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Differential cytotoxic T-lymphocyte (CTL) responses in HIV-1 immunised sibling chimpanzees with shared MHC haplotypes
Immunology Letters 66 (1999) 61 – 67
Differential cytotoxic T-lymphocyte (CTL) responses in HIV-1 immunised sibling chimpanzees with shared MHC haplotypes Sunita Balla-Jhagjhoorsingh, Petra Mooij, Gerrit Koopman, Tom Haaksma, Vera Teeuwsen, Jonathan Heeney *, Ronald Bontrop Departments of Virology and Immunobiology, Biomedical Primate Research Centre, P.O. 3306, 2280 HV, Rijswijk, Netherlands Received 19 October 1998; accepted 18 November 1998
Abstract Cell mediated immune responses to HIV-1 and CTL responses in particular differ dramatically in infected individuals. This may largely be influenced by the immunogenetic differences of different individuals such as those encoded by the MHC. These differences may be difficult to dissect due to the immunosuppressive nature of HIV-1 infection itself. In order to reduce the variables associated with effects of the virus, one recombinant viral antigen was chosen from a particular HIV-1 variant (rgp120 of the clinical isolate HIV-1W6.1D). To minimise differences between outbred hosts, we chose two sibling chimpanzees from which the family pedigree and genetic segregation with respect to polymorphic MHC molecules was known. Immunisation induced strong antigen specific antibody and T-helper immune responses. The magnitude and persistence of the humoral and T-helper immune responses were comparable in both chimpanzees. However, CTL responses were only observed in one sibling. These responses were subsequently mapped to several distinct epitopes. The CTL response to the immunodominant epitope was found to be presented in the context of a MHC molecule which was shared by both siblings. The absence of a CTL response in the other sibling is not yet understood, but could not be attributed to MHC alleles that were not shared by these two chimpanzees. These findings suggest that other polymorphic immunoregulatory mechanisms such as those involved in antigen processing and presentation influence host CTL responses to HIV-1. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Chimpanzees; HIV-1; CTL; MHC; Vaccine; Immune responses
1. Introduction Evidence supporting the critical role of CTL responses in controlling HIV-1 infection has accumulated from a variety of different types of studies. These include prophylactic vaccine studies where protection against infection or against disease progression was observed and studies where naturally acquired protection was seen against either infection or disease progression. There are several examples of studies where protection from infection has been observed. To evaluate the * Corresponding author: Tel.: +31-15-2842661; fax: + 31-152843986; e-mail: [email protected].
efficacy of unproven HIV-1 candidate vaccines non-human primates have proven to be the best models. In addition to humans, chimpanzees are one of the few species susceptible to persistent infection with HIV-1 [1]. They are genetically 98% similar to humans. In several chimpanzee studies HIV-1 vaccines induced HIV-1 neutralizing antibodies, the titer of which correlated with protection [2–4]. Emini et al. demonstrated that passive transfer of a particular monoclonal virus neutralizing antibody was able to block infection, thus providing specific proof that virus neutralizing antibodies could alone be protective [5]. Also in SIV vaccine studies with other non-human primates such as rhesus macaques, protection from infection has been observed. Gallimore et al. found an inverse correlation with the
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precursor frequency of vaccine induced nef CTL and virus load [6]. In a separate study we found that when vaccinated macaques were challenged with infected blood cells from a donor with AIDS, protection correlated with sharing of a particular MHC class I molecule with the infected donor. This suggested the role of MHC restricted CTL in protection from cell-associated infection [7]. A correlation with increased levels of b-chemokines and HIV-1 vaccine protection has also been observed in macaques challenged with chimeric SHIV [8]. It is interesting to speculate that these chemokines may have been released at least in part from cytolytic granules in HIV-1 specific CTL[9]. More recently, in two separate HIV-1 vaccine efficacy studies in chimpanzees, evidence has emerged to suggest a role of CTL in protection of chimpanzees from infection [10,11]. Naturally acquired protection has also been observed in several studies. HIV-1 specific immune responses were detected in individuals although they remained HIV-1 negative after repeated exposure to the virus and high-risk activity. These include a specific group of prostitutes in Africa or partners of HIV-1 positive individuals [12–15]. Furthermore children born to HIV-1 infected mothers have been studied. In children who were born HIV-1 positive, some sero-reverted a few months after infection [16]. Some children who were born HIV-1 negative also had detectable HIV-1 specific immune responses [17 – 20]. Whether this means that the infection was cleared or whether the immune responses were a reaction to the infection of the mother remains under discussion. In ‘naturally protected’ individuals the HIV specific immune responses found mainly consist of cellular immune responses in most cases. In particular the presence of specific CTL responses have been repeatedly observed. If infection