CD8+ Lymphocytes from Simian Immunodeficiency Virus-Infected Rhesus Macaques Recognize 14 Different Epitopes Bound by the Major Histocompatibility Complex Class I Molecule Mamu-A*01: Implications for Vaccine Design and Testing

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CD8+ Lymphocytes from Simian Immunodeficiency Virus-Infected Rhesus Macaques Recognize 14 Different Epitopes Bound by the Major Histocompatibility Complex Class I Molecule Mamu-A*01: Implications for Vaccine Design and Testing
    2001, 75(2):738. DOI: 10.1128/JVI.75.2.738-749.2001. J. Virol. SetteAlessandroThomson, John D. Altman, David I. Watkins and H. O'Connor, Xiaochi Wang, Michael C. Wussow, James A.Jing, John L. Dzuris, Max E. Liebl, Thorsten U. Vogel, David Todd M. Allen, Bianca R. Mothé, John Sidney, Peicheng  TestingImplications for Vaccine Design and Complex Class I Molecule Mamu-A*01:Bound by the Major Histocompatibility Macaques Recognize 14 Different EpitopesImmunodeficiency Virus-Infected Rhesus Lymphocytes from Simian+CD8 information and services can be found at: These include:  REFERENCES This article cites 76 articles, 43 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new Information about commercial reprint orders: To subscribe to to another ASM Journal go to:  onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om  onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   J OURNAL OF  V IROLOGY ,0022-538X/01/$04.00  0 DOI: 10.1128/JVI.75.2.738–749.2001Jan. 2001, p. 738–749 Vol. 75, No. 2Copyright © 2001, American Society for Microbiology. All Rights Reserved. CD8  Lymphocytes from Simian Immunodeficiency Virus-InfectedRhesus Macaques Recognize 14 Different Epitopes Bound bythe Major Histocompatibility Complex Class I MoleculeMamu-A*01: Implications for VaccineDesign and Testing TODD M. ALLEN, 1 BIANCA R. MOTHE´, 1,2 JOHN SIDNEY, 3 PEICHENG JING, 1 JOHN L. DZURIS, 3 MAX E. LIEBL, 1 THORSTEN U. VOGEL, 1 DAVID H. O’CONNOR, 1 XIAOCHI WANG, 4 MICHAEL C. WUSSOW, 1 JAMES A. THOMSON, 1 JOHN D. ALTMAN, 4 DAVID I. WATKINS, 1,2 *  AND  ALESSANDRO SETTE 3 Wisconsin Regional Primate Research Center, University of Wisconsin, Madison, Wisconsin 53715 1  ; Epimmune, San Diego,California 92121 3  ; Emory Vaccine Center, Emory University School of Medicine, Atlanta, Georgia 4  ; and Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53706-1532 2 Received 6 June 2000/Accepted 18 October 2000 It is becoming increasingly clear that any human immunodeficiency virus (HIV) vaccine should induce astrong CD8  response. Additional desirable elements are multispecificity and a focus on conserved epitopes.The use of multiple conserved epitopes arranged in an artificial gene (or EpiGene) is a potential means toachieve these goals. To test this concept in a relevant disease model we sought to identify multiple simianimmunodeficiency virus (SIV)-derived CD8  epitopes bound by a single nonhuman primate major histocom-patibility complex (MHC) class I molecule. We had previously identified the peptide binding motif of Mamu- A*01 2 , a common rhesus macaque MHC class I molecule that presents the immunodominant SIV   gag  -derivedcytotoxic T lymphocyte (CTL) epitope Gag_CM9 (CTPYDINQM). Herein, we scanned SIV proteins for thepresence of Mamu-A*01 motifs. The binding capacity of 221 motif-positive peptides was determined usingpurified Mamu-A*01 molecules. Thirty-seven peptides bound with apparent  K   d   values of 500 nM or lower, with21 peptides binding better than the Gag_CM9 peptide. Peripheral blood mononuclear cells from SIV-infectedMamu-A*01  macaques recognized 14 of these peptides in ELISPOT, CTL, or tetramer analyses. This studyreveals an unprecedented complexity and diversity of anti-SIV CTL responses. Furthermore, it represents animportant step toward the design of a multiepitope vaccine for SIV and HIV. With more than 30 million human