European Journal of Immunology

Volume 36, Issue 10 p. 2574-2584

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Induction of long-lasting multi-specific CD8⁺T cells by a four-component DNA-MVA/HIVA-RENTA candidate HIV-1 vaccine in rhesus macaques

Eung-Jun Im,

Eung-Jun Im

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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Joseph P. Nkolola,

Joseph P. Nkolola

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Avenue, FXB-310, Boston, MA 02115, USA

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Kati di Gleria,

Kati di Gleria

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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Andrew J. McMichael,

Andrew J. McMichael

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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Tomáš Hanke Dr.,

Corresponding Author

Tomáš Hanke Dr.

[email protected]

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hosptial, Oxford OX3 9DS, UK, Fax: +44-1865-222502Search for more papers by this author

Eung-Jun Im,

Eung-Jun Im

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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Joseph P. Nkolola,

Joseph P. Nkolola

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Avenue, FXB-310, Boston, MA 02115, USA

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Kati di Gleria,

Kati di Gleria

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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Andrew J. McMichael,

Andrew J. McMichael

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

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Tomáš Hanke Dr.,

Corresponding Author

Tomáš Hanke Dr.

[email protected]

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hosptial, Oxford OX3 9DS, UK, Fax: +44-1865-222502Search for more papers by this author

First published: 02 October 2006

https://doi.org/10.1002/eji.200636482

Citations: 19

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Abstract

As a part of a long-term effort to develop vaccine against HIV-1 clade A inducing protective T cell responses in humans, we run mutually complementing studies in humans and non-human primates (NHP) with the aim to maximize vaccine immunogenicity. The candidate vaccine under development has four components, pTHr.HIVA and pTH.RENTA DNA, and modified vaccinia virus Ankara (MVA).HIVA and MVA.RENTA, delivered in a heterologous DNA prime-MVA boost regimen. While the HIVA (Gag/epitopes) components have been tested in NHP and over 300 human subjects, we plan to test in humans the RENTA (reverse transcriptase, gp41, Nef, Tat) vaccines designed to broaden HIVA-induced responses in year 2007. Here, we investigated the four-component vaccine long-term immunogenicity in Mamu-A*01-positive rhesus macaques and demonstrated that the vaccine-induced T cells were multi-specific, multi-functional, readily proliferated to recall peptides and were circulating in the peripheral blood of vaccine recipients over 1 year after vaccine administration. The consensus clade A-elicited T cells recognized 50% of tested epitope variants from other HIV-1 clades. Thus, the DNA-MVA/HIVA-RENTA vaccine induced memory T cells of desirable characteristics and similarities to those induced in humans by HIVA vaccines alone; however, single-clade vaccines may not elicit sufficiently cross-reactive responses.

Abbreviations:

B-LCL:: B lymphoblastoid cell line
ICS:: intracellular cytokine staining
MVA:: modified vaccinia virus Ankara
NHP:: non-human primates
SFU:: spot-forming units

Introduction

Development of an effective HIV-1 vaccine is the best hope for curbing the HIV-1 pandemic. Most currently licensed vaccines work by induction of neutralizing antibodies; however, developing such vaccines for HIV-1 has proven extremely difficult 1. Although the design of vaccines that induce broadly neutralizing antibodies remains one of the main goals of HIV-1 research, a body of data suggests that induction of T cells might also confer a degree of protection 2. While HIV-1-specific T cells elicited by prophylactic vaccine cannot prevent infection of host cells by an incoming virus, they may substantially decrease tissue damage incurred during the acute phase of HIV-1 infection. In already infected individuals, stimulation of HIV-1-specific T cells may help to control HIV-1 replication, which may in turn delay progression to AIDS and reduce transmission to other individuals. Therefore, development of a safe, efficient and reliable vaccine inducing T cells is likely to both benefit healthy high-risk populations and increase the quality of lives for those already infected. In doing so, such a vaccine may slow the rate of the global HIV-1 pandemic.

