Table Of ContentMethods in
Molecular Biology 2407
Guido Poli
Elisa Vicenzi
Fabio Romerio Editors
HIV Reservoirs
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
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School of Lifeand MedicalSciences
University ofHertfordshire
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HIV Reservoirs
Methods and Protocols
Edited by
Guido Poli
School of Medicine and Human Immuno-Virology (H.I.V.) Group, San Raffaele University and Scientific
Institute, Milano, Italy
Elisa Vicenzi
Viral Pathogenesis and Biosafety Group, San Raffaele Scientific Institute, Milano, Italy
Fabio Romerio
Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Editors
GuidoPoli ElisaVicenzi
SchoolofMedicineandHuman ViralPathogenesisandBiosafetyGroup
Immuno-Virology(H.I.V.)Group SanRaffaeleScientificInstitute
SanRaffaeleUniversity Milano,Italy
andScientificInstitute
Milano,Italy
FabioRomerio
DepartmentofMolecularand
ComparativePathobiology
JohnsHopkinsUniversitySchoolofMedicine
Baltimore,MD,USA
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-1870-7 ISBN978-1-0716-1871-4 (eBook)
https://doi.org/10.1007/978-1-0716-1871-4
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Preface
In the early 1980s, a new deadly disease struck the world. Initially it affected gay commu-
nitiesintheUSA,butsoonitspreadtootherriskgroupsandemergedworldwide.Sinceits
mainclinicalfeatureswereassociatedwithaprofoundimmunologicaldepressionoccurring
inpeoplewithanotherwisehealthyimmunesystem,thenewpathologywasquicklycalledas
“AcquiredImmuno-DeficiencySyndrome(AIDS),”anoperationaldefinitionthathasnever
been amended thereafter. The etiologic agent causing AIDS was soon identified in a
previously unknown virus belonging to the retroviridae, shortly after named “Human
Immunodeficiency Virus (HIV).” As all retroviruses, HIV integrates the DNA version of
its genome into host cell chromosomes and in such form (termed “provirus”) persists
indefinitely. Although most infected cells die as a consequence of the cytopathic effects of
HIVorbecauseofarelatedimmuneresponse,someofthemsurvive,returntoaquiescent
stateinwhichthevirusliesdormant(provirallatency),andpersistindefinitelyaslongasthe
cells live forming what is nowadays commonly referred to as the “viral reservoir (VR).” As
immunecells,mostnotablyrestingmemoryCD4+Tcells,undergohomeostaticprolifera-
tion, the VR persists for the entire life of the individual even under fully suppressive
antiretroviraltherapy.
The relevance of the VR has been paradoxically highlighted by the great success of
combination antiretroviral therapy (cART) that was introduced in clinical practice in the
mid-1990sbycombiningpharmacologicinhibitorsoftwoviralenzymes:reversetranscrip-
tase (that converts the viral genomic RNA into a DNA version capable of integrating into
the host cell DNA) and protease (required to process the p55 Gag polyprotein following
viral budding to obtain mature, infectious virions). Nowadays, cART regimens have been
supplemented with other classes of antiretroviral drugs including integrase inhibitors and
entry/fusioninhibitors,andthevastmajorityoftreatedpatientsobservethedisappearance
of detectable virus in their peripheral blood (viremia), thus becoming non-contagious to
others (U¼U, i.e., undetectable equals untransmissible). Nonetheless, cART interruption
almostinvariablyresultsintherapidresurgenceofviremiaanddiseaseprogressioniftherapy
is not quickly restarted. This rapid viral rebound is mostly, if not exclusively, caused bythe
presenceoftheVR,althoughonlyaminorfraction(ca.10%)ofthepersistentlyinfectedcells
harborsreplication-competentproviruses,whereastheremaindercarrydefectiveproviruses
ofunclearclinicalrelevance.ThisabsolutelimitationofcARThasfueledanewresearchfield
aimed at the understanding of the cellular composition of the VR, the development of
surrogateinvitroandanimalmodelstodissectoutitsmoleculardeterminants,thediscovery
of more sophisticated methods of quantification of the VR, and the identification of
pharmacological and biologicagentsthat couldeither eradicate or permanently silencethe
integratedgenomeofreplication-competentproviruses.