immunodeficiency vi-rus (HIV)-infected individuals (World Health Organization[WHO] web site estimates), there can be few other more pressing bio-medical priorities than to produce an effective vaccine forHIV. Given the important role that CD8  lymphocytes play incontrolling viral replication (11, 32, 43, 49, 58), it is critical thatthis vaccine stimulate strong cytotoxic T-lymphocyte (CTL)responses. Simian immunodeficiency virus (SIV) infection of macaques provides the best nonhuman primate model to de-termine whether the generation of virus-specific CTLs canalter the course of disease after infection (33, 65). The nucle-otide sequences of the SIVs are closely related to those of HIV-1 and -2 (12, 24). SIV and HIV have similar tropisms forCD4 (16, 36), and infection with SIV causes an AIDS-likedisease in the majority of infected macaques by 1 year postin-oculation (35). Since macaques and humans have very similarimmune systems (10, 31, 63, 76), SIV infection of macaques isalso an excellent model to study the immunology of HIV in-fection of humans.SIV infection of macaques is currently the only cost-effectiveanimal model to test vaccine efficacy in vivo. Several vaccinestudies in macaques have already suggested that a strong im-mune response to SIV can be generated in appropriately im-munized monkeys (15, 19, 29, 42, 46, 47) and that this responsecan, in some cases, protect against the development of AIDS.In particular, cell-mediated responses to SIV appear to repre-sent a crucial component of vaccine protective efficacy. CD8  lymphocytes recognize pathogen-infected cells, are involved inthe host’s defensive response to intracellular pathogens (34),and may play an important role in the containment of the AIDS virus in infected individuals (74). This is especially evi-dent during the first few weeks postinfection (8, 39, 53, 57) andduring most phases of disease by mechanisms which includekilling of infected cells and suppression of replication (69, 75).It has recently been shown that depletion of CD8  cells usingmonoclonal antibodies (MAbs) resulted in increases in virusloads in SIV-infected animals (32, 43, 58). Besides this role incontainment of disease, CTLs may also be involved in provid-ing protection from infection with HIV (17, 54, 55). Thus,these observations collectively provide the rationale to explore whether CTLs can protect from AIDS virus infection in ananimal model.Currently, a single useful major histocompatibility complex (MHC) class I molecule (Mamu-A*01) in the rhesus macaque * Corresponding author. Mailing address: Wisconsin Regional Pri-mate Research Center, University of Wisconsin, 1220 Capitol Court,Madison, WI 53715-1299. Phone: (608) 265-3380. Fax: (608) 265-8084.E-mail:   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   has been well characterized. This allele is present in approxi-mately 25% of rhesus macaques of Indian descent (38, 73), andtetramers and ELISPOT assays for the single Mamu-A*01-restricted CTL epitope Gag_CM9 (CTPYDINQM; p11C,C 3  M) have been developed (2, 3, 28, 41). However, thus faronly a limited number of SIV-derived, Mamu-A*01-restrictedepitopes have been defined (2, 4, 22, 25, 44). Therefore, we wanted to examine whether additional Mamu-A*01-restrictedCTL epitopes derived from other regions of SIV could beidentified. Vaccination with multiple epitopes is likely of im-portance since escape from CTL induced against a singleepitope is possible (9, 23, 26, 45, 51, 64). CTL against epitopesin different proteins may also have very different effects onreducing viral burden. Finally, definition of multiple epitopes will allow more precise characterization and quantitation of immune responses against SIV, either during the course of natural infection or following immunization with experimental vaccines. MATERIALS AND METHODSMotif scanning of SIV proteins and peptide synthesis.  