cannot be blocked or prevented by vaccines it will be important to induce immune responses capable of preventing progression to disease (AIDS). Vaccine induced protection from disease or from high levels of viraemia (as a marker of disease progression) has been achieved in several non-human primate studies [2,6]. Protection from disease may in certain cases be naturally acquired. In the human population a group of untreated individuals have not developed evidence of progressing to AIDS despite being infected for more than 10 years. In such long term survivors (LTS) persistent HIV specific CTL responses can be detected against conserved epitopes [21 – 24]. A small percentage of these individuals have genetic resistance which is not linked with the MHC but rather linked to mutations of HIV-1 co-receptors or related molecules [13,25 – 28]. In chimpanzees the CCR5 HIV-1 co-receptor is intact and infected animals do not have the mutations associated with resistance to AIDS in humans [29]. The elimina-
tion of infected cells by cytotoxic immune responses is of importance for the host to eliminate and control the intracellular viral reservoir [30–32]. It has been described that certain HIV-1 peptides are preferentially presented to CTL by certain MHC class I molecules [21,33,34]. Increased frequencies of these MHC molecules, especially HLA-B*27 and − B*57, are observed in various LTS cohorts [21,33,35]. Since CTL are directed at peptides presented by a specific MHC class I molecule, these MHC alleles are also a strong indication of the importance of CTL in controlling viral infection. Furthermore it has been reported that certain CTL responses may be protective by reducing viral load [36–39]. Interestingly, in certain HIV-1 infected chimpanzees CTL were detected which were directed at epitopes in HIV-1 gag [40]. These sequences were found to be highly conserved throughout the majority of HIV-1 clades. The same epitopes are also reported in humans and in particular in the LTS [21,34,36– 39,41,42]. Since CTL play a pivotal role in protection against infection as well as protection against disease, an optimal HIV-1 vaccine candidate should be able to induce strong cellular immune responses. CTL responses against epitopes that are conserved throughout the HIV-1 clades may be useful for vaccination programs. In order to minimize the number of individual host variables involved in the induction of CTL responses we immunized two sibling chimpanzees with a subunit vaccine (rgp 120 antigen of the clinical isolate HIV1W6.1D). The aim of this study was to determine if CTL responses could be uniformly induced in two siblings from the same parents.
2. Materials and methods
2.1. Animals studied In this study two adult sibling West African chimpanzees (Pan troglodytes 6erus), aged 14 and 15 years (Ch-Do and Ch-So, respectively), were immunised i.m. at week 0, 4, 24 and 44. Each vaccination dose consisted of 200 mg rgp 120 of the clinical isolate HIV1W6.ID formulated with the adjuvant SBAS2 in a total volume of 1 ml as previously described [43]. The control chimpanzee Ch-Pe, aged 16 years, was immunised with 200 mg of the control antigen gD of HSV-2 formulated in the same adjuvant.
2.2. CTL responses CTL responses were assayed as described previously [7,40], using en6 overlapping 20mer peptides. Overlapping peptides spanning the entire rgp120 of HIV-1IIIB (ARP-740, MRC) and HIV-lW6.ID (ARP-7035, MRC)
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were obtained from MRC AIDS Reagent project and consisted of 47 20mers for HIV-lIIIB or 48 20mers for HIV-lW6.ID overlapping by 10 amino acids spanning en6 amino acid residues 28 – 457. As control overlapping peptides spanning the gag of HIV-lSF2 were obtained from MRC AIDS Reagent project (ADP 788, MRC) and consisted of 22 20mers overlapping by 10 amino acids spanning gag amino acid residues 135 – 364. In brief, PBMC were cultured with irradiated autologous T cell blasts presenting the peptides at a ratio of 0.5-1:1. After two days 20 U/ml IL-2 (MRC) was added and after 8–14 days the cells were cultured again with irradiated autologous T cell blasts presenting the peptide as stimulator cells after removal of dead cells from the culture by LSM density gradient centrifugation. The cells were then tested in a conventional radioactive chromium release assay. Autologous B cells were used as target cells at 5000 cells per well. After 5 h incubation at 37°C supernatants were harvested and counted in a gamma counter (Cobra 5, Packard). Percentages of specific 51Cr release were calculated as 100×(experimental release− spontaneous release)(maximum release− spontaneous release). Cells were tested in duplicate or triplicate. Spontaneous releases were tested in quadruplicate. Assays were based on the use of three different E:T ratios. Responses were considered positive when the specific lysis detected was greater than 10% and specific lysis declined with declining E:T ratios.
labeled CD8 (DK25) or CD4 (OKT4), peroxidase labeled Rabbit anti-FITC (DAKO) and peroxidase labeled Swine anti-Rabbit (DAKO). All incubation steps were performed at room temperature for 30 min. Endogenous peroxidases were blocked with 0.1% NaN3 plus 0.3% H2O2 in PBS after the incubation with the first antibody. AP activity was detected with naphthol– AS–MX phosphate (Sigma, St. Louis, MO), and Fast Blue BB (Sigma) in 0.1 M Tris–HCl pH 8.5 (20 min in the dark) yielding a blue colour HRP activity was detected using H2O2 (0.03%) and 3-amino-9-ethylcarbazole (AEC, Sigma) yielding a red color.
