Hierarchical Targeting of Subtype C Human Immunodeficiency Virus Type 1 Proteins by CD8+ T Cells: Correlation with Viral Load

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Hierarchical Targeting of Subtype C Human Immunodeficiency Virus Type 1 Proteins by CD8+ T Cells: Correlation with Viral Load
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    10.1128/JVI.78.7.3233-3243.2004. 2004, 78(7):3233. DOI: J. Virol. and Clive M. GrayJames McIntyre, Salim Abdool Karim, Haynes W. SheppardSusan Allen, Newton Kumwenda, Taha Taha, Glenda Gray, Lynn Zijenah, David Katzenstein, Rosemary Musonda,Mohube, Pauline Mokgotho, Efthyia Vardas, Mark Colvin, Agatha Masemola, Tumelo Mashishi, Greg Khoury, Phineas  T Cells: Correlation with Viral Load+CD8byImmunodeficiency Virus Type 1 Proteins Hierarchical Targeting of Subtype C Human http://jvi.asm.org/content/78/7/3233Updated information and services can be found at: These include:  REFERENCES http://jvi.asm.org/content/78/7/3233#ref-list-1at: This article cites 48 articles, 27 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  on S  e p t   em b  er 2 4  ,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  on S  e p t   em b  er 2 4  ,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 , Apr. 2004, p. 3233–3243 Vol. 78, No. 70022-538X/04/$08.00  0 DOI: 10.1128/JVI.78.7.3233–3243.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved. Hierarchical Targeting of Subtype C Human Immunodeficiency VirusType 1 Proteins by CD8  T Cells: Correlation with Viral Load  Agatha Masemola, 1 † Tumelo Mashishi, 1 † Greg Khoury, 1 Phineas Mohube, 1 Pauline Mokgotho, 1 Efthyia Vardas, 2,3 Mark Colvin, 2 Lynn Zijenah, 4 David Katzenstein, 5 Rosemary Musonda, 6 Susan Allen, 6 Newton Kumwenda, 7 Taha Taha, 7 Glenda Gray, 3 James McIntyre, 3 Salim Abdool Karim, 2,8 Haynes W. Sheppard, 9 Clive M. Gray, 1 *and the HIVNET 028 Study Team‡  National Institute for Communicable Diseases 1  and Perinatal HIV Research Unit, University of the Witwatersrand, 3  Johannesburg, and HIV Vaccine and Prevention Trials Unit, MRC, 2  and University of KwaZulu Natal, 8  Durban, South Africa; Department of  Immunology, University of Zimbabwe, Harare, Zimbabwe 4  ; Center for AIDS Research, Stanford University Medical Center, Stanford, 5  and California Department of Health Sciences, Richmond, 9 California; Zambia-UAB HIV Research Project, Lusaka, Zambia 6  ; and Johns Hopkins Research Project, Blantyre, Malawi 7 Received 15 August 2003/Accepted 21 November 2003  An understanding of the relationship between the breadth and magnitude of T-cell epitope responses and viral loads is important for the design of effective vaccines. For this study, we screened a cohort of 46 subtypeC human immunodeficiency virus type 1 (HIV-1)-infected individuals for T-cell responses against a panel of peptides corresponding to the complete subtype C genome. We used a gamma interferon ELISPOT assay toexplore the hypothesis that patterns of T-cell responses across the expressed HIV-1 genome correlate with viralcontrol. The estimated median time from seroconversion to response for the cohort was 13 months, and theorder of cumulative T-cell responses against HIV proteins was as follows: Nef > Gag > Pol > Env > Vif > Rev> Vpr > Tat > Vpu. Nef was the most intensely targeted protein, with 97.5% of the epitopes being clustered within 119 amino acids, constituting almost one-third of the responses across the expressed genome. Thesecond most targeted region was p24, comprising 17% of the responses. There was no correlation between viralload and the breadth of responses, but there was a weak positive correlation (  r  0.297;  P  0.034) between viral load and the total magnitude of responses, implying that the magnitude of T-cell recognition did notcontribute to viral control. When hierarchical patterns of recognition were correlated with the viral load,preferential targeting of Gag was significantly (  r  0.445;  P  0.0025) associated with viral control. These