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Where Have All The Monkeys Gone?: Evaluating SIV-Specific CTL in the Post-Mamu-A*01 Era

 

David H. O'Connor1, Todd M. Allen2, and David I. Watkins1

 

1Wisconsin Regional Primate Research Center, 1220 Capitol Court, Madison, WI USA 53715 2Massachusetts General Hospital, Bldg. 149, 13th Street,Charlestown, MA USA 02113

Simian immunodeficiency virus (SIV) infected rhesus macaques are currently the most widely used animal model for evaluating different vaccine modalities. Most candidate vaccines now seek to engender cytotoxic T-lymphocyte (CTL) responses, either singly or in concert with other immune responses, as CTL are clearly important in the naturally occurring immune responses to HIV and SIV [1-4]. The lack of well-characterized CTL epitopes in SIV remains a serious bottleneck in vaccine research. Most vaccines evaluate the quality of the CTL response by determining the frequency of CTL directed against a single epitope, the Mamu-A*01 restricted Gag181-189CM9 epitope. The focus on this epitope is understandable in light of the facts that Mamu-A*01 positive animals are common in captive bred macaque populations [5] and that Gag181-189CM9 is conserved in several commonly used SIV challenge strains, including SIVmac239, SIVmac251, and SHIV89.6P. However, the intense selection of these animals for inclusion in vaccine studies has created an acute shortage of Mamu-A*01-positive animals [6]. Therefore, it remains important to identify and characterize SIV-specific CTL responses restricted by other common rhesus MHC class I alleles to expand the number of animals accessible to vaccine research.

Fortunately, Mamu-A*01 is not the only common MHC class I allele in captive bred macaques. Sequence-based genotyping of macaques has identified four additional alleles (Mamu-A*02, -A*08, -B*01, and -B*17) that are present in >10% of macaques (unpublished data). The peptide binding motif for Mamu-B*17 has already been determined, and this information is currently being used to identify putative CTL epitopes in commonly used SIV strains [7]. This work has been facilitated by the development of techniques such as intracellular cytokine staining (ICS) [8-10] and IFN-gamma EliSpot that allow rapid ex vivo detection of responding CTL without time-consuming in vitro restimulations. Within the next few years, rhesus with other common MHC alleles will likely supercede Mamu-A*01 animals as the preferred model for testing SIV vaccines.

Another avenue for increasing the number of animals available for vaccine studies is to utilize Chinese rhesus macaque in addition to the more commonly utilized Indian rhesus macaques. Both Chinese and Indian macaques are readily infected with common SIV challenge strains including SIVmac251 and SIVmac239 [13, 14]. However, the immunogenetics of Chinese macaques differ substantially from Indian macaques (unpublished data). In an analysis of over 30 Chinese macaques, no animals expressing Mamu-A*01 were detected [15], though this allele is present in over 25% of Indian rhesus macaques [5]. These differences at the MHC class I loci are supported by evidence of genetic and morphological differences between the groups [16, 17]. Therefore, using Chinese macaques for SIV research will likely necessitate a duplication of the immunogenetic work that has already been performed for Indian macaques; identifying common MHC class I alleles, defining the peptide binding motifs for these alleles, predicting CTL epitopes based on the peptide binding motifs, and finally verifying these CTL responses ex vivo from SIV-infected Chinese macaques.

Despite the need to expand the SIV-infected rhesus macaque model, the description of new CTL epitopes is impeded by financial and practical considerations. The value of ICS for epitope identification has been tempered by the cost of applying this technique to comprehensively monitor immune responses to whole viral genomes. For example, simply generating a set of overlapping 15-mers spanning each of the proteins in SIVmac239 costs approximately $80,000. This initial expenditure, plus access to flow cytometers and SIV-infected animals, places CTL epitope identification beyond the means of most non-specialist laboratories. The routine costs of CTL epitope mapping are a burden even to specialist labs that have the capacity for high-throughput ICS. A single analysis of the entire cellular immune response against SIV costs over $700 and identifies only peptide pools (approximately 10 15-mer peptides each) that are reactive. To deconvolute the pool and identify the minimal, optimal CTL epitope, another $600 in ICS tests is required, plus the synthesis of $3500 in 8-mers, 9-mers, and 10-mers that span the reactive 15-mer. Finally, the restricting element for a response can be determined by testing antigen presenting cells expressing well-defined MHC class I alleles with the reactive CTL. However, these specialized antigen presenting cell lines are time-consuming to generate and have limited utility beyond epitope mapping. In sum, the cost for mapping a single novel SIV CTL epitope is upwards of $5000 (excluding initial peptide and animal husbandry costs). The reward for this expenditure can be uncertain, as peer-reviewed journals such as the Journal of Virology have stated that "[we] will not publish papers that simply ... identify new immunodominant peptides representing T- or B- cell epitopes ... Such information or reagents must instead be used in further experimentation to test an idea or relate a clear set of novel conclusions that derive from the data [18]." Though this guideline is intended to prevent the repetitive publication of manuscripts containing new CTL epitopes, it also actively discourages the identification of new CTL epitopes by favoring in-depth analysis of previously described epitopes.