The search for novel approaches evoking protective T cell responses against HIV-1 infection strongly relies on animal models. Although a great initial insight about vaccine performance and various novel vaccine vector combinations can be obtained in mice 3, 4, non-human primate (NHP) models were better predictors of the T cell immunogenicity in humans 5–7. A long list of similarities detected between the NHP and human data 6 argues that the NHP model can be used to better inform the clinicians on vaccine potential. However, even the best animal model cannot substitute for a clinical evaluation; pre-clinical and clinical vaccine studies should be carried out in parallel and accelerate together vaccine improvements.

In the absence of a clear single correlate of protection against HIV-1 infection 8, the quest for an HIV-1 vaccine remains at best semi-empirical. Therefore, evaluation of vaccine candidates should include multiple parameters such as frequency of vaccine-induced CD4⁺ and CD8⁺ T cells, T cell functionality in terms of their lytic activity, secretion of cytokines and ability to proliferate on antigen re-exposure, and the longevity of vaccine-induced memory T cells and their anatomical distribution. The frequency of vaccine responders indicates performance consistency.

HIV-1 is a highly variable virus 9 and a vaccine has to take this into account. There is an on-going debate whether or not CD8⁺ T cells raised against a single epitope variant will be able to recognize epitope variants of other clades, and if so, how efficiently. A number of studies have addressed this point with mixed results. While some studies clearly detected cross-clade-reactive HIV-1-specific CD8⁺ T cell responses 10–17, there is substantial evidence that CD8⁺ T cells can be highly sequence-specific 18–22. This was best demonstrated in systematic studies employing all possible single amino acid substitutions in each position of an MHC class I epitope, which suggested that as few as one in three epitope variants was recognized by a given T cell receptor 18, 23. This correlated well with theoretical predictions proposed for cross-recognition of MHC class I-restricted peptides by T cell receptors 24.

To assess the protective role of vaccine-induced T cells in the absence of any HIV-1-binding antibody, neutralizing or not, we constructed a DNA prime-modified vaccinia virus Ankara (MVA) boost candidate HIV-1 vaccine expressing a common immunogen HIVA derived from consensus HIV-1 clade A Gag p24/p17 sequences and a string of CD8⁺ T cell epitopes 25. Through gradual pre-clinical and clinical protocol improvements, we were able to demonstrate priming of HIV-1-specific T cell responses in 8/8 healthy and boost pre-existing HIV-1-specific responses in 16/16 HIV-1-infected vaccine recipients 26, 27.

While the HIVA immunogen has been an excellent model immunogen for both pre-clinical and clinical comparative studies of a number of vaccine vectors and vaccination regimens, for assessing the vaccine efficacy it is important to find formulations capable of inducing T cell responses specific for multiple HIV-1 proteins. Thus, to broaden responses induced by the HIVA vaccines, we designed a second immunogen RENTA derived from the reverse transcriptase, Env, Nef and Tat proteins of the consensus HIV-1 clade A sequences 28. We inserted the RENTA gene into plasmid DNA and MVA vectors, and demonstrated induction of RENTA-specific T cell responses in addition to those elicited by HIVA upon HIVA and RENTA vaccine co-delivery 28. Here, we characterized the DNA-MVA/HIVA-RENTA-elicited immune responses in rhesus macaques in terms of the T cell response longevity, breadth, functionality and ability to recognize CD8⁺ T cell epitopes from other HIV-1 clades. The vaccine performance in NHP is discussed in the context of our clinical trial results.

Results

Early vaccine-induced T cell responses

Five Mamu-A*01-positive macaques received two doses of the pTHr.HIVA/pTHr.RENTA DNA followed by two doses of the MVA.HIVA/MVA.RENTA vaccines (Fig. 1). These doses and timing mimicked those of the HIVA vaccines used in clinical trial IAVI 006 (manuscript in preparation). To facilitate detailed analyses of vaccine-induced CD8⁺ T cell responses in pre-clinical NHP studies, two immunodominant SIV-derived Mamu-A*01-restricted epitopes, CTPYDINQM (Gag) and STPESANL (Tat), were incorporated into the HIVA and RENTA immunogens, respectively 25, 28. Employing the corresponding peptides and overlapping peptide pools across the two immunogens, vaccine-elicited T cell responses recognizing multiple epitopes were readily detected after priming with the DNA vaccines (Fig. 2A). Following rMVA boost, at least the Gag- and Tat-specific T cells were capable of proliferation upon antigenic re-exposure (Fig. 2B).

