Simian Immunodeficiency Virus SIVmac239 Infection of MajorHistocompatibility Complex-Identical Cynomolgus
Macaques from Mauritius�
Roger W. Wiseman,1† Jason A. Wojcechowskyj,1† Justin M. Greene,1 Alex J. Blasky,1 Tobias Gopon,1Taeko Soma,1 Thomas C. Friedrich,1 Shelby L. O’Connor,2 and David H. O’Connor1,2*
Wisconsin National Primate Research Center, University of Wisconsin—Madison, Madison, Wisconsin 53706,1 and Department ofPathology and Laboratory Medicine, University of Wisconsin—Madison, Madison, Wisconsin 537062
Received 23 August 2006/Accepted 2 October 2006
Nonhuman primates are widely used to study correlates of protective immunity in AIDS research. Successfulcellular immune responses have been difficult to identify because heterogeneity within macaque major histo-compatibility complex (MHC) genes results in quantitative and qualitative differences in immune responses.Here we use microsatellite analysis to show that simian immunodeficiency virus (SIV)-susceptible cynomolgusmacaques (Macaca fascicularis) from the Indian Ocean island of Mauritius have extremely simple MHCgenetics, with six common haplotypes accounting for two-thirds of the MHC haplotypes in feral animals.Remarkably, 39% of Mauritian cynomolgus macaques carry at least one complete copy of the most frequentMHC haplotype, and 8% of these animals are homozygous. In stark contrast, entire MHC haplotypes are rarelyconserved in unrelated Indian rhesus macaques. After intrarectal infection with highly pathogenic SIVmac239virus, a pair of MHC-identical Mauritian cynomolgus macaques mounted concordant cellular immune re-sponses comparable to those previously reported for a pair of monozygotic twins infected with the same strainof human immunodeficiency virus. Our identification of relatively abundant SIV-susceptible, MHC-identicalmacaques will facilitate research into protective cellular immunity.
Nonhuman primates are important models for major humaninfectious diseases, including AIDS (6). As vaccine candidatesincreasingly focus on eliciting cell-mediated immunity againsthuman immunodeficiency virus (HIV) and simian immunode-ficiency virus (SIV), there is intense interest in the genes of themajor histocompatibility complex (MHC) that define the spec-ificity of the cellular immune response. In humans, the geneticsof the MHC are well defined, with only a single polymorphicHLA-A, HLA-B, and HLA-C locus per chromosome. In con-trast, MHC haplotypes in macaques contain a variable numberof expressed polymorphic class I (7, 9, 21, 29, 39, 40) and classII (10, 12) loci.
AIDS research has motivated study of MHC genetics innonhuman primates, most notably in rhesus macaques of In-dian origin. More than 130 MHC class I alleles and 160 MHCclass II alleles as well as two genomic sequences of the MHCregion from rhesus macaques are currently in GenBank. Thelarge number of defined MHC alleles highlights the heteroge-neity of these animals. Unfortunately for SIV research, thisdiversity generally limits investigators to MHC matching ani-mals for single class I alleles, such as Mamu-A*01, rather thanentire MHC haplotypes (6, 14). Shared MHC haplotypes, com-prising MHC class IA and IB genes, MHC class II genes, andtightly linked genes involved in antigen processing and inflam-mation (16), have been identified only in rhesus macaquesrelated by descent (31, 42). Therefore, it is exceedingly difficult
to study the influence of the entire gene-dense MHC region onSIV pathogenesis in unrelated rhesus macaques.
We became interested in Mauritian cynomolgus macaques(MCM) as a model of SIV pathogenesis because of theirunique natural history. Historical records suggest that Euro-pean seafarers introduced cynomolgus macaques to the smallIndian Ocean island of Mauritius within the last 500 years (36).Mitochondrial and Y chromosome DNA analyses indicate thatthe current MCM population of between 25,000 and 35,000monkeys descended from a very small founder population thatis most likely to have originated from Sumatra and has re-mained isolated for approximately 80 to 100 generations (22;A. J. Tosi and C. S. Coke, submitted for publication). In thecontemporary Finnish human population, which descendsfrom a limited number of ancestors within approximately thesame number of generations as MCM, entire shared MHChaplotypes are common (17). Thus, we hypothesized that theunusual natural history of MCM might portend the presence ofhigh-frequency MHC haplotypes (20). We discovered that sixhigh-frequency haplotypes encompassing both the MHC classI and class II loci account for almost all MHC diversity inMCM. We also demonstrated broadly similar cellular immuneresponses in MHC-identical MCM infected with SIVmac239.
MATERIALS AND METHODS
Animals and SIVmac239 challenge. Blood samples from feral MCM werepurchased directly for genetic analyses (Charles River BRF, Houston, TX). Ourinitial microsatellite analysis focused on five MCM that were examined previ-ously in an MHC class I allele discovery study (20). Subsequently, blood fromanother 112 feral MCM was obtained in two independent shipments fromCharles River BRF for MHC class I genotyping.
A pair of male MHC-identical MCM (CY0111 and CY0113) was selectedbased on microsatellite and reference strand conformation analysis. Both ani-
* Corresponding author. Mailing address: University of Wisconsin—Madison, 555 Science Drive, Madison, WI 53711. Phone: (608) 890-0845. Fax: (608) 265-8084. E-mail: [email protected].
† R.W.W. and J.A.W. contributed equally to this study.� Published ahead of print on 11 October 2006.
mals were challenged intrarectally with a single dose of 5 � 104 TCID50 (tissueculture dose sufficient to infect 50% of cells) of molecularly cloned SIVmac239Nef open virus (19). SIV-infected animals were cared for according to theregulations and guidelines of the University of Wisconsin Institutional AnimalCare and Use Committee.
Microsatellite analysis. Multiplex PCR assays were developed for 18 micro-satellite loci spanning the MHC region (Table 1). Four of these loci wereadapted for cynomolgus macaques from a previous study with rhesus macaques(31). Additional microsatellites were identified by screening human MHC primerpairs (15) for specificity against the rhesus MHC genomic sequence (9) withBLASTn (2). Three human primer pairs were used directly, while another 10microsatellite primer sequences were modified to reflect differences in the rhesusgenomic MHC sequence. Finally, the 9268 locus was identified by searching fornovel microsatellites in the rhesus genomic MHC sequence using ETANDEM(EMBOSS suite of software) (34), and primers were designed with Primer3software (http://frodo.wi.mit.edu).
