Novel simian immunodeficiency virus CTL epitopes restricted by MHC class I molecule Mamu-B*01 are highly conserved for long term in DNA/MVA-vaccinated, SHIV-challenged rhesus macaques
Jin Su1,
Mark A. Luscher1,
Yelin Xiong1,
Tarick Rustam1,
Rama Rao Amara2,
Eva Rakasz3,
Harriet L. Robinson2 and
Kelly S. MacDonald1,4
1 Department of Medicine, University of Toronto, Toronto, Canada
2 Vaccine Research Center, Yerkes Primate Research Center and Department of Microbiology and Immunology, Emory University, School of Medicine, Atlanta, GA 30322, USA
3 Wisconsin Regional Primate Research Center, Madison, WI, USA
4 Department of Microbiology, Mount Sinai Hospital, 1484-600 University Avenue, Toronto, ON M5G 1X5, Canada
Correspondence to: K. S. MacDonald; E-mail: kmacdonald{at}mtsinai.on.ca
 |
Abstract
|
---|
Simian immunodeficiency virus (SIV) infection of rhesus macaques provides an excellent model for investigating the basis of protective immunity against human immunodeficiency virus (HIV). One limitation of this model, however, has been the availability of a small number of known MHC class I-restricted CTL epitopes for investigating virus-specific immune responses. We assessed CTL responses against SIV Gag in a cohort of DNA/modified vaccinia virus Ankara (MVA)-vaccinated/simian-human immunodeficiency virus (SHIV)-challenged rhesus macaques. Here, we report the identification of five novel SIV CTL epitopes in Gag for the first time (Gag3946 NELDRFGL, Gag169177 EVVPGFQAL, Gag198206 AAMQIIRDI, Gag257265 IPVGNIYRR and Gag296305 SYVDRFYKSL) that are restricted by the common MHC class I molecule Mamu-B*01. CTL responses to these epitopes were readily detected in cryopreserved PBMC in multiple animals up to 62 weeks post-infection, both by IFN-
enzyme-linked immunospot assay and intracellular IFN-
staining. Importantly, viral sequencing results revealed that these epitopes are highly conserved in the SIV-challenged macaques over a long period of time, indicating functional constraints in these regions. Moreover, the presence of CTL responses targeting these epitopes has been confirmed in two independent cohorts of rhesus macaques that have been challenged by SHIV or SIV. Our findings provide valuable candidates for poly-epitope vaccines and for long-term quantitative monitoring of epitope-specific CD8+ responses in the context of this common Mamu class I allele. It may thus help increase the supply of rhesus macaques in which epitope-specific immunity can be studied in the context of SIV vaccine design.
Keywords: CTL, MHC, rhesus macaque, SIV, vaccine
 |
Introduction
|
---|
The indian-origin rhesus macaque infected with simian immunodeficiency virus (SIV) or SHIV has become the most commonly used primate models for assessing the immunogenicity and protective efficacy of AIDS vaccines (1, 2). CD8+ T cell responses play a key role in the containment of immunodeficiency viruses and strong CTL responses have been correlated with reduced plasma viral RNA loads in both SIV-infected macaques and human immunodeficiency virus (HIV)-infected humans (3). It is therefore critical that multiple vaccine-elicited SIV/SHIV-specific CTL responses in macaques can be monitored with quantitative precision and ease. Currently, only a few SIV/SHIV CTL epitopes are defined in the context of their restricting macaque MHC class I molecules (46). The detection of epitope-specific SIV/SHIV CTL responses in macaques has been mostly limited to a single dominant Gag epitope, Gag181189 (CM9, CTPYDINQM), restricted by the MHC class I allele Mamu-A*01, which is expressed in
20% of Indian-origin rhesus macaques (7, 8). Furthermore, there is evidence that Mamu-A*01 can influence the results of vaccine studies in SIV-challenged models since this allele has been found significantly associated with lower set-point viral load and prolonged survival time (911). Similarly, another MHC class I allele, Mamu-B*17, has also been correlated with slower disease progression recently (12). To avoid confounding results for vaccine or treatment trials using the macaque model, animals with other MHC alleles should be included in such studies. For this reason, it would be of enormous benefit to have knowledge of further SIV CTL epitopes that are reliably presented to CD8+ T lymphocytes by other common rhesus monkey MHC class I alleles. Moreover, increasing the number of MHC haplotypes in which such studies can be performed should help to alleviate supply and breeding issues in rhesus macaques available for vaccine research (13).
We assessed a cohort of 24 DNA/MVA-vaccinated, SHIV-challenged rhesus macaques (14) that included a number of Mamu-B*01-positive animals, an allele for which the definition of SIV epitopes has previously proven difficult. Here, we report the definition of five highly conserved Gag CTL epitopes presented by Mamu-B*01 and confirm a Mamu-A*02 epitope that has recently been described. Furthermore, these epitopes have been validated in a second independent cohort of rhesus macaques that have been challenged by SIV.
 |
Methods
|
---|
Animals, vaccination and challenge
Young adult rhesus macaques were cared for under protocols approved by the Emory University Institutional Animal Care and Use Committee. A total of 24 animals were primed with DNA vaccine and boosted with MVA constructs followed by SHIV challenge (14, 15). For further analysis, seven rhesus macaques from another vaccine trial, J471, J547, J746, J562, J383, J211 and J231, were included subsequently. These seven animals were maintained in accordance with the National Institutes of Health guidelines with the approval of the University of Wisconsin Research Animal Resource Center review committee. The animals in cohort 2, except for the controls J211 and J746, were alloimmunized with DNA vectors encoding Mamu-A*01 or DRB*w201 MHC antigens without added antigenic peptides, boosted with vaccinia (strain NYVAC) virus containing the same MHC antigens and with recombinant Mamu-A*01 and DRB*w201 protein, as part of an SIV challenge trial (Y. Guan, M.A. Luscher, E. Rakasz et al. manuscript in preparation). All animals in cohort 2 were subsequently infected with SIVmac239.