datasuggest that preferential targeting of Gag epitopes, rather than the breadth or magnitude of the responseacross the genome, may be an important marker of immune efficacy. These data have significance for the designof vaccines and for interpretation of vaccine-induced responses. It is estimated that 42 million people are infected with hu-man immunodeficiency virus type 1 (HIV-1), with 29.4 millioninfected individuals, constituting 70% of the global epidemic,living in sub-Saharan Africa (http://unaids.org/). The worst hitareas of the HIV/AIDS pandemic are in southern Africa, andin South Africa  25% of the adult population is infected (17), with high incidence rates (47). Molecular epidemiological stud-ies have shown that the HIV-1 epidemic is heterosexuallytransmitted and dominated by subtype C (22, 37, 44, 45). Thus,developing an effective HIV-1 vaccine to curtail this epidemicis increasingly important, and the identity of possible correlatesof immune protection against HIV infection or disease isthought to be fundamental to this process. Since one of themain outcomes of a possible efficacious vaccine would be thestimulation of anti-HIV T-cell immunity, it is important todefine the type and scope of T-cell immune responses thatcorrelate with control of the virus. Accordingly, the aim of thisstudy was to determine what aspects of CD8  T-cell immunityare associated with the control of subtype C HIV-1 in naturalinfections.Class I major histocompatibility complex-restricted CD8  Tlymphocytes play a central role in the host immune response toHIV infection (6, 11, 12, 14, 24, 25, 30). Compelling evidencefor the role of these cells has been shown by depleting CD8  T cells in SIV-infected macaques, resulting in enhanced virusreplication and accelerated pathogenesis (26, 34, 40). Vaccinestrategies capable of eliciting virus-specific CD8  T-cell re-sponses have been shown to control virus replication and toprevent the onset of disease in monkeys (4, 5, 41). With humanstudies, the emergence and preservation of cytotoxic T-lym-phocyte (CTL) responses in individuals with acute HIV infec-tion have been shown to coincide with a rapid decline inplasma viremia (29, 49); strong and robust HIV-specific CTL responses are maintained in pediatric and adult long-term non-progressors (13, 20, 27, 39), and disease progression is accom-panied by a decline in CTL responses (27). While CD8  T cellsappear to play an important role in anti-HIV immunity, thereare conflicting clinical data on the association of CD8  T-cellresponses with plasma viremia. Some studies have observed an * Corresponding author. Mailing address: National Institute forCommunicable Diseases, Private Bag X4, Sandringham 2131, South Africa. Phone: 27 11386 6372. Fax: 27 11386 6310. E-mail: cgray@nicd.ac.za.† A.M. and T.M. contributed equally to this work.‡ Contributing members of the HIVNET 028 Study Team are listedin Acknowledgments.3233   on S  e p t   em b  er 2 4  ,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   inverse correlation between plasma viral load and HIV-speci fi cT-cell responses (9, 10, 14, 21, 36, 38), and other studies havedemonstrated a positive correlation (7, 11). In contrast, recentreports have found no correlation between plasma viral loadand HIV-speci fi c CD8  T-cell responses (1, 36).The identi fi cation of CTL epitopes during the early stage of HIV-1 infection is thought to be important for vaccine design,since vaccines may elicit epitope-speci fi c responses that arerelevant for contemporary circulating viruses. These responsesare considered important as a de fi ning property of immuno-genicity in HIV-1 vaccine trials and are a desired outcome of  vaccine-induced T-cell immunity. In addition to the identi fi ca-tion and de fi nition of signi fi cant epitopes, it is also importantto de fi ne the qualitative nature of anti-HIV T-cell immunity.With this objective, we investigated CD8  T-cell responsesagainst a series of overlapping subtype C-based peptides