Microsatellite PCRs were carried out with PTC-225 thermocyclers (MJ Re-search) as 10-�l reactions containing 1� Phusion master mix (New EnglandBioLabs, Ipswich, MA), 10 ng genomic DNA, and 0.08 to 0.3 �M primers (Table1). The following touchdown PCR program was used: 98°C for 30 s; 3 cycles of98°C for 5 s, 64°C for 5 s, 72°C for 20 s; 3 cycles of 98°C for 5 s, 62°C for 5 s, 72°Cfor 20 s; 3 cycles of 98°C for 5 s, 60°C for 5 s, 72°C for 20 s; 6 cycles of 98°C for5 s, 58°C for 5 s, 72°C for 20 s; 25 cycles of 98°C for 5 s, 50°C for 5 s, 72°C for20 s; and a final extension at 72°C for 5 min. Fragment analysis of PCR productswas performed with an ABI 3730 DNA analyzer (Applied Biosystems, FosterCity, CA). One microliter of PCR product and 0.4 �l of ROX-ET550 DNAladder (GE Health Care, Piscataway, NJ) were diluted in 8.6 �l HiDi formamide(Applied Biosystems) and denatured for 1 min at 98°C. Samples were electro-kinetically injected at 2.5 kV for 15 s and run at 15 kV for 2,000 s using POP7polymer (Applied Biosystems). Data were analyzed using DAx data acquisitionanalysis software (Van Mierlo Software Consultancy, Eindhoven, The Nether-lands).
MHC class I RSCA. Transcribed MHC class I alleles were genotyped byreference strand conformation analysis (RSCA) of cDNA heteroduplexes fromperipheral blood mononuclear cells or whole blood essentially as describedpreviously (20) with the following modification. A 304-bp amplicon was amplifiedby PCR from cDNA in order to scan additional polymorphic sites in the highlyvariable peptide binding domains encoded by exons 2 and 3 using the 5� phos-phate (Phos)-modified primer 5�P-Refstrand (5�-[Phos]GCTACGTGGACGACACGC-3�) and Short3�RSCA (5�-TTCAGGGCGATGTAATCC-3�). The refer-ence strand providing optimal resolution of MCM heteroduplexes was Mamu-B*-07. A Mamu-B*07 clone was amplified using the dye-labeled primer 6FAM-5�-Refstrand (5�-6-carboxyfluorescein [FAM]CTACGTGGACGACACGC-3�)
and the 5� phosphate-modified primer Short3�RSCA-P (5�-[Phos]TTCAGGGCGATGTAATCC-3�). Heteroduplex mobilities were determined relative to aROX-ET900 size standard (GE Health Care) using DAx data acquisition anal-ysis software (Van Mierlo Software Consultancy).
MHC class I allele cloning and sequencing. MHC class I cDNAs were ampli-fied by PCR using a high-fidelity polymerase (Phusion; New England BioLabs),cloned into pCR-Blunt (Invitrogen, Carlsbad, CA), and sequenced essentially aspreviously described (20). In order to obtain sequences containing completepredicted open reading frames, cDNAs were amplified using PCR primers op-timized for known rhesus macaque MHC class I sequences. Each cDNA pool wasamplified with consensus primers (5�MHC UTR, 5�-GGACTCAGAATCTCCCCAGACGCCGAG-3�; and 3�MHC UTR A, 5�-CAGGAACAYAGACACATTCAGG-3�, or an alternate reverse primer 3�MHC UTR B, 5�-GTCTCTCCACCTCCTCAC-3�). Sequences were compiled for a minimum of 192 cDNA clonesfrom a representative homozygote of each MCM haplotype. Sequences wereanalyzed using Aligner software (CodonCode Corp.).
DRB genotyping. Sequence-specific PCR assays for 14 DRB alleles identifiedin MCM by Leuchte et al. (23) were optimized using the following conditions: 1�Phusion master mix, 0.05 �M concentration of each forward and reverse primer,and 10 ng of genomic DNA. Samples were amplified on MJ Research PTC-225thermocyclers at 98°C for 30 s; 35 cycles of 98°C for 5 s, 62°C to 72°C for 5 s, 72°Cfor 20 s; and a final extension at 72°C for 5 min (specific annealing temperaturesare available upon request). PCR products were then resolved on a 2.5% agarosegel and visualized with ethidium bromide and UV light.
Plasma virus analysis. The plasma virus concentration was determined usinga modification of methods described previously (41). Viral RNA was reversetranscribed and amplified using a SuperScript III Platinum one-step quantitativereverse transcription-PCR system (Invitrogen, Carlsbad, CA) in a LightCycler1.2 (Roche Diagnostics, Indianapolis, IN). The final reactions (20 �l) contained0.2 mM each deoxynucleoside triphosphate, 3 mM MgSO4, 0.015% bovine serumalbumin, 150 ng random hexamers (Promega, Madison, WI), 0.8 �l SuperScriptIII reverse transcriptase and Platinum Taq DNA polymerase in a single enzymemix, 600 nM each amplification primer (5�-GTCTGCGTCATCTGGTGCATTC-3� and 5�-CACTAGCTGTCTCTGCACTATGTGTTTTG-3�), and 100 nMprobe (5�-[FAM] CTTCCTCAGTGTGTTTCACTTTCTCTTCTGCG-3�). Thereverse transcriptase reaction was performed at 37°C for 15 min and then 50°Cfor 30 min. An activation temperature of 95°C for 2 min was followed by 50amplification cycles of 95°C for 2 min and 62°C for 1 min, with ramp times set to3 degrees per second. Serial dilutions of an SIV gag in vitro transcript were usedto generate a standard curve for each run. Copy numbers were determined byinterpolation onto the standard curve with the LightCycler software, version 4.0.
IFN-� ELISPOT analysis. Peripheral blood mononuclear cells were isolatedby Ficoll-Hypaque gradient centrifugation. A total of 1 � 105 to 2 � 105 cells
TABLE 1. MHC microsatellite primers and multiplex PCR amplification conditionsa
Locus Forward primer Reverse primer Dye PanelPrimerconcn(�M)
a Sequences of forward and reverse primers used for microsatellite analysis are given. Forward primers were labeled with the listed dyes, and multiplex panels wereestablished by varying primer concentrations. HEX, 6-carboxy-2�, 4, 4�, 5�, 7, 7�-hexachlorofluorescein; TAMRA, 6-carboxytetramethylrhodamine.
b Sequences of human microsatellite primers for these loci were modified to reflect rhesus macaque MHC genomic sequences.c Dashes indicate single-locus PCR amplification.
were incubated in duplicate or triplicate overnight with pools of overlapping15-mer peptides in gamma interferon (IFN-�) enzyme-linked immunospot(ELISPOT) plates (Mabtech, Columbus, OH). Plates were developed per themanufacturer’s instructions. Spots were imaged with an ELISPOT reader (AID,Strassberg, Germany) and counted by an ELISPOT reader, version 3.1.1, to limitbias. The mean number of spot-forming units (SFU) of background wells (with-out peptide) was subtracted from the mean of the sample wells. Responses were
considered positive if the difference between the sample and background wellswas above 2 standard deviations at two or more time points.