The aim of this book is to summarize the intense efforts of the international scientific
community toward the development of methods and models to study the VR with the
ultimate goal of achieving a “Functional Cure” of HIV infection. Part I will describe the
most relevant cell lines that have represented the earliest models to investigate proviral
latency and reactivation and that are still being used as convenient models to identify
novel potential determinants of viral latency. Next, the book will describe in vitro and
ex vivo primary cell models (Part II) and tissue-derived models (Part III) of persistent
v
vi Preface
infectionthat—incomparisontocelllines—morecloselyresemblethesituationinvivo.In
vivomodels that utilize nonhuman primatesor congenitally immune-deficientmice recon-
stitutedwithhumanimmunecellsarepresentedinPartIV,andmethodsfor thedetection
andanalysisofthereservoirwillbeexploredinPartV.Finally,PartVIfocusesonthecurrent
approachestoachieveeithera“FunctionalCure”oratleastcART-freelong-termremission;
these include therapeutic latency reversal, durable proviral silencing, and genome editing
approaches.
Our ambition is not only to offer the reader a comprehensive, updated collection of
state-of-the-artmethodologiesandmodelstotackletheVRbutalsotoenticetheminthis
fascinating“last battle”againstoneofthemostformidablekillersofmankind:theHuman
ImmunodeficiencyVirus.
Milano,Italy GuidoPoli
Milano,Italy ElisaVicenzi
Baltimore,MD,USA FabioRomerio
Contents
Preface ..................................................................... v
Contributors................................................................. ix
PART I CELL LINES WITH MINIMAL BUT INDUCIBLE PROVIRAL EXPRESSION
1 Jurkat-Derived(J-Lat,J1.1,andJurkatE4)andCEM-DerivedTCellLines
(8E5andACH-2)asModelsofReversibleProviralLatency.................. 3
AnthonyRodari,GuidoPoli,andCarineVanLint
2 U1andOM10.1.MyeloidCellLinesasSurrogateModels
ofReversibleProviralLatency ............................................ 17
GuidoPoli
PART II IN VITRO AND EX VIVO PRIMARY CELL MODELS OF MINIMAL
BUT INDUCIBLE PROVIRAL EXPRESSION
3 AnInVitroSystemtoModeltheEstablishmentandReactivationofHIV-1
LatencyinPrimaryHumanCD4+TCells ................................. 31
RuiLiandFabioRomerio
4 TheCulturedT ModelofHIVLatency................................. 45
CM
AlbertoBosque
5 AReliablePrimaryCellModelforHIVLatency:TheQUECEL
(QuiescentEffectorCellLatency)Method................................. 57
MeenakshiShukla,FredrickKizito,UriMbonye,KienNguyen,
CurtisDobrowolski,andJonathanKarn
6 TGF-βSignalingSupportsHIVLatencyinaMemoryCD4+TCellBased
InVitroModel ......................................................... 69
SydneyBergstresserandDeannaA.Kulpa
7 FlowCytometrySortingofMemoryCCR6+CD4+T-CellsforHIV
ReservoirQuantification................................................. 81
Ame´lieCattin,AugustineFert,DelphinePlanas,andPetronelaAncuta
8 HumanMonocyte-DerivedMacrophages(MDM):
Model1(GM-CSF)..................................................... 91
ClaudiaAlteri,LorenzoPiermatteo,FrancescaCeccheriniSilberstein,
ValentinaSvicher,andCarloFedericoPerno
9 HumanMonocyte-DerivedMacrophages(MDM):
Model2 ............................................................... 97
