HIV Reservoirs: Methods and Protocols

Preface
Contents
Contributors
Part I: Cell Lines with Minimal but Inducible Proviral Expression
	Chapter 1: Jurkat-Derived (J-Lat, J1.1, and Jurkat E4) and CEM-Derived T Cell Lines (8E5 and ACH-2) as Models of Reversible Pr...
		1 Introduction
			1.1 CEM-Derived T Cell Lines (8E5 and ACH-2)
				1.1.1 8E5 Cell  Line
				1.1.2 ACH-2 Cell  Line
			1.2 Jurkat-Derived T Cell Lines (J-Lat, J1.1, and Jurkat E4)
				1.2.1 J1.1 Cell  Line
				1.2.2 J-Lat Cell Lines
				1.2.3 Jurkat E4 Cell  Line
			1.3 HIV-1 Transcriptional Reactivation: How to Measure  it?
		2 Materials
			2.1 LRA Stimulation
			2.2 Well-Established LRA Active on ACH-2 Cells
			2.3 RT-qPCR Assay
			2.4 FACS Assay
			2.5 p24 ELISA
		3 Methods
			3.1 ACH-2 Cells
			3.2 LRA Stimulation of Jurkat-Derived Cell Lines
			3.3 RNA Extraction
			3.4 Turbo DNase Treatment
			3.5 Reverse Transcription
			3.6 Real-Time Quantitative PCR
			3.7 FACS Assay
			3.8 p24 ELISA
		4 Notes
		References
	Chapter 2: U1 and OM10.1. Myeloid Cell Lines as Surrogate Models of Reversible Proviral Latency
		1 Introduction
			1.1 U1 Cell  Line
			1.2 OM-10.1 Cell  Line
		2 Materials
			2.1 Complete Medium
			2.2 Well-Established Latency-Reversing Agents Active on U1 and OM-10.1 Cells
		3 Methods
		References
Part II: In Vitro and Ex Vivo Primary Cell Models of Minimal but Inducible Proviral Expression
	Chapter 3: An In Vitro System to Model the Establishment and Reactivation of HIV-1 Latency in Primary Human CD4+ T Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture Media and Reagents
			2.2 Cell Isolation Kits, Supplies, and Buffers
			2.3 Flow Cytometry
			2.4 HIV-1 p24 ELISA
		3 Methods
			3.1 Day 0: Generation of Immature Monocyte-Derived Dendritic Cells (iMDDC)
			3.2 Day 5: Activation of Naïve CD4+ T Cells
			3.3 Day 9: HIV-1 Infection and Expansion of Activated CD4+ T Cells
			3.4 Day 24: Isolation of Memory CD4+ T Cells and Generation of Resting Cells
			3.5 Day 53: Reactivation of HIV-1 Replication from Resting Latently Infected CD4+ T Cells
		4 Notes
		References
	Chapter 4: The Cultured TCM Model of HIV Latency
		1 Introduction
		2 Materials
			2.1 Media, Cytokines and Antiretroviral Drugs
			2.2 Cell Purification and Staining
			2.3 Tissue Culture
		3 Methods
			3.1 PBMC Isolation and Naïve CD4T Purification Using Density Gradient Centrifugation
			3.2 Generation of Latently Infected TCM Cells
			3.3 Staining for Surface CD4 and Intracellular HIV Gag
		4 Notes
		References
	Chapter 5: A Reliable Primary Cell Model for HIV Latency: The QUECEL (Quiescent Effector Cell Latency) Method
		1 Introduction
		2 Materials
			2.1 Cytokines and Antibodies for Generating Effector CD4 T Cells
			2.2 Cell Growth Media
			2.3 Source of Cells and Virus
			2.4 Cell Isolation  Kit
			2.5 Cell Polarization and Reactivation Reagents
			2.6 Tissue Culture Supplies and Instrument
			2.7 Flow Antibody and Reagents
			2.8 RNA FISH Reagents and Supplies
		3 Methods
			3.1 Day 0: Activation and Initial Polarization
			3.2 Day 3: Continuing Polarization
			3.3 Day 6: Infection with Pseudotyped Reporter Virus
			3.4 Day 7: Dilution of Virus
			3.5 Days 8-13: Cell Husbandry and Expansion
			3.6 Day 14: CD8a+ Cell Isolation
			3.7 Days 14 to 28: Cell Quiescence