cor-responding to the nine expressed HIV-1 gene regions by usinga gamma interferon (IFN-  ) ELISPOT assay. We measuredthe frequency of T-cell responses from individuals recentlyinfected with subtype C HIV-1 and identi fi ed associations be-tween patterns of CD8  T-cell recognition and viral control.There was no signi fi cant association between the breadth of response and the plasma viral load, although there was a sig-ni fi cant positive correlation between total cumulative re-sponses. When the order of recognition of protein regions wasplaced in a hierarchy for each individual, we found a signi fi cantpositive correlation between the preferential targeting of Gagand the plasma viral load. These data represent importantinformation regarding mechanisms associated with viral con-trol and how multigenic vaccine candidates may be evaluated. MATERIALS AND METHODSPatient cohort.  Forty-six patients were recruited within approximately 2 yearsof seroconversion, with an estimated median time of 13 months (Table 1). Thetime from seroconversion was estimated as the midpoint between the last anti-body-negative assay result and the  fi rst HIV-1-positive enzyme-linked immu-nosorbent assay result. All individuals were na ï  ve for antiretroviral drugs. Theseindividuals were recruited as part of the HIVNET 028 Study, and patients wereenrolled from  fi  ve clinic sites in four southern African countries: Malawi (  n  8),Zimbabwe (  n    10), Zambia (  n    8), and South Africa (  n    20). Theseindividuals displayed a wide range of viral loads, with some individuals showingcontrol of viremia and others showing no control (Table 1). There were nosigni fi cant associations between the country of srcin and viremia, and for thepurposes of this study, individuals were grouped as one cohort. Synthetic peptides.  Synthetic peptides spanning nine subtype C HIV-1 generegions (with the exception of integrase) were made by using 9- fl uorenylmethoxycarbonyl-based solid-phase chemistry (Natural and Medical Sciences Institute,University of Tu ¨ bingen, Tu ¨ bingen, Germany). All peptides were checked for thecorrect molecular weight by Elektrospray QTOF-mass spectrometry. Peptidepurities ranged from 70 to 80%. A total of 396 overlapping peptides weresynthesized, some of which were designed to match gene regions selected forinclusion in subtype C vaccine candidates. Pol, Rev, Tat, Nef, and gp160 werebased on Du151 and Du179 (gp160) (48), and Gag, Vpu, Vif, and Vpr werebased on consensus C (a generous gift from Marcus Altfeld, MassachusettsGeneral Hospital). Nef peptides were synthesized as 15-mers overlapping at 11amino acids (aa), and the remaining peptides varied from 15- to 18-mers over-lapping at 10 residues and were designed by use of PeptGen (http://www.hiv.lanl.gov/content/hiv-db/PEPTGEN/PeptGenSubmitForm.html). Peptides were dis-solved in 100% dimethyl sulfoxide at an initial concentration of 10 mg/ml and were pooled at 40   g/ml/peptide stock in phosphate-buffered saline (PBS) in which the  fi nal dimethyl sulfoxide concentration was always  0.5%. Design of peptide pools.  The 396 peptides used for this study were arranged ina pool with a matrix design allowing a single screen of responses across theHIV-1 genome on one 96-well plate. Each of the nine gene regions was pooled with no more than 24 peptides/pool. Table 2 shows the details of the pools foreach of the nine expressed gene regions.Forty-eight more pools were constructed in a matrix design (M1 to M48)containing 12 or 5 peptides/pool. By employing a pool and matrix approach, it was possible to identify individual peptide responses from multiple pool re-sponses. For example, if a response was identi fi ed in Gag pool 1, the Tat pool,Env pools 2 and 5, and Nef pools 2 and 3 and the matrix pool responses werefound in M2, M21, M27, M39, and M48, this resulted in peptide selections of Gag 3, Tat 1 and 11, Env 51 and 104, and Nef 20 and 21. Thus, from the 396peptides in the initial screen, the selection was eventually narrowed to sevenindividual peptides for con fi rmation. PBMC preparation.  