A DNA/MVA-based candidate human immunodeficiency virus vaccine for Kenya induces multi-specific T cell responses in rhesus macaques

Journal of General Virology (2002), 83, 75 80. Printed in Great Britain... SHORT COMMUNICATION A DNA/MVA-based candidate human immunodeficiency virus vaccine for Kenya induces multi-specific T cell responses in rhesus macaques Edmund G.-T. Wee, Sandip Patel, Andrew J. McMichael and Toma s Hanke MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford OX3 9DS, UK The minimum requirement for candidate human immunodeficiency virus (HIV) vaccines to enter clinical evaluation in humans should be their demonstrable immunogenicity in non-human primates: induction of antibodies neutralizing primary HIV isolates or elicitation of broad T cellmediated immune responses. Here, we showed in rhesus macaques that the very same vaccines that had entered clinical trials in Oxford and Nairobi, plasmid pthr.hiva DNA and recombinant modified vaccinia virus Ankara MVA.HIVA in a prime-boost protocol (Hanke & McMichael, Nature Medicine 6, 951 955, 2000), induced cellular immune responses specific for multiple HIV-derived epitopes. This was demonstrated by using the intracellular cytokine staining and ELISPOT assays detecting interferon-γ and pools of peptides employed in the clinical studies. These results have both boosted our expectations for the performance of these vaccines in humans and increased our confidence about the choice of these assays as the primary readouts in the on-going human trials. Close to 40 million people have been infected with human immunodeficiency virus type 1 (HIV-1), many of whom will develop AIDS. At least two out of three of these individuals live in sub-saharan Africa, where education programs have had virtually no effect in slowing the spread of HIV, and the highly active antiretroviral therapy available in industrialized countries is too costly and complex and in any case fails to clear the virus from the body. Moreover, 90% of the infected people are not aware of their HIV status. Under these circumstances, the best hope for controlling the HIV pandemic is effective prophylactic vaccination. While the inaccessibility of neutralizing epitopes on the Author for correspondence: Toma s Hanke. Fax 44 1865 222502. e-mail thanke molbiol.ox.ac.uk 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., 0001-8058 2002 SGM HF

E. G.-T. Wee and others 1999). Thus, a protective HIV vaccine should induce HIVspecific 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 highrisk 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 5 10 p.f.u. of MVA.HIVA intradermally (i.d.) in 0 3 ml of 140 mm NaCl, 10 mm Tris HCl 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 Tris HCl ph 7 7 and 0 05 mm EDTA, followed for all three monkeys by 5 10 p.f.u. of MVA.HIVA i.d. in 0 1 ml of 140 mm NaCl and 10 mm Tris HCl ph 7 7. These doses were designated HXR and Him, respectively. Macaques Gloria and Gerry received an additional boost of 5 10 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-γ+ cells and cells secreting IFN-γ, 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 IFNγ 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 1 4 of 15- amino-acid (aa)-long peptides overlapping by 11 aa across the p24 p17 region of the HIVA immunogen plus a mixture of costimulatory anti-cd28 and anti-cd49d antibodies for an initial 2 h period at 37 C, 5% CO. 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-γ FITCconjugated (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-γ-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. The IFN-γ release upon an HIVA-specific restimulation was assessed in an ELISPOT assay. The procedures and reagents of the MABTECH kit (cat. no. 3420M-2A) were used throughout. Briefly, PBMC were isolated on a Lymphoprep cushion and incubated at 37 C, 5% CO for 24 h with peptide p11c, C- M, of which the aa sequence is contained in the polyepitope portion of the HIVA immunogen (Hanke & McMichael, 2000). HG