Viral sequence analysis. Cell-free plasma was obtained by Ficoll density gra-dient centrifugation of EDTA anticoagulated whole blood, and viral RNA wasisolated as for measurements of plasma virus concentration. Amplification ofviral sequences was performed using a QIAGEN one-step reverse transcription-PCR kit (QIAGEN, Valencia, CA). For time points with plasma virus concen-trations of �103 viral RNA copies/ml of plasma, amplicons of �500 to 1,000 basepairs were generated throughout the SIV genome as previously described (28).For time points with plasma virus concentrations of �103 viral RNA copies/ml ofplasma, amplicons of �150 to 200 base pairs were generated around targetedsites of interest within the SIV genome. Primer sequences and PCR conditionsare available upon request. Amplicons were purified using a QIAquick gelextraction kit (QIAGEN, Valencia, CA) and then directly sequenced using aDYEnamic ET Terminator cycle sequencing kit (GE Health Care). Sequencingreactions were resolved on an ABI 3730 (Applied Biosystems, Foster City,CA) and edited using CodonCode Aligner software (CodonCode Corp., Ded-ham, MA).
Nucleotide sequence accession numbers. Novel MHC class I sequences weredeposited in GenBank (accession numbers DQ979878 to DQ979886).
RESULTS
Microsatellite analysis of Mauritian cynomolgus macaques.In order to define MHC haplotypes in MCM, we identified apanel of 18 microsatellite markers spanning the entire 5-MbMHC region of macaques; 10 of these loci lie within the MHCclass I region (Fig. 1). The majority of the primers used toamplify these markers were adapted to reflect rhesus genomicsequences (Table 1) (9, 15, 31). First, we used these markers togenotype DNA from five MCM previously shown to possessthe MHC class I alleles Mafa-B*430101, Mafa-B*440101, andMafa-B*460101 (20). One of these animals, A4M, was homozy-gous at all 18 microsatellite loci (Fig. 1 and 2), while the otherfour animals had one copy of the same putative haplotype thatwe termed H1. These results demonstrate that the Mafa-
FIG. 1. Localization and properties of microsatellite markers in theMHC region of cynomolgus macaques. The schematic map is extra-polated from the MHC genomic sequence of rhesus macaques (9).Approximate positions of microsatellites and shaded boxes for theclass IA, IB, and II gene clusters are given on a kilobase scale, orientedwith the telomere at the top. Microsatellite properties, including ob-served heterozygosity [Het (obs)], nucleotides comprising the repeatunit, and number of alleles observed, are given to the right of eachmarker.
FIG. 2. Microsatellite analysis of cynomolgus macaques (A1M,A2M, A4M, A5M, and A6M) carrying high-frequency MHC class Ialleles. Microsatellite allele sizes (in base pairs) are shown for eachanimal. The shaded box indicates the common haplotype (H1) that isshared between each of these MCM.
VOL. 81, 2007 SIVmac239 AND MHC-IDENTICAL MAURITIAN MACAQUES 351
B*430101, Mafa-B*440101, and Mafa-B*460101 cluster is acomponent of an expansive, well-conserved haplotype that en-compasses the entire 5-Mb MHC region.
We then extended our MHC microsatellite analysis to acohort of 112 additional feral MCM. Allele frequencies forthese 18 loci in MCM are given in Table 2. The number ofallele sizes per locus ranged from two (C4_2_25) to nine(D6S2691). Overall, 46/117 (39%) of this cohort carried atleast one complete copy of the H1 haplotype (Fig. 3 and 4),and 9/117 (8%) animals were homozygous for H1. Addition-ally, we identified 14 more MCM that were homozygous at all18 microsatellite loci (Fig. 4). From these 14 animals, we de-fined an additional five haplotypes (H2 to H6) (Fig. 3). Takentogether, two-thirds of the 234 chromosomes examined boremicrosatellite signatures of one of these six common haplo-types (Fig. 3), and simple recombination events could generallyaccount for the remaining haplotypes (Fig. 4). This extensivesharing of MHC haplotypes is unprecedented among ma-caques (6, 29, 31).
RSCA of selected MCM confirms MHC haplotypes. To ver-ify that the haplotypes inferred from microsatellite mappingare linked with specific MHC alleles, we performed MHC classI RSCA. RSCA is a modified heteroduplex assay that is par-ticularly well suited for characterizing complex gene families,such as MHC class I and class II (3, 18, 20). After heteroduplexformation with a fluorescently labeled reference strand, indi-vidual alleles are distinguished from one another on a nonde-naturing polyacrylamide gel. As expected, RSCA from repre-sentative homozygotes resulted in distinct peak profiles,reflecting the differing MHC class I allele repertoires on eachhaplotype (Fig. 5). Moreover, each of the homozygous haplo-type profiles was additive in the respective heterozygous ani-mals (Fig. 5).
In addition, MHC class I RSCA was used to test our hy-pothesis that almost all MCM MHC haplotypes either areintact or result from simple recombination events between H1through H6. In animals where the predicted recombinationregion is distal to the MHC class I loci, transcribed allelepatterns matched the relevant haplotype in the class I region.When the putative recombination breakpoint occurred withinthe MHC class I loci, chimeric allele profiles were observed(data not shown).
Identification of transcribed MHC class I alleles for eachcommon haplotype. Next, we identified the specific transcribedMHC class I alleles associated with the H1 through H6 hap-lotypes. Cloning and sequencing of PCR-amplified cDNAsfrom representative homozygous animals unambiguouslylinked specific MHC class I alleles with each of the six haplo-types (Table 3). The H1 haplotype carries Mafa-B*430101,Mafa-B*440101, and Mafa-B*460101, a result that confirmedour previous speculation that these alleles are inherited on acommon haplotype (20). This haplotype also carries two MHCclass IA alleles, Mafa-A*290101 and Mafa-A*250301. Surpris-ingly, these MHC class IA alleles are conserved between thethree most common haplotypes, H1, H2, and H3. All threehaplotypes carry identical Mafa-A*290101 alleles and eitherMafa-A*250201 or Mafa-A*250301, which differ by only a sin-gle amino acid in the signal peptide. Therefore, more than90% of MCM are predicted to possess these class IA alleles(Fig. 4). In contrast, “high-frequency” MHC class I alleles in
TABLE 2. Microsatellite allele frequencies and observed andexpected heterozygosities in MCMa
201 0.56 D6S2771 392 0.16204 0.30 396 0.38207 0.06 397 0.46He 0.59 He 0.62Ho 0.56 Ho 0.62
a Microsatellite allele sizes are given along with the frequency of each alleleand the expected (He) and observed (Ho) heterozygosity values for each locus inthis MCM cohort (n 234 chromosomes). Several examples of null microsat-ellite alleles were noted for specific haplotypes, e.g., the 9268 locus for H3 andH4. These are likely to result from mismatches between the target locus and theprimer binding sequences.