			3.8 RNA  FISH
		4 Notes
		References
	Chapter 6: TGF-β Signaling Supports HIV Latency in a Memory CD4+ T Cell Based In Vitro Model
		1 Introduction
		2 Materials
			2.1 General Laboratory Equipment
			2.2 General Laboratory Supplies
			2.3 Reagents
			2.4 LARA Culture Media
		3 Methods
			3.1 Day 0 Memory CD4+ T cell Enrichment from HIV-Naïve Peripheral Blood Mononuclear Cell (PBMC)
			3.2 Day 3 In Vitro Infection of Memory CD4+ T Cells with Replication Competent  HIV
			3.3 Day 6. Initiation of Latency Culture
			3.4 Day 10. Replenish Latency Culture Medium
			3.5 Day 13. Conclusion of Latency Culture
		4 Notes
		References
	Chapter 7: Flow Cytometry Sorting of Memory CCR6+CD4+ T-Cells for HIV Reservoir Quantification
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 Reagents
			2.3 Antibodies and Dyes
			2.4 Equipment
		3 Methods
			3.1 Negative Selection of Total CD4 + T-Cells from PBMC Using MACS (Miltenyi Biotec; See Note 1)
			3.2 Cell Staining
			3.3 FACSorting
			3.4 Downstream Applications
		4 Notes
		References
	Chapter 8: Human Monocyte-Derived Macrophages (MDM): Model 1 (GM-CSF)
		1 Introduction
		2 Materials
			2.1 Isolation of Monocytes from Buffy Coats of HIV-Seronegative Blood Donors
			2.2 Differentiation of Monocytes in Macrophages
			2.3 Macrophages Infection
		3 Methods
			3.1 Isolation of Monocytes from Buffy Coats of HIV-Seronegative Blood Donors
			3.2 Differentiation of Monocytes into Macrophages
			3.3 Assessment of MDM Purity by Flow Cytometry
			3.4 Infection of Macrophages with HIV-1
		4 Notes
		References
	Chapter 9: Human Monocyte-Derived Macrophages (MDM): Model 2
		1 Introduction
		2 Materials
			2.1 Isolation of PBMC from Buffy Coats of HIV-Seronegative Blood Donors
			2.2 Differentiation of Monocytes into Monocyte-Derived Macrophages (MDM)
			2.3 MDM Infection
		3 Methods
			3.1 Differentiation of Monocytes into Monocyte-Derived Macrophages (MDM)
			3.2 Polarization of MDM into M1 vs. M2 Cells
			3.3 M12-MDM as Surrogate Model of Reversible Proviral Latency in Primary Macrophages
		4 Notes
		References
	Chapter 10: Modeling HIV Latency in Astrocytes with the Human Neural Progenitor Cell Line HNSC.100
		1 Introduction
		2 Materials
			2.1 HNSC.100 Cell Culture
			2.2 Reactivation of HNSCLatGFP1.2 cells
		3 Methods
			3.1 Coating of Culturing Vessel with Poly-l-Lysine (See Note 3)
			3.2 Propagation of HNSC.100 Cells
			3.3 Seeding of Cells
			3.4 Differentiation of HNSC.100
			3.5 Re-Activation of HNSCLatGFP1.2 Cells
			3.6 Usage of HNSCLatGFP1.2 Cells to Analyze HIV-1 Reactivation Inhibitors
		4 Notes
		References
	Chapter 11: Hematopoietic Stem and Progenitor Cells (HSPCs)
		1 Introduction
			1.1 Isolation of CD133+ HSPCs from Umbilical Cord Blood
			1.2 In Vitro HSPC Latency Model
			1.3 Bone Marrow Aspiration
			1.4 Bone Marrow Progenitor Cell Isolation
			1.5 Genomic DNA Extraction
			1.6 Single Genome Amplification (SGA) PCR Assay
			1.7 Sequence Data Analyses
		2 Materials
			2.1 Isolation of CD133+ HSPCs from Umbilical Cord Blood
			2.2 In Vitro HSPC Latency Model
			2.3 Bone Marrow Aspiration
			2.4 Bone Marrow Progenitor Cell Isolation
			2.5 Genomic DNA Extraction
			2.6 Single Genome Amplification (SGA) PCR Assay
			2.7 Sequence Data Analyses
		3 Methods