Regardless, eight new SIV and SHIV CTL epitopes have been mapped in the two years since the last sequence compendium review on this topic [19]. Epitopes that have been fully characterized, including MHC class I restriction, are subdivided into those restricted by A-loci alleles (Table I) and B-loci alleles (Table II). Responses that have not been minimally mapped or whose restriction is uncertain are shown in Table III.

Mark O'Connor

Todd M. Allen

David I. Watkins

Bette T. Korber

phone:608-265-3379

617-726-7846

608-265-3380

505-665-4453

fax:&608-265-8084

617-726-5411

608-263-4031

505-665-3493

doconnor@primate.wisc.edu

tallen2@partners.org

watkins@primate.wisc.edu

btk@t10.lanl.gov

Table I.

Virus

Species

Protein

Epitope

Restricting
Alleles

GenBank

References

SIVmac239

Rhesus

Gag 149-157

LSPRTLNAW

Mamu-A*01

U50836

11

SIVmac251

Rhesus

Gag 181-189

CTPYDINQM

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Gag 254-262

QNPIPVGNI

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Gag 340-349

VNPTLEEMLT

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Gag 372-379

LAPVPIPF

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Pol 10-20

EAPQFPHGSSA

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Pol 106-115

LGPHYTPKIV

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Pol 110-118

YTPKIVGGI

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Pol 322-331

GSPAIFQYTM

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Pol 437-446

IYPGIKTKHL

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Pol 551-559

QVPKFHLPV

Mamu-A*01

U50836

11

SIVmac251

Rhesus

Pol 584-592

STPPLVRLV

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Pol 655-663

SGPKTNIIV

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Env 233-240

CAPPGYAL(L)