Indian rhesus macaques, such as Mamu-A*01, are rarely foundin more than 25% of captive-bred monkeys.
Common MHC haplotypes extend through the DRB locus inMCM. To verify that the microsatellite haplotype signaturesassociate with discrete MHC class II genotypes, we examinedthe highly polymorphic MHC class II-DRB locus (4, 23, 31).We used allele-specific PCR with representative homozygousMCM genomic DNAs to assign 11 of 15 known Mauritian DRBalleles to haplotypes H1 through H6 (Table 3). Both the alleliccomposition and relative frequencies of our microsatellite-basedMHC haplotypes are consistent with short-range (�150 kb) DRBhaplotypes defined previously in two independent cohorts ofMCM (4, 23). High-resolution cloning and sequencing from ho-mozygous MCM will be necessary to more rigorously define thecomplete gene content of the MHC class II region.
High frequency of MHC class I- and MHC class II-identicalMCM. As illustrated in Fig. 6, more than one-quarter of thisferal MCM cohort (32/117) comprises clusters of 7 or moreMHC-identical individuals. If the MHC class I region is con-sidered alone, 72/117 (62%) MCM have one or more fullymatched individuals distributed among 14 distinct homozygousand heterozygous haplotype combinations (Fig. 4 and 6). Thisunique population of animals provides opportunities to per-form a wide variety of studies in which genetic control over theMHC of the subjects might be important and that have beenpreviously unattainable with nonhuman primates.
SIVmac239 challenge of MCM. We infected two MHC classI-identical MCM with SIVmac239 to examine the predictabil-ity and reproducibility of SIV pathogenesis and cellular immu-nity in animals with identical MHC genetics. This pair of MCM
FIG. 3. Microsatellite haplotypes for the MHC region of Mauritian cynomolgus macaques. (a) Microsatellite allele sizes (in base pairs)characteristic for each microsatellite locus were associated with each MHC haplotype. The six common haplotypes were designated H1 to H6 andassigned colors (H1, black; H2, red; H3, blue; H4, green; H5, yellow; and H6, gray) for illustrative purposes throughout the figures. In severalinstances, multiple microsatellite alleles for a specific locus were associated with an MHC haplotype, e.g., the D6S2691 locus for H2 and H3. null,undetectable amplification due to primer mismatch or absence of target locus. (b) Microsatellite analysis was used to determine the frequency ofcommon and recombinant MHC haplotypes (n 234 chromosomes).
VOL. 81, 2007 SIVmac239 AND MHC-IDENTICAL MAURITIAN MACAQUES 353
FIG. 4. Microsatellite MHC haplotypes of Mauritian cynomolgus macaques. Six common MHC haplotypes were inferred based on microsat-ellite analysis of 117 feral MCM obtained from Charles River BRF. Solid colored bars indicate intact MHC haplotypes, while mixed colorsrepresent recombinant chromosomes. For example A3M and A4M are homozygous for H4 (green) and H1 (black), respectively, while A6M is asimple heterozygote for the H1 and H6 haplotypes. In contrast, A8M carries H4 plus a recombinant haplotype with the H2 class IA region andH6 for the rest of the MHC region. Hatched boxes define ambiguous regions resulting from identical microsatellite allele sizes between neighboringhaplotype blocks. Individual boxes indicate variant microsatellite allele sizes relative to the expected common haplotype; these rare variantsgenerally differ by the addition or loss of a single repeat unit.
was originally selected based on RSCA that demonstrated thatthey share identical profiles of transcribed MHC class I alleles(data not shown). As illustrated in Fig. 7A, microsatellite anal-ysis revealed that CY0113 carries an H2/H3 recombinant hap-
lotype with a Mafa-A*250301 allele that differs by only a singleresidue in the signal peptide compared to the H3 haplotype inCY0111. After challenge with SIVmac239, these animals ex-hibited very similar plasma virus levels for the first 16 weeks of
FIG. 4—Continued.
VOL. 81, 2007 SIVmac239 AND MHC-IDENTICAL MAURITIAN MACAQUES 355
infection (Fig. 7B) before beginning to diverge. After 40 weeksof infection, plasma virus concentrations differed by approxi-mately 2 log units.
We predicted that these two MHC-identical animals wouldexhibit similar CD8�-T-lymphocyte (CTL) responses and thatthese responses, in turn, would select similar viral escape vari-ants (28). CTL responses were measured in this pair of animalsby IFN-� ELISPOT. During the chronic phase of infection, weconsistently detected 11 CTL responses against regions of Rev,Nef, Gag, Tat, Env, and Pol in CY0113 (Fig. 7C). Six of theseresponses were also detected in CY0111, though the magni-tudes of these responses were often lower. Interestingly,CY0111 did not mount any unique responses that were notalso detected in CY0113. Unfortunately, sample limitationsprecluded whole-proteome analyses of acute-phase cellularimmune responses in these animals.
Given the similarities in immunological responses in the twoanimals, we hypothesized that their immune responses wouldselect similar viral variants. The higher plasma virus concen-trations (greater than 1,000 copies/ml) in CY0113 allowedanalyses of a majority of the viral genome at multiple timepoints spanning the course of infection. In CY0111, the lowchronic-phase plasma virus concentrations (fewer than 1,000copies/ml) precluded sequencing of the entire SIV genome.Therefore, we used our analysis of viral sequences fromCY0113 to focus on a subset of regions for examination in virusisolated from CY0111 (Fig. 7D). We designed small amplicons(150 to 200 bp) to specifically amplify and sequence thesetargeted regions. Four regions of the genome with viral varia-tion consistent with CTL escape were identified in CY0113 andsubsequently evaluated in CY0111 (Fig. 7D). Remarkably,both MHC-identical animals exhibited mutations in these re-gions, though the affected amino acids were distinct in eachanimal. With the exception of the Tat26–36 region, strong CTLresponses were detected at 3 weeks postinfection in at least
one of the two animals (data not shown), strongly suggestingthat the shared variability results from immune escape.