The Mamu-A*01 pep-tide binding motif is defined by the requirement for proline (P) in position 3 (2).Live-cell binding assays indicated that in addition to the requirement for P inposition 3, Mamu-A*01 preferentially bound peptides bearing a small residue inposition 2 (A, V, S, T, or P) and hydrophobic (A, L, I, V, and M) or aromatic(F, W, and Y) residues at the C terminus.This motif was utilized to scan the SIVmac251 sequence to identify potentialMamu-A*01 binding peptides between 8 and 11 residues in length, and 111peptides were identified. Additionally, 50 9-mer and 50 10-mer sequences wereselected by removing the restriction for small residues in position 2, for a total of 211 peptides. The corresponding peptides were then synthesized as crude ma-terial by Chiron Mimotopes (San Diego, Calif.). Lyophilized material was resus-pended at 20 mg/ml in 100% dimethyl sulfoxide and then diluted to requiredconcentrations in phosphate-buffered saline (PBS).Radiolabeled probe peptides and peptides subsequently determined to bindMamu-A*01 with high affinity (500 nM or less) were resynthesized at Epimmuneon a larger scale using standard  tert -butoxycarbonyl or 9-fluorenylmethoxy car-bonyl solid-phase methods, as previously described (56). These were purified to  95% homogeneity by reverse-phase high-pressure liquid chromatography, andcomposition was ascertained by amino acid analysis, sequencing, and/or massspectrometry analysis. Mamu-A*01 purification.  721.221 cells transfected with the Mamu-A*01cDNA were utilized as the source of Mamu-A*01 molecules. Cells were main-tained in vitro by culture in RPMI 1640 medium (Flow Laboratories, McLean,Va.) supplemented with 2 mM  L  -glutamine (Gibco, Grand Island, N.Y.), 100 U(100   g/ml) of penicillin-streptomycin solution (Gibco), and 10% heat-inacti- vated fetal calf serum (FCS; Hazleton Biologics) and grown for large-scalecultures in roller bottle apparatuses.Mamu-A*01 was purified from cell lysates as previously described (62). Briefly,cells were lysed at a concentration of 10 8 cells/ml in 50 mM Tris-HCl (pH 8.5)containing 1% NP-40 (Fluka Biochemika, Buchs, Switzerland), 150 mM NaCl,5 mM EDTA, and 2 mM phenylmethylsulfonyl fluoride. Lysates were thenpassaged through 0.45-  m filters and cleared of nuclei and debris by centrifu-gation at 10,000    g   for 20 min, and MHC molecules were purified by affinitychromatography.For affinity purification, columns of inactivated Sepharose CL4B and protein A-Sepharose were used as precolumns. Mamu-A*01 was captured by repeatedpassage over protein A-Sepharose beads conjugated with the anti-HLA(A,B,C)antibody W6/32 as previously described (2). After two to four passages, theW6/32 column was washed with 10 column volumes of 10 mM Tris-HCl (pH 8.0) with 1% NP-40, 2 column volumes of PBS, and 2 column volumes of PBScontaining 0.4%  n -octylglucoside. Finally, Mamu-A*01 molecules were eluted with 50 mM diethylamine in 0.15 M NaCl containing 0.4%  n -octyglucoside (pH11.5). A 1/25 volume of 2.0 M Tris (pH 6.8) was added to the eluate to reducethe pH to   8.0. The eluate was then concentrated by centrifugation in Cen-triprep 30 concentrators at 2,000 rpm (Amicon, Beverly, Mass.). Protein purity,concentration, and effectiveness of depletion steps were monitored by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis. Mamu-A*01 binding assay.  