Mamu-A*01

U50836

11

SHIV-89.6

Rhesus

Env 435-443

YAPPISGQI

Mamu-A*01

U50836

20

SIVmac239

Rhesus

Env 502-510

ITPIGLAPT

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Env 620-628

TVPWPNASL

Mamu-A*01

U50836

11

SIVsmE660

Rhesus

Env 620-628

TVPWPNETL

Mamu-A*01

U50836

21

SIVmac239

Rhesus

Env 726-734

SSPPSYFQT

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Env 727-737

SPPSYFQTHT

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Env 761-769

SWPWQIEYI

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Tat 28-35

STPESANL

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Vif 14-22

RIPERLERW

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Vif 144-152

QVPSLQYLA

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Vpx 8-18

IPPGNSGEETI

Mamu-A*01

U50836

11

SIVmac239

Rhesus

Vpx 39-48

HLPRELIFQV

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Vpx 102-111

GPPPPPPPGL

Mamu-A*01

U50836

Unpublished

SIVmac239

Rhesus

Rev 86-95

DPPTNTPEAL

Mamu-A*01

U50836

Unpublished

SIVmac251

Rhesus

Nef 159-167

YTSGPGIRY

Mamu-A*02

U50837

22

SHIVHXBc2

Rhesus

Env 99-106

KPCVKLTP

Mamu-A*08

23

SIVmac251

Rhesus

Env 305-312

YNLTMKCR

Mamu-A*02

U50837

24

SIVmac239

Rhesus

Env 495-502

GDYKLVEI

Mamu-A*11

25-27

SIVmac32H-J5

Cynomolgus

Gag 242-250

SVDEQIQWM

Mafa-A*02

28


Table II

Virus

Species

Protein

Epitope

RestrictingAlleles

GenBank

References

SIVmac251

Rhesus

Env 501-509

EITPIGLAP

Mamu-B*01

U42837

29

SIVmac239

Rhesus

Nef 136-146

ARRHRILDMYL

Mamu-B*03

U41825

25-27

SIVmac239

Rhesus

Env 573-581

KRQQELLRL

Mamu-B*03

U41825

25-27

SIVmac239

Rhesus

Nef 62-69

QGQYMNTP

Mamu-B*04

U41826

25-27

SHIVHXBc2

Rhesus

Env 553-561

NNLLRAIEA

Mamu-B*12

23

SIVmac239

Rhesus

Nef 165-173

IRYPKTFGW

Mamu-B*17

25-27

 

Table III

Virus

Species

Protein

Epitope

References

SIVmac251

Rhesus

Gag 35-59

VWAANELDRFGLAESLLENKEGCQK

30

SIVmac251

Rhesus

Gag 246-281

QIQWMYRQQNPIPVGNIYRRWIQLGLQKCVRMYNPT

31-34

SIVmac251

Cynomolgus

Gag 296-315

SYVDRFYKSLRAEQTDAAYK

35

SIVmac251

Rhesus

Env 21-30

YCTLYVTVFY

Unpublished

SIVmac239

Rhesus

Env 113-121

CNKSETDRW

36

SIVmac251

Rhesus

Env 262-281

SCTRMMETQTSTWFGFNGTR

Unpublished

SIVmac251

Rhesus

Env 292-301

GRDNRTIISL

Unpublished

SIVmac251

Rhesus

Env 312-331

RRPGNKTVLPVTIMSGLVFH

Unpublished

SIVmac251

Rhesus

Nef 108-123

LRAMTYKLAIDMSHFI

31-34, 42

SIVmac251

Rhesus

Nef 128-137

GLEGIYYSAR

31-34

SIVmac251

Rhesus

Nef 155-169

DWQDYTSGPGIRYPK

31-34

SIVmac251

Rhesus

Nef 164-178

GIRYPKTFGWLWKLV

26, 31-34

SIVmac251

Rhesus

Nef 171-179

FGWLWKLVP

27

SIVmac251

Rhesus

Nef 201-225

SKWDDPWGEVLAWKFDPTLAYTYEA

31-34

SIVmac239

Rhesus

Nef 157-167

QDYTSGPGIRY

37

SIVmac239

Sooty mangabey

Nef 20-28

LLRARGETY

37

SIVmac239

Rhesus

Vpr 74-81

RGGCIHSR

Unpublished

SIVmac239

Rhesus

Nef 45-53

GLDKGLSSL

Unpublished

SIVmac251

Rhesus

Nef 169-178

KTFGWLWKLV

38

SIVmac251

Rhesus

Nef 211-225

LAWKFDPTLAYTYEA

38

SIVmac251

Rhesus

Nef 112-119

SYKLAIDM

38, 42

SIVmac251

Rhesus

Nef 120-135

SHFKEKGGLEGIYYS

42

SIVmac251

Rhesus

Nef 125-138

EKGGLELIYYSARR

42

SHIV-HXBc2

Rhesus

Gag 321-340

TLLIQNANPDCKLVLKGLGV

39

SHIV-HXBc2

Rhesus

Gag 421-440

DHVMAKCPDRQAGFLGLGPW

39

SIVNef 239

Rhesus

Env 484-492

AEVAELYRL

40

SIVmac239

Sooty mangabey

Gag 196-205

HQAAMQIIRD

41

SIVmac239

Sooty mangabey

Env 429-437

YVPCHIRQI

41

Naturally-infected

Sooty mangabey

Env 428-437

NYVPCHIRQI

41

SIVmac239

Sooty mangabey

Env 339-363

PKQAWCWFGGKWKDAIKEVKQTIVK

41

SIVmac239

Sooty mangabey

Nef 21-30

LRARGETYGR

41

SIVmac239

Sooty mangabey

Nef 20-30

LLRARGETYGR

41

Naturally-infected

Sooty mangabey

Nef 21-32

LRARGETYGRLL

41

 

 

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last modified: Wed Mar 17 15:07 2010


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