1 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK
2 Microbiology and Tumorbiology Center, Karolinska Institutet, Box 280, S17177 Stockholm, Sweden
Correspondence
Tomas Hanke
thanke{at}molbiol.ox.ac.uk
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ABSTRACT |
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Published ahead of print on 30 October 2002 as DOI 10.1099/vir.0.18738-0.
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Introduction |
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An effective HIV vaccine may have to stimulate a range of host defences, including mucosal and innate immunities, neutralizing antibodies and cell-mediated immune responses. The variability of the HIV envelope and inaccessibility of potentially neutralizing epitopes on primary isolates (Kwong et al., 1998; Wyatt et al., 1998
) continues to hamper the development of vaccines that induce neutralizing antibodies. This shifted the focus of many vaccinologists towards the induction of CD8+ cytotoxic T lymphocytes (CTLs), which have been shown to play an important role in the control of HIV infection (Borrow et al., 1994
, 1997
; Goulder et al., 1997
; Haas et al., 1996
; Jin et al., 1999
; Kent et al., 1997
; Koenig et al., 1995
; Koup et al., 1994
; Phillips et al., 1991
; Price et al., 1997
, 1998
; Rowland-Jones & McMichael, 1995
; Schmitz et al., 1999
; Wagner et al., 1998
; Wilson et al., 1999
, 2000
; Wolinsky et al., 1996
; Yang et al., 1997
; Zhang et al., 1996
). However, determining the level of protection that vaccine-induced CTLs can confer against HIV exposure and in already infected individuals on anti-retrovirus therapy will be possible only through development of strategies that reliably elicit strong and durable CTL responses in humans.
Conceptually, gene-based vaccines consist of an immunogen, vaccine vector and an optional immunomodulator. While the immunogen defines vaccine specificity and provides a basic level of intrinsic immunogenicity, the choice of a vaccine vector determines the strength and longevity of the elicited immune responses. These can be further enhanced by particular combinations of heterologous vectors expressing a common immunogen in a primeboost application (Allen et al., 2000; Amara et al., 2001
; Hanke et al., 1998
, 1999
; Heeney et al., 2000
; Kent et al., 1998
; Nilsson et al., 2001
; Osterhaus et al., 1999
; Robinson et al., 1999
; Schneider et al., 1998
). Our finding that a successive immunization with DNA- and modified vaccinia virus Ankara (MVA)-based vaccines is particularly immunogenic for CD8+ CTLs (Hanke et al., 1998
; Schneider et al., 1998
) is being evaluated in phase I/II clinical trials in Oxford (UK) (unpublished observations) and Nairobi (Kenya). In these trials, an immunogen, which is derived from HIV-1 clade A, termed HIVA (Hanke & McMichael, 2000
), is used.
Semliki Forest virus (SFV) as a vaccine vector has a number of selling features. First of all it is very safe. While even the highly pathogenic strains in mice are non-pathogenic in humans, the experimental SFV vaccines are derived from strains that are, in mice, highly attenuated (Atkins et al., 1999). SFV replicates in the cytoplasm through amplification of its RNA genome, i.e. resulting in a high copy number of mRNA, the translation of which is not limited by processing. Cytoplasmic replication also removes the risk of chromosomal integration. In addition, SFV induces apoptosis of infected cells; therefore, the virus genome does not persist in the tissue. Recombinant SFV (rSFV) vaccines can be delivered in three forms: RNA, DNA or virus particles. For the stock production of particles, three mRNAs are co-transfected into packaging cells to reduce the possibility of recombination, which could reconstitute replication-competent particles: one mRNA with the packaging signal encoding the SFV polymerase and an immunogen, and two other mRNAs supplying the capsid and envelope proteins in trans (Smerdou & Liljeström, 1999b
). As a vaccine, rSFV induced better protective responses than a plasmid DNA in mice (Fleeton et al., 2000
) and was immunogenic in primates alone (Berglund et al., 1997
) and in a combined immunization protocol (Mossman et al., 1996
; Heeney, 2000
; Nilsson et al., 2001
). In addition, most people do not have a pre-existing immunity to SFV. These properties make SFV a suitable and potentially very attractive vector for human subunit vaccines.