Quantitative assays for the binding of peptides tosoluble Mamu-A*01 molecules on the basis of the inhibition of binding of aradiolabeled standard probe peptide to detergent-solubilized MHC molecules were performed utilizing the protocol previously described for the binding of peptides to HLA class I molecules (62). Briefly, 1 to 10 nM radiolabeled probepeptide, iodinated by the chloramine T method (27), was coincubated at roomtemperature with various amounts of purified Mamu-A*01 in the presence of 1   M human   2 -microglobulin (Scripps Laboratories, San Diego, Calif.) and acocktail of protease inhibitors. Following a 2-day incubation, the percent of MHC bound radioactivity was determined by size exclusion gel filtration chro-matography on a TSK 2000 column. A position 1 C 3   A analog of the SIV Gag 181-190 peptide (ATPYDINQML) was used as the radiolabeled probe. In the case of competitive assays, theconcentration of peptide yielding 50% inhibition of the binding of the radiola-beled probe peptide was calculated. Peptides were initially tested at one or twohigh doses. The 50% inhibitory concentration (IC 50 ) of peptides yielding positiveinhibition was then determined in subsequent experiments, in which two to six further dilutions were tested, as necessary. Since under the conditions used theconcentration of label is less than that of MHC and the IC 50  is equal to the MHCconcentration, the measured IC 50  values are reasonable approximations of thetrue  K   d  values. Each competitor peptide was tested in two to four completelyindependent experiments. As a positive control, in each experiment the unla-beled version of the radiolabeled probe was tested. IFN-   ELISPOT assay.  Ninety-six-well flat-bottomed plates (U-Cytech-BV, Amsterdam, The Netherlands) were coated with 5  g of anti-gamma interferon(IFN-  ) MAb MD-1 (U-Cytech-BV) overnight at 4°C. The plates were then washed 10 times with PBST (PBS [Gibco-BRL] containing 0.05% Tween 20[Sigma Chemical, St. Louis, Mo.]), and then the plates were blocked with 2%PBSA (PBS containing 2% bovine serum albumin [BSA; Sigma Chemical]) for1 h at 37°C. The 2% PBSA was discarded from the plates, and freshly isolatedperipheral blood mononuclear cells (PBMC) were added. Cells were resus-pended in RPMI 1640 (Mediatech) supplemented with penicillin, streptomycin,and 5% fetal bovine serum (FBS; Biocell) (R05). The R05 also contained either5   g of concanavalin A (Sigma Chemical) per ml, 1 to 10   M various Mamu- A*01-bound peptides, 1 to 10   M irrelevant SIV envelope peptide E 3  V(ELGDYKLV), or no peptide. Input cell numbers were 2.0    10 5 peripheralblood lymphocytes in 100  l/well in triplicate wells.Cells were then incubated for 16 h at 37°C in 5% CO 2 , after which the cells were removed from the plates by shaking and 200  l of ice-cold deionized water was added per well to lyse the remaining PBMC. Plates were incubated on ice for15 min and then washed 20 times with PBST. Next, 1   g of rabbit anti-IFN-  polyclonal biotinylated detector antibody solution (U-Cytech-BV) per well wasadded, and the plates were incubated for 1 h at 37°C. The plates were washed 10times with PBST, after which 50  l of a gold-labeled anti-biotin immunoglobulinG solution (U-Cytech BV) was added. The plates were incubated for 1 h at 37°Cand washed 10 times with PBST. Thirty microliters of activator mix (U-CytechBV) per well was added, and the plates were developed for about 30 min. Theactivator mix consists of a silver salt solution that precipitates at the sites of goldclusters (from the gold-labeled antibiotin solution), visualizing the sites wherethe IFN-   was secreted. When black spots appeared in the wells under aninverted microscope, the wells were washed with distilled water to stop devel-opment and then air dried.Wells were imaged with IP Lab Spectrum 3.23 software using a HamamatsuC4880 series camera attached to a Nikon TE 300 inverted microscope. Spots were counted manually. A spot-forming cell (SFC) was defined as a large blackspot with a fuzzy border (37). To determine significance levels, a baseline foreach peptide was first established using the average and standard deviation of thenumber of SFCs in three independent assays as performed on Mamu-A*01  butSIV-naive animals. A threshold significance value corresponding to this averageplus two standard deviations was then determined. In our analysis of samplesfrom SIV-infected Mamu-A*01  animals, a response was considered positive if the number of SFCs exceeded the threshold significance level for that specificpeptide. Generation of in vitro-cultured CTL effector cells.  