Cross‐clade recognition of p55 by cytotoxic T lymphocytes in HIV‐1 infection
McAdam, Stephen1,4; Kaleebu, Pontiano2; Krausa, Peter1,5; Goulder, Philip1; French, Neil2; Collin, Beth1; Blanchard, Tom1; Whitworth, Jimmy2; McMichael, Andrew1; Gotch, Frances3,6
1Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK
2Medical Research Council Programme on AIDS in Uganda, Uganda Virus Research Institute, Entebbe, Uganda
3Department of Immunology, Imperial College School of Medicine, Chelsea and Westminster Hospital, London, UK.
4Present address: Institute of Transplantation Immunology, Rikshospitalet, Oslo, Norway
5Present address: Applied Biosystems Division, Perkin Elmer, Foster City, California, USA.
6Requests for reprints to: Dr Frances Gotch, Department of Immunology, Imperial College School of Medicine, Chelsea and Westminster Hospital, Fulham Road, London SW10 9NH, UK.
Sponsorship: The work was supported by the Medical Research Council of Great Britain (grant no. G9406578) and Crusaid-Star.
Date of receipt: 20 August 1997; revised: 16 January 1998; accepted: 29 January 1998.
Objectives: To evaluate cross-clade recognition of p55 antigen by cytotoxic T lymphocytes (CTL) in persons infected with diverse clades of HIV-1; to facilitate the development of a CTL-inducing vaccine to prevent transmission of multiple clades of HIV-1.
Design: Experiments were designed to evaluate whether persons in Uganda and the United Kingdom, infected with diverse clades of HIV-1, have CTL capable of recognizing and killing autologous target cells infected with recombinant vaccinia viruses (rVV) expressing the Gag protein from A, B, C and D clade HIV-1. The extent of cross-reactivity within such individuals, each infected with characterized virus, might reflect the type of cross-reactive immune response inducible by a monovalent vaccine.
Methods: Asymptomatic HIV-positive individuals were fully tissue-typed by ARMS (amplification of refractory mutation system) polymerase chain reaction. rVV expressing the Gag protein from identified A, B, C and D viruses were prepared. CTL were cultured and tested for cytolytic activity on autologous rVV-infected or peptide-pulsed B cells.
Results: Ugandan patients had inducible CTL responses recognizing A, B, C and D clade HIV-1 Gag. The majority of UK patients had inducible CTL responses that recognized two or more clades. No patient showed any HIV-2 cross-reactivity. Cross-reactive responses were characterized in three Ugandan patients.
Conclusions: Most patients tested mounted cross-reactive CTL responses that recognized Gag proteins from clades of HIV-1 other than those with which they were infected.
HIV-specific cytotoxic T lymphocyte (CTL) recognition of short viral peptide fragments bound to major histocompatibility complex (MHC) class I molecules on an infected cell can result in lysis of the infected cell, activation of the T cell and release of interferon-γ and tumour necrosis factor-α . Correlations have been shown between the rapid decrease in plasma viraemia following primary infection with HIV and the emergence of high levels of virus-specific human leukocyte antigen (HLA)-restricted CTL very early in disease [2,3]. A negative correlation has been shown between HIV-2-specific CTL activity and HIV-2 viral load, whereas in HIV-1 infection high levels of CTL have been associated with non-progression to disease [4–6]. Several groups have reported HIV-specific CTL activity in HIV-negative babies born to seropositive mothers [7–9]. Similarly, others have reported HIV-specific CTL in highly exposed seronegative adults [10,11], which has led to suggestions that CTL may have a protective role. Indeed, Gallimore et al.  found that following vaccination with recombinant vaccinia virus (rVV) expressing simian immunodeficiency virus Nef, macaques showed a negative correlation between the Nef-specific CTL precursor frequency measured prior to challenge, and viral load following challenge. These results have fuelled interest in the development of CTL-based prophylactic vaccines for HIV.