2.3. Phenotypic characterization
3.1. Cytolytic responses
The phenotype of cytolytic T cell lines was assessed by triple color flow cytometry analysis (FACSort, Becton Dickinson, Morentos View USA), using fluorescein –isothiocyanate labeled anti-CD8 (Becton Dickinson); phycoerythin labeled anti-CD4 and phycoerythin-Cy5 labeled anti-CD3 (Becton Dickinson).
Throughout the immunisation period HIV-1 envelope specific CTL responses were only detected in one (Ch-So) out of the two rgp120 immunised chimpanzees. In PBMC from Ch-Do CTL to rgp120 were not observed, nor in the control chimpanzee Ch-Pe (Fig. 1). Gag specific CTL responses were not detected when tested at several time points in all three animals. In Ch-So a env specific CTL response was first detected 20 weeks post the second immunisation when tested on autologous B cells. The cytolytic responses were found to be elicited by CD3 + CD8 + effector cells as demonstrated by FACS analysis (Fig. 2). These effector cells were also positive for the cytotoxic granule associated enzymes GrA and B, TIA-1 and perforin (data not shown).
2.4. Immunocytochemistry The granzymes GrA and GrB, the RNA binding protein TIA-1 and perforin expression on CD8 and CD4 positive T cells was studied using a double staining technique. Cytospin preparations were fixed in acetone for 9 min, washed in PBS, and preincubated with normal goat serum (10% in PBS). Subsequently, the slides were incubated with anti-GrA (GA11) or -GrB (GB11), both kindly provided by Dr. E. Hack (CLB, Amsterdam, The Netherlands), anti-TIA-1 (Hialeah, FL, USA) or anti-perforin mAb (T cell Diagnostics, Wobum MA, USA), secondary biotinylated antibody (Goat anti-Mouse, DAKO, Glostrup, Denmark), a streptavidine–biotin – alkaline phosphatase complex (DAKO), normal mouse serum (5% in PBS, Jackson Immunoresearch Laboratories, West Grove, PN), FITC
2.5. MHC class I typing and characterization of restriction elements Chimpanzees were initially typed for their Patr class I antigens by serological methods [44]. The corresponding Patr class I nucleotide sequences were determined using the method described by Ennis et al. [45]. The correlation of serotypes and nucleotide sequences was established based on performing extensive segregation studies [46]. These restriction elements were further identified when testing cytotoxic reactivity of CTL of Ch-So on a panel of allogeneic Patr-A, -B and -C locus typed B cells as targets.
3. Results
3.2. Epitope mapping The CTL response in Ch-So was found to be directed against the pool of peptides covering the COOH terminus half of the HIV-1 envelope. Subsequent testing with smaller pools of peptides showed that the cytolytic activity was directed against three regions in en6 (Fig. 3). Positive CTL responses were seen against peptides
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Fig. 1. Cytolytic responses against pools of 20mer peptides with a 10mer overlap covering gag of HIV-1SF2 and en6 of HIV-1W6.ID Responses were measured in the two immunized chimpanzees, Ch-Do and Ch-So and the control animal Ch-Pe on autologous peptide pulsed B cells. The E:T ratio used was 5:1 after two rounds of stimulation with PBMC isolated 20 weeks after the 2nd immunization.
31, 43 and 44 of H1V-1W6.ID en6. The response, however, was not always found to be directed at all three peptides at all time points. The immunodominant CTL response was directed against the epitope in peptide 31 and found at all time points after this up to 40 weeks post the fourth immunisation. The other two CTL responses against epitopes in peptide 43 and peptide 44 were detected less frequently and were more difficult to characterise. From the immunodominant epitope in the 20mer peptide 31, 10mer peptides were synthesised with an 8mer overlap. The epitope in peptide 31 was found to be NTLKQIVIKL spanning the amino acid residues 318 to 327 (Fig. 3).
3.3. Characterisation of MHC restriction elements To determine the restricting MHC molecules for the immunodominant CTL response, an allogeneic B cell
Fig. 2. FACS scattergram of a CTL culture of Ch-So. Cultured cells were stained with PE labelled anti-CD4, FITC labelled anti-CD8 and RPE-Cy5 labeled anti-CD3 monoclonal antibodies. Percentage of the different T cell populations in culture is depicted in the quadrants.
panel was used. These cells were typed for the different Patr molecules and selected for the presence of known MHC class I and II nucleotide sequences. The autologous and (partly) matched allogeneic target B cells were pulsed with the peptide NTLKQIVIKL and subsequently tested with the effector cells of Ch-So (Fig. 4). The target B cells that were lysed were expected to share the same Patr allele that functions as restriction element. As expected, CTL from Ch-So against NTLKQIVIKL effectively lysed autologous target B cells and also cells from ChFr, Ch-Wo and Ch-Do. All these cells share the Patr-B*14 molecules indicating that the selected CTL from Ch-So recognize the en6 epitope NTLKQIVIKL in the context of Patr-B*14 molecules.