			3.1 Isolation of CD133+ HSPCs from Umbilical Cord Blood
			3.2 In Vitro HSPC Latency Model
			3.3 Bone Marrow Aspiration
			3.4 Bone Marrow Progenitor Cell Isolation
			3.5 Genomic DNA Extraction
			3.6 SGA PCR Assay
			3.7 Sequence Data Analyses
		4 Notes
		References
Part III: In Vitro and Ex Vivo Tissue-Derived Models of Reversible Proviral Latency
	Chapter 12: Ex Vivo HIV Infection Model of Cervico-Vaginal and Rectal Tissue
		1 Introduction
		2 Materials
			2.1 BSL2+ Lab Equipment
			2.2 Media, Reagents and Virus
		3 Methods
			3.1 Human Cervicovaginal Tissue Preparation, Culture, and Infection: Immersion Model
			3.2 Human Cervicovaginal Tissue Culture and Infection: Polarized Model
			3.3 Human Rectal Tissue Preparation, Culture and Infection in Immersion Model
			3.4 Human Rectal Tissue Preparation, Culture and Infection in Polarized Model
			3.5 HIV Quantification
			3.6 Experimental Example of CV Tissue Infection and Evaluation of Compound Antiviral Activity
			3.7 Data Analysis: HIV Infection and Inhibition
		4 Notes
		5 Conclusions
		References
	Chapter 13: More than a Gender Issue: Testis as a Distinctive HIV Reservoir and Its Implication for Viral Eradication
		1 Introduction
		2 Materials
			2.1 Tissue Processing
			2.2 Solutions
			2.3 Flow Cytometry
		3 Methods
			3.1 Tissue Supply
			3.2 Cell Extraction with Enzymatic Treatment
			3.3 Flow Cytometry Cell  Sort
		4 Notes
		References
Part IV: Animal Models
	Chapter 14: Experimental Models to Study HIV Latency Reversal from Male Genital Myeloid Cells
		1 Introduction
		2 Materials
			2.1 For Mucosa Reconstructed and Infected In Vitro
			2.2 For Ex Vivo Reactivation of Latent HIV
		3 Methods
			3.1 HIV Latency Reversal in Male Genital Mucosa Reconstructed and Infected In Vitro
			3.2 Ex Vivo HIV Latency Reversal of Tissue Reservoirs
		4 Notes
		5 Conclusions
		References
	Chapter 15: Decidua Basalis: An Ex Vivo Model to Study HIV-1 Infection During Pregnancy and Beyond
		1 Introduction
		2 Materials
			2.1 Decidual Tissue Collection, Culture and Digestion
			2.2 Macrophage Isolation and Culture
			2.3 Viral Replication
			2.4 Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling (TUNEL) Assay
		3 Methods
			3.1 Human Decidual Tissue Collection
			3.2 Decidual Histocultures
			3.3 Tissue Viability with a TUNEL Assay
			3.4 Isolation of Decidual Macrophages
			3.5 HIV-1 Infection of Decidual Histocultures
			3.6 HIV-1 Infection of Decidual Macrophages
		4 Notes
		References
	Chapter 16: HIV Reservoirs: Modeling, Quantification, and Approaches to a Cure
		1 Materials
			1.1 Infection of NHPs with Virus of Interest and Quantification of Plasma Viral  Load
			1.2 Antiretroviral Therapy for Latency Studies in  NHPs
			1.3 Assessing Longitudinal Health in SIV Latency Studies
			1.4 Sampling and Evaluating the Response to Cure-Directed Interventions
		2 Methods
			2.1 Selection of NHPs for Cure Studies
			2.2 Virus Selection and Route of Infection
			2.3 Antiretroviral Therapy to Suppress Virus Replication in  NHPs
			2.4 Assessing NHP Health During Longitudinal Cure Studies
			2.5 Samples and Sample Processing
			2.6 Experimental Groups and Outcome Measures
		3 Concluding Remarks
		References
	Chapter 17: Human Hematopoietic Stem Cell (HSC)-Engrafted NSG Mice for HIV Latency Research