Here, we describe the construction of rSFV particles expressing the HIVA protein and assess their immunogenicity in mice on their own and in a combined SFV.HIVA primeMVA.HIVA boost regimens.
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Methods |
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Cell lines.
Baby hamster kidney (BHK)-21 cells were maintained in complete BHK medium supplemented with 5 % foetal calf serum, 10 % tryptose phosphate broth, 2 mM glutamine, 20 mM HEPES and antibiotics (10 µg streptomycin ml-1 and 100 IU penicillin ml-1).
Preparation of stock SFV.HIVA particles.
The sequence encoding HIVA was isolated from pTHr.HIVA (Hanke & McMichael, 2000) as a HindIIINotI fragment and ligated into the pET-43 vector (Novagen). A PmlISmaI fragment containing the HIVA open reading frame (ORF) was then inserted into the SmaI site of pSFVb12a, which attached a 34 aa enhancer sequence of the capsid and the foot-and-mouth disease virus 2a cleavage site to the HIVA gene (Smerdou & Liljeström, 2000). Packaging of recombinant RNA encoding HIVA into rSFV particles was done using a two-helper RNA system (Smerdou & Liljeström, 1999a
). In brief, BHK cells were co-transfected with the recombinant and two additional helper mRNAs, one of which coded for the SFV capsid and the other for the envelope proteins. After 48 h of incubation, medium containing recombinant virus stock was harvested and purified (Fleeton et al., 1999
). Indirect immunofluorescence of infected BHK cells was performed to determine the titre of the recombinant virus stocks (Liljeström & Garoff, 1994
).
Analysis of expression of HIVA antigen from rSFV and rMVA particles.
Metabolic labelling of SFV.HIVA- or MVA.HIVA-infected cells with [35S]methionine has been described previously (Liljeström & Garoff, 1994). Briefly, BHK cells were infected with SFV.HIVA or MVA.HIVA at an m.o.i. of 5. After 15 h, growth medium was replaced with methionine-free minimum essential medium for 30 min prior to the addition of fresh medium containing 75 µCi (2·7 MBq) [35S]methionine ml-1. After a 15 min labelling period, the cells were incubated further for various times in medium containing unlabelled methionine. Supernatants were collected and the cells lysed with Nonidet P-40 buffer containing 100 mM iodoacetamide.
Protein sample preparation and analysis.
Cell lysates were analysed by immunoprecipitation followed by SDS-PAGE, as described previously (Liljeström & Garoff, 1994). Cell lysates were immunoprecipitated with protein ASepharose and an anti-Pk-tag monoclonal antibody (mAb) (Serotec) overnight at 4 °C. Cell pellets were washed, resuspended in SDS sample buffer and heated at 95 °C for 5 min prior to SDS-PAGE on a 10 % acrylamide reducing gel.
Immunofluorescence for the detection of HIVA expression.
Indirect immunofluorescence of SFV.HIVA- or MVA.HIVA-infected BHK cells was carried out to detect the expression of the HIVA protein. BHK cells were infected with SFV.HIVA or MVA.HIVA at an m.o.i. of 5. After a 15 h growth period, cells were fixed in methanol and protein expression was detected by incubation of the cells with anti-Pk-tag mAb at a concentration of 0·1 µg ml-1 followed by anti-mouse IgG conjugated to FITC (Sigma).
Vaccines and immunizations.