4. Discussion The HIV-1 rgp120 vaccine used in this study has been shown to induce strong humoral and cellular immune responses in rhesus macaques in which 10 out of 12 vaccinated rhesus macaques were protected from SHIV infection [43]. The two chimpanzees studied had the same parents and shared a particular MHC haplotype (Fig. 5). However, CTL responses were only seen in Ch-So. The assays revealed that there was a difference in CTL response in these two sisters. The most plausable explanation is that the CTL precursor frequency in Ch-Do was too low to detect with the methods used. The lack of peptide specific CTL in Ch-Do may have been due to a restricting MHC molecule not shared between the two siblings. Since Ch-So inherited a unique Patr-B*10 molecule from her mother Ch-Di and Ch-Do did not, we expected this to be the restricting MHC molecule for the CTL responses. Since Ch-Do would not be able to present the epitopes in context of the correct MHC molecules, a CTL response would not be induced. But when B cells of Ch-Do were pulsed with the immunodominant epitope NTLKQIVIKL, the cells were able to present the peptide in the context of the right MHC molecule. This demonstrated that B cells of Ch-Do are able to present the peptide to the T cells in the context of the Patr-B*14 molecule. Another consideration was the T cell recptor (TCR) repertoire of Ch-Do. The TCR repertoire is unique for every individual due to the large number of independent TCR gene re-arrangements which could well account for the different CTL responses between these closely related chimpanzees. Other viral infections that these chimpanzees have separately experienced may have influenced the TCR repertoire differently in these two animals. Alternatively, the lack of a HIV-1 specific CTL response could be due to a defect at the level of peptide processing or a TAP (transporter associated with anti-
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Fig. 3. Epitope mapping of the CTL responses against the CTL epitopes in en6 of HIV-1W6.ID in Ch-So against 20mer peptides with a 10mer overlap presented by autologous B cells. The bottom six bars represent the cytolytic activity against pools of peptides. The next five bars represent the cytolytic activity against individual peptides and then the next six bars represent the responses measured using custom made 10mer peptides with an 8mer overlap covering peptide 31. The response measured against the pool containing peptides 43 – 48 was defined with the individual peptides which are depicted in the top six bars.
gen) deficiency [47], preventing peptide transport and loading on the MHC class I molecules in this particular animal. This would then inhibit the presentation of the antigen to CTL. But since the target B cells could also process 20mers and present the correct epitope to CTL of Ch-So, this remains an unlikely explanation. Another factor playing a role could be the presence of different Killer cell inhibitory receptors (KIRs) in the two animals [48]. These KIRs are thought to have a regulatory role [49] by providing an inhibitory signal to cytotoxic cells in general preventing the release of the cytotoxic granules [50]. In Ch-Do the right epitope could be presented to the CTL. The KIRs in Ch-Do however could give a stronger inhibitory signal than the KIRs in Ch-So. This stronger signal would switch off the cytotoxic response of the CTL of Ch-Do. In Ch-So the cytotoxic response is obviously not blocked. It remains to be elucidated whether differences in KIR signals play a role in the observed discordance in CTL response in Ch-Do and Ch-So. Furthermore, it remains to be resolved whether a defect in certain co-stimulatory factors, like certain chemokines in Ch-Do, plays a role in the lack of HIV-l specific CTL responsiveness. In summary, rgp120 antigen formulated with the SBAS2 adjuvant was shown to induce strong and per-
sistent CTL responses in Ch-So. The CTL responses in Ch-So were found to be directed at three different epitopes in the HIV-1 env. The immunodominant CTL epitope was NTLKQIVIKL. Ch-Do had the same parents as Ch-So and their humoral as well as their T-helper immune responses were of the same magnitude and persistence (data not shown). The only observed difference in immune responses of these two siblings was the lack of HIV-1 specific CTL responsiveness in Ch-Do. This observation of a discordance in CTL response in related individuals may be important for our future understanding of how certain individuals respond differently to viral infections and vaccines.
Acknowledgements We thank Drs Ivonne G. Nieuwenhuis for her technical assistance and Jeannette Schouw for her secretarial assistance. Reagents were generously supplied by programme EVA (European Vaccine against AIDS). We are grateful for the fruitful collaboration with Dr C. Bruck, Dr G. Voss and colleagues at SBBio who have povided us with antigen and adjuvant formulations. This research was supported by the ‘European Cen-
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Fig. 4. MHC class I restriction for the epitope NTLKQIVIKL recognised by CTL of Ch-So. CTL of Ch-So were tested on a B cell panel of matched and mismatched targets. Shown are the typings of the Patr-A, -B and -C molecules, ?? and -- represent unknown or blank specificity’s. +: targets were recognised and lysed by CTL, -: targets were not recognised and lysed by CTL. In italics are the restricting Patr molecules shared by the targets recognised.
Fig. 5. Family tree of Ch-Do and Ch-So.
tralised Facility (ECCF) for Preclinical HIV-1 vaccine development’ (BMH4-CT97-2067). Part of the research reported was supported by the EC grant ‘Correlates of protection from HIV infection and AIDS’ (BMH4CT97-2055).