The genetic diversity of HIV-1 is a major obstacle to overcome if an effective prophylactic vaccine, which can be used worldwide, is to be developed. Nucleotide sequence analysis of the env or gag genes of HIV-1 isolates reveals two distinct groups, O and M. Viruses within the M group can be further subdivided into at least 10 subtypes or clades (A–J) [13–15]. Although the ability of patient sera to neutralize isolates in vitro does not appear to be influenced by viral subtypes [16,17], very little is known about CTL responses to non-B clade viruses. Examination of the Los Alamos database reveals that most regions of HIV that contain known CTL epitopes display at least some variation between isolates which may be of immunological consequence. Even single conservative amino-acid substitutions within the peptide epitope can be sufficient to abolish recognition by some CTL or result in an altered recognition and an inappropriate response in others [18,19].
It is of considerable importance to evaluate whether vaccines generated from subtype B viruses might induce cross-reactive CTL capable of recognizing cells infected with virus of another clade. We therefore wished to evaluate whether persons in Uganda and the United Kingdom infected with A, B, C or D clade virus had, within their CD8+ T-cell population, cytolytic cells capable of recognizing and killing autologous target cells infected with rVV expressing the Gag protein from A, B, C and clade D HIV-1. The extent of cross-reactivity within the population of individuals, infected with different clades of virus, might reflect the type of cross-reactive immune response that could be induced by a monoclade vaccine.
Genetic diversity in different populations, as exemplified by the presence of diverse HLA class I molecules, has also to be taken into account in studies such as these. Specific peptide epitopes from clade B viruses have been shown to be presented by common Caucasian class I HLA molecules, and such peptides may be used to restimulate low levels of CTL precursor cells in suitably vaccinated individuals . It will therefore be necessary to identify viral epitopes presented by class I molecules that are common in Uganda, in order for us to assess the immunogenicity of any putative CTL-inducing vaccine in Uganda.
Materials and methods
Ugandan donors were selected from asymptomatic HIV-infected individuals attending the Uganda Virus Research Institute and the AIDS Support Organization clinics, in Entebbe. UK donors were selected from HIV-infected individuals attending genitourinary clinics in Oxford and London.
Viral genetic subtyping
Viruses infecting individual patients were subtyped where possible using the envelope heteroduplex mobility assay (HMA) . Patients T014, T029 and T010 were infected with A clade virus. Patients U003 and T007 were infected with either A or C clade virus, and further analysis with Gag HMA and sequencing is currently being conducted. Patient T009 was infected with D clade. Patients pt84, pt91, 36M, 065, 0120, 008 and 868 were all Caucasians infected with B clade virus and were from the United Kingdom. Patient 46D was of unknown ethnic origin and the infecting clade of virus was not identified. Patient EW was infected in Mombassa, Kenya and therefore may have been infected with non-B clade virus.
Tissue typing was performed at HLA class I and class II by ARMS (amplification of refractory mutation system) polymerase chain reaction (PCR) using sequence-specific primers [22,23].
Recombinant vaccinia viruses
For viruses A2, C and D, p55 of gag was amplified by PCR from plasmids kindly supplied by F. McCutchan (Henry M. Jackson Foundation, Rockville, Maryland, USA) using the Pyrococcus furioufus enzyme with the following primers: 5′-GTAACCATGGGTGCGAG AGCG-3′ and 5′-GGCCAGATCTCATGATTTGAG GGAA-3′. PCR products were then digested with the BglII and NcoI restriction enzymes and cloned into pSC11 vector, which was precut with NcoI and BglII. The p55 DNA for virus A1 was originally amplified from a Ugandan patient and cloned into a modified pCDNA3 plasmid (a kind gift from P. Balfe, University College Hospital, London, UK). This fragment was then excised by digestion with BamHI and NcoI and subcloned into pSC11, which was precut with NcoI and BglII. Preparations of 500 ng of plasmid DNA of each clone were then prepared using a CsCl protocol and then sequenced using the Sequenase Version 2.0 protocol (United States Biochemical Corporation, Cleveland, Ohio, USA).
rVV were prepared and purified according to well established protocols . These novel vaccinia constructs were tested for their ability to present antigens to two established CTL lines and a CTL clone.