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Rhesus macaque immunization schedule. The immunogenicity of four-component vaccine DNA-MVA/HIVA-RENTA was assessed in five Mamu-A*01-positive rhesus macaques. The animals received a 1-mg i.m. dose of each pTHr.HIVA and pTHr.RENTA DNA twice 4 wk apart followed by 4- or 16-wk interval before two intradermal doses of 5×10⁷ PFU of each MVA.HIVA and MVA.RENTA 4 wk apart. The HIVA and RENTA vaccines were needle-injected into the animals’ arms and thighs, respectively.

Identification of novel Mamu-A*01-binding peptides

To increase the power of our analysis and taking advantage of the macaque known tissue type, we identified novel Mamu-A*01-restricted T cell epitopes. The previously defined Mamu-A*01 binding motif 29 was used to scan the HIVA and RENTA amino acid sequences for potential binders (Fig. 3A). A total of five peptides were selected as candidate Mamu-A*01 binders: LSPRTLNAWV (LSP), FSPEVIPMF (FSP) and YSPVSILDI (YSP) from HIVA p24, and KTPKFRLPI (KTP) and GTPISPIETV (GTP) from RENTA reverse transcriptase (Fig. 3B). The LSP epitope is shared between the HIVA and SIV sequences and as a 9-mer LSPRTLNAW was identified previously as a Mamu-A*01-restricted CD8⁺ T cell epitope 30. The FSP epitope is part of a previously defined CD8⁺ T cell epitope restricted to human HLA-B57 associated with long-term non-progression 31. Interestingly, the GTP epitope of RENTA is an artificially formed epitope, of which the ‘Glu-Thr’ is coded for by restriction enzyme site Kpn I inserted to join two coding regions of the RENTA chimeric gene.

In order to assess the binding of each candidate peptides to the Mamu-A*01 molecule, a small-scale peptide-MHC class I complex refold was performed with each candidate peptide. Protein refolds in the presence of all predicted peptides yielded stable complexes, which upon mass spectrometry analysis produced peaks corresponding to the expected peptide relative molecular masses (Fig. 3C). Thus, all five newly identified peptides bound Mamu-A*01.

Vaccine-induced memory T cells can proliferate and produce cytokines

Next, we assessed whether the Mamu-A*01-binding peptides induced T cell responses in the vaccine recipients. Frozen PBMC samples from animals Joe, Jane and Judd from up to 36 wk after the last immunization were first expanded by peptide stimulation in culture for 12 days and production of cytokines was then measured using an intracellular cytokine staining (ICS) assay. The magnitude of expansion varied for individual animals and peptides, but among the animals, all identified epitopes of HIVA and RENTA corresponding to Mamu-A*01-binding peptides elicited memory T cells, which were able to proliferate and produce IFN-γ (Fig. 4A), suggesting strongly that these peptides represent bona fide epitopes. These newly defined epitopes were then used to assess induction of memory T cells at later time points. All available tested PBMC up to wk 54 of the schedule could expand and produce IFN-γ and TNF-α upon re-exposure to the appropriate peptide, with TNF-α production being generally more pronounced (Fig. 4B).

Induction of long-lasting CD8⁺ T cell responses

It is very rare that vaccinated animals are kept for over 1-year-long periods without further procedures or a pathogenic virus challenge. Therefore, having identified novel Mamu-A*01-restricted epitopes, we decided to measure vaccine-induced CD8⁺ T cell responses at these very late time points. Utilizing these epitope peptides in an ex vivo IFN-γ ELISPOT assay, weak, but consistent HIV-1-specific responses were detected at four different time points between wk 70–76 of the vaccination schedule (Fig. 5A).

While responses to the marker epitopes of each immunogen HIVA and RENTA, which were selected for their immunodominance during SIV infection, were weak or borderline according to our criteria of positivity of at least twice and over 50 spot-forming units (SFU)/10⁶ PBMC above the no-peptide background, all tested animals had definite T cell responses to at least two other epitopes, namely FSP and GTP. These dominating responses had been detected as early as at wk 24 (not shown), suggesting that the relative contributions of individual epitopes to the overall responses remained stable over 1 year. At least for animals Jill and Jig, circulating T cells at wk 70 of the schedule were capable of in vitro expansion upon peptide stimulation and lysis of peptide-sensitised targets (Fig. 5B).

