Construction and immunogenicity in a prime–boost regimen of a Semliki Forest virus-vectored experimental HIV clade A vaccine

Tomás Hanke1, Christina Barnfield2, Edmund G.-T. Wee1, Lena Ågren2, Rachel V. Samuel1, Natasha Larke1 and Peter Liljeström2

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, S–17177 Stockholm, Sweden

Correspondence
Tomas Hanke
thanke{at}molbiol.ox.ac.uk


   ABSTRACT
Top
ABSTRACT
Introduction
Methods
Results and Discussion
REFERENCES
 
A novel, experimental subunit human immunodeficiency virus (HIV) vaccine, SFV.HIVA, was constructed. This consists of Semliki Forest virus (SFV), which is a suitable vaccine vector for use in humans, and a passenger gene encoding HIVA, which is an immunogen derived from HIV-1 clade A that is being currently tested in clinical trials of combined DNA- and modified vaccinia virus Ankara (MVA)-vectored vaccines in Oxford (UK) and Nairobi (Kenya). In the mouse, the SFV.HIVA vaccine was highly immunogenic for T cell-mediated immune responses and induced T cell memory that lasted for at least 6 months. SFV.HIVA was also compared to the vaccines currently used in the clinical trials and was shown to be as effective in T cell induction as pTHr.HIVA DNA but less immunogenic than MVA.HIVA. When tested in a prime–boost regimen, SFV.HIVA-induced responses could be boosted by MVA.HIVA. This work is a part of a long-term effort to build a panel of subunit vaccines expressing a common immunogen, which will allow both a direct comparison of various vaccine vectors and combined vaccination regimens in humans and provide more flexibility and/or a potential optimization of vaccinations for individuals based on their pre-existing anti-vector immunity.

Published ahead of print on 30 October 2002 as DOI 10.1099/vir.0.18738-0.


   Introduction
Top
ABSTRACT
Introduction
Methods
Results and Discussion
REFERENCES
 
Human immunodeficiency virus (HIV) infection continues to spread at an alarming rate. Education has been slow to make an impact and highly active anti-retrovirus therapy is, for the majority of people, too expensive and complex and in any case fails to clear the virus from the body. Moreover, the vast majority of HIV-infected people do not know that they are infected. Under these circumstances, development of a safe and effective prophylactic vaccine is the best hope for controlling the HIV epidemic. If successful, very similar vaccination strategies might be used in a therapeutic setting to benefit people who are already infected.

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 prime–boost 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 prime–MVA.HIVA boost regimens.


   Methods
Top
ABSTRACT
Introduction
Methods
Results and Discussion
REFERENCES
 
Recombinant DNA.
All enzymes used for recombinant DNA work were purchased from New England Biolabs or Boehringer Mannheim and used under the reaction conditions recommended by the vendors.

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 HindIII–NotI fragment and ligated into the pET-43 vector (Novagen). A PmlI–SmaI 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 A–Sepharose 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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Table 1. Immunization schedules

 
Preparation of MHC–peptide tetrameric complexes.
The MHC–peptide tetrameric complexes were prepared as described before (Altman et al., 1996).The gene encoding the H-2Dd heavy chain was modified so that the expressed protein missed the transmembrane and cytosolic tail segments and contained a BirA biotinylation site at its C terminus. For the human {beta}2-microglobulin gene, the fragment encoding the leader sequence was deleted (Garboczi et al., 1992). Both chains were expressed in Escherichia coli strain BL-21 (DE3) pLysS (Novagen) as inclusion bodies, which were purified, refolded by dilution in the presence of the peptide RGPGRAFVTI and biotinylated. The 45 kDa refolded complex was purified on FPLC and ion-exchange columns. An ELISA using alkaline phosphatase-conjugated streptavidin (Sigma) followed by a colorimetric reagent was used to measure the concentration of the biotinylated MHC–peptide complex monomer. To induce the formation of tetrameric complexes, Streptavidin–phycoerythrin conjugate (ExtrAvidin, Sigma) was added to the refolded MHC–peptide complex monomer solution in a 1 : 4 molar ratio.

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 MHC–peptide 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)-{gamma} ELISPOT assay and the rest was used for a bulk CTL culture.

Enumeration of IFN-{gamma}-secreting splenocytes by ELISPOT assay.
The ELISPOT assay was carried out using the Mouse IFN-{gamma}-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-{gamma}, 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-{gamma} 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.


   Results and Discussion
Top
ABSTRACT
Introduction
Methods
Results and Discussion
REFERENCES
 
Construction of rSFV particles expressing the HIVA immunogen
The HIVA ORF was inserted into the pSFV vector and rSFV particles were produced as described in Methods. Using the C-terminal mAb epitope Pk, the expression of the HIVA protein in SFV.HIVA-infected BKH cells was confirmed by indirect immunofluorescence (Fig. 1A). When compared with MVA.HIVA-infected cells, similar expression levels of the HIVA antigen were observed. Production of the HIVA antigen was also demonstrated by [35S]methionine pulse–chase experiments followed by immunoprecipitation (Fig. 1B). All of the radioactively labelled HIVA protein was found in the cell lysate and a majority of it was degraded within 3 h after synthesis. No HIVA was detected in the tissue culture supernatant.



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Fig. 1. Expression of HIVA. (A) Immunofluorescence of cells infected with either SFV.HIVA (left) or MVA.HIVA (right) using the C-terminal mAb epitope Pk for detection of the HIVA protein. (B) Analysis of HIVA antigen expressed by rSFV in transfected BHK-21 cells. After labelling with [35S]methionine, cells were chased with cold methionine for the indicated times. Both the cell lysates and supernatants were analysed by immunoprecipitation, 10 % SDS-PAGE and autoradiography.