		1 Introduction
		2 Materials
			2.1 Generation of HSC-NSG Mice
			2.2 Screening of HSC-NSG Mice by Flow Cytometry Analysis of Blood Samples
			2.3 Infection of HSC-NSG Mice and Quantification of HIV RNA in Plasma
			2.4 Suppression of Plasma HIV RNA for Establishment of HIV Latency
		3 Methods
			3.1 Generation of HSC-NSG Mice by Intrahepatic Injection of Human CD34+ Cells into Newborn NSG Mice
			3.2 Screening of HSC-NSG Mice for Human Cell Reconstitution
			3.3 Infection of HSC-NSG Mice with HIV, Quantification of Plasma HIV RNA, and CD4+ T Cells
			3.4 Suppression of Plasma HIV RNA for Establishment of HIV Latency
		4 Notes
		References
	Chapter 18: Inducing Long-Term HIV-1 Latency in the TKO-BLT Mouse Model
		1 Introduction
		2 Materials
		3 Methods
			3.1 Production of TKO-BLT  Mice
				3.1.1 Presurgical Preparation
				3.1.2 Tissue Implantation Surgery
				3.1.3 Isolation CD34+ Hematopoietic Progenitor Cells (HPC) from Fetal Liver
				3.1.4 Preparation of TKO Mouse Bone Marrow
				3.1.5 Injection of the CD34+ Stem Cells
				3.1.6 Splenocyte Injection, Staple Removal and Assessment of Reconstitution
			3.2 HIV-1 Infection
			3.3 Pretreatment Evaluation of Plasma p24 Antigenemia, CD4+ T Cell Counts, and CD4/CD8 Ratio
				3.3.1 Bleeding TKO-BLT  Mice
				3.3.2 Plasma Collection and Determination of Plasma p24 Antigenemia
				3.3.3 Processing TKO-BLT Blood
				3.3.4 Determination of CD4+ T Cell Counts and CD4/CD8 Ratio
			3.4 Induction of Latency Using Combination Antiretroviral Therapy (cART)
				3.4.1 Administering Injectable cART Regimens
				3.4.2 Administering Free-Fed cART Mouse Chow Regimens
			3.5 Obtaining Samples to Evaluate HIV-1 Viral and Proviral Burden
				3.5.1 Evaluation of Plasma Viral RNA (vRNA)
				3.5.2 Evaluation of the HIV-1 Proviral Reservoir
		4 Notes
		References
Part V: Methods for Detection and Analysis of the Reservoir
	Chapter 19: In Situ Multiplexing to Identify, Quantify, and Phenotype the HIV-1/SIV Reservoir Within Lymphoid Tissue
		1 Introduction
		2 Materials
			2.1 Tissue Collection and Slide Generation
			2.2 In Situ Hybridization and Immunofluorescence
		3 Methods
			3.1 Tissue Collection and Slide Generation
			3.2 In Situ Hybridization-DNAScope
			3.3 Immunofluorescence
			3.4 Optional Modification 1: Stripping and Multiplexing with a Tyramide Signal Amplification-Based Reaction
			3.5 Optional Modification 2: Additional Immunofluorescence-Based Phenotyping
			3.6 Optional Modification 3: In Situ Hybridization Multiplexing-RNAscope
		4 Notes
		References
	Chapter 20: Single-Cell Multiparametric Analysis of Rare HIV-Infected Cells Identified by Duplexed RNAflow-FISH
		1 Introduction
		2 Materials
			2.1 Basic Equipment and Plasticware
			2.2 Solutions and Media
			2.3 Thawing of PBMCs
			2.4 CD4+ T Cell Enrichment
			2.5 Surface Protein Staining
			2.6 Intracellular Protein Staining
			2.7 RNAflow-FISH
		3 Methods
			3.1 CD4+ T Cell Calculation
			3.2 Thawing of PBMCs
			3.3 CD4+ T Cell Enrichment
			3.4 Surface Staining
			3.5 Intracellular Staining
			3.6 RNA Hybridization
			3.7 Analysis
		4 Notes
		References
	Chapter 21: Ex Vivo Differentiation of Resting CD4+ T Lymphocytes Enhances Detection of Replication Competent HIV-1 in Viral O...