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Differential cytotoxic T-lymphocyte (CTL) responses in HIV-1 immunised sibling chimpanzees with shared MHC haplotypes Sunita Balla-Jhagjhoorsingh, Petra Mooij, Gerrit Koopman, Tom Haaksma, Vera Teeuwsen, Jonathan Heeney *, Ronald Bontrop Departments of Virology and Immunobiology, Biomedical Primate Research Centre, P.O. 3306, 2280 HV, Rijswijk, Netherlands Received 19 October 1998; accepted 18 November 1998
Abstract Cell mediated immune responses to HIV-1 and CTL responses in particular differ dramatically in infected individuals. This may largely be influenced by the immunogenetic differences of different individuals such as those encoded by the MHC. These differences may be difficult to dissect due to the immunosuppressive nature of HIV-1 infection itself. In order to reduce the variables associated with effects of the virus, one recombinant viral antigen was chosen from a particular HIV-1 variant (rgp120 of the clinical isolate HIV-1W6.1D). To minimise differences between outbred hosts, we chose two sibling chimpanzees from which the family pedigree and genetic segregation with respect to polymorphic MHC molecules was known. Immunisation induced strong antigen specific antibody and T-helper immune responses. The magnitude and persistence of the humoral and T-helper immune responses were comparable in both chimpanzees. However, CTL responses were only observed in one sibling. These responses were subsequently mapped to several distinct epitopes. The CTL response to the immunodominant epitope was found to be presented in the context of a MHC molecule which was shared by both siblings. The absence of a CTL response in the other sibling is not yet understood, but could not be attributed to MHC alleles that were not shared by these two chimpanzees. These findings suggest that other polymorphic immunoregulatory mechanisms such as those involved in antigen processing and presentation influence host CTL responses to HIV-1. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Chimpanzees; HIV-1; CTL; MHC; Vaccine; Immune responses
1. Introduction Evidence supporting the critical role of CTL responses in controlling HIV-1 infection has accumulated from a variety of different types of studies. These include prophylactic vaccine studies where protection against infection or against disease progression was observed and studies where naturally acquired protection was seen against either infection or disease progression. There are several examples of studies where protection from infection has been observed. To evaluate the * Corresponding author: Tel.: +31-15-2842661; fax: + 31-152843986; e-mail: [email protected].
efficacy of unproven HIV-1 candidate vaccines non-human primates have proven to be the best models. In addition to humans, chimpanzees are one of the few species susceptible to persistent infection with HIV-1 [1]. They are genetically 98% similar to humans. In several chimpanzee studies HIV-1 vaccines induced HIV-1 neutralizing antibodies, the titer of which correlated with protection [2–4]. Emini et al. demonstrated that passive transfer of a particular monoclonal virus neutralizing antibody was able to block infection, thus providing specific proof that virus neutralizing antibodies could alone be protective [5]. Also in SIV vaccine studies with other non-human primates such as rhesus macaques, protection from infection has been observed. Gallimore et al. found an inverse correlation with the
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precursor frequency of vaccine induced nef CTL and virus load [6]. In a separate study we found that when vaccinated macaques were challenged with infected blood cells from a donor with AIDS, protection correlated with sharing of a particular MHC class I molecule with the infected donor. This suggested the role of MHC restricted CTL in protection from cell-associated infection [7]. A correlation with increased levels of b-chemokines and HIV-1 vaccine protection has also been observed in macaques challenged with chimeric SHIV [8]. It is interesting to speculate that these chemokines may have been released at least in part from cytolytic granules in HIV-1 specific CTL[9]. More recently, in two separate HIV-1 vaccine efficacy studies in chimpanzees, evidence has emerged to suggest a role of CTL in protection of chimpanzees from infection [10,11]. Naturally acquired protection has also been observed in several studies. HIV-1 specific immune responses were detected in individuals although they remained HIV-1 negative after repeated exposure to the virus and high-risk activity. These include a specific group of prostitutes in Africa or partners of HIV-1 positive individuals [12–15]. Furthermore children born to HIV-1 infected mothers have been studied. In children who were born HIV-1 positive, some sero-reverted a few months after infection [16]. Some children who were born HIV-1 negative also had detectable HIV-1 specific immune responses [17 – 20]. Whether this means that the infection was cleared or whether the immune responses were a reaction to the infection of the mother remains under discussion. In ‘naturally protected’ individuals the HIV specific immune responses found mainly consist of cellular immune responses in most cases. In particular the presence of specific CTL responses have been repeatedly observed. If infection cannot be blocked or prevented by vaccines it will be important to induce immune