Peptides
A 395-member set of 9- or 10-mer overlapping peptides corresponding to the sequence of SIVmac239 Gag was obtained from Mimotopes (Clayton, Victoria, Australia) and from Aventis Pasteur (Toronto, Canada). For fine mapping of CTL epitopes, 8-, 9- and 10-mer peptides were synthesized (Sigma-Genosys, Pittsburg, PA) corresponding to the SIVmac239 Gag regions of interest. All the peptides were dissolved in dimethyl sulfoxide (DMSO) (MP Biomedicals, Costa Mesa, CA).
MHC typing by PCR-sequence-specific primers
Genomic DNA was extracted from macaque PBMC using QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia CA). PCR was performed using sequence-specific primers (SSP) 5'-cagcgacgccgagagtcg-3' and 5'-ccgcggcggtccaggagt-3' for Mamu-B*01 (550-bp amplicon) and 5'-ggggccctggccctgact-3' and 5'-ctcgccctccaggtaggt-3' for Mamu-A*02 (920-bp amplicon). Mamu-DRB primers 5'-gccagtgtccccccagcacgtttc-3' and 5'-gcaagctttcacctcgccgctg-3' were used as an internal control (260-bp amplicon) (16). Thermal cycling conditions were as follows: denaturation for 1 min at 96°C was followed by five cycles of 96°C for 25 s, 70°C for 50 s and 72°C for 45 s; 21 cycles of 96°C for 25 s, 65°C for 50 s and 72°C for 45 s and four cycles of 96°C for 25 s, 55°C for 1 min and 72°C for 2 min. PCR amplicons were sequenced to confirm the identity of the MHC allele.
IFN-
enzyme-linked immunospot assay
A two-step approach was used to identify Gag epitopes using IFN-
enzyme-linked immunospot assay (ELISPOT) assay. First, the 395 peptides were organized in a matrix of 80 peptide pools, each pool containing 10 peptides (17). ELISPOTs were performed as previously described (18) with 2 x 105 cells per well in the presence of the peptide pools (10 µg ml1 for each peptide) to screen for candidate CTL epitopes. DMSO was used as a negative control and 0.2 µg ml1 staphylococcal enterotoxin B (SEB, Toxin Technology Inc., FL, USA) as a positive control. To avoid DMSO toxicity, the total amount of DMSO in culture was <0.5% (v/v) for all assays. Spot-forming cells (SFC) were counted using an image analyzer (KS ELISPOT, Zeiss, Oberköchen, Germany). A response was considered positive if the pool generated greater than double the DMSO background and had a minimum of 50 spots per 1 x 106 PBMC over the background. Second, each candidate epitope identified from the intersecting pools was tested individually by ELISPOT. DMSO negative controls usually gave little background, 520 spots per 1 x 106 PBMC, and this background was subtracted when calculating SFC for each sample. SEB positive controls usually yielded >2000 spots per 1 x 106 PBMC. A response was considered positive by the same criteria used in the first screen.
Intracellular IFN-
cytokine staining
IFN-
intracellular cytokine staining (ICS) by flow cytometry was performed to determine the MHC restriction to the new Gag epitopes. For Mamu-B*01, heterologous PBMC from five Mamu-B*01+ macaques cryopreserved prior to immunization and viral challenge, J211, J531, J525, J383 and J547, were used as target cells for antigen presentation. The MHC type of the target cells was confirmed by both PCR-SSP and sequencing. Mamu-B*01 target cells were chosen for these assays to otherwise maximize the extent of MHC mismatch with the responder cells. To control for antigen presentation by unknown allele, at least two different target cells were used to confirm the restriction. The frequencies of epitope-specific CD8+ cells were normalized since both effector and target cells were macaque PBMC in the same gate of FACS, in a ratio of effectors to targets of 2:1. For Mamu-A*02, a transfected B-cell line, 721.221.A*02, stably expressing Mamu-A*02 [kindly supplied by David Watkins (4)] was used. Heterologous macaque PBMC target cells were treated with 50 µg ml1 mitomycin C (SigmaAldrich Co., St Louis, MO, USA) and washed three times. Target cells (1 x 105 mitomycin C-treated heterologous PBMC or 721.221.A*02 cells) were pulsed with a single peptide at a concentration of 40 µg ml1, or in a range of concentrations 0.440 µg ml1 for fine mapping, at 37°C overnight and washed three times before being mixed with effector PBMC. Target cells pulsed with DMSO were used as a negative control and with SEB (0.2 µg ml1) as a positive control. Target cells pulsed with one Gag peptide that was shown negative by ELISPOT was used as a second negative control. Cryopreserved effector PBMC were thawed and cultured overnight at 37°C in RPMI culture medium with 10% FBS before use. Effector PBMC (4 x 105) were mixed with the pulsed target cells followed by incubation at 37°C for 6 h in the presence of 10 µg ml1 Brefeldin A (BD Biosciences, San Jose, CA, USA). Cells were permeabilized with FACS Permeabilizing Solution 2 (BD Biosciences) and stained with FITCanti-CD3, PEanti-IFN-
, PerCPanti-CD8 and allophycocyaninanti-CD69 (all reagents from BD Biosciences). Acquisition was performed on a FACSCalibur flow cytometer, collecting 100 000200 000 lymphocyte-gated events per sample and data were analyzed with the software FlowJo (version 3.6.1, Tree Star, San Carlos, CA, USA).