DISCUSSION
In this study, we discovered that six haplotypes account foralmost all of the MHC diversity in feral Mauritian cynomolgusmacaques. Combining genetic mapping with polymorphic mi-crosatellite markers, MHC class I RSCA, high-throughputcloning and sequencing, and MHC class II-DRB allele-specificPCR, we were able to infer the entire MHC class I and classII-DRB genotypes of more than 100 animals. Sizable groups ofcompletely MHC-identical animals, including a cluster ho-mozygous for the most frequent MHC haplotype, were iden-tified. We also successfully infected a pair of MCM with iden-tical MHC class I and class II genetics with SIVmac239. To ourknowledge, this is the first study to show successful SIVmac239infection of Mauritian cynomolgus macaques, though the sus-ceptibility of these animals is not surprising in light of recentdata showing susceptibility to other pathogenic SIVs, includingSIVmac251 and SHIV89.6P (32).
Our initial results suggest that SIV-specific cellular immuneresponses are generally uniform in specificity in MHC-identi-cal animals. This study mirrored two recent evaluations ofmonozygotic twins infected at the same time with the samestock of HIV (13, 44). We identified four regions of SIV thataccumulated variation by 16 weeks postinfection. CTL againstall four regions were detected during infection, suggesting thatthe variation results from CTL selective pressure. The patternof chronic-phase epitope recognition in our animals was verysimilar to the twins monitored by Yang and colleagues (44).Animal CY0113 mounted 11 CTL responses. Six of the sameresponses were detected in CY0111, though the magnitude ofthe responses was lower. Lower plasma virus concentrations inCY0111 may account for the weaker CTL responses, a phe-
nomenon which was also noted in the twins monitored by Yanget al. (44). Despite the similar CTL responses in these animals,plasma viremia became substantially higher in CY0113 afterthe first 16 weeks of infection. The different clinical outcomesin these two animals could result from subtle differences inepitope specificity not resolved with IFN-� ELISPOT, anti-body responses, innate immune responses, stochastic differ-ences in T-cell receptor utilization, and different patterns of
viral evolution. Unlike the two studies that relied on the ser-endipitous identification of twins infected with the same strainof HIV, it should be possible to infect additional MHC-iden-tical MCM with SIVmac239 to study why animals with identi-cal MHC genetics and similar CTL responses nonetheless ex-hibit differences in SIV pathogenesis.
The genetic simplicity of the MCM MHC is unprecedentedamong macaques and will fundamentally expand the scope of
FIG. 5. RSCA of transcribed MHC class I alleles. RSCA was performed with a Mamu-B*07 reference strand and cDNA PCR products fromhomozygous and heterozygous animals representing H1 through H5. RSCA assesses differences in electrophoretic mobility that result from theunique heteroduplex conformations that form between sequence-mismatched MHC alleles and a fluorescently labeled reference strand. Theseprofiles are characteristic for each homozygous haplotype, with three to six peaks per haplotype that correspond to individual class I alleles. Thehatched heteroduplex peaks with an apparent mobility of 525 bp are the Mafa-A*25 variant alleles that are shared between H1 through H3 (seeTable 3). Several samples contain a residual Mamu-B*07 homoduplex that migrates just before 300 bp.
VOL. 81, 2007 SIVmac239 AND MHC-IDENTICAL MAURITIAN MACAQUES 357
SIV studies that can be undertaken with nonhuman primates.MCM that share identical MHC haplotypes (are MHC hap-loidentical) or that carry completely distinct MHC haplotypescan be easily identified using polymorphic microsatellite map-ping and selected for further studies. Adoptive lymphocytetransfer studies, such as those with inbred strains of mice thathave defined the correlates of protective immunity in Friendretrovirus infections, will be possible with MCM that are com-pletely matched for both MHC haplotypes (11, 25). For thefirst time, it may be possible to study the in vivo correlates ofprotective cellular immunity by transferring SIV-specific lym-phocytes from a donor animal into naive recipients immedi-ately prior to SIV challenge. These studies could directly testthe hypothesis that the failure of cellular immunity to controlSIV infection results from an inability of CTL to mobilize tosites of viral replication early during infection (33). Addition-ally, in vitro data suggest that certain CTL specificities sup-press SIV and HIV replication far more effectively than others(24, 43). The use of MCM for adoptive transfers of individualCTL specificities could provide a useful method for both iden-
tifying and characterizing the shared biological attributes ofeffective CTL response.
MCM with defined MHC haplotypes may also be very usefulfor vaccine studies that seek to elicit cellular immunity. Mamu-A*01-positive Indian rhesus macaques are often used in SIVvaccine research, primarily because these animals consistentlymount an immunodominant Gag181-189CM9 CTL response thatprovides a convenient biomarker for assessing the induction ofcellular immune responses. The magnitude of Gag181-189CM9responses varies approximately 10-fold between Mamu-A*01-pos-itive animals receiving identical vaccine formulations (1, 8) andSIV challenges (26). The magnitude of Gag181-189CM9 re-sponses may be indirectly modified by alleles other thanMamu-A*01, since competition between expressed class I al-leles could lead to differential Mamu-A*01 cell surface expres-sion (37, 38). MCM that possess completely identical MHCgenes eliminate this source of variability and therefore mayimprove the consistency of vaccine-elicited cellular immuneresponses.
The evolutionary basis for the MHC genetic simplicity of
TABLE 3. MHC class I and II alleles detected for six common MCM haplotypesa
HaplotypeAllele
H1 H2 H3 H4 H5 H6
Class IA A*250301, A*290101 A*250301, A*290101 A*250201, A*290101 A*300101, A*310101 A*330101 A*320101
Class IB B*430101, B*440101,B*460101
B*480101, B*600101,B*630101
B*450101, B*510101,I*100101
B*470101, B*620101,I*110101
B*110102, B*12,B*500101, B*610101
B*490101, B*640101,B*650101, I*100201
Class II-DRB DRB6*0101, DRB*W2101,DRB*W501
DRB1*1001,DRB*W402
DRB1*1002 DRB1*0401, DRB5*0301,DRB4*0101
DRB1*0401, DRB5*0301 DRB1*0402, DRB*W401
a MHC class IA and IB alleles were identified for each haplotype by cDNA cloning and sequencing. cDNA cloning was performed with mRNA from the followinganimals homozygous for the class I region: A4M (H1), CR013 (H2), CR011 (H3), A3M (H4), CR001 (H5), and CR079 (H6). Class II-DRB alleles were determinedby sequence-specific PCR with genomic DNA from representative homozygous and heterozygous animals. The MHC haplotypes deduced from our MCM cohort bymicrosatellite analysis are consistent with MCM DRB haplotypes reported previously (4, 23).
