical genes, except for the nef gene, to the one in the vaccinated plasmid (Fig. 1
). SHIV-NM-3rN was grown in macaque monkey PBMCs and cell-free virus stocks were prepared. Four vaccinated macaques and two naive macaques were challenged intravenously with 1x102 TCID50 SHIV-NM-3rN.
Detection of plasma viral RNA.
Plasma virus loads were determined after challenge. Viral RNA was extracted from the plasma using the QIAamp Viral RNA Mini kit and their levels were determined by quantitative RT-PCR using a Taqman RT-PCR kit (Suryanarayana et al., 1998). Primers used in this assay were SIVII-696F and SIVII-784R and the probe was SIVII-731 (Perkin-Elmer). For a standard curve, quantitative assays for the measurement of viral RNA copy numbers in plasmas of a SHIV-infected monkey were performed by the Bayer Reference Testing Laboratory (Emeryville) with a branched DNA Signal Amplification assay (Pachl et al., 1995
; Tsai et al., 1997
). For each run, a standard curve was generated from dilutions of viral RNA extracted from the monkey's plasma. Under these conditions, the detection limit was 500 copies ml-1.
Virus isolation.
Virus was isolated as described previously (Ui et al., 1999). In brief, PBMCs were obtained from the monkeys at 2 and 4 weeks post-challenge (p.c.). Then, 1x106 PBMCs were co-cultured with 1x106 M8166 cells for 4 weeks in RPMI 1640 medium supplemented with 10 % FBS and 20 mM L-glutamine.
Detection of recombination.
DNA was extracted using the QIAamp DNA Mini kit. PCR was carried out using extracted DNA from 5x104 cells and the primer pair HnefF1 (5'-ACAGGGCTTGGAAAGGATTTTGCTA-3', nt 93509374) and SnefR1 (5'-CCCCGTAACATCCCCTTGTGGAAAGTCCC-3', nt 1017810206). The challenge virus, SHIV-NM-3rN, was detected as a 859 bp band and the vaccination plasmid, pSHIV-NM-3rn ZF1*, was detected as a 680 bp band. To determine whether the vaccination plasmid (which has an XhoI site) was present, the PCR products were incubated with XhoI. Cleavage at this site would create bands of 580 and 100 bp in size. The regions of the challenge viruses amplified do not have an XhoI site. Thus, if the PCR products were not digested with XhoI, then they would have been derived only from the challenge virus.
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RESULTS |
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Zinc-finger mutants of HIV-1 and Moloney murine leukaemia virus have been reported to be non-infectious (Gorelick et al., 1990; Meric & Goff, 1989
; Mizuno et al., 1996
). We next determined whether the pSHIV-NM-3rn ZF1* plasmid produced infectious virus. CD4+ M8166 cells were infected with the supernatants of transfected COS-1 cells containing equal amounts of viral RNAs, as measured by quantitative RT-PCR. Conspicuous syncytium formation was observed in M8166 cells infected with the supernatant from the SHIV-NM-3rn plasmid. The culture supernatant of these cells accumulated RT activity. In contrast, there was no indication of infectivity in the cells infected with the supernatants from pSHIV-NM-3rn ZF1* during the observation period of more than 1 month. Thus, the alteration in the N-terminal zinc finger resulted in the total loss of virus infectivity.
Virus particles produced by the mutant DNA had almost the same amount of p27CA, p17MA and unprocessed Gag precursor proteins as the wild-type. An immunoblot analysis together with electron microscopic observation showed that the plasmid pSHIV-NM-3rn ZF1* produced non-infectious virus particles with an average diameter of 100 nm (Fig. 3). Similar results were obtained using pSHIV-NM-3rn (data not shown).