Fine mapping of Mamu-B*01 epitopes
T cells from peripheral blood, lymph nodes and spleen were re-stimulated with the 8-, 9- and 10-mer peptides corresponding to the region of interest and ICS was performed for fine mapping of the CTL epitopes restricted by Mamu-B*01. Briefly, purified mononuclear cells from peripheral blood, lymph nodes and spleen were pulsed with peptide for 2 h at 37°C and thereafter cultured for 2 days in the presence of 2500 U ml1 IL-7 (R&D Systems, Minneapolis, MN, USA). At day 3, 20 U ml1 of IL-2 (R&D Systems) was added, and the culture medium containing 100 U ml1 of IL-2 was refreshed every second day. After 7 days, CD8-positive cells were enriched by using the MACS® system (Miltenyi Biotec, Inc., Auburn, CA, USA). Lymphocytes from the 7-day old in vitro stimulated cultures were washed twice with FACS buffer (PBS, 2% FBS) and incubated with 10 µl of anti-CD8FITC (BD Biosciences) for 30 min at 4°C in MACS® buffer (PBS, 0.5% BSA and 2 mM EDTA). Cells were then incubated with anti-FITC-labeled beads (Miltenyi Biotec) and enriched by using positive selection columns according to the manufacturer's protocol. These CD8-positive cells were again stimulated in vitro using mitomycin C-treated, peptide-pulsed, autologous PBMC cryopreserved prior to immunization as stimulator cells. After another 7 days, the cells were used as effectors in the ICS assays, as described above.
Amplification of viral RNA from plasma and sequencing
Sequencing of plasma viral RNA was performed as described by O'Connor et al. (19). In brief, cell-free plasma was collected at week 82 post-challenge by centrifugation of EDTA anti-coagulated whole blood on a Ficoll density gradient. Virus was extracted from the plasma using the QIAamp Viral RNA Mini Kit (Qiagen inc.) according to the manufacture's instructions for large volume samples. For each viral RNA samples, 510 reverse transcription (RT)PCR reactions were performed using the One-Step RTPCR kit (Qiagen), with two primers corresponding to the SIVmac239 sequences that flank the Gag open reading frame 5'-GGAAGAGGCCTCCGGTTGCA-3' and 5'-GGTGCGAGGGCCTCTTTCAG-3', at a final concentration of 0.6 µM. The RTPCR conditions were as follows: 50°C for 30 min, 95°C for 15 min, five cycles of 95°C for 30 s, 65°C for 30 s and 72°C for 2 min followed by 35 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 2 min. The PCR amplicons (
1200 bp) from multiple PCR reactions were cloned into the pCRII-TOPO vector using the TOPO-TA Cloning Kit (Invitrogen, Carlsbad, CA, USA) and 1824 clones from individual animals were sequenced using the CEQTM 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA, USA). All the clones were sequenced in both directions using overlapping primers corresponding to SIVmac239 Gag sequences, 5'-GTTACCACCTATTTGTTG-3', 5'-CATTCACGCAGAAGAGAAAG-3', 5'-GCATTTTGAATCAGCAGTG-3', 5'-CATACCAGTAGGCAACAT-3' and the two primers corresponding to the vector flanking sequences Sp6 (5'-ATTTAGGTGACACTATAG-3') and T7 (5'-TAATACGACTCACTATAGGG-3'). Sequence analysis was done with the software Vector NTI Contig Express Version 5.3 (Invitrogen InforMax Inc., Frederick, MD, USA).
 |
Results
|
---|
Among the 24 macaques in the first cohort, 11 animals expressed Mamu-B*01 (45%) and 4 expressed Mamu-A*02 (17%). Seven additional Mamu-B*01-positive animals in a second cohort were included for further fine mapping of CTL epitopes. Cryopreserved PBMC from week 20 and week 62 (cohort 1) or week 16 (cohort 2) post-challenge, with viability
95%, were tested for CTL responses by IFN-
ELISPOT and ICS. Multiple epitopes were identified in each animal including known epitopes such as the Mamu-A*01-restricted Gag181189 (20).