Groups of four 5- to 6-week-old female BALB/c mice were immunized at weeks 0, 2 or both with pTHr.HIVA DNA, SFV.HIVA or MVA.HIVA alone or in combinations (Table 1). For pTHr.HIVA and MVA.HIVA, clinical batches of vaccines produced by COBRA Therapeutics (Keele, UK) and Impfstoffwerk Dessau-Tornau (IDT, Germany), respectively, were used. Either a total of 50 µg pTHr.HIVA DNA in 0·1 ml 140 mM NaCl, 0·05 mM EDTA and 0·5 mM Tris/HCl, pH 7·7, solution or 106 p.f.u. MVA.HIVA in 0·1 ml 140 mM NaCl and 10 mM Tris/HCl, pH 7·7, solution was administered using needle injections of the tibial muscles of hind legs. All intramuscular (i.m.) injections were carried out under general anaesthesia. SFV.HIVA particles were administered subcutaneously (s.c.) at a dose of 106 IU. All procedures and care strictly conformed to the UK Home Office guidelines.
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Isolation of mouse peripheral blood mononuclear cells (PBMCs).
Approximately 100 µl blood was taken from individual mice by a venepuncture on the day of sacrifice. Blood was prevented from coagulation by the addition of 200 µl blood buffer (PBS, 10 mM EDTA and 100 U heparin ml-1). Red blood cells (RBCs) were lysed by the addition of 1·5 ml RBC lysis buffer (Puregene) followed by centrifugation at 3500 r.p.m. for 5 min. PBMCs were then washed once with R0 (RPMI 1640 supplemented with penicillin/streptomycin).
Flow cytometry.
About 106 mouse PBMCs were washed once with PBA (PBS, 1 % BSA and 0·1 % sodium azide) and incubated on ice with 1 µg MHCpeptide tetrameric complex for 20 min and for a further 20 min after the addition of an anti-mouse CD8 mAb conjugated to Tricolor (Caltag). Cells were then washed three times with PBA prior to a formaldehyde fixation (PBS, 2 % formaldehyde and 1 % BSA) and analysed on a Becton Dickinson FACScalibur flow cytometer using the CELLQUEST software (Becton Dickinson).
Isolation of splenocytes.
At 10 days or 6 months after the last immunization, spleens were removed and pressed individually through a cell strainer (Falcon) using the rubber plunger of a 2 ml syringe. Splenocytes were washed twice with R0 and suspended in 10 ml lymphocyte medium [RPMI 1640 supplemented with 10 % foetal bovine serum (FBS), penicillin/streptomycin, 20 mM HEPES and 15 mM 2-mercaptoethanol]. A 2 ml sample of splenocyte suspension was used for the interferon (IFN)- ELISPOT assay and the rest was used for a bulk CTL culture.
Enumeration of IFN--secreting splenocytes by ELISPOT assay.
The ELISPOT assay was carried out using the Mouse IFN--Secreting Cell kit (U-Cytech), according to the manufacturer's instructions. In brief, splenocytes isolated 10 days or 6 months following the last immunization of BALB/c mice were restimulated in 48-well plates at 8x106 cells per well in R10 (RPMI 1640 supplemented with 10 % FBS and penicillin/streptomycin) alone, supplemented with 4 µg concanavalin A ml-1 or specific peptide RGPGRAFVTI derived from HIV-1 and restricted by H-2Dd (Takahashi et al., 1988
) at 4 µg ml-1 for 15 h at 37 °C in 5 % CO2. The cells were then removed from the wells by careful washing in R0, set up in anti-IFN-
, pre-coated 96-well plates with the same stimulation as before at concentrations of 6, 3 or 1·5x105 cells per well in triplicates and incubated for a further 5 h at 37 °C. Following lysis of the cells by a 10 min incubation with water on ice, spots were visualized using a biotin-conjugated anti-IFN-
antibody and an enhancement system followed employing a dual activator system. Spots were counted using an ELISPOT reader (Autoimmun Diagnostika) and expressed as spot-forming units (s.f.u.) per 106 cells.
Bulk CTL cultures.