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HIV-1 Env- and Gag-specific killer cell activities
Table 1 summarizes HIV-1 Env- and Gag-specific killer cell activities in the monkeys between 14 and 24 weeks p.v. All monkeys vaccinated showed HIV-1 Env- or SIV Gag-specific killer cell activities. MM234 showed HIV-1 Env- and SIV Gag-specific killer cell activities at 14 weeks p.v. (against Env at 14 and 19 weeks p.v. and against Gag at 14 weeks p.v.). MM212, MM214 and MM231 showed Gag-specific killer cell activities at 24 weeks p.v., at 16 and 24 weeks p.v. and at 19 weeks p.v., respectively.
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Virus loads of challenge virus in plasma
Vaccinated monkeys were intravenously challenged with a 100 TCID50 dose of SHIV-NM-3rN at 24 weeks p.v. Two naive macaques, MM125 and MM126, were also inoculated as unvaccinated controls.
Plasma virus loads were monitored by quantitative RT-PCR (Fig. 5). Viral RNA was not detected in any of the macaque sera before challenge with the infectious virus. Virus loads in the two naive monkeys (MM126 and MM125) at 1 week p.c. were approximately 2·7x103 and 3x104 copies ml-1, respectively. Then, at 2 weeks p.c., the virus loads of these two monkeys reached their peak values (both approximately 107 copies ml-1). The virus loads of these monkeys remained above the detection limit until 12 weeks p.c. On the other hand, at 1 week p.c., the virus load of MM234 was below 1·5x103 copies ml-1 and the virus loads of the other vaccinated monkeys were below the detectable level. Peak virus loads at 2 weeks p.c. of all vaccinated monkeys were two to three orders of magnitude lower than those of the naives. It is noteworthy that it took only 4 weeks for the virus loads to decrease below the detectable level in the three monkeys vaccinated.
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DISCUSSION |
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Recently, some studies have reported DNA vaccination regimes that use a replication-defective full-genome plasmid for cats or non-human primates. The cats vaccinated with a full-genome plasmid showed a CTL response but did not show any antibody response (Hosie et al., 1998). In the 11 monkeys vaccinated with the full SIV genome, only three monkeys showed antibody responses (Gorelick et al., 2000
). Our previous report (Akahata et al., 2000
) showed that two of four monkeys vaccinated with the full genome of HIV-1NL432 had antibody responses. In the present study, all monkeys vaccinated showed a CTL response. However, three monkeys did not show any antibody response and one monkey showed only a weak anti-Gag antibody response. These recent studies (Hosie et al., 1998
; Gorelick et al., 2000
; Akahata et al., 2000
) reported that vaccinations that induced little or no antibody response still conferred moderate protection against challenge viruses. It seemed to be more important to induce a CTL response than an antibody response. DNA vaccinations with a full-genome plasmid tend to induce cell-mediated immunity but also tend to be not so effective at inducing humoral immunity during the period soon after the vaccination. Overexposure to the antigens may shift the immunity from a Th1-type immunity to a Th2-type immunity (Fuller & Haynes, 1994
; Haynes et al., 1994
). The difficulty of inducing antibody responses with the full-genome DNA vaccination could be due to insufficient antigen expression shifting to Th2-type immunity, as we suggested previously (Akahata et al., 2000
).
There is some reluctance to use full-genome plasmids as vaccines because of the possibility of the genome reverting to the pathogenic wild-type. When we designed the plasmid pSHIV-NM-3rn ZF1*, we changed 4 nt to alter two cysteine residues to two serine residues in an N-terminal zinc-finger motif to reduce the possibility of reversion. Thus, reversion to the wild-type seems very unlikely. To confirm the safety of our vaccination regime, we examined the recombination between the DNA vaccination plasmids and the challenge viruses. No other studies have yet reported a recombination between a DNA vaccination plasmid and a challenge virus. In the present study, no recombination was observed.
In this study, we showed that a DNA vaccination regime using a full-genome plasmid was potentially effective without acting in combination with any other booster. Further studies are needed to determine whether modification of this regime, such as by using the plasmid in combination with protein or recombinant virus vector boosters, can achieve better protection.
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ACKNOWLEDGEMENTS |
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Received 3 January 2003;
accepted 7 May 2003.