Cryopreserved peripheral blood mononuclear cells(PBMC) were thawed and allowed to rest overnight in R10 medium (RPMI 1640[Invitrogen, Paisley, United Kingdom]) supplemented with 10% fetal calf serum TABLE 1. Clinical characteristics of 46 patients screened for fullgenome expressed responses at speci fi c times after seroconversion Patientno.Monthspostsero-conversionCD4 count(per mm 3 )CD8 count(per mm 3 )CD4/CD8ratioNo.of RNA copies/ml 1 4.4 371 853 0.435 15,5652 4.4 610 727 0.839 15,5883 4.9 434 234 1.855 504 4.9 661 684 0.966 29,0135 5.1 512 714 0.717 1,5236 5.3 473 1,143 0.414 4,1087 5.5 424 846 0.501 4,1608 6.5 637 591 1.178 ND  b 9 6.7 538 796 0.676 1,19510 8.0 188 1,140 0.165 331,44111 8.0 486 720 0.675 9,74612 8.5 45 174 0.259 202,73513 8.5 75 311 0.241 239,82814 5.3 481 577 0.834 146,91615 10.5 1,307 669 1.954 1,10416 10.8 167 432 0.387 46,32817 11.2 242 907 0.267 73,75318 11.7 287 595 0.482 33,92119 12.0 780 739 1.055 6,18920 12.2 455 508 0.896 20,43921 12.4 300 479 0.626 11,46322 12.9 703 1,151 0.611 2,30023 13.1 361 2,000 0.18 46,98424 14.3 255 741 0.344 16,95225 14.3 460 483 0.952 5,01426 12.9 608 685 0.888 8,58327 14.4 670 2,307 0.29 77428 15.1 322 710 0.454 5,78829 16.1 522 507 1.03 14,19930 16.5 253 1,663 0.152 68,52131 17.6 143 641 0.223 23,27332 18.1 600 1,212 0.495 6,84133 19.0 397 1,432 0.277 1,86234 19.0 448 1,252 0.358 8,04835 20.1 663 1,551 0.427 56936 21.9 476 709 0.671 93,69737 22.5 855 927 0.922 10838 22.8 178 331 0.538 39,88739 23.0 250 395 0.633 3,88040 23.2 216 473 0.457 25,24941 24.1 308 451 0.683 33,57542 24.4 453 1,141 0.397 37,70643 24.5 560 883 0.634 71844 24.7 507 602 0.842 45,00745 25.0 337 542 0.622 10,72746 25.7 364 804 0.453 1,983Median 13.0 451 712 0.6 11,46325% IQR  a 8.1 290 517 0.4 3,88075% IQR  a 19.8 555 922 0.8 37,706  a IQR, interquartile range.  b ND, not done. 3234 MASEMOLA ET AL. J. V IROL  .   on S  e p t   em b  er 2 4  ,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   (Invitrogen) and 50 U of gentamicin (Invitrogen) at 2  10 6 to 4  10 6 PBMC/mlat 37 ° C in 5% CO 2  prior to use in the ELISPOT assay (15). After the incubation,cells were counted by trypan blue dye exclusion and were used in assays. ELISPOT assay.  ELISPOT assays were performed as previously described(31). Brie fl  y cryopreserved PBMC were thawed and plated in 96-well polyvinyl-idene di fl uoride plates (MAIP S45; Millipore, Johannesburg, South Africa) thathad been coated with 50  l of anti-IFN-  monoclonal antibody 1-D1k (2  g/ml)(Mabtech, Stockholm, Sweden) overnight at 4 ° C. Peptides were added directly tothe wells at a  fi nal concentration of 2  g/ml along with 1  10 5 to 2  10 5 cellsin 50  l of R10 medium and were incubated at 37 ° C in 5% CO 2 . After 16 to 18 h,plates were extensively washed with PBS and wash buffer (PBS, 1% fetal calf serum, and 0.001% Tween 20), followed by incubation with a biotinylated anti-IFN-  monoclonal antibody (2  g/ml) (clone 7-B6-1; Mabtech) at room temper-ature for 3 h. After six more washes with wash buffer, 2   g of streptavidin-horseradish peroxidase (Pharmingen)/ml was added to the wells, and the plates were incubated for another 1 h at room temperature. Spots were visualized byusing Novared substrate (Vector, Burlingame, Calif.) according to the manufac-turer ’ s instructions. Duplicate wells containing PBMC and R10 medium as wellas R10 medium alone were used as negative controls. Wells containing PBMCand phytohemagglutinin served as positive controls. As an additional control,duplicate wells containing PBMC and pools of cytomegalovirus, Epstein-Barr virus, and in fl uenza virus (CEF) epitopes were used (16). The number of spotsper well was counted with an Immunospot (Cellular Technology Ltd., Cleveland,Ohio) automated plate counter. Peptide responses were con fi rmed by usingindividual peptides in the ELISPOT assay, and in some experiments, CD8  T-cell dependence was con fi rmed by CD8 and immunoglobulin G (mock) de-pletion by use of magnetic beads (Dynal, Oslo, Norway) according to the man-ufacturer ’ s instructions.  