FIG. 6. Complete MHC identity among a cohort of 117 Mauritian cynomolgus macaques. Only instances where seven or more homozygotesor simple heterozygotes with identity across the entire 5-Mb MHC region were observed are illustrated. Our cohort contained 11 additional animalsrepresenting five distinct haplotype combinations with complete MHC identity. The physical order of loci within each gene cluster is unknown.
FIG. 7. SIVmac239 infection of MHC class I-identical Mauritian cynomolgus macaques. (a) Microsatellite haplotypes were determined andused to infer the complete complement of class I and class II-DRB transcribed alleles for CY0111 and CY0113. (b) Plasma virus concentrationsfor each animal were measured at various time points throughout infection. The plasma virus concentrations are quantified as the number of copiesof viral RNA per milliliter of plasma. (c) IFN-� ELISPOT analysis was used to measure SIV-specific CTL responses in the chronic phase. Eithersingle peptides or peptide pools spanning the indicated amino acid sequences of the specific SIV proteins were used. A � indicates 50 to 99 SFUper million cells, �� indicates 100 to 499 SFU per million cells, and ��� indicates �500 SFU per million cells; a indicates �50 SFU. (d) Viralsequences in four specific regions of the SIV genome were analyzed to determine whether similar mutation patterns occurred in both animals. TheSIV proteins and the wild-type amino acid sequences are indicated. Dots represent identity with the wild-type sequence. Amino acid replacementsthat resulted from a mixed population of nucleotides are indicated with a lowercase letter of the variant amino acid. Amino acid replacements thatresulted from a complete nucleotide replacement are indicated with an uppercase letter of the variant amino acid. “ND” indicates that thesequence was not determined; wk, weeks.
VOL. 81, 2007 SIVmac239 AND MHC-IDENTICAL MAURITIAN MACAQUES 359
MCM is unclear. The limited MHC repertoire of MCM mayreflect selective advantages of these haplotypes for the Mau-ritian environment. It appears more likely that the limitedMHC diversity described here is the result of a classic popu-lation bottleneck or founder effect (22, 30, 36; A. J. Tosi andC. S. Coke, submitted for publication). Consequently, there islittle reason to assume that the relative genetic homogeneity ofMCM is restricted to the MHC. Given the excitement sur-rounding gene mapping with isolated human populations (5,35), MCM may provide an outstanding resource for mappingand identifying non-MHC loci associated with differences inSIV pathogenesis. Studies with HIV-infected individuals haverevealed a number of such polymorphic non-MHC loci asso-ciated with AIDS restriction (27).
Fortunately, the population of MCM available for researchand the selection of genetically defined macaques are relativelyabundant. In 2005 alone, 1,670 MCM were imported to theUnited States by a single distributor (Tami Lass, Charles RiverBRF, personal communication). Based on our results, approx-imately 130 H1 homozygous animals should be available an-nually. Likewise, when all simple homozygotes and heterozy-gotes for the common haplotypes are included, the number ofMCM estimated to populate MHC-identical clusters exceeds600 per year. These numbers could likely be increased signif-icantly with only a modest effort at selective breeding usingMHC microsatellite markers such as those described here.
In conclusion, the high frequency of identical MHC haplo-types in MCM is extraordinary among nonhuman primatesused in experimental biology. MCM represent an exceptionalsource of MHC-identical nonhuman primates with broad ap-plications for AIDS vaccine and pathogenesis investigations.
ACKNOWLEDGMENTS
This work was supported by NIAID contract numberHHSN266200400088C/N01-AI-40088 and NIH grant 1R21AI068488-01A2. This publication was made possible in part by grant number P51RR000167 from the National Center for Research Resources(NCRR), a component of the National Institutes of Health (NIH), tothe Wisconsin National Primate Research Center, University of Wis-consin—Madison. This research was conducted in part at a facilityconstructed with support from Research Facilities Improvement Pro-gram grant numbers RR15459-01 and RR020141-01.
This publication’s contents are solely the responsibility of the authorsand do not necessarily represent the official views of NCRR or NIH.
We thank Eva Rakasz, Shari Piaskowski, Jessica Furlott, Kim Weis-grau, Gemma May, and Robert DeMars for helpful discussions. Wealso thank Jody Hegeland, Amy Schara, Eric Peterson, Mike Dobbert,Casey Fitz, and staff at the Wisconsin National Primate ResearchCenter for technical assistance and veterinary care.
REFERENCES
1. Allen, T. M., T. U. Vogel, D. H. Fuller, B. R. Mothe, S. Steffen, J. E. Boyson,T. Shipley, J. Fuller, T. Hanke, A. Sette, J. D. Altman, B. Moss, A. J.McMichael, and D. I. Watkins. 2000. Induction of AIDS virus-specific CTLactivity in fresh, unstimulated peripheral blood lymphocytes from rhesusmacaques vaccinated with a DNA prime/modified vaccinia virus Ankaraboost regimen. J. Immunol. 164:4968–4978.
2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.Basic local alignment search tool. J. Mol. Biol. 215:403–410.
3. Arguello, J. R., M. Perez-Rodriguez, A. Pay, G. Fisher, A. McWhinnie, andJ. A. Madrigal. 2003. HLA typing with reference strand-mediated confor-mation analysis. Methods Mol. Biol. 210:173–189.
4. Blancher, A., P. Tisseyre, M. Dutaur, P. A. Apoil, C. Maurer, V. Quesniaux,F. Raulf, M. Bigaud, and M. Abbal. 2006. Study of Cynomolgus monkey(Macaca fascicularis) MhcDRB (Mafa-DRB) polymorphism in two popula-tions. Immunogenetics 58:269–282.
5. Bonnen, P. E., I. Pe’er, R. M. Plenge, J. Salit, J. K. Lowe, M. H. Shapero,
R. P. Lifton, J. L. Breslow, M. J. Daly, D. E. Reich, K. W. Jones, M. Stoffel,D. Altshuler, and J. M. Friedman. 2006. Evaluating potential for whole-genome studies in Kosrae, an isolated population in Micronesia. Nat. Genet.38:214–217.
6. Bontrop, R. E., and D. I. Watkins. 2005. MHC polymorphism: AIDS sus-ceptibility in non-human primates. Trends Immunol. 26:227–233.