Identification of Mamu-B*01-restricted CTL epitopes
A 395-member set of 9- or 10-mer overlapping peptides corresponding to the sequence of SIVmac239 Gag was used to identify Gag epitopes by IFN-
ELISPOT assay. Among the Mamu-B*01+ macaques, responses to five peptides were identified that were not seen in Mamu-B*01-negative animals. They were Gag3948 (NELDRFGLAE), Gag169177 (EVVPGFQAL), Gag198206 (AAMQIIRDI), Gag257265 (IPVGNIYRR) and Gag296304 (SYVDRFYKS). The responses detected by ELISPOT assays were shown in Fig. 1(A) in a representative animal (RSn5) and were summarized in Fig. 1(B). One previously described Mamu-B*01 epitope in SIV Env protein (21) was also tested in six animals and response was detectable in three of them (
50%), with a geometric mean of 142 SFC (Fig. 1C, antigen H). IFN-
ICS by FACS was performed in multiple animals to determine the MHC restriction to the new Gag epitopes. The MHC types of the effector cells and target cells are listed in Table 1. Figure 2 shows the frequencies of the peptide-specific CD69+IFN-
+ population of CD8+ lymphocytes in the tested animals. The DMSO controls and the negative peptide controls usually gave a background in the range of 0.0050.016% and SEB positive controls usually exhibited responses of 25%. Isotype controls, as well as the staining of target cells alone, gave extremely low background (data not shown). All of the peptides were recognized by nine or more of the Mamu-B*01+ animals (Fig. 1B). Fifteen out of the 18 Mamu-B*01+ macaques (
83%) showed response to Gag3948 with a geometric mean of 282 SFC per 1 x 106 PBMC. Ten Mamu-B*01+ macaques (
56%) exhibited responses to Gag169177 with a geometric mean of 275 SFC. Nine animals (
50%) responded to Gag198206 with a geometric mean of 256 SFC per 1 x 106 PBMC. Twelve Mamu-B*01+ macaques (
67%) responded to Gag257265 and Gag296304, with geometric means of 244 SFC and 232 SFC per 1 x 106 PBMC, respectively. Eight animals showed responses to all of the five Gag peptides and another nine animals responded to at least one of the five Gag peptides. One Mamu-B*01+ animal had no response to any of the five Mamu-B*01 peptides, but it had responses to another two Gag peptides the restriction of which could not be determined because no more cryopreserved cells were available for this individual.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1. CTL responses toward Gag peptides determined by IFN- ELISPOT assays in macaque cryopreserved PBMC. Numbers of SFC/1 x 106 shown are average of duplicates subtracted background of DMSO negative controls. (A) CTL responses against individual peptides adjacent to a new Gag epitope in a representative animal RSn5 at week 62 post-challenge. (B) CTL responses against individual Gag epitopes in the tested animals; grey diamonds depict the animals from the first cohort and black triangles depict the animals from the second cohort. Bars represent the geometric means. The numbers below each peptide depict the percentage of the 18 Mamu-B*01+ macaques that responded to the peptide. (C) CTL responses against the Gag peptides (antigens F and G) in Mamu-A*02+ macaque PBMC and the response against the previously identified Env peptide (antigen H) in Mamu-B*01+ macaque PBMC. Bars depict the geometric means. The numbers below each peptide depict the percentage of the four Mamu-A*02+ (antigens F and G) or of the six tested Mamu-B*01+ (antigen H) macaques that responded to the peptide.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1. MHC typing of the macaques from which PBMC were used as effectors and targets in the Mamu-B*01 restriction assays
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 2. ICS restriction analyses for SIV Gag-specific CTL responses by FACS in PBMC of the macaques. The frequency of responding CD8+ cells against each peptide is shown as average of triplicates. Animals RPr5, RZe5, RDm5, RWd6, RDe5, RSn5, J471 and J547 are Mamu-B*01 positive. Animal RLp4 is Mamu-A*02 positive. A. Gag3948, NELDRFGLAE; B. Gag169177, EVVPGFQAL; C. Gag198206, AAMQIIRDI; D. Gag257265, IPVGNIYRR; E. Gag296304, SYVDRFYKS; F. Gag7079, TGSENLKSLY; G. Gag7180, GSENLKSLYN; H. A negative peptide as negative control. Error bars represent standard deviations.
|
|
Fine mapping of the SIV Gag CTL epitopes
To further determine the minimal optimal sequence of the newly identified SIV Gag epitopes, two animals from the second cohort, J211 and J231, were sacrificed at week 20 post-challenge. T cells from peripheral blood, lymph nodes and spleen of the two animals were re-stimulated with the 8-, 9- and 10-mer peptides corresponding to the epitope regions, respectively. After 14 days of culture, the 8-, 9- and 10-mer peptides were tested at 40 µg ml1 by ICS restriction assay to define the minimal optimal region of the epitopes (Fig. 3). Thereafter, dilutions of peptides of each length were tested by ICS to determine which peptide elicited optimal stimulation of the T cells (Fig. 4). The 8-mer peptide Gag3946 NELDRFGL, the 9-mer peptides Gag169177 EVVPGFQAL, Gag198206 AAMQIIRDI and Gag257265 IPVGNIYRR and the 10-mer peptide Gag296305 SYVDRFYKSL stimulated the Gag-specific T cells more effectively than all other peptides. We therefore conclude that Gag3946 NELDRFGL, Gag169177 EVVPGFQAL, Gag198206 AAMQIIRDI, Gag257265 IPVGNIYRR and Gag296305 SYVDRFYKSL represent the minimal optimal CTL epitopes recognized in vivo, as listed in Fig. 5(A).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3. Fine mapping of the SIV Gag epitopes by ICS restriction analyses using 40 µg ml1 8-, 9- and 10-mer peptides performed on re-stimulated T cells derived from lymph nodes in macaques J211 (A) and J231 (B). A-8mer, Gag3946, NELDRFGL; A-9mer, Gag3947, NELDRFGLA; A-10mer, Gag3948, NELDRFGLAE. B-8mer, Gag169176, EVVPGFQA; B-9mer, Gag169177, EVVPGFQAL; B-10mer, Gag169178, EVVPGFQALS. C-8mer, Gag198205, AAMQIIRD; C-9mer, Gag198206, AAMQIIRDI; C-10mer, Gag198207, AAMQIIRDII. D-8mer, Gag257264, IPVGNIYR; D-9mer, Gag257265, IPVGNIYRR; D-10mer, Gag257266, IPVGNIYRRW. E-8mer, Gag296303, SYVDRFYK; E-9mer, Gag296304, SYVDRFYKS; E-10mer, Gag296305, SYVDRFYKSL.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 4. Definition of minimal optimal CD8-positive T cell epitopes. Re stimulated T cells derived from spleen (J231) or PBMC (J211) were tested in ICS using a range of concentrations of 8- and 9-mer peptides or 9- and 10-mer peptides in order to determine which peptide results in maximal stimulation. A-8mer, Gag3946, NELDRFGL; A-9mer, Gag3947, NELDRFGLA; B-8mer, Gag169176, EVVPGFQA; B-9mer, Gag169177, EVVPGFQAL; C-8mer, Gag198205, AAMQIIRD; C-9mer, Gag198206, AAMQIIRDI; D-8mer, Gag257264, IPVGNIYR; D-9mer, Gag257265, IPVGNIYRR; E-9mer, Gag296304, SYVDRFYKS; E-10mer, Gag296305, SYVDRFYKSL.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 5. Alignments of the newly identified SIV Gag epitopes. (A) Alignment of the Mamu-B*01 epitopes shows the binding preference at positions 3, 6 and C-terminus. Asterisk denotes the previously described Env epitope (21). (B) Alignment of various SIV, HIV-2 and HIV-1 (clade B) amino acid sequences in regions of the new SIV Gag CTL epitopes. Gag3946, Gag169177, Gag198206, Gag257265 and Gag296305 are Mamu-B*01 epitopes and Gag7179 is Mamu-A*02 epitope. Residues in critical positions (positions 3, 6 and C-terminus for Mamu-B*01 and position 2 and C-terminus for Mamu-A*02) are shaded in gray. The epitope sequences are boxed.
