MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK1
Author for correspondence: Tomá Hanke. Fax +44 1865 222502. e-mail thanke{at}molbiol.ox.ac.uk
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Abstract |
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While the inaccessibility of neutralizing epitopes on the primary HIV isolates has been a major impediment to the development of envelope-based vaccines, considerable evidence has accumulated which indicates that CD8+ T cells are crucial for the control of HIV infection. First, the kinetics of the CD8+ cytotoxic T lymphocyte (CTL) response in acute and chronic HIV infections and inverse relationship with HIV virus load implied an anti-HIV effect in vivo (Borrow et al., 1994 ; Kent et al., 1997
; Koup et al., 1994
; Wilson et al., 2000
). Second, CTL selected virus escape mutants (Borrow et al., 1994
, 1997
; Goulder et al., 1997
; Haas et al., 1996
; Koenig et al., 1995
; Phillips et al., 1991
; Price et al., 1997
; Wilson et al., 1999
; Wolinsky et al., 1996
) and do so probably in most patients. Third, a temporary removal of CD8+ cells in acute and chronic simian immunodeficiency virus (SIV) infections by the in vivo use of anti-CD8 antibodies caused a marked rise in the virus load and failure to control acute viraemia (Jin et al., 1999
; Schmitz et al., 1999
). Fourth, CTL reduced and could eliminate HIV from cell cultures (Price et al., 1998
; Stranford et al., 1999
; Wagner et al., 1998
; Yang et al., 1997
; Zhang et al., 1996
). Fifth, several vaccines induced protection in monkeys independent of virus-neutralizing antibodies (Amara et al., 2001
; Barouch et al., 2000
; Gallimore et al., 1995
; Kent et al., 1998
; Robinson et al., 1999
). Sixth, the identification of exposed seronegative/uninfected individuals with CTL responses against HIV raised the possibility that CTL responses have at least contributed to the prevention of infection (Rowland-Jones & McMichael, 1995
). Finally, CTL have been shown to control many other virus infections in both mice and humans and could protect against new infections (Blaney et al., 1998
; Fu et al., 1999
; Schneider et al., 1998
). This growing list of experimental data together with new potent technologies capable of inducing high levels of circulating virus-specific CD8+ T cells have strengthened the belief that a successful HIV vaccine is possible and within reach (Hanke, 2001
).
Murine models of chronic viral infections strongly support the requirement of virus-specific CD4+ T helper cells for efficient generation of anti-viral immune responses, including CTL (Matloubian et al., 1994 ; von Herrath et al., 1996
). An analogous situation is seen in HIV-infected people, in whom a progressive depletion of CD4+ T cells weakens the responses to both HIV and opportunistic infections (Rosenberg et al., 1999
). Thus, a protective HIV vaccine should induce HIV-specific T helper cells as well as CTL.
Our original finding that a successive immunization with DNA- and modified vaccinia virus Ankara (MVA)-based vaccines expressing a common immunogen is a potent way of inducing CD8+ CTL (Hanke et al., 1998a ; Schneider et al., 1998
) has been since reinforced by us and others (Allen et al., 2000
; Amara et al., 2001
; Hanke et al., 1999a
, b
). The evaluations of the safety and immunogenicity of this approach have already commenced in healthy low-risk volunteers in Oxford and Nairobi, with the view of proceeding into a high-risk cohort in Kenya to test the vaccine efficacy. For this reason, the immunogen HIVA delivered by DNA and MVA was tailor-designed to match HIV clade A, the most prevalent local HIV strain. It consists of a consensus HIV clade A p24/p17 and a string of CTL epitopes (Hanke & McMichael, 2000
), which includes the SIV p11C, C-M sequence presented by the rhesus monkey Mamu-A*01 molecule (Allen et al., 1998
). Here, induction of multi-specific CD4+ and CD8+ T cell responses in rhesus macaques using clinical preparations of the pTHr.HIVA and MVA.HIVA vaccines in a prime-boost regimen was demonstrated. Furthermore, the elicited immune responses were measured using the same assays which are employed in the clinical trials, thus validating both the vaccine approach and detection of elicited immune responses in a pre-clinical setting in non-human primates.
Five rhesus macaques (Macaca mulatta) positive for the Mamu-A*01 allele of the major histocompatibility complex (MHC) received initially a total of four successive immunizations: two using the plasmid pTHr.HIVA DNA and two with recombinant MVA.HIVA at 3 week intervals. In particular, two animals (Gaffa and Gilda) received 8 µg pTHr.HIVA epidermally using the Dermal XR particle delivery device (designated XR; PowderJect Vaccines, Inc.) or gene gun followed by needle-injected 5x108 p.f.u. of MVA.HIVA intradermally (i.d.) in 0·3 ml of 140 mM NaCl, 10 mM TrisHCl pH 7·7 solution, which is a dose similar to the immunizations in our previous macaque experiments employing a prototype multi-CTL epitope, here designated MXR (Hanke et al., 1999b ). Three animals received human doses similar to those used or planned to be used in the clinical trials: one animal (Gloria) was vaccinated epidermally with 4 µg of pTHr.HIVA DNA and two animals (Gerry and Gege) were needle-injected intramuscularly (i.m.) with 500 µg of pTHr.HIVA in 0·5 ml of 140 mM NaCl, 0·5 mM TrisHCl pH 7·7 and 0·05 mM EDTA, followed for all three monkeys by 5x107 p.f.u. of MVA.HIVA i.d. in 0·1 ml of 140 mM NaCl and 10 mM TrisHCl pH 7·7. These doses were designated HXR and Him, respectively. Macaques Gloria and Gerry received an additional boost of 5x107 p.f.u. of MVA.HIVA i.d. on week 26. All immunizations and venipunctures were carried out under sedation with ketamine and the animals were clinically examined regularly. All procedures and care strictly conformed to the UK Home Office Guidelines.