An 8 ml sample of cell suspension containing 8/10 of the total number of splenocytes was incubated with 2 µg peptide ml-1 in an humidified incubator in 5 % CO2 at 37 °C for 5 days. On the day of the CTL assay, effector cells were washed three times with RPMI, resuspended at 107 cells ml-1 in R10 medium and used in a 51Cr-release assay, as described below.
Target cells and standard 51Cr-release assay.
Effector cells were diluted twofold in a 96-well, U-bottom plate (Costar) to yield after addition of the target cells 50 : 1, 25 : 1, 12 : 1 and 6 : 1 effector to target ratios. A total of 5000 51Cr-labelled P815 cells in a medium containing 10-7 M peptide was then added to the effector cells and incubated at 37 °C for 4 h. Spontaneous and total chromium releases were estimated from the wells, in which the target cells were kept in a medium alone or 5 % Triton X-100, respectively. Percentage specific lysis was calculated as [(sample release-spontaneous release)/(total release-spontaneous release)]x100. Spontaneous release was lower than 5 % of the total c.p.m.
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Results and Discussion |
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This paper reports on the construction of a novel experimental subunit HIVA vaccine, SFV.HIVA. Using three different mutually complementing T cell assays, the relatively high and long-lasting immunogenicity of the vaccine in mice was demonstrated and compared to two other vaccines currently tested in humans, pTHr.HIVA DNA and MVA.HIVA.
An MVA.HIVA p.f.u. of 106 per dose was used. Because of the high MVA immunogenicity, the MVA.HIVA results stand on their own in this work. In all three assays, one MVA.HIVA vaccination was as good as two MVA.HIVA and the heterologous primeboost immunizations (Figs 1 and 2, and Tables 3 and 4
). Perhaps, the benefit of the heterologous primeboost protocol over the one or two MVA.HIVA schedule might be better seen at lower doses of MVA.HIVA or in more stringent immunizations of non-human primates (Hanke et al., 1999
; Allen et al., 2000
; Heeney et al., 2000
) and man (unpublished observations).
Our long-term aim is to build a panel of vaccine vectors expressing a common immunogen. The rationale is at least fourfold: first, to directly compare these vectors for their effectiveness in induction of both CD8+ and CD4+ T cell responses in animal models and humans; second, to assess the immunogenicities of various combined regimes using sequential immunizations; third, to evaluate the effect of a parallel use of different vectors on the breadth of induced T cell responses; and fourth, to generate a means of overcoming both pre-existing and vaccine-induced anti-vector immunities, which can negatively affect the immunogenicity of the passenger immunogen. For these types of studies, the HIVA immunogen is particularly suitable because it contains well-characterized CTL epitopes recognized by murine, rhesus macaque and human CTLs. Furthermore, HIVA has a growing safety record in humans, the species in which the ultimate immunogenicity evaluation of HIV vaccines has to be carried out and which no animal model can substitute.
HIV-1 is a highly variable virus, which is classified into M, N and O major groups. The M group has spread around the world and is further diversified into clades A to K in different geographical regions. We have argued previously that a candidate vaccine should match the appropriate local clades (McMichael & Hanke, 2002). Because the HIVA immunogen uses consensus clade A HIV sequences, it is designed specifically for areas with high prevalence of clade A infections, such as subSaharan Africa, Thailand and Russia (Neilson et al., 1999
), all of which are in a desperate need of an HIV vaccine. Therefore the addition of SFV.HIVA onto the list of clade A vaccines acceptable for use in humans might increase the chances that in the future, an effective T cell vaccination for these regions becomes available.
In conclusion, it cannot be stressed enough that only clinical trials aimed to optimize the elicitation of T cell responses in humans will provide a basis for the eventual proof or disproof of the at-present-frequently pursued hypothesis that CTLs can prevent establishment of an HIV infection and/or significantly delay the onset of AIDS in individuals who have become infected. This work represents one little step towards this goal.
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ACKNOWLEDGEMENTS |
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Received 30 July 2002;
accepted 21 October 2002.