|
|
Conserved features in the Mamu-B*01 epitopes suggest a tentative binding motif involving positions 3, 6 and the C-terminus (Fig. 5A). Preferred residues at position 3 were bulky non-polar hydrophobic amino acids (M) and aliphatic residues (L and V). Position 6 was associated with bulky non-polar hydrophobic amino acids as well, both aromatic (F) and aliphatic (I). In C-terminal position, both hydrophobic aliphatic residues (I and L) and basic residues (R) were tolerated and the length of the peptides vary from 8 to 10 amino acids due to the preference of the C-terminal residue. The previously described Env epitope (21), which was determined with 12-mer peptides overlapping by nine amino acids, can also be fitted in this model if the sequence is shifted with one amino acid and shortened with two amino acids (Fig. 5A). More detailed biochemical binding analysis is needed to confirm the binding motif of Mamu-B*01.
Confirmation of Mamu-A*02-restricted CTL epitope
Among the four Mamu-A*02+ macaques, responses toward two adjacent peptides, Gag7079 (TGSENLKSLY) and Gag7180 (GSENLKSLYN), were positive in three animals and the responses were at a level similar to that determined by ELISPOT (geometric mean of 300 SFC per 1 x 106 PBMC, Fig. 1C). Gag6978 and Gag7281 were negative, thus suggesting that the responses were directed to Gag7179 (GSENLKSLY). This Mamu-A*02 restriction was confirmed in the animal RLp4 (Fig. 2) by ICS, in which the DMSO and negative peptide controls usually gave a background in the range of 0.0210.028%. The single Mamu-A*02+ macaque which did not respond to Gag7179 did respond to several other Gag epitopes at week 62. The Gag7179 epitope has been recently described by Vogel et al. (22) but was unknown to us at the time of our study. Thus, this work represents a blinded independent confirmation of this epitope in an unrelated animal cohort.
The newly identified SIV CTL epitopes are in highly conserved regions
Sequence alignment reveals that the regions encoding the newly identified CTL epitopes are highly conserved among the commonly used SIV strains (Fig. 5B). Only minor substitutions are seen in Gag198206 and none in the positions 3, 6 or C-terminus. Moreover, these epitope sequences are also mostly identical in the HIV-2 strains that are closely related to SIV. Although a considerable number of amino acid substitutions are exhibited within most of these epitopes among HIV-1 viruses which are more remote from SIV, amino acids at critical positions, the position 3, 6 and C-terminus for the Mamu-B*01 epitopes and the position 2 and C-terminus for the Mamu-A*02 epitope, are either identical to the SIV sequences or substituted to residues with close chemical characteristics.
We further investigated whether the epitope sequences were conserved among the SIV quasi-species in our challenged Mamu-B*01+ animals. Plasma viral load and CD4+ T cell counts are depicted as geometric means for all the B*01 animals (Fig. 7A) and individually for each of the animals (Fig. 7BF). Plasma samples were collected at week 82 post-challenge from five animals of cohort 1 and viral RNAs were sequenced and analyzed for the whole Gag region. Sequences within the five epitopes are highly conserved in these animals over time, as shown in Fig. 6. Except for the Gag3946 epitope sequence of the virus from animal RZe5, all the epitope regions exhibited 08% sequence variation, of which most occurred at the unimportant positions for Mamu-B*01 binding. The virus from animal RZe5 showed variation in all 18 sequenced clones in the epitope Gag3946 at the C-terminal position, a critical binding site for Mamu-B*01 molecule. However, this C-terminal residue was substituted to I (17 out of the total 18 clones) or V (1 out of the total 18 clones), which are aliphatic amino acids as is the original residue L. It is not known what effect this substitution may have on antigenicity since we could not determine the CTL activity against this variant due to a lack of cells from this animal. Another single clone (out of a total 22 sequenced clones) with a variation at position 6, an important binding site for Mamu-B*01, was found in animal RDm5 in the epitope Gag3946 sequence with the substitution of the aromatic residue F to the aliphatic residue L. The low frequency of this substitution in the analysis, in addition to the stable clinical status including very low plasma viral loads and sustained CD4 counts (Fig. 7C), suggests that it does not represent an established escape mutation at this time point in infection.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 7. Plasma viral load (filled circle) and CD4+ T cell count (filled diamond) of the Mamu-B*01+ animals. CD4+ counts 4 weeks prior to the viral challenge is included. Panel A: summary of 11 Mamu-B*01+ animals from cohort 1. The geometric means of viral loads and CD4+ counts of the 11 animals are shown at all the time points. Panels BF: viral load and CD4+ counts of the five animals from which the viral sequencing was performed at week 82 post-challenge.