Vaccinia expressing the B subtype Gag has been described elsewhere . Vaccinia expressing HIV-2 Gag was supplied by Transgene (Strasbourg, France) and expressing influenza A PB2 (basic polymerase 2) was a kind gift from Dr B. Moss (National Institutes of Health, Bethesda, Maryland, USA). Vaccinia expressing influenza A nucleoprotein (NP) was prepared in our laboratory.
Overlapping peptide sets from HIV-1SF2 p24 and HIV-1SF2 p17 were obtained from the UK Medical Research Council AIDS Reagent Project. These were all 20-mers with a 10 amino-acid overlap. Six pools of peptides were prepared and tested at a final concentration of 10−5 mol/l for each individual peptide. Each pool consisted of between five and seven individual peptides. Single peptides were tested at a concentration of 10−6 mol/l.
Generation of bulk-cultured CTL
Bulk-cultured CTL were generated as previously described . CTL activity was first tested after 14 days of culture.
Chromium release assays
All targets used were Epstein-Barr virus-transformed B cell lines (BCL). Target cells were infected with three plaque-forming units of vaccinia per cell for 2 h and then incubated in R-10 overnight. Targets were labelled with 150 µCi 51Cr (Amersham International, Amersham, Buckinghamshire, UK) before being washed three times and plated into U-bottomed 96-well plates at 5 × 103 cells/well in 50 µl. Effectors were added to 100 µl of RPMI medium plus 10% fetal calf serum and incubated with the targets for 6 h. The approximate efficiency of infection of the targets was checked where appropriate by resuspending the remaining targets in 500 µl of 300 µmol/l X-gal in double-distilled H20, before incubating at 37°C for 30 min. Percentage specific lysis was calculated using the following formula: 100 × (E – M)/(T – M), where E is the Cr released into 20 µl supernatant from wells containing targets and effectors, M is the Cr released into wells containing targets and medium only, and T is the Cr released from wells containing targets lysed by the addition of 5% Triton-X100. Spontaneous release [100 × (M/T)] was always < 30%. For peptide-pulsed targets, B cells were pulsed with peptide (or pools of peptides) for 1 h after Cr-labelling before being washed as above. A positive CTL assay was defined as one in which the lysis of specific target cells was more than 10% greater than the lysis of irrelevant target cells (autologous B cells infected with rVV influenza NP or rVV influenza PB2 or incubated with medium instead of peptide).
Recognition of non-clade B peptides by a CTL clone derived from a clade B-infected patient
Many of the HLA-B8-positive patients infected with clade B viruses that we have previously investigated make strong CTL responses to an octamer peptide in p17 of Gag . To test whether B clade p17-specific CTL were likely to recognize viral isolates circulating within Uganda, we sequenced proviral DNA from 10 randomly selected Ugandan patients and synthesized the peptides encoded in these sequences. These were then tested for recognition by cloned CTL, from a patient infected with a clade B virus, that were stimulated using a peptide that corresponds to the B clade consensus sequence. Figure 1 shows that none of the Ugandan variant sequences showed significant cross-reactivity even at very high peptide concentrations (although one Ugandan patient had provirus encoding for the index sequence). Although this degree of CTL specificity has been described for many other CTL lines and clones investigated, with different HLA restriction patterns and with different specificities, such data may give a misleading impression and the polyclonal response that a patient actually makes in vivo to a whole antigen or series of antigens may be far more heterogeneous. We therefore decided to prepare rVV expressing Gag p55 from HIV isolates collected from diverse geographical regions and representative of different HIV subtypes (Table 1) in order to evaluate polyclonal cross-clade recognition of p55 by CTL.
