1 MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK
2 University of St Andrews, School of Biology, St Andrews, UK
3 Nuffield Department of Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK
4 International AIDS Vaccine Initiative, 110 William Street, New York, NY 10038, USA
5 Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya
Correspondence
Tomá Hanke
tomas.hanke{at}imm.ox.ac.uk
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ABSTRACT |
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Present address: Wellcome Trust Kilifi Labs, PO Box 230, Kilifi, Kenya.
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INTRODUCTION |
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It is generally believed that an effective HIV vaccine may have to stimulate both neutralizing antibodies and cell-mediated immune responses and do so both systemically and at mucosal sites (McMichael & Hanke, 2003). While the instability and inaccessibility of potentially neutralizing epitopes on primary HIV isolates have hampered the development of envelope-based vaccines (Kwong et al., 1998
, 2002
; Wyatt et al., 1998
), a few new potent technologies capable of inducing high levels of circulating virus-specific CD8+ cytotoxic T lymphocytes (CTLs) are emerging (Hanke, 2003
).
CTLs recognize, through their T-cell receptor, short HIV-derived peptides (810 aa long) presented by a self major histocompatibility complex molecules called HLA in humans. Recognition of these HLApeptide complexes in the context of the right accessory signals stimulates naive and precursor memory CTLs to proliferate and express effector functions. These include lysis of HIV-infected cells, which limits the production of new HIV virions, and production of soluble cytokines and chemokines that can directly or indirectly contribute to the control of HIV replication. The advantage of CTLs over antibodies is that the recognized peptides can originate from both surface and inner HIV proteins. The disadvantage is that CTLs cannot prevent the first wave of cell-free HIV from infecting host cells, i.e. induce sterilizing immunity. However, once CTLs become stimulated by antigen-presenting cells, they produce soluble factors relatively quickly that can, at least in vitro, prevent HIV from entering cells (Zhang et al., 1996; Price et al., 1998
; Wagner et al., 1998
; Stranford et al., 1999
). Thus, providing that an individual is exposed to a low HIV dose, CTLs can theoretically prevent HIV from spreading and establishing a generalized infection by eliminating HIV-infected cells early in infection. Alternatively, CTLs can keep the virus at levels so low that both transmission to other individuals and progression to AIDS are prevented. The net effect of these actions might be that CTLs increase the threshold for infection and therefore decrease the HIV transmission rate (Hanke, 2003
). However, these predictions can be only proven or disproven by first developing methodology for induction of T-cell responses in humans followed by a controlled efficacy clinical trial.
HIV-1 is a highly variable virus, for which intra-subtype variation could be as high as 20 % and inter-subtype diversification can reach 35 % of the amino acid sequence (Thomson et al., 2002). Clade B is the most common in Europe and North America, clades A, C and D dominate in Africa, and C is now the most widespread clade. In Nairobi, clade A currently causes about 70 % of HIV infections (Neilson et al., 1999
). Although some reports have demonstrated that cross-clade CD8+ T-cell responses can be detected (Cao et al., 1997
; Ferrari et al., 1997
; Walker & Korber, 2001
), other studies have not shown this (Dorrell et al., 1999
, 2001
). Such studies require careful peptide titration experiments designed to reach physiologically relevant concentrations. Thus, unless vaccines can stimulate very strong T-cell responses to multiple epitopes, it is only prudent to match the vaccine immunogens to the circulating HIV clades in the target population (McMichael & Hanke, 2002
).
Previously, we have constructed a DNA primemodified vaccinia virus Ankara (MVA) boost candidate HIV vaccine expressing a common immunogen, designated HIVA (Hanke & McMichael, 2000). It is derived from consensus HIV-1 clade A Gag p24/p17 sequences and a string of clade A CTL epitopes; it does not contain the envelope gene and focuses solely on the induction of cell-mediated immune responses. In pre-clinical studies, both the pTHr.HIVA DNA and the MVA.HIVA vaccine components were highly immunogenic in mice and macaques (Hanke & McMichael, 2000
; Wee et al., 2002
). Reported here are small phase I clinical trials of this vaccine in healthy uninfected human volunteers in Oxford run under the auspices of the Medical Research Council of the United Kingdom (MRC UK) and the International AIDS Vaccine Initiative (IAVI).