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 6. Alignment of the viral sequences from the Mamu-B*01+ animals at week 82 post-challenge. Amino acid sequences in regions of the new SIV Gag CTL epitopes, Gag3946, Gag169177, Gag198206, Gag257265 and Gag296305, are compared with the sequence of the vaccine immunogen. Dashes indicate identical amino acids to the immunogen sequence and the bold amino acids represent the variations. Numbers of sequenced clones and clones with a variation in each animal are shown, as well as, the percentage of variants (boxed) for each epitope in the individual animal. Residues in critical positions (positions 3, 6 and C-terminus) are shaded in gray.
|
|
 |
Discussion
|
---|
Here we report identification of five new SIV CTL epitopes in Gag for the first time (Gag3946 NELDRFGL, Gag169177 EVVPGFQAL, Gag198206 AAMQIIRDI, Gag257265 IPVGNIYRR and Gag296305 SYVDRFYKSL) that are highly conserved and restricted by the common MHC class I molecule Mamu-B*01, and confirmed one recently described Mamu-A*02 epitope, Gag7179 GSENLKSLY. These novel Gag epitopes will be particularly useful for evaluating Gag as a CTL target, given that currently most of the vaccines include Gag sequence and Gag is well expressed in the viral replication cycle. The peptide-binding preference for Mamu-B*01 that has been revealed in this study will also facilitate the identification of further CTL epitopes in other SIV genes. Tetramers can now be synthesized for the peptideMamu-B*01 pairs, enabling more quantitative and sensitive analysis for vaccine-elicited or naturally occurred SIV-specific CTL after vaccination or infection.
The putative peptide-binding motif suggests that positions 3, 6 and the C-terminus are the anchor residues for Mamu-B*01. Interestingly, Mamu-B*01 is the only known macaque MHC B locus allele that has been reported to phylogenetically cluster with the human, gorilla and chimpanzee B locus alleles (23). Its most similar human allele is HLA-B*2702 but the homology is limited (differs by 25 amino acids in
1 and
2 domains) and they do not share a common peptide-binding motif. Although the majority of MHC class I molecules uses position 2 and C-terminal amino acid as anchor positions, exceptions are commonly seen among the motifs of human and other species. For example, HLA-A*0101 makes use of positions 3 and 9 (HIV-1 Gag p177078) and HLA-B8 uses positions 3, 5 and 9 (HIV-1 Gag p172330, gp120210, nef94101 and RT185193). More alleles exhibit auxiliary anchors at positions 2, 3, 5 and 6, especially the B alleles, such as B14, B*3801, B40 and B*7801. So do some of the mouse MHC alleles (for details see SYFPEITHI database at http://syfpeithi.bmi-heidelberg.com). In general, it is hard to draw a line between anchors and auxiliary anchors and some peptide motifs contain unusual characteristics. In our study, although V, L and M are present at position 3 and F and I at position 6, they all possess similar chemical properties and such substitutes are commonly seen in other peptide motifs. Similarly, the preference of L, I or R at the C-terminal position follows the very common attribute that allows either hydrophobic or basic amino acids. The uncommon peptide-binding preference may be one of the explanations why the Mamu-B*01 epitope has been difficult to identify despite vigorous attempts in recent years. We have demonstrated that the CTL responses against the overlapping 9- or 10-mer peptides adjacent to the epitope sequence (with one amino acid shift) were in all cases weaker than those against the minimal epitope itself. Hence, it supports the alignment with positions 3, 6 and C-terminus as the principal anchors for Mamu-B*01 instead of the common positions 2 and C-terminus. The optimal length of the peptides varies from 8 to 10 amino acids very likely due to the preference of the C-terminal residue for binding. A previously described Env epitope sequence can also be fitted in our putative binding motif by shifting one amino acid. Unlike the present study where there is consistent evidence indicating the minimal epitope sequence, this epitope was identified using only 12-mer peptides overlapping with nine amino acids, there is no data available for the responses toward the adjacent overlapping peptides with one amino acid shift to this Env epitope. However, we noted that the frequency and strength of the response against this Env epitope detected in our animal cohorts was lower than the other Gag epitopes we identified (see Fig. 1C). One possible reason may be that the epitope sequence is not optimal and this needs to be further confirmed.
The restriction analysis for Mamu-B*01 in this study was performed using non-autologous mismatched PBMC due to the lack of Mamu-B*01 transfectants. Hence, the possibility that the same peptide is presented by multiple MHC molecules cannot be completely ruled out. However, we tested a total of 18 Mamu-B*01 animals, with target cells from five different animals for restriction (see Table 1), it should be very unlikely that the same peptide can be presented by an unknown allele that happens to exist in multiple pairs of effector and target individuals. Moreover, the results from fine-mapping restriction analysis were completely concordant in the two tested animals with different MHC types, tested with two different target cells also having different MHC types, with regard to the optimal length of peptides and peptide-titration curves (Figs 3 and 4). This further diminishes the possibility that the newly identified epitopes are presented by certain unknown MHC alleles besides Mamu-B*01.