Presentation of rVV to HIV-specific CTL
The presentation of new recombinants (rVV-gagA1, rVV-gagA2, rVV-gagC, rVV-gagD) to CTL and the resulting lysis as measured in Cr-release assays was checked using two cell lines and the clone shown in Fig. 2. The 24-14 cell line (derived from patient 868) was specific for an HLA-B*27-restricted epitope (amino acids 263–272), which was invariant between the different isolates, and resulted in comparable levels of lysis in targets infected with the different recombinants (Fig. 2a). An HLA-B*0801-restricted 24–13 (amino acids 259–267) variant-specific clone from patient 008 caused similar levels of lysis when incubated with HLA-B*0801-positive targets infected with the rVV constructs (Fig. 2b). This clone has been shown to recognize variant 24–13 peptides with either D or E at position 2. In contrast, a CTL line specific for an HLAA*0201-restricted Gag epitope (amino acids 77–85; also from patient 868) failed to recognize targets infected with either of the constructs expressing Gag p55 cloned from A clade isolates. Sequence analysis of this epitope in different clades suggested that the lack of recognition may have been due to a phenylalanine substituted for tyrosine at the third position of this epitope in Gag p55 expressed by both of the A clade rVV. The variation within this epitope in the D clade rVV (isoleucine at position 5) was well recognized by patient 868 CTL and corresponded to a natural variant found in his provirus (unpublished data). The above data confirms that target cells infected with rVV expressing p55 from A, C and D clades of HIV-1 were able to present Gag antigens in an appropriate fashion to CTL.
Cross-clade recognition of p55 by CTL
To test whether the response to Gag was cross-reactive between clades we cultured CTL from individuals infected with different clades of HIV-1 restimulated with autologous virus, and then tested for recognition of autologous Epstein-Barr virus-transformed B cells (BCL) infected with the different rVV. Although this restimulation system may not have been the most sensitive method of inducing CTL in culture, it did avoid introducing any specificity bias that may have been induced by the use of peptides or vaccinia for restimulation, and should therefore better reflect CTL specificity in vivo. Out of six Ugandan patients tested, five (U003, T009, T010, T014 and T029) had inducible CTL responses that were able to recognize the rVV expressing all four different clades of HIV-1 Gag (Fig. 3a). The remaining patient (T007) made a response that was specific for rVV-gagA2 (HIV-1). The response in T007 was more than clade-specific as autologous target cells infected with rVV-gagA1 were not recognized. This patient appeared to have a response that was completely specific for A2 Gag p55, and his CTL were therefore probably recognizing an epitope that was different in A1 and A2 Gag. This patient was shown by HMA to be infected with A or C clade virus. One patient (U003), also infected with A or C clade virus, although having CTL showing some cross-reactivity between clades, preferentially recognized A1 Gag.
None of the patients showed any HIV-2 Gag cross-reactivity when lysis of HIV-2 rVV-gag-infected target cells was compared with lysis of autologous B cells infected with rVV-PB2 or uninfected cells.
Out of seven patients tested in the United Kingdom (five out of seven infected with B clade HIV-1), the majority had CTL responses (> 10% greater lysis than that of the same target cells infected with an irrelevant rVV) that recognized two or more clades (Fig. 3b). Patient EW, who was infected with HIV-1 in East Africa and therefore may have been infected with non-B clade virus, made a CTL response recognizing A and C clade Gag only. Patient 36M had cross-reactive CTL that were capable of recognizing all clades except D. Patient 46D had CTL which primarily recognized autologous target cells expressing rVV A2 Gag with a weak response to clade D Gag. Patient pt84 had cross-reactive CTL but target cells infected with rVV-gagD were seen best. In patient pt91, high background lysis of uninfected target cells or target cells infected with rVV-PB2 was seen but specific recognition of target cells expressing rVV B Gag was observed. Both patients 065 and 0120 made cross-reactive CTL responses to autologous target cells expressing p55 from all the HIV-1 clades tested.
None of these patients showed any HIV-2 Gag cross-reactivity when lysis of rVV HIV-2 Gag target cells was compared with lysis of autologous cells infected with rVV-PB2 or uninfected cells.