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METHODS |
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HIV-uninfected males and females between 18 and 60 years of age who gave informed consent were screened, enrolled and immunized after giving further written informed consent to participate (further recruitment details can be found at http://www.oxavi.org/). All volunteers were assessed by experienced counsellors to be at low risk for HIV infection. For the pTHr.HIVA DNA-alone arm, six and 12 volunteers were randomized into two dosage groups to receive 100 µg (nos 0106) or 500 µg (nos 0718) of plasmid DNA, respectively, on days 0 and 21 by injection into alternate deltoid muscles. The volunteers and the clinical and immunological laboratory staff were blinded for the received DNA dose until all volunteers had passed day 189. The MVA.HIVA-alone arm was an open-label study, in which eight volunteers (nos 2128) were injected intradermally with 5x107 p.f.u. of MVA.HIVA on days 0 and 21 into alternate arms. In the primeboost study, nine volunteers (nos 02, 03, 04, 09, 11, 12, 14, 16 and 18) from the DNA study were boosted using the same dose of MVA, timing and route as in the MVA-alone trial at 914 months after their last DNA dosing. The volunteers who were boosted with MVA were chosen on the basis of willingness to re-enrol, not according to their immunological responses. All blood samples coming to the laboratory were coded.
Production of the pTHr.HIVA and MVA.HIVA vaccines.
The pTHr.HIVA DNA and MVA.HIVA vaccines (Hanke & McMichael, 2000) were produced under Good Manufacturing Practice (GMP) conditions by COBRA Therapeutics (Keele, UK) (Williams et al., 1998
) and IDT (Roßlau, Germany), respectively. The GMP vaccine batches were tested on their own and in the primeboost protocol for toxicity, biodistribution and persistence in BALB/c mice in the Good Laboratory Practice facilities of Huntingdon Life Sciences (Huntingdon, UK) and showed no toxicity or evidence of persistence in any tissue other than at the site of injection at 5 weeks after the completion of dosing (Hanke et al., 2002
). Thus, both vaccines were acceptable for use in phase I clinical trials as required by the UK Medicines Control Agency.
Isolation of peripheral blood mononuclear cells (PBMCs).
Blood was drawn from healthy volunteers in vaccutainers containing preservative-free heparin. PBMCs were isolated from the whole blood by Ficoll Hypaque cushion centrifugation using Lymphoprep 1.077 (Nycomed Pharma) as described previously. Cells were washed twice in RPMI [RPMI 1640 (Sigma-Aldrich) supplemented with glutamine and penicillin/streptomycin], counted and resuspended in R10 [RPMI supplemented with 10 % fetal bovine serum (Sigma-Aldrich)] at 2x106 cells ml-1. Isolated PBMCs were kept at 37 °C and 5 % CO2 until use.
Preparation of peptide pools.
HPLC-purified peptides were purchased from Sigma-Genosys, with a purity of at least 80 % by mass spectroscopy. Individual peptides were dissolved in DMSO (Sigma-Aldrich) to yield a stock of 10 mg ml-1 and stored at -80 °C. To prepare pools of 2023 peptides (Fig. 1a), 20 µl of each peptide was combined and phosphate-buffered saline (PBS) was added to 5 ml. The pool was filtered, aliquoted, stored at 4 °C and used within 1 week. For the assay, 10 µl of pooled peptides were added per well, so that each peptide was at a final concentration of 4 µg ml-1.
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ELISPOT data analysis.