It is noted that not all of the studied animals showed detectable CTL responses toward all of the newly identified epitopes, and the frequencies of the epitope-specific CTL were not always high in the peripheral blood compared with responses often encountered with the highly immunodominant Gag181189. Some reduction in the apparent response may be a result of the use of cryopreserved PBMC. In addition, in cohort 1, these responses were detected in the context of vaccinated animals that were clinically non-progressive with low viral load after SHIV challenge. Thus, the responding cell frequencies at the time of collection may have been reduced due to very low viral loads at these time points [geometric means of plasma viral load in those Mamu-B*01 and Mamu-A*02 animals were
700 copies ml1 at week 20 and 200 copies ml1 at week 62 post-challenge (15)]. In cohort 2, animals were evaluated at week 16 post-challenge with SIVmac239 and two animals were sacrificed at week 20 for epitope fine-mapping studies. These animals had relatively high viral loads at the time samples were collected; geometric mean of plasma viral RNA was 1.5 x 106 copies ml1 (M.A. Luscher, Y. Guan, N. Khanna., et al. manuscript in preparation). Not surprisingly, the CTL responses detected were relatively higher as well (see Fig. 1B). Furthermore, two control animals in cohort 2, J211 and J741, were evaluated and the responses toward the newly identified epitopes were detected. Hence, these epitope responses can be stably detected in both vaccinated/challenged and naturally infected rhesus macaques. Overall, responses to the Mamu-B*01 epitopes are less dominant than the response to Gag181189, and their detection in these experiments highlights the advantage of using optimal 9- to 10-mer peptides in the detection of sub-dominant responses. Since Mamu-A*01 animals with the highly immunodominant responses to Gag181189 react to SIV/SHIV infection differently compared with animals with other MHC alleles (911), it is important to be able to accurately assess animals of diverse MHC in the context of vaccine studies, especially the presence of multiple sub-dominant responses.
The observation that these epitopes were readily detected in a cohort of vaccinated/challenged macaques that were clinically and virologically stable up to 90 weeks post-infection (Fig. 7) suggests that CTL escape from these epitopes may not be occurring. Not all SIV/SHIV epitope responses are conserved over time. A dominant Tat-specific CTL response in Mamu-A*01 macaques that is detected during the initial days following SIV infection has been shown to be subject to epitope escape and to disappear early in the chronic phase. The utility of these newly defined Mamu-B*01 Gag epitopes is determined, in part, by the degree to which these particular Gag sequences are conserved among various SIV and SHIV isolates in common experimental use. Most of the SHIV chimeras incorporate the SIVmac239 Gag and therefore express these epitopes. Sequence alignment reveals that all of these newly identified epitopes are in the highly conserved regions of commonly used SIV strains and the HIV-2 strains that are closely related to SIV. Although amino acid substitutions are more common among HIV-1 viruses, amino acids at critical positions are either identical to the SIV sequences or substituted by residues with close chemical characteristics. Viral RNA sequencing of the Mamu-B*01 animals also revealed that these epitopes are highly conserved, with a few random substitutions at the unimportant peptide-binding positions. In animal RZe5, the two varients, I (17/18) and V (1/18), at the important position C-terminus of the epitope Gag3946 have similar chemical properties to the original residue L (Fig. 6) and the residue I is indeed preferred at this position as shown in Gag198206. Hence, neither the peptide-binding capability (as for escape mutation) nor the functional structure of the protein (as for fitness cost) is expected to be changed significantly, since viral replication was still under efficient control in this animal over a long period of time. However, it is noted that this animal had several intermittent blips of viral load before the time of sequencing and apparently it was experiencing another viral blip at the time of sequencing (Fig. 7B). We were unable to determine whether the variation in this Mamu-B*01 epitope contributed to the increase of viral replication since we had no additional stored samples from this animal. Nevertheless, all the critical residues have been conserved to possess similar chemical properties, implying that these amino acids may be functionally constrained. In addition, stable clinical and virologic status was maintained in the Mamu-B*01 animals for nearly 2 years after viral challenge. CTL responses have been implicated in such viral control, and it is possible that responses to the newly identified B*01 epitopes participate in this process. Thus, the Gag epitopes identified here may be useful candidates for poly-epitope vaccines or long-term monitoring of Gag-specific CTL responses.
To date, Mamu-A*01 is the best-studied rhesus macaque class I molecule. However, since it has been reported to have positive influence in preventing disease progression, it is apparent that studies in Mamu-A*01 animals need to be appropriately evaluated against Mamu-A*01 controls. This need only compounds the issue of relative short supply of these animals. One aim of our study was to identify SIV-specific CTL epitopes restricted by other common macaque MHC class I alleles which have no obvious genetic impact on disease progression, unlike Mamu-A*01 or Mamu-B*17. Both Mamu-B*01 and Mamu-A*02 are commonly seen MHC class I alleles, with frequencies 1236% and 1828%, respectively, among Indian-origin rhesus macaques (6), and neither of them has been found having any influence on disease progression (10). Therefore, our findings of these new Gag epitopes will considerably advance the utility of Mamu-B*01- and Mamu-A*02-positive animals in SIV vaccine studies and may contribute to increasing the supply of animals that can effectively be used in such experiments where the ability to follow epitope-specific CD8+ T cell responses is desirable.
 |
Acknowledgements
|
---|
We would like to thank David I. Watkins for making his facilities available to us and David O'Connor for his helpful advice on viral RNA sequencing. This work was supported by grants from the National Institutes of Health (no. 5 P01 AI43045-05) and J.S. and K.S.M. receive career support from the Ontario HIV Treatment Network.
 |
Abbreviations
|
---|
DMSO | dimethyl sulfoxide |
ELISPOT | enzyme-linked immunospot assay |
HIV | human immunodeficiency virus |
ICS | intracellular cytokine staining |
MVA | modified vaccinia virus Ankara |
RT | reverse transcription |
SEB | staphylococcal enterotoxin B |
SFC | spot-forming cells |
SHIV | simian-human immunodeficiency virus |
SIV | simian immunodeficiency virus |
SSP | sequence-specific primers |
 |
Notes
|
---|
Transmitting editor: C. Paige
Received 20 September 2004,
accepted 21 February 2005.