CTL cultures were estab-lished from EDTA-treated peripheral blood samples as previously described (2).Briefly, Ficoll-Hypaque-separated PBMC were stimulated 1:1 with 5    10 6  -irradiated (3,000 rad) autologous B lymphoblastoid cell line cells (B-LCLs)pulsed with the appropriate peptide (5   M) in R10 medium. Cultures weresupplemented with R10 containing 20 U of recombinant interleukin-2 (rIL-2), agift from Hoffman-LaRoche (Nutley, N.J.), per ml. On day 7, viable cells wererestimulated and again expanded in the presence of rIL-2. CTL activity wasassessed after 14 days of culture in a standard  51 Cr release assay. V OL  . 75, 2001 MULTIPLE SIV CTL EPITOPES IN MACAQUES 739   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   CTL analysis.  SIV-specific CTL activity was assessed using a standard  51 Crrelease assay (2).  51 Cr-labeled Mamu-A*01  B-LCL targets were pulsed withSIV peptides or an irrelevant influenza virus NP peptide (SNEGSYFF). Targetcells (5  10 3 ) were incubated for 5 h with CTL effectors at effector-to-target cellratios ranging from 20:1 to 50:1. CTL activity was calculated from the counts perminute present in harvested supernatants using the formula % specific release  (experimental release  spontaneous release)/(maximal release  spontaneousrelease)  100. The reported percent specific lysis represents the  51 Cr releasedfrom the Mamu-A*01 peptide-pulsed targets minus the  51 Cr released from targetcells pulsed with the irrelevant influenza virus NP peptide (SNEGSYFF). Spon-taneous release was always less than 20% of maximal release. Mamu-A*01 tetramers.  Soluble tetrameric Mamu-A*01 MHC class I/SIVGag_CM9 peptide complexes were constructed as previously described (3, 5). Tetramer staining.  Fresh unstimulated PBMC (10 6 ) were washed twice influorescence-activated cell sorting (FACS) buffer (PBS [Gibco] with 2% FCS[BioCell]) in a 96-well U-bottomed plate. In a 100-  l volume, cells were stainedin the dark for 40 min at room temperature with the tetramer (1   g/ml for in vitro cultures, 5  g/ml for fresh PBMC), anti-rhesus CD3 fluorescein isothiocya-nate (FITC) MAb (10   l; BioSource), and anti-CD8  -PerCP antibody (3   l;Becton Dickinson). Cells were washed four times with FACS buffer and fixed byadding 450  l of 2% paraformaldehyde (PFA). Sample data were acquired on aBecton Dickinson FACSCalibur instrument and analyzed using CellQuest soft- ware (Becton Dickinson Immunocytometry Systems, San Jose, Calif.). Back-ground tetramer staining of fresh, unstimulated PBMC from naive Mamu-A*01  animals was routinely less than 0.08%. Intracellular IFN-   staining.  A total of 2  10 5 cells from in vitro-stimulatedCTL cultures were incubated at 37°C for 1 h with phorbol myristate acetate-ionomycin (50 ng/ml and 1   g/ml, respectively), 5   M Gag-CM9 peptide, or acontrol influenza virus peptide (SNEGSYFF) in the presence of Mamu-A*01  B-LCL (10 5 ) as antigen-presenting cells (APC). Cells were then treated with 10  g of brefeldin A per ml to inhibit protein trafficking and incubated a further 4to 5 h at 37°C. Cells were then washed twice with FACS buffer (PBS plus 2%FCS) and stained with CD8  -PerCP and Mamu-A*01-phycoerythrin (PE) tet-ramers. After fixation with PFA overnight, cells were washed twice with FACSbuffer and treated with 150  l of permeabilization buffer (0.1% saponin in FACSbuffer) for 5 min at room temperature. Cells were washed once more with 0.1%saponin and then incubated in the dark for 50 min with 1   l of anti-humanIFN-  -FITC MAb (Pharmingen; clone 4S.B3; catalog no. 18904A). Finally, cells were washed four times with 0.1% saponin buffer, and a 100-  l cell suspension was fixed with 450  l of 2% PFA.  