FrancescaGraziano,ElisaVicenzi,andGuidoPoli
10 ModelingHIVLatencyinAstrocyteswiththeHumanNeural
ProgenitorCellLineHNSC.100 ......................................... 103
AmelieBauerandRuthBrack-Werner
vii
viii Contents
11 HematopoieticStemandProgenitorCells(HSPCs) ........................ 115
ValeriH.Terry,GretchenE.Zimmerman,MariaC.Virgilio,
MarkM.Painter,DaleBixby,andKathleenL.Collins
PART III IN VITRO AND EX VIVO TISSUE-DERIVED MODELS
OF REVERSIBLE PROVIRAL LATENCY
12 ExVivoHIVInfectionModelofCervico-VaginalandRectalTissue .......... 157
LouiseA.Ouattara,NikolasC.Vann,andGustavoF.Doncel
13 MorethanaGenderIssue:TestisasaDistinctiveHIVReservoirandIts
ImplicationforViralEradication.......................................... 173
Jean-PierreRouty,FranckP.Dupuy,JohnLin,andSte´phaneIsnard
PART IV ANIMAL MODELS
14 ExperimentalModelstoStudyHIVLatencyReversalfromMaleGenital
MyeloidCells .......................................................... 189
FernandoReal,YonatanGanor,andMorganeBomsel
15 DeciduaBasalis:AnExVivoModeltoStudyHIV-1InfectionDuring
PregnancyandBeyond .................................................. 205
NabilaJabrane-FerratandHichamElCosta
16 HIVReservoirs:Modeling,Quantification,andApproaches
toaCure .............................................................. 215
AmirDashti,VidishaSingh,andAnnChahroudi
17 HumanHematopoieticStemCell(HSC)-EngraftedNSGMice
forHIVLatencyResearch ............................................... 229
TrianaRivera-Megias,NhutM.Le,andAlonsoHeredia
18 InducingLong-TermHIV-1LatencyintheTKO-BLT
MouseModel .......................................................... 253
YunyunDiandKerryJ.Lavender
PART V METHODS FOR DETECTION AND ANALYSIS OF THE RESERVOIR
19 InSituMultiplexingtoIdentify,Quantify,andPhenotype
theHIV-1/SIVReservoirWithinLymphoidTissue......................... 277
KathleenBusman-Sahay,MichaelD.Nekorchuk,CarlyElizabethStarke,
ChiNgaiChan,andJacobD.Estes
20 Single-CellMultiparametricAnalysisofRareHIV-InfectedCells
IdentifiedbyDuplexedRNAflow-FISH ................................... 291
MathieuDube´andDanielE.Kaufmann
21 ExVivoDifferentiationofRestingCD4+TLymphocytesEnhancesDetection
ofReplicationCompetentHIV-1inViralOutgrowthAssays................. 315
ElizabethR.Wonderlich,MonicaD.Reece,andDeannaA.Kulpa
22 QuantitativeViralOutgrowthAssaytoMeasuretheFunctional
SIVReservoirinMyeloidCells ........................................... 333
C.M.Abreu,R.T.Veenhuis,E.N.Shirk,S.E.Queen,B.T.Bullock,
J.L.Mankowski,L.Gama,andJ.E.Clements
Contents ix
23 Near-Full-LengthSingle-GenomeHIV-1DNASequencing ................. 357
GuinevereQ.LeeandMathiasLichterfeld
24 TILDA:Tat/RevInducedLimitingDilutionAssay ......................... 365
CynthiaLunguandFrancescoA.Procopio
PART VI CURE AND LONG-TERM REMISSION STRATEGIES
25 LatencyReversalandClearanceofPersistentHIVInfection.................. 375
DavidM.Margolis
26 CureandLong-TermRemissionStrategies................................. 391
LuisaMoriandSusanaT.Valente
27 PathwaysTowardaFunctionalHIV-1Cure:BalancingPromise
andPerilsofCRISPRTherapy............................................ 429
JonathanHerskovitz,MahmudulHasan,MilankumarPatel,
BhaveshD.Kevadiya,andHowardE.Gendelman
Index ...................................................................... 447