Induction of broad T cell responses in monkeys Table 1. Antigen-specific induction of intracellular IFN-γ in PBMC CD4 PBMC CD8 PBMC Animal/dose* No Ag SEB p24 Pool 1 Pool 2 Pool 3 Pool 4 No Ag SEB p24 Pool 1 Pool 2 Pool 3 Pool 4 Week 12 Gaffa MXR 0 10 1 52 0 11 0 04 0 01 0 02 0 04 0 08 2 73 0 22 0 28 0 06 0 13 0 06 Gilda MXR 0 00 0 56 ND 0 00 0 00 0 01 0 01 0 09 2 13 ND 1 25 0 17 0 20 0 22 Gloria HXR 0 00 0 64 0 00 0 02 0 00 0 00 0 02 0 02 1 39 0 34 0 30 0 06 0 00 0 00 Gerry Him 0 00 0 50 0 03 0 00 0 00 ND 0 00 0 11 3 37 0 19 0 52 0 19 ND 0 09 Gege Him 0 00 1 63 0 00 0 00 0 00 0 00 0 00 0 08 2 64 0 13 0 30 0 18 0 14 0 18 Week 30 Gaffa MXR 0 08 1 09 0 00 0 02 0 00 0 00 0 22 0 11 2 39 0 09 0 07 0 03 0 04 0 37 Gilda MXR 0 08 1 48 0 26 0 22 0 20 0 29 0 16 0 00 2 19 0 25 0 19 0 14 0 08 0 14 Gloria HXR 0 00 1 79 0 01 0 00 0 04 0 00 0 03 0 01 2 08 0 16 0 15 0 11 0 02 0 06 Gerry Him 0 02 1 79 0 00 0 02 0 01 0 04 0 07 0 04 2 44 0 18 0 21 0 07 0 05 0 05 Gege Him 0 01 1 18 0 01 0 04 0 01 0 01 0 00 0 04 2 48 0 11 0 19 0 05 0 03 0 04 * Animals received a total of four immunizations with pthr.hiva (weeks 0 and 3) and MVA.HIVA (weeks 6 and 9). Data are shown as the percentage of CD4+ or CD8+ PBMC positive for CD69 and IFN-γ. Animals received additional boost with 5 10 p.f.u. of MVA.HIVA at week 26. ND, Not done. Table 2. Frequencies of IFN-γ-releasing PBMC upon specific peptide stimulation determined in an ELISPOT assay Week 24 In vitro Week 10 Animal/dose* restimulation Whole Whole CD8-depleted Gaffa MXR p11c, C-M 164 6 ND Pool 1 ND 200 ND Pool 2 ND 628 ND Pool 3 ND 473 ND Pool 4 ND 0 ND Gilda MXR p11c, C-M 132 10 0 Pool 1 ND 262 3 Pool 2 ND 652 20 Pool 3 ND 614 20 Pool 4 ND 103 0 Gloria HXR p11c, C-M 207 15 ND Pool 1 ND 135 ND Pool 2 ND 319 ND Pool 3 ND 1404 ND Pool 4 ND 1345 ND Gerry Him p11c, C-M 341 40 ND Pool 1 ND 40 ND Pool 2 ND 741 ND Pool 3 ND 704 ND Pool 4 ND 13 ND * Animals received a total of four immunizations with pthr.hiva (week 0 and 3) and MVA.HIVA (weeks 6 and 9). Assay was carried out on either whole or CD8+ cell-depleted PBMC. Frequencies are expressed as no. of SFU per 10 PBMC above no-peptide background. ND, Not done. HH

E. G.-T. Wee and others The released IFN-γ was captured by a monoclonal antibody immobilized on the bottom of assay wells, visualized by combination of a second monoclonal antibody coupled to an enzyme and a chromogenic substrate, and the spots were counted using the AID ELISpot Reader System (Autoimmun Diagnostika). It was found that 1 week after the last MVA.HIVA immunization four out of five vaccinated macaques responded to this Mamu-A*01-restricted epitope (Table 2). However, technical difficulties with the isolation of PBMC from Gege s blood might have occluded the detection of responses in this monkey. On week 34, only four animals were tested and all four showed responses against multiple epitopes. Magnetic-bead depletion of CD8+ cells from the PBMC of Gilda almost completely abolished the IFN-γ release suggesting that, at least in this macaque, the majority of IFNγ producing cells were CD8 positive. There was no incentive to further map HIV-1-derived epitopes in these macaques in which Mamu-A*01 was the only known MHC molecule. Thus, the vaccination induced multi-specific CD8+ T cell responses that lasted at least 21 weeks. No significant reactivity above background of PBMC with Mamu-A*01 p11c, C-M peptide tetrameric complexes was observed (not shown). However, 300 SFU per 10 PBMC seen in our macaques 1 week after the MVA.HIVA vaccination, i.e. at the peak of tetramer responses (Hanke et al., 1999b), would translate into 0 1% of CD8+ cells (Tan et al., 1999) which, for macaques, is at the limit of tetramer sensitivity. Note that this and our previous studies (Allen et al., 2000; Hanke et al., 1999b) showing high tetramer reactivity after the DNA MVA vaccination employed different immunogens [see Hanke et al. (1998b) and Hanke & McMichael (2000)], in which the Mamu- A*01-restricted p11c, C-M epitope is inserted between different adjacent aa sequences opening possibilities for differential processing and presentation. Indeed, in the present study, the p11c, C-M response was not dominant (Table 2) as it was shown for Mamu-A*01+ SIV-infected monkeys (Miller et al., 1991). 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-γ, 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-γ. In contrast, the ELISPOT assay stimulated purified PBMC with peptide alone and the results included CD69-negative IFN-γ-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 ongoing 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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