Animals, viruses, and infections.  Rhesus macaques used in this study wereidentified as Mamu-A*01  by PCR-SSP and direct sequencing as previouslydescribed (38). All rhesus macaques used in this study were Mamu-A*01   withthe exception of animal 95003. Rhesus macaques 96078 and 96087 are naivemacaques. Animals 94004 and 96031 were vaccinated 10 weeks previously with aDNA-modified vaccinia virus Ankara (MVA) regimen expressing the Gag_CM9peptide (3). Animal 95024 was infected intravenously with 40 50% tissue cultureinfectious doses of a heterogeneous SIV stock (srcinally provided by R. C.Desrosiers, Harvard University and New England Regional Primate ResearchCenter). The stock was amplified by growth on rhesus PBMC with a final passageon CEMx174 cells to increase titers (50, 68). Rhesus macaques 95114, 95115,96031, and 95003 were infected intrarectally with a molecularly cloned virus,SIVmac239. This stock was amplified on rhesus PBMC only. SIV-infected ani-mals were cared for according to an experimental protocol approved by theUniversity of Wisconsin Research Animal Resource Committee. RESULTSIdentification of 37 SIV-derived peptides which bind toMamu-A*01.  To explore whether multiple CTL epitopes inMamu-A*01  rhesus macaques could be identified, we usedthe previously defined motif for Mamu-A*01 to scan all SIVproteins (2). A total of 211 peptides were identified which wereanalyzed using in vitro peptide-binding experiments utilizingpurified Mamu-A*01 molecules. Each potential binder wasused to outcompete the radiolabeled probe peptide in ourpeptide binding assay. Under the stoichometric conditionsused in the assay, IC 50  is a reasonable approximation of   K   d . It was found that 37 peptides bound with an IC 50  of less than 500nM (Table 1). The 500 nM affinity threshold has previouslybeen shown to be associated with recognition in vivo in bothmurine and human systems (59, 60, 70, 72). Seventeen of thepeptides identified herein bound Mamu-A*01 with IC 50  valuesof 50 nM or less and therefore would be classified as high-affinity binders. The remaining 20 peptides bound in the 51 to500 nM range and would be classified as intermediate binders(56). It is noteworthy that 21 peptides bound with greateraffinity than the known Gag_CM9 epitope. Interestingly, nopotential Mamu-A*01-restricted peptides that bound with IC 50  values of less than 500 nM were identified in Nef or Vpr. Elispot identifies 14 Mamu-A*01-bound peptides in SIV-infected macaques.  We then analyzed whether the 37 selectedpeptides (IC 50   500 nM) were actually recognized in vivo byfresh PBMC derived from SIV-infected Mamu-A*01  animals(Table 2). Two naive, uninfected Mamu-A*01  animals (96078and 96087) were initially tested in Elispot assays. None of the37 peptides induced significant responses at either 1 or 10  Mpeptide concentrations in either of these control animals (datanot shown). TABLE 1. SIV-derived peptides that bind to Mamu-A*01 with IC 50  values below 500 nM  a Peptide no.or virusProtein andamino acids Peptide Sequence IC 50 (nM)Refer-ence 1 Env 235–242 Env_CL8 CAPPGYAL 1.92 Pol 143–152 Pol_LV10 LGPHYTPKIV 3.03 Pol 51–61 Pol_EA11 EAPQFPHGSSA 3.74 Env 235–243  Env_CL9  CAPPGYALL 5.55 Env 729–738 Env_ST10 SPPSYFQTHT 8.26 Pol 621–629  Pol_SV9 621  STPPLVRLV 8.77 Pol 588–596 Pol_QV9 QVPKFHLPV 9.18 Env 622–630  Env_TL9  TVPWPNASL 109 Vpx 102–111 Vpx_GL10 GPPPPPPPGL 2310 Pol 474–483 Pol_IL10 IYPGIKTKHL 2311 Pol 621–628 Pol_SL8 STPPLVRL 2612 Pol 147–155 Pol_YI9 YTPKIVGGI 2613 Gag 372–379 Gag_LF8 LAPVPIPF 2914 Rev 87–96 Rev_DL10 DPPTNTPEAL 3915 Pol 359–368 Pol_GM10 GSPAIFQYTM 4816 Gag 372–380 Gag_LA9 LAPVPIPFA 5017 Vpx 39–48 Vpx_HV10 HLPRELIFQV 5018 Env 763–771 Env_SI9 SWPWQIEYI 6719 Pol 957–964 Pol_MI8 MTPAERLI 7720 Pol 34–43 Pol_QF10 QMPRQTGGFF 7821 Pol 359–367 Pol_GT9 GSPAIFQYT 