 
SFV.HIVA particles induced HIV-specific immune responses that lasted for at least 6 months
The induction of HIV-specific T cell responses by SFV.HIVA particles was assessed and compared to the pTHr.HIVA DNA and MVA.HIVA vaccines (Hanke & McMichael, 2000) in the mouse. A sensitive readout was facilitated by employing epitope RGPGRAFVTI (Takahashi et al., 1988), which is included in the multi-CTL epitope string of HIVA. Thus, groups of BALB/c mice were immunized either with 106 IU SFV.HIVA particles s.c., 50 µg pTHr.HIVA DNA i.m. or 106 p.f.u. MVA.HIVA i.m. once or twice at a 2 week interval. Mice were sacrificed for immunological analysis 10 days after the last immunization (Table 1, experiment 1). Both the 51Cr-release and ELISPOT assays carried out on splenocytes from individual mice showed that a single delivery of SFV.HIVA particles induced in all animals CTL responses similar to those elicited by pTHr.HIVA DNA but lower compared to the more complex and immunogenic MVA.HIVA vaccine (Fig. 2A and Table 2). T cell induction was also confirmed by detection of PBMCs reactive with the H-2Dd–RGPGRAFVTI tetrameric complexes, although this assay seemed to be, in this case, less informative (Table 3).



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Fig. 2. Short-term analysis of CTL precursors. The immunogenicities of three vectors expressing HIVA alone (A) or in a heterologous prime–boost regimen (B) were assessed by employing an epitope recognized by mouse CTLs included into the polyepitope string. Mice were sacrificed 10 or 28 days after the last immunization (see Table 1), splenocytes from individual mice were isolated, separately peptide-restimulated in vitro for 5 days and tested in a 51Cr-release assay against peptide pulsed (full) or unpulsed (open) targets. Lines represent an average lysis±SD of each immunization group.

 

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Table 2. IFN-{gamma} ELISPOT assay on ex vivo splenocytes 10 or 28 days after the last immunization

Freshly isolated splenocytes were restimulated with peptide for 16 h and the number of IFN-{gamma}-producing cells was determined (s.f.u. per 106 splenocytes). Values for individual animals in each group are shown.

 

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Table 3. Reactivities of MHC–peptide tetrameric complexes with PBMCs 10 or 28 days after the last immunization

Blood was drawn from the immunized mice on the day of sacrifice and PBMCs were tested for their reactivities (% CD8+ PBMCs) with the RGPGRAFVTI/H-2Dd tetramer. Values for individual animals in each group are shown.

 
To assess the longevity of the induced immune responses and also as a more stringent comparison of immunogenicities of these three vaccine vectors, CTLs were analysed 6 months after immunization (Table 1, experiment 2). The 51Cr-release assay involving a 5 day in vitro peptide restimulation readily detected persisting CTL precursors (Fig. 3A). These lytic activities were again similar between the SFV.HIVA and pTHr.HIVA vaccines and stronger for MVA.HIVA. The frequencies of IFN-{gamma}-releasing cells over 6 months approximately halved (Table 4).



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Fig. 3. Analysis of CTL precursors 6 months after immunization. The immunogenicities of three vaccines expressing HIVA alone (A) or in a heterologous prime–boost regimen (B) were immunized (Table 1) and assessed 6 months after immunizations, as described in the legend of Fig. 2. Lines represent an average lysis±SD of each immunization group of either peptide pulsed (full) or unpulsed (open) targets.

 

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Table 4. IFN-{gamma} ELISPOT assay on ex vivo splenocytes 6 months after the last immunization

Freshly isolated splenocytes were restimulated with peptide for 16 h and the number of IFN-{gamma}-producing cells was determined (s.f.u. per 106 splenocytes). Values for individual animals in each group are shown.

 
Augmentation of immune responses by SFV.HIVA prime–MVA.HIVA boost regimen
Data are emerging that combined heterologous prime–boost vaccinations are superior to repeated immunizations with a single vaccine modality (Hanke, 2001). To test the suitability of SFV for priming of CTLs, mice were immunized with SFV.HIVA and boosted with MVA.HIVA 2 weeks later. As a reference, a sequential immunization with pTHr.HIVA and MVA.HIVA currently tested in the clinic was used. For both the DNA and SFV vaccinations, MVA increased the lytic activities of restimulated splenocyte cultures compared to the two single vaccine applications, although these responses were not significantly higher than MVA.HIVA alone (Fig. 1B and 2B). Similarly, for the IFN-{gamma} ELISPOT assay carried on ex vivo splenocytes, the heterologous immunizations were more potent than two sequential doses of SFV.HIVA or pTHr.HIVA (Table 2). Although the tetramer reactivities on PBMCs were not significantly different (Table 3), the first two assays indicated that the immune responses induced by SFV.HIVA could be augmented by MVA.HIVA. Good memory levels of CTLs were detected 6 months after the last immunization, indicating that the responses were relatively long lasting (Fig. 3 and Table 4).

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 prime–boost immunizations (Figs 1 and 2, and Tables 3 and 4). Perhaps, the benefit of the heterologous prime–boost 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.


   ACKNOWLEDGEMENTS
 
This work was supported by the MRC, UK, Vetenskapsrådet, Sweden, and the European Community Fifth Framework Programme.


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Received 30 July 2002; accepted 21 October 2002.