7. Boyson, J. E., C. Shufflebotham, L. F. Cadavid, J. A. Urvater, L. A. Knapp,A. L. Hughes, and D. I. Watkins. 1996. The MHC class I genes of the rhesusmonkey. Different evolutionary histories of MHC class I and II genes inprimates. J. Immunol. 156:4656–4665.
8. Casimiro, D. R., F. Wang, W. A. Schleif, X. Liang, Z. Q. Zhang, T. W. Tobery,M. E. Davies, A. B. McDermott, D. H. O’Connor, A. Fridman, A. Bagchi,L. G. Tussey, A. J. Bett, A. C. Finnefrock, T. M. Fu, A. Tang, K. A. Wilson,M. Chen, H. C. Perry, G. J. Heidecker, D. C. Freed, A. Carella, K. S. Punt,K. J. Sykes, L. Huang, V. I. Ausensi, M. Bachinsky, U. Sadasivan-Nair, D. I.Watkins, E. A. Emini, and J. W. Shiver. 2005. Attenuation of simian immu-nodeficiency virus SIVmac239 infection by prophylactic immunization withDNA and recombinant adenoviral vaccine vectors expressing Gag. J. Virol.79:15547–15555.
9. Daza-Vamenta, R., G. Glusman, L. Rowen, B. Guthrie, and D. E. Geraghty.2004. Genetic divergence of the rhesus macaque major histocompatibilitycomplex. Genome Res. 14:1501–1515.
10. de Groot, N., G. G. Doxiadis, N. G. de Groot, N. Otting, C. Heijmans,A. J. M. Rouweler, and R. E. Bontrop. 2004. Genetic makeup of the DRregion in rhesus macaques: gene content, transcripts, and pseudogenes.J. Immunol. 172:6152–6157.
11. Dittmer, U., and K. J. Hasenkrug. 2000. Different immunological require-ments for protection against acute versus persistent Friend retrovirus infec-tions. Virology 272:177–182.
12. Doxiadis, G. G., A. J. Rouweler, N. G. de Groot, A. Louwerse, N. Otting, E. J.Verschoor, and R. E. Bontrop. 2006. Extensive sharing of MHC class IIalleles between rhesus and cynomolgus macaques. Immunogenetics 58:259–268.
13. Draenert, R., T. M. Allen, Y. Liu, T. Wrin, C. Chappey, C. L. Verrill, G.Sirera, R. L. Eldridge, M. P. Lahaie, L. Ruiz, B. Clotet, C. J. Petropoulos,B. D. Walker, and J. Martinez-Picado. 2006. Constraints on HIV-1 evolutionand immunodominance revealed in monozygotic adult twins infected withthe same virus. J Exp. Med. 203:529–539.
14. Evans, D. T., L. A. Knapp, P. Jing, J. L. Mitchen, M. Dykhuizen, D. C.Montefiori, C. D. Pauza, and D. I. Watkins. 1999. Rapid and slow progres-sors differ by a single MHC class I haplotype in a family of MHC-definedrhesus macaques infected with SIV. Immunol. Lett. 66:53–59.
15. Gourraud, P. A., S. Mano, T. Barnetche, M. Carrington, H. Inoko, and A.Cambon-Thomsen. 2004. Integration of microsatellite characteristics in theMHC region: a literature and sequence based analysis. Tissue Antigens64:543–555.
16. Horton, R., L. Wilming, V. Rand, R. C. Lovering, E. A. Bruford, V. K.Khodiyar, M. J. Lush, S. Povey, C. C. J. Talbot, M. W. Wright, H. M. Wain,J. Trowsdale, A. Ziegler, and S. Beck. 2004. Gene map of the extendedhuman MHC. Nat. Rev. Genet. 5:889–899.
17. Karell, K., N. Klinger, P. Holopainen, A. Levo, and J. Partanen. 2000. Majorhistocompatibility complex (MHC)-linked microsatellite markers in afounder population. Tissue Antigens 56:45–51.
18. Kennedy, L. J., S. Quarmby, N. Fretwell, A. J. Martin, P. G. Jones, C. A.Jones, and W. E. Ollier. 2005. High-resolution characterization of the canineDLA-DRB1 locus using reference strand-mediated conformational analysis.J. Hered. 96:836–842.
19. Kestler, H. W., III, Y. Li, Y. M. Naidu, C. V. Butler, M. F. Ochs, G. Jaenel,N. W. King, M. D. Daniel, and R. C. Desrosiers. 1988. Comparison of simianimmunodeficiency virus isolates. Nature 331:619–622.
20. Krebs, K. C., Z. Jin, R. Rudersdorf, A. L. Hughes, and D. H. O’Connor. 2005.Unusually high frequency MHC class I alleles in Mauritian origin cynomol-gus macaques. J. Immunol. 175:5230–5239.
21. Kulski, J. K., T. Anzai, T. Shiina, and H. Inoko. 2004. Rhesus macaque classI duplicon structures, organization and evolution within the alpha block ofthe major histocompatibility complex. Mol. Biol. Evol. 21:2079–2091.
22. Lawler, S. H., R. W. Sussman, and L. L. Taylor. 1995. Mitochondrial DNAof the Mauritian macaques (Macaca fascicularis): an example of the foundereffect. Am. J. Phys. Anthropol. 96:133–141.
23. Leuchte, N., N. Berry, B. Kohler, N. Almond, R. LeGrand, R. Thorstensson,F. Titti, and U. Sauermann. 2004. MhcDRB-sequences from cynomolgusmacaques (Macaca fascicularis) of different origin. Tissue Antigens 63:529–537.
24. Loffredo, J. T., E. G. Rakasz, J. P. Giraldo, S. P. Spencer, K. K. Grafton, S. R.Martin, G. Napoe, L. J. Yant, N. A. Wilson, and D. I. Watkins. 2005. Tat28–35SL8-specific CD8� T lymphocytes are more effective than Gag181–189CM9-specificCD8� T lymphocytes at suppressing simian immunodeficiency virus replicationin a functional in vitro assay. J. Virol. 79:14986–14991.
25. Messer, R. J., U. Dittmer, K. E. Peterson, and K. J. Hasenkrug. 2004.Essential role for virus-neutralizing antibodies in sterilizing immunity againstFriend retrovirus infection. Proc. Natl. Acad. Sci. USA 101:12260–12265.