HIV-1 clade A vaccine-elicited CD8⁺ T cells displayed limited cross-reactivity to other epitope variants

There is uncertainty whether CD8⁺ T cells elicited by a single clade/sequence vaccine can recognize epitope variants of other clades. Peptides corresponding to 24 variants of the LSP, FSP, YSP and KTP epitopes relevant for HIV-1 infection (Los Alamos Laboratory HIV database) were synthesized and tested in an ELISPOT assay for stimulation of IFN-γ secretion by HIVA-RENTA vaccine-raised T cells. Overall, 50% of peptide variants were recognized (Fig. 6A). Although all three variants of the immunodominant FSP epitope triggered IFN-γ secretion, recognition of 5/6, 3/7 and 1/8 epitope variants were confirmed on both occasions for the LSP, YSP and KTP epitopes, respectively (Fig. 6B). Positive scoring of variants would not change even if more stringent criteria of three times and more than 50 SFU/10⁶ PBMC over the background were used. Note that variants FSP-2N, YSP-4A and YSP-2R were more efficient in the induction of specific IFN-γ release than their vaccine counterparts. Limited cell samples did not allow for testing cross-reactivity of consensus clade A vaccine-induced T cells against pooled peptides corresponding to other HIV-1 clades.

Discussion

In this work, we demonstrated that the pTHr-MVA/HIVA-RENTA vaccines efficiently induced HIV-1-specific CD8⁺ T cell responses in NHP. These responses were multi-specific, multi-functional, capable of proliferation and detectable for at least 1 year after vaccination. These are desirable features of vaccine-induced T cell memory.

Here, we added to the already long list of identified rhesus macaque epitopes 29, 30, 32 five novel HIV-1-derived T cell epitopes restricted by the Mamu-A*01 MHC molecule using a novel method for identification of MHC-binding peptides. The epitope identification enabled a detection of long-lasting vaccine-induced T cell responses and contributed to the usefulness of the rhesus macaque as a model for assessing candidate HIV-1 vaccine immunogenicity. Indeed, if the new epitope peptides were not utilized for the evaluation of long-term immunogenicity, some detected responses might have been missed. Measurements of vaccine-stimulated T cells in NHP 68 wk after vaccination are rare, if not unique, as animals are usually challenged 6 months after vaccination 6, 33, 34 and, consequently, long-term immunogenicity data in NHP are generally missing. Here, we demonstrated a generation of a long-lasting functional immunological memory by the DNA-rMVA heterologous regimen.

Vaccines for HIV-1 will have to be designed to deal with the enormous variability of HIV-1. The extent of CD8⁺ T cell cross-reactivity remains controversial with evidence both for 10–17 and against 18–22 its broad effectiveness. At least in the one animal tested on two occasions, we observed cross-reactivity of CD8⁺ T cells with 50% of tested epitope variants not present in the vaccine (Fig. 6). Although cross-reactivity of macaque Mamu-A*01-restricted T cell repertoire does not directly translate to humans, the underlying principles of the molecular interactions involved in the MHC-peptide/T cell receptor recognition are the same in the macaque and man. Thus, before vigorous T cells capable of recognition of multiple HIV-1 clades can be induced by active vaccination, it is prudent for the candidate HIV-1 vaccines to match the predominant clade in the target population and the breadth of T cell cross-reactivity should be closely monitored when the vaccines are used.

We have successfully used rhesus macaques to assess T cell immunogenicity of candidate vaccines and demonstrated several important parallels between this NHP model and humans. Thus, in our initial rhesus macaque study using polyepitope immunogen HW and the same pTH and MVA vectors as used here, we showed that the DNA prime-rMVA boost regimen was more potent for induction of T cells than MVA alone 35, 36, and that the tetramer frequencies of vaccine-induced T cells peaked 1 wk after the rMVA dosing and decreased thereafter 36. These results are similar to our recent data in humans 26, 27, with the difference in healthy HIV-1-uninfected individuals that in NHP an efficient boost was delivered even with the second and third rMVA dosing 36. Here, we showed that vaccine-induced responses were detectable over 1 year after vaccination. We are currently re-bleeding vaccine recipients from our early trials, i.e. 1–5 years after vaccination, to assess the longevity of responses induced by the pTHr.HIVA-MVA.HIVA regimen in humans and are finding responses in about two thirds of volunteers (Nicola Winstone, unpublished).