responses capable of preventing progression to disease (AIDS). Vaccine induced protection from disease or from high levels of viraemia (as a marker of disease progression) has been achieved in several non-human primate studies [2,6]. Protection from disease may in certain cases be naturally acquired. In the human population a group of untreated individuals have not developed evidence of progressing to AIDS despite being infected for more than 10 years. In such long term survivors (LTS) persistent HIV specific CTL responses can be detected against conserved epitopes [21 – 24]. A small percentage of these individuals have genetic resistance which is not linked with the MHC but rather linked to mutations of HIV-1 co-receptors or related molecules [13,25 – 28]. In chimpanzees the CCR5 HIV-1 co-receptor is intact and infected animals do not have the mutations associated with resistance to AIDS in humans [29]. The elimina-
tion of infected cells by cytotoxic immune responses is of importance for the host to eliminate and control the intracellular viral reservoir [30–32]. It has been described that certain HIV-1 peptides are preferentially presented to CTL by certain MHC class I molecules [21,33,34]. Increased frequencies of these MHC molecules, especially HLA-B*27 and − B*57, are observed in various LTS cohorts [21,33,35]. Since CTL are directed at peptides presented by a specific MHC class I molecule, these MHC alleles are also a strong indication of the importance of CTL in controlling viral infection. Furthermore it has been reported that certain CTL responses may be protective by reducing viral load [36–39]. Interestingly, in certain HIV-1 infected chimpanzees CTL were detected which were directed at epitopes in HIV-1 gag [40]. These sequences were found to be highly conserved throughout the majority of HIV-1 clades. The same epitopes are also reported in humans and in particular in the LTS [21,34,36– 39,41,42]. Since CTL play a pivotal role in protection against infection as well as protection against disease, an optimal HIV-1 vaccine candidate should be able to induce strong cellular immune responses. CTL responses against epitopes that are conserved throughout the HIV-1 clades may be useful for vaccination programs. In order to minimize the number of individual host variables involved in the induction of CTL responses we immunized two sibling chimpanzees with a subunit vaccine (rgp 120 antigen of the clinical isolate HIV1W6.1D). The aim of this study was to determine if CTL responses could be uniformly induced in two siblings from the same parents.
2. Materials and methods
2.1. Animals studied In this study two adult sibling West African chimpanzees (Pan troglodytes 6erus), aged 14 and 15 years (Ch-Do and Ch-So, respectively), were immunised i.m. at week 0, 4, 24 and 44. Each vaccination dose consisted of 200 mg rgp 120 of the clinical isolate HIV1W6.ID formulated with the adjuvant SBAS2 in a total volume of 1 ml as previously described [43]. The control chimpanzee Ch-Pe, aged 16 years, was immunised with 200 mg of the control antigen gD of HSV-2 formulated in the same adjuvant.
2.2. CTL responses CTL responses were assayed as described previously [7,40], using en6 overlapping 20mer peptides. Overlapping peptides spanning the entire rgp120 of HIV-1IIIB (ARP-740, MRC) and HIV-lW6.ID (ARP-7035, MRC)
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were obtained from MRC AIDS Reagent project and consisted of 47 20mers for HIV-lIIIB or 48 20mers for HIV-lW6.ID overlapping by 10 amino acids spanning en6 amino acid residues 28 – 457. As control overlapping peptides spanning the gag of HIV-lSF2 were obtained from MRC AIDS Reagent project (ADP 788, MRC) and consisted of 22 20mers overlapping by 10 amino acids spanning gag amino acid residues 135 – 364. In brief, PBMC were cultured with irradiated autologous T cell blasts presenting the peptides at a ratio of 0.5-1:1. After two days 20 U/ml IL-2 (MRC) was added and after 8–14 days the cells were cultured again with irradiated autologous T cell blasts presenting the peptide as stimulator cells after removal of dead cells from the culture by LSM density gradient centrifugation. The cells were then tested in a conventional radioactive chromium release assay. Autologous B cells were used as target cells at 5000 cells per well. After 5 h incubation at 37°C supernatants were harvested and counted in a gamma counter (Cobra 5, Packard). Percentages of specific 51Cr release were calculated as 100×(experimental release− spontaneous release)(maximum release− spontaneous release). Cells were tested in duplicate or triplicate. Spontaneous releases were tested in quadruplicate. Assays were based on the use of three different E:T ratios. Responses were considered positive when the specific lysis detected was greater than 10% and specific lysis declined with declining E:T ratios.
labeled CD8 (DK25) or CD4 (OKT4), peroxidase labeled Rabbit anti-FITC (DAKO) and peroxidase labeled Swine anti-Rabbit (DAKO). All incubation steps were performed at room temperature for 30 min. Endogenous peroxidases were blocked with 0.1% NaN3 plus 0.3% H2O2 in PBS after the incubation with the first antibody. AP activity was detected with naphthol– AS–MX phosphate (Sigma, St. Louis, MO), and Fast Blue BB (Sigma) in 0.1 M Tris–HCl pH 8.5 (20 min in the dark) yielding a blue colour HRP activity was detected using H2O2 (0.03%) and 3-amino-9-ethylcarbazole (AEC, Sigma) yielding a red color.