26. Mothe, B. R., H. Horton, D. K. Carter, T. M. Allen, M. E. Liebl, P. Skinner,
T. U. Vogel, S. Fuenger, K. Vielhuber, W. Rehrauer, N. Wilson, G. Franchini,J. D. Altman, A. Haase, L. J. Picker, D. B. Allison, and D. I. Watkins. 2002.Dominance of CD8 responses specific for epitopes bound by a single majorhistocompatibility complex class I molecule during the acute phase of viralinfection. J. Virol. 76:875–884.
27. O’Brien, S. J., and G. W. Nelson. 2004. Human genes that limit AIDS. Nat.Genet. 36:565–574.
28. O’Connor, D. H., A. B. McDermott, K. C. Krebs, E. J. Dodds, J. E. Miller,E. J. Gonzalez, T. J. Jacoby, L. Yant, H. Piontkivska, R. Pantophlet, D. R.Burton, W. M. Rehrauer, N. Wilson, A. L. Hughes, and D. I. Watkins. 2004.A dominant role for CD8�-T-lymphocyte selection in simian immunodefi-ciency virus sequence variation. J. Virol. 78:14012–14022.
29. Otting, N., C. M. Heijmans, R. C. Noort, N. G. de Groot, G. G. Doxiadis, J. J.van Rood, D. I. Watkins, and R. E. Bontrop. 2005. Unparalleled complexityof the MHC class I region in rhesus macaques. Proc. Natl. Acad. Sci. USA102:1626–1631.
30. Peltonen, L., A. Palotie, and K. Lange. 2000. Use of population isolates formapping complex traits. Nat. Rev. Genet. 1:182–190.
31. Penedo, M. C., R. E. Bontrop, C. M. Heijmans, N. Otting, R. Noort, A. J.Rouweler, N. de Groot, N. G. de Groot, T. Ward, and G. G. Doxiadis. 2005.Microsatellite typing of the rhesus macaque MHC region. Immunogenetics57:198–209.
32. Reimann, K. A., R. A. Parker, M. S. Seaman, K. Beaudry, M. Beddall, L.Peterson, K. C. Williams, R. S. Veazey, D. C. Montefiori, J. R. Mascola, G. J.Nabel, and N. L. Letvin. 2005. Pathogenicity of simian-human immunodefi-ciency virus SHIV-89.6P and SIVmac is attenuated in cynomolgus macaquesand associated with early T-lymphocyte responses. J. Virol. 79:8878–8885.
33. Reynolds, M. R., E. Rakasz, P. J. Skinner, C. White, K. Abel, Z. M. Ma, L.Compton, G. Napoe, N. Wilson, C. J. Miller, A. Haase, and D. I. Watkins.2005. CD8� T-lymphocyte response to major immunodominant epitopesafter vaginal exposure to simian immunodeficiency virus: too late and toolittle. J. Virol. 79:9228–9235.
34. Rice, P., I. Longden, and A. Bleasby. 2000. EMBOSS: the European Molec-ular Biology Open Software Suite. Trends Genet. 16:276–277.
35. Service, S., J. DeYoung, M. Karayiorgou, J. L. Roos, H. Pretorious, G.Bedoya, J. Ospina, A. Ruiz-Linares, A. Macedo, J. A. Palha, P. Heutink, Y.Aulchenko, B. Oostra, C. van Duijn, M. R. Jarvelin, T. Varilo, L. Peddle, P.Rahman, G. Piras, M. Monne, S. Murray, L. Galver, L. Peltonen, C. Sabatti,A. Collins, and N. Freimer. 2006. Magnitude and distribution of linkage
disequilibrium in population isolates and implications for genome-wide as-sociation studies. Nat. Genet. 38:556–560.
36. Sussman, R. W., and I. Tattersall. 1986. Distribution, abundance, and pu-tative ecological strategy of Macaca fascicularis on the island of Mauritius,southwestern Indian Ocean. Folia Primatol. 46:28–43.
37. Tourdot, S., and K. G. Gould. 2002. Competition between MHC class Ialleles for cell surface expression alters CTL responses to influenza A virus.J. Immunol. 169:5615–5621.
38. Tourdot, S., M. Nejmeddine, S. J. Powis, and K. G. Gould. 2005. DifferentMHC class I heavy chains compete with each other for folding independentlyof B2-microglobulin and peptide. J. Immunol. 174:925–933.
39. Uda, A., K. Tanabayashi, O. Fujita, A. Hotta, K. Terao, and A. Yamada.2005. Identification of the MHC class I B locus in cynomolgus monkeys.Immunogenetics 57:189–197.
40. Uda, A., K. Tanabayashi, Y. K. Yamada, H. Akari, Y. J. Lee, R. Mukai, K.Terao, and A. Yamada. 2004. Detection of 14 alleles derived from the MHCclass I A locus in cynomolgus monkeys. Immunogenetics 56:155–163.
41. Wilson, N. A., J. Reed, G. S. Napoe, S. Piaskowski, A. Szymanski, J. Furlott,E. J. Gonzalez, L. J. Yant, N. J. Maness, G. E. May, T. Soma, M. R.Reynolds, E. Rakasz, R. Rudersdorf, A. B. McDermott, D. H. O’Connor,T. C. Friedrich, D. B. Allison, A. Patki, L. J. Picker, D. R. Burton, J. Lin, L.Huang, D. Patel, G. Heindecker, J. Fan, M. Citron, M. Horton, F. Wang, X.Liang, J. W. Shiver, D. R. Casimiro, and D. I. Watkins. 2006. Vaccine-induced cellular immune responses reduce plasma viral concentrations afterrepeated low-dose challenge with pathogenic simian immunodeficiency virusSIVmac239. J. Virol. 80:5875–5885.
42. Wojcechowskyj, J. A., L. J. Yant, R. W. Wiseman, S. L. O’Connor, and D. H.O’Connor. 2007. Control of simian immunodeficiency virus SIVmac239 isnot predicted by inheritance of Mamu-B*17-containing haplotypes. J. Virol.81:406–410.
43. Yang, O. O., P. T. Sarkis, A. Trocha, S. A. Kalams, R. P. Johnson, and B. D.Walker. 2003. Impacts of avidity and specificity on the antiviral efficiency ofHIV-1-specific CTL. J. Immunol. 171:3718–3724.
44. Yang, O. O., J. Church, C. M. Kitchen, R. Kilpatrick, A. Ali, Y. Geng, M. S.Killian, R. L. Sabado, H. Ng, J. Suen, Y. Bryson, B. D. Jamieson, and P.Krogstad. 2005. Genetic and stochastic influences on the interaction ofhuman immunodeficiency virus type 1 and cytotoxic T lymphocytes in iden-tical twins. J. Virol. 79:15368–15375.
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