 |
References
|
---|
- Barouch, D. H., Santra, S., Schmitz, J. E. et al. 2000. Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokine-augmented DNA vaccination. Science 290:486.[Abstract/Free Full Text]
- Hirsch, V. M., Fuerst, T. R., Sutter, G. et al. 1996. Patterns of viral replication correlate with outcome in simian immunodeficiency virus (SIV)-infected macaques: effect of prior immunization with a trivalent SIV vaccine in modified vaccinia virus Ankara. J. Virol. 70:3741.[Abstract]
- Letvin, N. L., Barouch, D. H. and Montefiori, D. C. 2002. Prospects for vaccine protection against HIV-1 infection and AIDS. Annu. Rev. Immunol. 20:73.[CrossRef][ISI][Medline]
- Evans, D. T., Jing, P., Allen, T. M. et al. 2000. Definition of five new simian immunodeficiency virus cytotoxic T-lymphocyte epitopes and their restricting major histocompatibility complex class I molecules: evidence for an influence on disease progression. J. Virol. 74:7400.[Abstract/Free Full Text]
- Mothe, B. R., Sidney, J., Dzuris, J. L. et al. 2002. Characterization of the peptide-binding specificity of Mamu-B*17 and identification of Mamu-B*17-restricted epitopes derived from simian immunodeficiency virus proteins. J. Immunol. 169:210.[Abstract/Free Full Text]
- Robinson, S., Charini, W. A., Newberg, M. H. et al. 2001. A commonly recognized simian immunodeficiency virus Nef epitope presented to cytotoxic T lymphocytes of Indian-origin rhesus monkeys by the prevalent major histocompatibility complex class I allele Mamu-A*02. J. Virol. 75:10179.[Abstract/Free Full Text]
- Allen, T. M., Sidney, J., del Guercio, M. F. et al. 1998. Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus. J. Immunol. 160:6062.[Abstract/Free Full Text]
- Miller, M. D., Yamamoto, H., Hughes, A. L., Watkins, D. I. and Letvin, N. L. 1991. Definition of an epitope and MHC class I molecule recognized by gag-specific cytotoxic T lymphocytes in SIVmac-infected rhesus monkeys. J. Immunol. 147:320.[Abstract/Free Full Text]
- Mothe, B. R., Weinfurter, J., Wang, C. et al. 2003. Expression of the major histocompatibility complex class I molecule Mamu-A*01 is associated with control of simian immunodeficiency virus SIVmac239 replication. J. Virol. 77:2736.[Abstract/Free Full Text]
- Muhl, T., Krawczak, M., Ten Haaft, P., Hunsmann, G. and Sauermann, U. 2002. MHC class I alleles influence set-point viral load and survival time in simian immunodeficiency virus-infected rhesus monkeys. J. Immunol. 169:3438.[Abstract/Free Full Text]
- Pal, R., Venzon, D., Letvin, N. L. et al. 2002. ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency. J. Virol. 76:292.[Abstract/Free Full Text]
- O'Connor, D. H., Mothe, B. R., Weinfurter, J. T. et al. 2003. Major histocompatibility complex class I alleles associated with slow simian immunodeficiency virus disease progression bind epitopes recognized by dominant acute-phase cytotoxic-T-lymphocyte responses. J. Virol. 77:9029.[Abstract/Free Full Text]
- Schapiro, S. J. 2000. A few new developments in primate housing and husbandry. Scand. J. Lab. Anim. Sci. 27:103.[ISI]
- Amara, R. R., Villinger, F., Altman, J. D. et al. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292:69.[Abstract/Free Full Text]
- Tang, Y., Villinger, F., Staprans, S. I. et al. 2002. Slowly declining levels of viral RNA and DNA in DNA/recombinant modified vaccinia virus Ankara-vaccinated macaques with controlled simian-human immunodeficiency virus SHIV-89.6P challenges. J. Virol. 76:10147.[Abstract/Free Full Text]
- Knapp, L. A., Lehmann, E., Piekarczyk, M. S., Urvater, J. A. and Watkins, D. I. 1997. A high frequency of Mamu-A*01 in the rhesus macaque detected by polymerase chain reaction with sequence-specific primers and direct sequencing. Tissue Antigens 50:657.[ISI][Medline]
- Kern, F., Bunde, T., Faulhaber, N. et al. 2002. Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals. J. Infect. Dis. 185:1709.[CrossRef][ISI][Medline]
- Rowland-Jones, S. L., Dong, T., Fowke, K. R. et al. 1998. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J. Clin. Investig. 102:1758.[Abstract/Free Full Text]
- O'Connor, D. H., Allen, T. M., Vogel, T. U. et al. 2002. Acute phase cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus infection. Nat. Med. 8:493.[CrossRef][ISI][Medline]
- Chen, Z. W., Kou, Z. C., Lekutis, C. et al. 1995. T cell receptor V beta repertoire in an acute infection of rhesus monkeys with simian immunodeficiency viruses and a chimeric simian-human immunodeficiency virus. J. Exp. Med. 182:21.[Abstract/Free Full Text]
- Yasutomi, Y., McAdam, S. N., Boyson, J. E., Piekarczyk, M. S., Watkins, D. I. and Letvin, N. L. 1995. A MHC class I B locus allele-restricted simian immunodeficiency virus envelope CTL epitope in rhesus monkeys. J. Immunol. 154:2516.[Abstract/Free Full Text]
- Vogel, T. U., Friedrich, T. C., O'Connor, D. H. et al. 2002. Escape in one of two cytotoxic T-lymphocyte epitopes bound by a high-frequency major histocompatibility complex class I molecule, Mamu-A*02: a paradigm for virus evolution and persistence? J. Virol. 76:11623.[Abstract/Free Full Text]
- Boyson, J. E., Shufflebotham, C., Cadavid, L. F. et al. 1996. The MHC class I genes of the rhesus monkey: different evolutionary histories of MHC class I and II genes in primates. J. Immunol. 156:4656.[Abstract/Free Full Text]