2.3. Phenotypic characterization
3.1. Cytolytic responses
The phenotype of cytolytic T cell lines was assessed by triple color flow cytometry analysis (FACSort, Becton Dickinson, Morentos View USA), using fluorescein –isothiocyanate labeled anti-CD8 (Becton Dickinson); phycoerythin labeled anti-CD4 and phycoerythin-Cy5 labeled anti-CD3 (Becton Dickinson).
Throughout the immunisation period HIV-1 envelope specific CTL responses were only detected in one (Ch-So) out of the two rgp120 immunised chimpanzees. In PBMC from Ch-Do CTL to rgp120 were not observed, nor in the control chimpanzee Ch-Pe (Fig. 1). Gag specific CTL responses were not detected when tested at several time points in all three animals. In Ch-So a env specific CTL response was first detected 20 weeks post the second immunisation when tested on autologous B cells. The cytolytic responses were found to be elicited by CD3 + CD8 + effector cells as demonstrated by FACS analysis (Fig. 2). These effector cells were also positive for the cytotoxic granule associated enzymes GrA and B, TIA-1 and perforin (data not shown).
2.4. Immunocytochemistry The granzymes GrA and GrB, the RNA binding protein TIA-1 and perforin expression on CD8 and CD4 positive T cells was studied using a double staining technique. Cytospin preparations were fixed in acetone for 9 min, washed in PBS, and preincubated with normal goat serum (10% in PBS). Subsequently, the slides were incubated with anti-GrA (GA11) or -GrB (GB11), both kindly provided by Dr. E. Hack (CLB, Amsterdam, The Netherlands), anti-TIA-1 (Hialeah, FL, USA) or anti-perforin mAb (T cell Diagnostics, Wobum MA, USA), secondary biotinylated antibody (Goat anti-Mouse, DAKO, Glostrup, Denmark), a streptavidine–biotin – alkaline phosphatase complex (DAKO), normal mouse serum (5% in PBS, Jackson Immunoresearch Laboratories, West Grove, PN), FITC
2.5. MHC class I typing and characterization of restriction elements Chimpanzees were initially typed for their Patr class I antigens by serological methods [44]. The corresponding Patr class I nucleotide sequences were determined using the method described by Ennis et al. [45]. The correlation of serotypes and nucleotide sequences was established based on performing extensive segregation studies [46]. These restriction elements were further identified when testing cytotoxic reactivity of CTL of Ch-So on a panel of allogeneic Patr-A, -B and -C locus typed B cells as targets.
3. Results
3.2. Epitope mapping The CTL response in Ch-So was found to be directed against the pool of peptides covering the COOH terminus half of the HIV-1 envelope. Subsequent testing with smaller pools of peptides showed that the cytolytic activity was directed against three regions in en6 (Fig. 3). Positive CTL responses were seen against peptides
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Fig. 1. Cytolytic responses against pools of 20mer peptides with a 10mer overlap covering gag of HIV-1SF2 and en6 of HIV-1W6.ID Responses were measured in the two immunized chimpanzees, Ch-Do and Ch-So and the control animal Ch-Pe on autologous peptide pulsed B cells. The E:T ratio used was 5:1 after two rounds of stimulation with PBMC isolated 20 weeks after the 2nd immunization.
31, 43 and 44 of H1V-1W6.ID en6. The response, however, was not always found to be directed at all three peptides at all time points. The immunodominant CTL response was directed against the epitope in peptide 31 and found at all time points after this up to 40 weeks post the fourth immunisation. The other two CTL responses against epitopes in peptide 43 and peptide 44 were detected less frequently and were more difficult to characterise. From the immunodominant epitope in the 20mer peptide 31, 10mer peptides were synthesised with an 8mer overlap. The epitope in peptide 31 was found to be NTLKQIVIKL spanning the amino acid residues 318 to 327 (Fig. 3).
3.3. Characterisation of MHC restriction elements To determine the restricting MHC molecules for the immunodominant CTL response, an allogeneic B cell
Fig. 2. FACS scattergram of a CTL culture of Ch-So. Cultured cells were stained with PE labelled anti-CD4, FITC labelled anti-CD8 and RPE-Cy5 labeled anti-CD3 monoclonal antibodies. Percentage of the different T cell populations in culture is depicted in the quadrants.
panel was used. These cells were typed for the different Patr molecules and selected for the presence of known MHC class I and II nucleotide sequences. The autologous and (partly) matched allogeneic target B cells were pulsed with the peptide NTLKQIVIKL and subsequently tested with the effector cells of Ch-So (Fig. 4). The target B cells that were lysed were expected to share the same Patr allele that functions as restriction element. As expected, CTL from Ch-So against NTLKQIVIKL effectively lysed autologous target B cells and also cells from ChFr, Ch-Wo and Ch-Do. All these cells share the Patr-B*14 molecules indicating that the selected CTL from Ch-So recognize the en6 epitope NTLKQIVIKL in the context of Patr-B*14 molecules.