Spot-forming units (s.f.u.) in the ELISPOT wells were counted by an automated ELISPOT reader (AutoImmun Diagnostika). Assay plates that did not meet quality criteria were discarded, i.e. if (i) there was no positive control response or (ii) the background in the no-peptide wells exceeded 100 s.f.u. per million PBMCs (20 s.f.u. per 200 000-cell well), although over 95 % of backgrounds were under 65 s.f.u. per million PBMCs. For the DNA, MVA and DNAMVA boost trials, 75, 78 and 83 % of plates were accepted and the mean backgrounds in these assays were 36, 33 and 37 s.f.u. per million PBMCs, respectively. For calculations of peptide-stimulated responses, s.f.u. were determined from the 200 000-cell wells only. A response was considered positive if the peptide-pulsed cells gave frequencies of s.f.u. above the mean no-peptide background plus 2 standard deviations, i.e. positive with at least 95 % confidence, and the response was at least 50 s.f.u. per million PBMCs above the background. To calculate the mean responses for each time point, all responses (both positive and negative) for Gag overlapping peptide pools 14 and for known HIVA CTL epitope pools 57 were summed after subtracting the backgrounds to simplify the data presentation. Pools 14 and 57 were not added, to avoid possible counting of the same Gag-specific responses twice (see Fig. 1a).
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RESULTS |
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T-cell responses to intramuscular pTHr.HIVA DNA
Eighteen volunteers were vaccinated with either 100 or 500 µg of DNA and their T-cell responses were measured by frequency of fresh PBMC releasing IFN- in response to peptide stimulation in vitro. The overall pattern of responses is shown in Fig. 2
(a). The results for summed responses specific for Gag (peptide pools 14) and known CTL epitopes in all of the HIVA immunogen, i.e. in the Gag and polyepitope regions (peptide pools 57; see Fig. 1a
), are presented as an arithmetic mean ±standard error of all volunteer responses, positive and negative, at a particular time of the protocol (Fig. 2a
) and varied in strength, ranging from 0 to over 900 s.f.u. per million PBMCs, hence the large standard errors of the mean. Maximum summed responses for each volunteer irrespective of time are also shown (Table 1
). The HIV-specific responses were compared to no-peptide, and influenza virus/EBV/CMV peptide pool and PHA were used as negative and positive controls, respectively (not shown, for an example see Fig. 1b
). Defining a positive response as one which is above the plate no-peptide background mean plus at least 2 standard deviations and at least 50 s.f.u. per million PBMCs above the background, the numbers of responders at each time point and the cumulative number of responders are shown (Fig. 2b
). The responses to the DNA vaccine in responders were detected on average on 1·7 separate occasions and each volunteer responded to an average of 2·5 Gag pools (out of a maximum of four pools). For these small volunteer numbers, no differences were observed between the two doses of DNA. Surprisingly, HIV-specific responses were detected at 1 year after vaccination in seven volunteers, of which the stronger ones were confirmed on frozen cell samples. Otherwise, responses tended to peak 1428 days after immunization. We were also able to identify several of the single peptides recognized by T cells of volunteers 05, 06 and 14 (not shown). According to our positivity criteria, a total of 14 volunteers responded to the DNA vaccination, of whom four and 10 received 100 and 500 µg doses, respectively.
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T-cell responses to the pTHr.HIVA primeMVA.HIVA boost regimen
Nine of the DNA-primed volunteers were vaccinated with the MVA.HIVA 914 months after their last DNA immunization. The arithmetic means ±standard errors of all positive and negative responses detected by IFN- release at a given day after the MVA.HIVA vaccine are presented in Fig. 4
(a). Note that at the time of the MVA vaccination, only one volunteer had detectable DNA-induced T-cell responses. Cumulatively, eight out of nine volunteers had responses higher than the plate background mean plus 2 standard deviations and at least 50 s.f.u. per million PBMCs above the background (Fig. 4b
) with the highest frequency of over 2000 s.f.u. per million PBMCs (Table 1
). On average, 3·2 responding blood samples per responder were identified and the average breadth of Gag-specific responses was 3·3 out of four Gag peptide pools. Given the fact that each pool may contain several peptides recognized by the vaccine-induced T cell, these results are encouraging.