8222 Gag 181–189  Gag_CM9  CTPYDINQM 8623 Vif 75–82 Vif_LL8 LTPERGWL 9024 Gag 170–177 Gag_VL8 VVPGFQAL 9925 Vif 100–107 Vif_VI8 VTPDYADI 10326 Vif 144–152 Vif_QA9 QVPSLQYLA 14127 Tat 28–35 Tat_TL8 TTPESANL 14828 Vif 14–22 Vif_RW9 RIPERLERW 17029 Env 133–140 Env_AV8 AAPTSAPV 17530 Gag 254–262 Gag_QI9 QNPIPVGNI 19631 Env 431–439 Env_YI9 YVPCHIRQI 21032 Env 728–736 Env_ST9 SSPPSYFQT 22033 Vpx 8–18 Vpx_II11 IPPGNSGEETI 24134 Gag 340–349 Gag_VT10 VNPTLEEMLT 26735 Env 504–512 Env_IT9 ITPIGLAPT 28636 Gag 149–157 Gag_LW9 LSPRTLNAW 35537 Pol 692–700 Pol_SV9 692  SGPKTNIIV 366SIV Env 235–243  Env_CL9  CAPPGYALL 25SIV Pol 621–629  Pol_SV9  STPPLVRLV 22SIV Env 622–630  Env_TL9  TVPWPNETL 25SIV Gag 181–189  Gag_CM9  CTPYDINQM 2, 44SHIV Env 431–439  Env_YI9  YAPPISGQI 22  a Previously defined Mamu-A*01-restricted SIV/SHIV CTL epitopes areshown in boldface. 740 ALLEN ET AL. J. V IROL  .   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om   Using IFN-   ELISPOT analysis of fresh PBMC derivedfrom four SIV-infected Mamu-A*01  macaques, we were ableto demonstrate that 14 of these newly defined peptides, inaddition to the previously identified Gag_CM9 epitope (2, 44), were well recognized (Fig. 1). The number of SFCs detectedagainst each peptide in these animals ranged from 11 to 114per 200,000 PBMC plated. While considerable variability ex-isted from animal to animal with respect to peptides that wererecognized, with few exceptions replicate assays conducted onPBMC from each animal gave reproducible responses. Stimu-lation with 12 of these peptides gave positive responses inanimal 96031 (Fig. 1A). While this animal demonstrated a verybroad immune response, the strongest response was to theGag_CM9 peptide against which this animal had been previ-ously vaccinated (Table 2). When PBMC from animal 96031 were stimulated with a lower concentration of peptide (1  M), while many of the epitopes still induced equally strong re-sponses, some of the weakly responding epitopes were nolonger stimulatory (Fig. 1A). Unlike animal 96031, animal95024 (25 months post-SIV infection) responded to only a fewof the peptides (Fig. 1A). Interestingly, three new peptides(Env_CL8, Pol_LV10, and Env_TL9) gave better responsesthan the Gag_CM9 epitope. Animal 95114 also demonstrateda very broad Mamu-A*01-restricted immune response afterSIV infection (Fig. 1B). In this animal, a total of 22 peptides were recognized. In the first assay conducted on this animal,eight of the peptides gave SFC values greater than that for theGag_CM9 epitope. Finally, in animal 95115, while a few low-responding peptides were detected in the initial assay, with theexception of responses against the Gag_CM9 epitope, theseresponses appeared to subside over time (Fig. 1C). A summaryof the ELISPOT responses in the four SIV-infected animals ispresented in Table 3. In total, 14 peptides which gave signifi-cant ELISPOT responses in at least two independent assays were considered positive. Since the Env_CL8 and Env_CL9peptides overlap, we are considering this to represent a singlepositive response.To rule out the possibility that the reactivity against thesedifferent peptides is actually the result of cross-reactivity of Tcells generated against the Gag_CM9 peptide, we carried outELISPOT assays using the complete set of peptides in aMamu-A*01  animal (94004) that had been vaccinated onlyagainst the Gag_CM9 peptide. This animal had previouslyshown good reactivity against this peptide, with up to 20% of this animal’s CD3 CD8   lymphocytes being positive for tet-ramer staining against the Gag_CM9 epitope 1 week followingits first MVA (3). Analysis of Gag_CM9-reactive lymphocytesfrom this animal 10 weeks after receiving MVA revealed 91SFCs per 200,000 cells (data not shown). No reactivity was seenagainst any of the other peptides, indicating that the responsesin the SIV-infected animals