4. Discussion The HIV-1 rgp120 vaccine used in this study has been shown to induce strong humoral and cellular immune responses in rhesus macaques in which 10 out of 12 vaccinated rhesus macaques were protected from SHIV infection [43]. The two chimpanzees studied had the same parents and shared a particular MHC haplotype (Fig. 5). However, CTL responses were only seen in Ch-So. The assays revealed that there was a difference in CTL response in these two sisters. The most plausable explanation is that the CTL precursor frequency in Ch-Do was too low to detect with the methods used. The lack of peptide specific CTL in Ch-Do may have been due to a restricting MHC molecule not shared between the two siblings. Since Ch-So inherited a unique Patr-B*10 molecule from her mother Ch-Di and Ch-Do did not, we expected this to be the restricting MHC molecule for the CTL responses. Since Ch-Do would not be able to present the epitopes in context of the correct MHC molecules, a CTL response would not be induced. But when B cells of Ch-Do were pulsed with the immunodominant epitope NTLKQIVIKL, the cells were able to present the peptide in the context of the right MHC molecule. This demonstrated that B cells of Ch-Do are able to present the peptide to the T cells in the context of the Patr-B*14 molecule. Another consideration was the T cell recptor (TCR) repertoire of Ch-Do. The TCR repertoire is unique for every individual due to the large number of independent TCR gene re-arrangements which could well account for the different CTL responses between these closely related chimpanzees. Other viral infections that these chimpanzees have separately experienced may have influenced the TCR repertoire differently in these two animals. Alternatively, the lack of a HIV-1 specific CTL response could be due to a defect at the level of peptide processing or a TAP (transporter associated with anti-
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Fig. 3. Epitope mapping of the CTL responses against the CTL epitopes in en6 of HIV-1W6.ID in Ch-So against 20mer peptides with a 10mer overlap presented by autologous B cells. The bottom six bars represent the cytolytic activity against pools of peptides. The next five bars represent the cytolytic activity against individual peptides and then the next six bars represent the responses measured using custom made 10mer peptides with an 8mer overlap covering peptide 31. The response measured against the pool containing peptides 43 – 48 was defined with the individual peptides which are depicted in the top six bars.
gen) deficiency [47], preventing peptide transport and loading on the MHC class I molecules in this particular animal. This would then inhibit the presentation of the antigen to CTL. But since the target B cells could also process 20mers and present the correct epitope to CTL of Ch-So, this remains an unlikely explanation. Another factor playing a role could be the presence of different Killer cell inhibitory receptors (KIRs) in the two animals [48]. These KIRs are thought to have a regulatory role [49] by providing an inhibitory signal to cytotoxic cells in general preventing the release of the cytotoxic granules [50]. In Ch-Do the right epitope could be presented to the CTL. The KIRs in Ch-Do however could give a stronger inhibitory signal than the KIRs in Ch-So. This stronger signal would switch off the cytotoxic response of the CTL of Ch-Do. In Ch-So the cytotoxic response is obviously not blocked. It remains to be elucidated whether differences in KIR signals play a role in the observed discordance in CTL response in Ch-Do and Ch-So. Furthermore, it remains to be resolved whether a defect in certain co-stimulatory factors, like certain chemokines in Ch-Do, plays a role in the lack of HIV-l specific CTL responsiveness. In summary, rgp120 antigen formulated with the SBAS2 adjuvant was shown to induce strong and per-
sistent CTL responses in Ch-So. The CTL responses in Ch-So were found to be directed at three different epitopes in the HIV-1 env. The immunodominant CTL epitope was NTLKQIVIKL. Ch-Do had the same parents as Ch-So and their humoral as well as their T-helper immune responses were of the same magnitude and persistence (data not shown). The only observed difference in immune responses of these two siblings was the lack of HIV-1 specific CTL responsiveness in Ch-Do. This observation of a discordance in CTL response in related individuals may be important for our future understanding of how certain individuals respond differently to viral infections and vaccines.
Acknowledgements We thank Drs Ivonne G. Nieuwenhuis for her technical assistance and Jeannette Schouw for her secretarial assistance. Reagents were generously supplied by programme EVA (European Vaccine against AIDS). We are grateful for the fruitful collaboration with Dr C. Bruck, Dr G. Voss and colleagues at SBBio who have povided us with antigen and adjuvant formulations. This research was supported by the ‘European Cen-
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Fig. 4. MHC class I restriction for the epitope NTLKQIVIKL recognised by CTL of Ch-So. CTL of Ch-So were tested on a B cell panel of matched and mismatched targets. Shown are the typings of the Patr-A, -B and -C molecules, ?? and -- represent unknown or blank specificity’s. +: targets were recognised and lysed by CTL, -: targets were not recognised and lysed by CTL. In italics are the restricting Patr molecules shared by the targets recognised.
Fig. 5. Family tree of Ch-Do and Ch-So.
tralised Facility (ECCF) for Preclinical HIV-1 vaccine development’ (BMH4-CT97-2067). Part of the research reported was supported by the EC grant ‘Correlates of protection from HIV infection and AIDS’ (BMH4CT97-2055).
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