Assessment of cutoffs in the ELISPOT assay.  An important criterion forde fi ning what constitutes a positive response in the ELISPOT assay is the lowerthreshold, or cutoff, above which a response is classi fi ed as positive. For deter-mination of the background reactivity to the HIV-1 peptides, PBMC from 25HIV-1-seronegative individuals were used to de fi ne the spread of negative re-sponses. Figure 1 shows the spread of spot-forming units (SFU)/10 6 PBMC, where the mean SFU/10 6 PBMC for each pool ranged from 13 to 19, with anoverall mean value of 16    22 SFU/10 6 PBMC. For the purpose of de fi ning acutoff, 4 standard deviations (88 SFU/10 6 PBMC) above the mean, or   104SFU/10 6 PBMC, was considered positive. Additionally, if the number of spots inthe negative controls exceeded 20 SFU/10 6 PBMC per well, the ELISPOT assay was repeated. A further criterion used for de fi ning positive responses was amatch between a pool response and a matrix response. Statistical and data analysis.  All data were expressed as medians with inter-quartile ranges and were analyzed by use of nonparametric statistics. Kruskal-Wallis nonparametric analysis of variance was used to test for signi fi cant differ-ences across gene regions, and Dunn ’ s pairwise analysis was used to identifydifferences between gene regions and regions recognized within each gene re-gion. For tests between two groups, the Wilcoxon signed rank test was used. Forassessments of the relationships between responses, Spearman rank correlations were used and corrected for multiple tests. Statistical analysis was performed byuse of SigmaStat 2.0 (SPSS Science, Chicago, Ill.).Cumulative responses identi fi ed for each protein region were adjusted forprotein length by dividing the total combined pool response identi fi ed for eachindividual by the number of amino acids making up the protein region. Thebreadth of responses for each participant was calculated by (i) counting thenumber of positive peptide pools and (ii) counting the number of protein re-gions. Hierarchical responses were identi fi ed by ordering each of the combinedpeptide pool responses (corresponding to Gag, Pol, Vif, Vpr, Tat, Rev, Vpu,Env, and Nef) from one to nine, with one being the highest SFU/10 6 PBMC valueand nine being the lowest response. RESULTSDistribution of HIV-1-speci fi c T-cell responses across sub-type C.  Forty-four of 46 (95.6%) subtype C HIV-1-infectedindividuals responded to one or more of the 396 peptides usedin this study. Figure 2 shows the distribution of SFU/10 6 PBMC for the 44 responders across the expressed genome, with 87% recognizing Nef, 83% recognizing Gag, 74% recog-nizing Pol, 63% recognizing Env, 28% recognizing Vif, 22% TABLE 2. Arrangement of each of the nine expressedgene regions into pools GeneregionNo. of poolsNo. of peptides/ poolPeptidelength (aa)Overlap(aa)Strain thatpeptides werebased on Pol 4 24 15 – 18 10 Du 151Tat 1 12 15 – 18 10 Du 151Rev 1 14 15 – 18 10 Du 151Env 5 24 15 – 18 10 Du 179Nef 5 10 15 11 Du 151Gag 5 14 15 – 18 10 ConsensusVif 2 12 15 – 18 10 ConsensusVpr 1 11 15 – 18 10 ConsensusVpu 1 9 15 – 18 10 Consensus FIG. 1. Distribution of responses from HIV-1-seronegative individ-uals to each of the peptide pools (Gag to Nef), showing the boundariesof the mean SFU/10 6 PBMC and 4 standard deviations (dotted linesdemarcated by arrow), with the upper threshold being 104 SFU/10 6 PBMC.FIG. 2. Distribution of responses across complete peptide poolsets, showing medians with the 10th and 90th percentiles as well asindividual plots. The proportion of individuals responding to eachpeptide set is shown above the plot, and the signi fi cance levels betweenregions are shown below the plot. Signi fi cance was measured byKruskal-Wallis nonparametric analysis of variance and Dunn ’ s pair- wise analysis. n.s., not signi fi cant.V OL  . 