We and others demonstrated multi-specific multi-functional T cell responses induced by the DNA-rMVA regimen in NHP (here and 3, 37, 38), again similar to results in humans, with the exception that human responses were biased towards the CD4⁺ T cell compartment 27, 39–41. This CD4⁺ T cell bias might be due to differences in the relative vaccine doses used in NHP compared to those used in humans. For example, if the same vaccine dose is used in a 180-cm/80-kg man as in a 2-kg monkey, the man receives approximately 20× lower dose per body surface area 42. Another similarity between macaques and humans is that no discernible differences were observed between the two immunization schedules: short and long gap between the DNA and rMVA dosing did not dramatically affect the quality or longevity of responses, although small subject numbers were involved (manuscripts in preparation). Finally, because there is no adequate challenge model for the HIVA-RENTA vaccines other than using chimpanzee infection with HIV-1, the four-component vaccination has not been tested in NHP for protective efficacy.

In conclusion, we have demonstrated in NHP that the four-component pTHr-MVA/HIVA-RENTA candidate vaccine induced multifunctional and long-lasting memory T cells. These results together with parallel data from clinical tests and previous studies in NHP support further development of this vaccine approach and provide additional encouragement for our planned MVA/HIVA-RENTA efficacy studies in HIV-1-infected individuals during antiretroviral treatment secession.

Materials and methods

The vaccines

The preparation of the pTHr.HIVA, pTHr.RENTA, MVA.HIVA and MVA.RENTA vaccines was described previously 25, 28. The plasmid DNA and rMVA were prepared in compliance with Good Manufacturing Practice by Cobra Biomanufacturing (UK) and IDT (Germany), respectively.

Monkeys, vaccination and isolation of peripheral blood mononuclear cells

Rhesus macaques (Macaca mulatta), which were screened for the Mamu-A*01 allele of MHC class I (D. I. Watkins, Wisconsin Regional Primate Research Center), received four successive immunizations, two using the plasmid pTHr.HIVA and pTHr.RENTA DNA and two delivering MVA.HIVA and MVA.RENTA (Fig. 1). The animals received 1 mg of each DNA in 0.5 mL of 140 mM NaCl, 0.5 mM Tris-HCl pH 7.7 and 0.05 mM EDTA i.m. or 5×10⁷ PFU of each MVA in 0.1 mL of 140 mM NaCl and 10 mM Tris-HCl pH 7.7 intradermally. Monkey PBMC were isolated using the Lymphoprep cushion centrifugation (Nycomed Pharma A/S) and either used fresh, or were frozen and stored in liquid nitrogen until use. All immunizations and venipunctures were carried out without sedation and the animals were regularly clinically examined. All procedures and care strictly conformed to the UK Home Office Guidelines.

Peptides and preparation of peptides pools

High-performance liquid chromatography-purified peptides were purchased from Sigma-Genosys (Cambridge, UK), with a purity of at least 80% by mass spectroscopy. Individual peptides were dissolved in dimethylsulfoxide (Sigma-Aldrich) to yield a stock of 10 mg/mL and stored at –80°C. Peptides used for mini-refolds were synthesized using the Advanced Chemtech automated synthesizer and yielded a purity of >95%.

Refold of Mamu-A*01/β₂-microglobulin/peptide complexes

The genes coding for the Mamu-A*01 heavy chain (kindly provided by Dr. D. I. Watkins) and human β₂-microglobulin light chain were modified as described previously 43. Both heavy and light chains of MHC were expressed in Escherichia coli strain BL-21, purified from inclusion bodies, denatured in 8 M urea, refolded in the presence of peptide and purified on FPLC and monoQ ion exchange columns.