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DISCUSSION |
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The DNA vaccine provoked positive responses in 78 % (n=18) volunteers. In general, the T-cell responses were fairly weak and transient. No differences were discernible between the 100 and 500 µg doses of DNA, both of which are regarded as low. Remarkably, using the same criteria of positivity, seven DNA-immunized volunteers showed T-cell responses after 1 year. These late responses were completely unexpected, as they had not been reported in previous DNA vaccine studies, but they have rarely been sought before. Using standard HIV diagnostic tests, we ruled out HIV infection as the cause of the late T-cell responses in DNA vaccine recipients. In addition, given the very low prevalence of HIV infection in the Oxford region and the rigorous selection of volunteers to be at negligible risk of HIV exposure, we think it is extremely unlikely that the responders had been exposed to HIV and somehow immunized. Also, because the responses were to more than one peptide pool, it is very unlikely that this represents a cross-reaction to some other intercurrent infection. The numbers of responders were small, however, and these late responses need to be sought and confirmed in future trials.
MVA.HIVA vaccination elicited HIV-specific T-cell responses in 88 % (n=8) volunteers, of whom five had responses detectable at 1 year after vaccination. As anticipated, the MVA-induced responses were stronger and more frequently detected than those elicited by DNA. Similar results were seen in macaques immunized with recombinant MVA (Seth et al., 1998) and in human volunteers immunized with a recombinant canarypox vaccine (Cao et al., 2003
).
The pTHr.HIVA primeMVA.HIVA boost protocol induced responses in 89 % (n=9) volunteers. As far as it can be inferred from these small numbers of volunteers, responses elicited by the DNA primeMVA boost regimen were very similar in the whole PBMCs to those induced by MVA alone. Preliminary studies showed a difference in the frequencies of non-CD8+, presumably CD4+ T-cell responses, which were estimated by a magnetic-dynabead depletion of CD8+ cells (not shown). These experiments suggested that better non-CD8+ responses were observed for the DNA and especially primeboost vaccinations compared to MVA alone. Similar observations were made in the trial of candidate vaccines for malaria (McConkey et al., 2003). Generation of HIV-specific CD4+ T-cell responses would be encouraging, because it could facilitate stimulation of a better quality immune response than that elicited during natural HIV infection, and needs to be explored in future studies. In healthy HIV-uninfected volunteers, CD8+ T cells are primed in the presence of intact CD4+ help and therefore may be more efficient when facing the incoming HIV (Janssen et al., 2003
).
Higher frequencies of responders to the vaccination were detected in these three trials than seen previously (Ugen et al., 1998; Wang et al., 1998
; Boyer et al., 2000
; MacGregor et al., 2002
; McConkey et al., 2003
). A number of circumstances could have contributed to this. First, fresh rather than frozen-and-thawed cells were used throughout the trials and during the validation of the IFN-
ELISPOT assay fresh cells gave two- to threefold higher frequencies (Mwau et al., 2002
). Second, different and possibly less-sensitive assays were employed in previous trials. Third, the IFN-
ELISPOT assay can be desensitized in an attempt to decrease the background no-peptide counts (E. G.-T. Wee, A. J. McMichael & T. Hanke, unpublished data), which may be genuine and caused by a reactivated CMV infection (Dunn et al., 2002
). It was noted that the IFN-
spots generated by the vaccine-induced T cells were much smaller compared to the ones produced by PHA and well-established responses to chronic viral infections such as the influenza virus/EBV/CMV (Fig. 1b
) or indeed HIV (Mwau et al., 2002
). This might indicate that, in vaccine trials, responses that are being freshly induced and measured during the course of the trial are qualitatively different from those observed in HIV-infected people which are often used for validation of trial assays. This could also explain the apparent transient nature of these responses, their different resistance to freezingthawing procedures and sometimes marginal functionality. Experiments addressing these issues are under way. The significance of IFN-
production for the defence against HIV infection is not clear; other T-cell functions may be more relevant and should be explored in future. Ultimately, efficacy can only be shown in a phase III trial in humans. Therefore, the most encouraging observation in these trials is the high frequency of individuals whose T-cell responses were primed to a number of HIV epitopes.
At this stage, it is important to advance the progress of vaccines that can stimulate strong and specific T-cell responses to a proof-of-principle or efficacy trial as quickly and safely as possible. The pTHr.HIVA and MVA.HIVA vaccines by virtue of their initial immunogenicity in human trials described here look promising, but there is still some way to go.
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ACKNOWLEDGEMENTS |
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Received 6 October 2003;
accepted 4 December 2003.