were likely not a result of cross-reactivity to the Gag_CM9 peptide. To investigate whetherSIV infection alone was responsible for these reactivities, weused all 37 peptides in an ELISPOT assay in an SIV-infectedMamu-A*01-negative animal (95003). None of the peptides were recognized in this animal (data not shown).To determine whether the peptide-specific production of IFN-   was an MHC class I-restricted response, a replicateELISPOT assay (200,000 cells/well, 10   M peptide) was con-ducted using CD8  -depleted PBMC from animal 95114. Inthis assay, positive responses were no longer detected frompeptides which had induced positive responses in bulk PBMC(data not shown), confirming the role of CD8  T cells inmediating these responses.  Activity of newly identified epitopes in recall CTL assaysfrom SIV-infected animals.  We then used  51 Cr release CTL assays to determine whether these peptides could recall in vitromemory CTL activity from SIV-infected animals. When testedin a chronically SIV-infected Mamu-A*01  macaque (95024),several of the peptides recalled good CTL activity after a2-week culture period (Table 4). Additional cultures were alsoinitiated from two other SIV-infected Mamu-A*01  animals(95114 and 95115). As with ELISPOT, not all peptides thatinduced a CTL response in a particular animal did so in allanimals. Seventeen peptides were reproducibly consideredpositive by  51 Cr release assays under the criteria listed in Table4. Fourteen of these peptides were recognized in more thanone animal, while the three remaining peptides were repro-ducibly detected only in a single animal.Compared to the 14 peptides positively identified by theELISPOT assays, recall in vitro memory CTL activity wasdetected against 7 of these peptides. Of the remaining sevenELISPOT-positive peptides, three yielded a positive CTL re-sponse in a single CTL assay (Table 4). Unfortunately, repli-cate CTL assays were not conducted to confirm these re-sponses. In addition, nine peptides that were not consistentlyrecognized in ELISPOT demonstrated positive recall memoryCTL activity. Therefore, recall memory CTL activity was de-tectable against a significant number of the peptides which had yielded positive responses by ELISPOT. Tetramer analysis of antigen-specific CD8  cells.  We nextdetermined whether we could detect antigen-specific CD8  responses against four of the newly defined peptides in freshPBMC using Mamu-A*01 tetramers refolded with each of these peptides (Table 5). Responses were examined in three of the SIV-infected macaques (96031, 95114, and 95115) whichhad demonstrated positive ELISPOT responses to these pep-tides, as well as in a naive Mamu-A*01  animal (96078). TheGag_CM9 tetramer detected levels of antigen-specific CD3CD8  T lymphocytes ranging from 0.35 to 2.68% in the SIV-infected animals. Positive responses detected using the otherfour tetramers, however, were lower and ranged between 0.14and 0.48%. The Env_TL9 (TVPWPNASL) and Env_CL8(CAPPGYAL) tetramers detected good responses in all three TABLE 2. MHC type, vaccination, and infection status of animals used to detect multiple CTL epitopes  a  Animalno.MHCtype Vaccination Infection 96078 A*01 No No96087 A*01 No No96031 A*01 Yes, 2 wk prior to infection Yes, SIVmac239 i.r.94004 A*01 Yes, 10 wk previously No95024 A*01 No Yes, SIVmac biologicalisolate i.v.95114 A*01 No Yes, SIVmac239 i.r.95115 A*01 No Yes, SIVmac239 i.r.95003 — No Yes, SIVmac239 i.r.  a  Animals were vaccinated with DNA-MVA containing the Gag_CM9 peptide(3). Infection with SIVmac239 or SIVmac biological isolate was done intrave-nously (i.v.) or intrarectally (i.r.). V OL  . 75, 2001 MULTIPLE SIV CTL EPITOPES IN MACAQUES 741   onM ar  c h 1 2  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j  v i  . a s m. or  g /  D  ownl   o a d  e d f  r  om 
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