Further characterization of the cross-reactive CTL response in three Ugandan patients
In three Ugandan patients (U003, T009 and T029), further characterization of the cross-reactive CTL response was carried out using individual peptides and peptide pools from HIV-1SF2 p24 and HIV-1SF2 p17 described above. Patient U003 had made a cross-reactive CTL response with a preference for A1 (Fig. 3a). When tested with pools of peptides this patient made a highly specific response to the pool of peptides p24-12 to p24-17 (Fig. 4a). When CTL from patient U003 were tested with autologous target cells pulsed with individual peptides, a clear response was seen to peptide p24-17 (Fig. 4b). When sequence analysis was performed, several amino-acid changes were seen within this 20-mer in the different clades (Fig. 4c). Since CTL from this individual appeared to have a preference for A1 Gag, this suggests that either the cross-reactive epitope recognized was at the aminoterminus of the peptide, which is relatively invariant in HIV-1 (but with two major substitutions in HIV-2), and that there was a second A1-specific response that was not seen in our assays because we were using SF2-based peptides, or that the epitope was at the carboxy-terminus of the peptide (which is more variable) with the A1 specificity determined by the change of AE to KL. In either case, HIV-2 non-cross-reactivity was accounted for by the changes seen in the HIV-2 sequence. This patient had class I HLA-A*1/A*3001, B*71/B*50, C*03/C*04 and it must be assumed that an epitope contained within p24-17 was being presented by one of these molecules. To define the MHC restriction of this epitope, matched BCL that share single class I molecules with U003, were tested for their ability to present p24-17 to U003 CTL. It was observed that only autologous BCL and a BCL sharing HLA-B*71 were recognized when prepulsed with p24-17. It is therefore likely that HLA-B*71 was the restricting element for p24-17.
CTL from patient T009 were cross-reactive and able to recognize autologous target cells infected with rVV expressing p55 from all clades tested (Fig. 3a). When CTL from this patient were tested with autologous target cells pulsed with pools of peptides, a clear response was seen to pool p24-18 to p24-22 (Fig. 4d). Furthermore, a specific response to peptide p24-22 was seen (Fig. 4e). When sequence analysis of this epitope within the different clades was performed, the peptide was seen to be invariant at the amino-terminal and it seemed likely that the peptide epitope recognized was within this area of the peptide (Fig. 4f). Such positioning would suggest that the non-recognition of HIV-2 Gag by CTL from this patient was due to the M→L change at position 4 of the 20-mer. Alternatively, the epitope could be at the carboxy-terminus and T009 CTL could tolerate the glutamic acid and serine substitutions at positions 13 and 15, respectively. The failure to recognize HIV-2 Gag rVV could then be accounted for by the glutamine and leucine substitutions at positions 16 and 20. This patient was tissue-typed and had class I HLA-A*30.02/A*6802, B*7/B*13, C*0602/ C*0702v, and it was again assumed that the epitope contained within p24-22 was being presented by one of these molecules. A panel of class I-matched BCL were used to define the MHC restriction of this epitope. Autologous BCL and a cell line sharing only HLA-B7 were able to present p24-22 to T009 CTL, and it was concluded that HLA-B7 was the restricting element for this peptide.
CTL from patient T029 were cross-reactive and able to recognize autologous target cells infected with rVV expressing p55 from all clades tested (Fig. 3a). When CTL from this patient were tested with autologous target cells pulsed with pools of peptides, a clear response was seen to pool p24-12 to p24-17 (Fig. 4g).
Furthermore, a specific response to p24-16 was seen (Fig. 4h). A comparison of this SF2 sequence with the p55 sequences from rVV showed that the central residues of the peptide were invariant between the HIV-1 sequences (Fig. 4i). If the CTL from T029 were specific for an epitope in the centre of this 20-mer, then the substitution of a phenylalanine and arginine at positions 11 and 12 of the peptide with a glutamine and serine in the HIV-2 Gag sequence would explain why this rVV was not recognized. This patient had class I HLA-A*30.02/A*6602, B*5801/B*81, C*04/C*0701, and it was assumed that the epitope contained within peptide p24-16 was being presented by one of these molecules.
Thus, the above experiments demonstrated preliminary characterization of cross-reactive peptide epitopes in relatively conserved parts of the Gag protein, which should ideally be included in a future vaccine.