The combination of the pTHr.HIVA and MVA.HIVA vaccines focuses on the induction of cell-mediated immunity (Hanke & McMichael, 2000 ) and their immunogenicities in human volunteers are assessed in validated intracellular cytokine and ELISPOT assays estimating the frequencies of CD69+ IFN-
+ cells and cells secreting IFN-
, respectively.
The intracellular cytokine assay is designed for detection of functional primarily CD4+, but also CD8+ cell populations, which upon antigen and antibody co-stimulation express IFN- and activation marker CD69 (Maino & Picker, 1998
; Suni et al., 1998
). The assay was carried out according to the recommendations of the FastImmune Kit vendor (Becton Dickinson, cat. no. 340970) with a few modifications. Briefly, whole heparinized blood drawn from immunized macaques was incubated without any antigen, with staphylococcal enterotoxin B (SEB), HIV-1 p24 (IIIB) or pools 14 of 15-amino-acid (aa)-long peptides overlapping by 11 aa across the p24/p17 region of the HIVA immunogen plus a mixture of co-stimulatory anti-CD28 and anti-CD49d antibodies for an initial 2 h period at 37 °C, 5% CO2. Brefeldin A was then added to inhibit cytokine secretion and the samples were incubated for an additional 14 h before terminating the reaction with EDTA and the FACS fix solution. At this point, the blood was placed at -80 °C until analysis. At convenience, samples were thawed, peripheral blood mononuclear cells (PBMC) permeabilized and incubated with mouse anti-huIFN-
FITC-conjugated (Biosource International, cat. no. AHC4338), mouse anti-CD69 APC-conjugated (Becton Dickinson, cat. no. 340560) and mouse anti-huCD4 R-PE-conjugated (BD PharMingen, cat. no 556616) monoclonal antibodies. Addition of anti-huCD8 PerCP-conjugated (Becton Dickinson, cat. no.347314) antibodies allowed a parallel analysis of the CD8+ cellular responses. Arbitrarily, samples were considered positive if the percentage of stained cells increased after stimulation at least 3-fold compared to the no-antigen baseline. Thus, although the FACS analysis of blood from the immunized monkeys failed to demonstrate CD4+, but revealed CD8+ cell responses at week 12, all animals had in their circulation CD69-positive IFN-
-producing CD4+ or CD8+ cells at week 30 (Table 1
). Furthermore, the use of four peptide pools suggested that two animals responded to more than one and in one case at least four different epitopes (Table 1
, Gilda, week 30). Note that approximately the first 2 and 1/2 of peptide pools correspond to p24 and that these pool responses on several occasions exceeded those of the p24 stimulation. This may be accounted for by differential processing of peptides versus protein antigens and the fact that p24 was of HIV-1 clade B.
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Exactly matching CD8+ T cell responses were not detected by the intracellular cytokine and ELISPOT assays (Table 1 versus Table 2
). However, although both these assays measure IFN-
, they are not identical. Thus, the intracellular cytokine assay used a stimulation through CD28/CD49d plus the T cell receptor, the stimulation was carried out on whole blood, which although optimized by the vendor is likely to be less efficient than on isolated PBMC (hence the need for antibody co-stimulation), and the results are expressed as only CD69-positive cells showing intracellular IFN-
. In contrast, the ELISPOT assay stimulated purified PBMC with peptide alone and the results included CD69-negative IFN-
-releasing cells. Moreover, the assays were carried out at different time-points.
In conclusion, we have confirmed in non-human primates the CD4+ and CD8+ T cell immunogenicities of the very vaccines that have entered clinical trials. Particularly encouraging is the fact that in all animals responses were elicited which were targeted at multiple HIV-derived epitopes and that these were detected by the readout assays employed in the on-going human trials. The current work is being followed by a larger macaque study addressing the dosing and timing requirements of the two vaccine components for an efficient T cell induction. This line of experimentation is consistent with the principle of testing thoroughly candidate HIV vaccines in non-human primates before and/or in parallel with their clinical evaluation.
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Acknowledgments |
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Grant supports from MRC UK and International AIDS Vaccine Initiative are fully acknowledged.
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References |
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Received 23 August 2001;
accepted 25 September 2001.