78, 2004 VIRAL LOAD AND CD8  T-CELL RESPONSES TO HIV-1 3235   on S  e p t   em b  er 2 4  ,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   recognizing Vpr, 17% recognizing Tat, 15% recognizing Rev,and 2% recognizing Vpu. When median SFU/10 6 PBMC val-ues were compared across different regions of the genome, thatfor Nef was highest. No signi fi cant differences existed betweenGag, Pol, Env, and Nef SFU/10 6 PBMC values. However, theseresponses were all signi fi cantly higher than those for Vif, Vpr,Tat, Rev, and Vpu (Fig. 2). Distribution of HIV-1-speci fi c T-cell responses within sub-type C proteins.  The extent of T-cell targeting within each generegion was further delineated by examining the cumulativefrequencies of SFU/10 6 PBMC in speci fi c pools, demarcated byamino acid numbers in Fig. 3. Overall, the highest cumulativemagnitude of HIV-1-speci fi c cells was found directed to thecentral region of Nef, between aa 52 and 171 (Fig. 3A). This119-aa stretch made up 97.5% of the anti-Nef responses andconstituted 32% of the total cumulative SFU/10 6 PBMC rela-tive to the rest of the genome. Within Gag, responses to aa 219to 322 in p24 were signi fi cantly (  P   0.05) higher than those toany other subregion of Gag (Fig. 3A). Relative to the totalcumulative response across the genome, responses to p24made up 17.4%. For Pol, the SFU/10 6 PBMC values for re- verse transcriptase (aa 355 to 533) and RNase H (aa 534 to689) peptides were signi fi cantly higher than those for otherregions within Pol, and they collectively amounted to 9.6% of the total responses across the genome. Because integrase wasmissing from the pool makeup, it was not possible to gaugehow this region was targeted or how it in fl uenced the spread of responses within Pol. Within Env, there appeared to be auniform spread of SFU/10 6 PBMC values within gp41 andgp120 (aa 192 to 694). Of the accessory gene products, there was a higher response to the N-terminal half of Vif (aa 1 to102) than to the C terminus, although the difference was notsigni fi cant. Relative to the total positive response across thegenome, the SFU/10 6 PBMC values for Rev and Tat consti-tuted 4.4 and 2%, respectively, and the least recognized pep-tides were those corresponding to Vpr and Vpu.We next analyzed the frequencies of individual peptide poolresponses targeted by subtype C HIV-1-infected individuals.Our data indicated that the most recognized region was thecentral region of Nef, followed by p17 (aa 1 to 111), p24 (aa219 to 322), RNase H (aa 534 to 689), and pools within Env(Fig. 3B). Although there was a higher magnitude of responseto epitopes in p24 Gag, epitopes in p17 were more frequentlyrecognized. Identity of epitopic regions within Gag, Pol, Vif, Env, andNef.  Some of the responses identi fi ed in the initial full-genomescreen were con fi rmed by using single peptides in subsequentELISPOT assays. Single-peptide con fi rmations allowed us toidentify possible epitopes within 15- to 18-mers. Table 3 showsthe amino acid sequences of con fi rmed subtype C peptideresponses to Gag, Pol, Vif, Env, and Nef, and 24 of 47 (51%)responses were also identi fi ed as being CD8  T-cell mediatedafter the depletion of CD8  cells in the ELISPOT assay. There were insuf  fi cient samples to con fi rm the remaining responsesas being CD8  T-cell mediated. Seventy-four percent (35 of 47) of con fi rmed subtype C peptide responses were matched FIG. 3. Responses to peptide pools. (A) Cumulative SFU/10 6 PBMC responses to each of the peptide pools. (B) Frequencies of individualsrecognizing each of the peptide pools, depicted as amino acid numbers, to show more detailed recognition of sites within pools. Consecutive aminoacid numbers are shown across all pools, amounting to a total of 3,091 aa, corresponding to the complete expressed genome.3236 MASEMOLA ET AL. J. V IROL  .   on S  e p t   em b  er 2 4  ,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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