We have shown extensive cross-reactivity in CTL responses in HIV-infected individuals between four of the major HIV-1 clades. Three of these clades are the predominant subtypes in regions of the world where there is greatest need for prevention of infection/disease, and where a vaccine is most likely to be tested. The first vaccines that are tested are likely to be based on B clade HIV-1, since most experimental laboratory work has been based on the B clade because it is prevalent in the developed world.
The data presented here demonstrate that responses of clonal CTL populations or of cultured long-term CTL lines that have been propagated using single specific peptides, may be very specific in their activity and may not reflect the broader response in vivo. However, it seems that most patients infected with diverse clades of HIV-1 have the capability to mount cross-reactive CTL responses that recognize the Gag proteins of clades of HIV-1 other than those with which they were infected. A few patients appear to make responses that are specific for Gag from a single clade. However, it has been reported  that most individual patients infected with HIV-1 make CTL responses specific for several peptide epitopes derived from, for example, Nef, Pol, and Env, as well as Gag. It seems likely, therefore, that the cross-reactivity that we are measuring is the minimum that might be found in any one patient. We may also be missing isolate-specific CTL responses. For example, if we had used a single rVV expressing p55 Gag from clade A1 virus when testing patient T007, we would not have seen any Gag-specific CTL response. Clearly this patient had CTL that recognized an epitope that was present in autologous virus and rVV A2 but was absent in rVV A1. No recombinant construct is going to be able to reflect absolutely the virus with which a patient is infected unless recombinants specific for each patient are prepared. Patients with a particular HLA class I type (such as HLA-B8) may make CTL responses that are specific for peptides from variable regions of the virus , and such individuals might be expected to show less cross-reactivity.
Despite initial reports , we failed to demonstrate the presence of CTL that were cross-reactive between HIV-1 and HIV-2. We were unable to test expression of HIV-2 Gag in cells infected with HIV-2 rVV-gag in the same way as the HIV-1 rVV-gag constructs were tested. However, the same stocks of HIV-2 rVV-gag are being used in ongoing experiments in West Africa where CTL from a large number of individuals infected with HIV-2 have been shown to recognize autologous target cells infected with HIV-2 rVV-gag. Some cross-reactive CTL have recently been reported in exposed but uninfected individuals who may have been exposed to both HIV-1 and HIV-2 . In this case, CTL were restimulated using specific peptides and may, or may not, reflect a normal in vivo response. Our results make the described, although not confirmed, putative protection offered by prior infection with HIV-2 to infection with HIV-1  unlikely to be mediated by CTL.
Many of our Ugandan patients may have been exposed to A, D and C clade virus (or may have been infected with recombinant viruses), but both patients EW and 065 had a documented single exposure to virus, so it seems unlikely that cross-reactivity is a function of multiple exposure. This is however a preliminary report, and we need to study many more patients to estimate the frequencies of cross-reactive CTL, to examine specificities other than to p55 Gag and to examine CTL activity in individuals infected with defined recombinants. We also need to elucidate whether these CTL responses are cross-reactive at the clonal level or whether, as seems more likely, we are measuring polyclonal responses that reflect the in vivo situation. In this respect we feel that a natural infection with a quasispecies of a dynamic virus such as HIV-1 may lead to good polyclonal cross-reactive cellular immune responses, whereas a vaccine based on a single sequence (in a recombinant vector or a DNA vaccine for example), may induce less cross-reactivity. A vaccine based on a live attenuated virus, as has been suggested by Desrosiers and coworkers [29,30], would be expected to induce cross-reactive responses.
In order to evaluate the CTL responses induced by vaccine preparations we need to continue to identify cross-reactive peptides restricted through common HLA class I molecules in genetically diverse populations that can be used in vaccine trials. Such peptides should be should be included in vaccine preparations and will be used in restimulation strategies in order to quantify CTL responses.
The authors thank the patients and staff at the Uganda Virus Research Institute and the AIDS Support Organization clinics for their cooperation and M. Bunce and M. Barnado from the Transplant Centre, Churchill Hospital, Oxford for help with the molecular tissue typing; also B. Biryahwaho and P. Tuguma for support in Uganda.
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This article has been cited 3 time(s).
Africa; CD8; cellular immunity; vaccine
© Lippincott-Raven Publishers.
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