Matrix-assisted laser desorption ionisation mass spectrometry of peptides

Bruker Daltonics Ultraflex matrix-assisted laser desorption ionisation time-of-flight was used to identify individual peptides. One microliter of saturated matrix α-cyano-4-hydroxycynammic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was added to 1 µL sample of Mamu-A*01/β₂-microglobulin/peptide complexes (approximately 1 µg of protein) dissolved in 0.1% trifluoroacetic acid, mixed and immediately applied onto a stainless metal target plate and left to dry at room temperature. The plate was then inserted into the matrix-assisted laser desorption ionisation mass spectrometer and the sample was analysed in reflector mode. Relative molecular masses were obtained in monoprotonated form adding 1 Da extra to the expected relative molecular mass.

In vitro restimulation of PBMC

Frozen PBMC were thawed and restimulated with 10 µM peptide in 200 µL R10 (RPMI 1640 supplemented with 10% FCS and penicillin/streptomycin) medium in a round-bottomed 96-well plate in 5% CO₂ at 37°C for 3 days. On day 3, cells were transferred to a 24-well plate and 2.5 mL R10 medium containing 500 U/mL human IL-2 was added to each well. On days 6 and 9, 1.5 mL of supernatants were removed and R10 containing IL-2 was added. For multiple rounds of restimulation, 5 µM peptide-pulsed irradiated autologous B lymphoblastoid cell lines (B-LCL) were added on days 13 and 23.

ICS and FACS analysis

After a prolonged peptide stimulation in culture, PBMC were washed twice with R0 (RPMI 1640 supplemented with penicillin/streptomycin) medium and rested in R10 containing no IL-2 or peptide for further 2 days. On the day of assay, 1×10⁶ PBMC were washed, incubated with 5×10³ B-LCL pre-pulsed with 5 µg/mL peptides in 5% CO₂ at 37°C for 90 min and Golgiplug (BD Biosciences) containing brefeldin A was added. After a further 6-h incubation, cells were washed with FACS wash buffer (PBS and 1% FCS), stained with CD8-PE (clone RPA-T8; BD Biosciences) at 4°C for 30 min and permeabilized with Cytofix/Cytoperm^TM solution (BD Biosciences) at room temperature for 20 min. Cells were then washed with Perm wash buffer (PBS and 1% FCS, 10% BD Perm/wash) and stained with anti-IFN-γ-FITC mAb (clone 4S.B3; BD Biosciences) or anti-TNF-α-FITC mAb (clone MAb11; BD Biosciences) at 4°C for 30 min. After the final washing step, cells were fixed with 10% CellFIX^TM (BD Biosciences) and all chromogen-labelled cells were analysed by flow cytometry using the CellQuest software (BD Biosciences).

IFN-γ ELISPOT assay

The frequencies of cells releasing IFN-γ upon restimulation using HIVA and RENTA-derived peptides or peptide pools was assessed in an ELISPOT assay. The procedures and reagents of the MABTECH kit (cat. No. 3420M-2A) were used throughout. Briefly, freshly isolated PBMC were rested without peptides at 37°C in 5% CO₂ for 8 h and incubated with peptides at concentration 5 μg/mL for further 20 h. After a culture stimulation, the cells were rested for 2 days before being tested for production of cytokines. The released IFN-γ was captured by mAb GZ-4 immobilized on the bottom of assay wells, visualized by combination of second mAb 7-B6-1 coupled to an enzyme and a chromogenic substrate (Bio-Rad), and the spots were counted using the AID ELISpot Reader System (Autoimmun Diagnostika).

In vitro CFSE killing assay

B-LCL target cells were unpulsed or pulsed with 10 μM peptide and labelled with 80 nM or 320 nM CFSE, respectively. These target cells were then combined in round-bottomed 96-well plate assay wells with 2-wk in vitro stimulated PBMC effector cells at effector-to-target ratio of 30:1, incubated at 37°C in 5% CO₂ for 6 and 16 h and analysed using a flow cytometry. Percent specific lysis was calculated as 100 – (number of pulsed / number of unpulsed surviving cells with effectors divided by number of pulsed / number of unpulsed surviving cells without effectors × 100) 44.

Acknowledgements

This work was supported by the Medical Research Council UK and the International AIDS Vaccine Initiative.

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Citing Literature

Volume36, Issue10

October 2006

Pages 2574-2584

Induction of long-lasting multi-specific CD8⁺T cells by a four-component DNA-MVA/HIVA-RENTA candidate HIV-1 vaccine in rhesus macaques