		1 Introduction
		2 Materials
			2.1 General Laboratory Equipment
			2.2 General Laboratory Supplies
			2.3 Reagents
			2.4 dQVOA Culture Media
		3 Methods
			3.1 Day -8
			3.2 Day -7. rCD4+ Enrichment, Dilution, and Differentiation from HIV-1+  PBMC
			3.3 Day -3. Replenish Differentiation Medium
			3.4 Day 0. Mitogen Activation and Costimulation of Differentiated rCD4+ T Cells in the Presence of Irradiated Allogenic  PBMC
			3.5 Day +1 Dilution of the Mitogen Activation Signal and Induction of Viral Outgrowth
			3.6 Day +5. Replenish Feeding Medium
			3.7 Day +8. Replenish Feeding Medium
			3.8 Day +11. End dQVOA, Harvest the Culture Supernatant for the Identification of HIV-1 Gag+ Wells, and Calculate HIV-1 Reserv...
		4 Notes
		References
	Chapter 22: Quantitative Viral Outgrowth Assay to Measure the Functional SIV Reservoir in Myeloid Cells
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Media
			2.3 Other Reagents
		3 Methods
			3.1 Coat Plates
			3.2 Isolate Single Cell Suspensions from Blood and Tissues (See Notes 7 and 8 and Fig. 1)
			3.3 Preselection
			3.4 Positive Selection of Myeloid Cells from Blood, Spleen, BAL and Brain: See Notes 10-12
			3.5 Plate Myeloid Cells: See Note 13
			3.6 Wash, Activate and Collect QVOAs
				3.6.1 Prepare CEMx174 Expander Cells in R10 Media
				3.6.2 Activate QVOAs: See Note 16
				3.6.3 Collect QVOA Samples: See Note 17
			3.7 RNA Vacuum Isolation
			3.8 M훟-QVOA Analysis: SIV gag RNA Quantification by RT-qPCR and IUPM Calculation-See Note 19
			3.9 M훟-QVOA Controls
				3.9.1 To Evaluate Myeloid Cell Selection Purity by Flow Cytometry
				3.9.2 Quantitation of CD3+ T Cells Contamination in TCR Wells Using qPCR for TCRß RNA (See Note 23)
				3.9.3 Quantitation of  DNA
				3.9.4 Calculation of CD4 Contamination in M훟-QVOA Using Data from Table 3 (See Notes 24-26 for Details)
		4 Notes
		References
	Chapter 23: Near-Full-Length Single-Genome HIV-1 DNA Sequencing
		1 Introduction
		2 Materials
		3 Methods
			3.1 Nucleic Acid Extraction
			3.2 Droplet Digital PCR (ddPCR) Quantification of Total HIV  DNA
			3.3 PCR Amplification of Near Full-Genome HIV  DNA
			3.4 Beads Purification of PCR Amplicons
			3.5 Illumina Library Preparation and Sequencing
			3.6 Viral Genome Bioinformatics Analysis
		4 Notes
		References
	Chapter 24: TILDA: Tat/Rev Induced Limiting Dilution Assay
		1 Introduction
		2 Materials
			2.1 Cell Isolation and Stimulation
			2.2 Preamplification  Step
			2.3 Seminested Real-Time  PCR
		3 Methods
			3.1 Cell Preparation (2 h)
			3.2 Preamplification Step (3 h)
			3.3 Seminested Real-Time PCR Step (2 h)
		4 Notes
		References
Part VI: Cure and Long-Term Remission Strategies
	Chapter 25: Latency Reversal and Clearance of Persistent HIV Infection
		1 Introduction
		2 Latency Reversal: Creating a Window of Vulnerability in the Persistent Viral Reservoir
		3 Challenges for Latency Reversal
		4 Induced and Engineered Immune Responses to Eradicate
		5 Human Testing of Eradication Approaches
		6 And a New Approach to Shrink the Reservoir
		References
	Chapter 26: Cure and Long-Term Remission Strategies
		1 Introduction
		2 The Nature of the HIV Reservoir and Viral Latency
			2.1 A Definition of the Viral Reservoir
			2.2 Mechanisms of Latency Establishment and Maintenance
		3 HIV Transcription
		4 The Nature of Latency During Antiretroviral Therapy
		5 Silencing HIV Transcription
			5.1 Targeting Viral Factors
				5.1.1 Trans-activator of Transcription (Tat) Protein
				5.1.2 The Transactivation Response Element (TAR)  RNA
			5.2 Targeting Host Factors
				5.2.1 Positive Elongation Factor b (P-TEFb)
				5.2.2 Xeroderma pigmentosum Type B (XPB)
				5.2.3 Heat-Shock Protein 90 (Hsp90)
				5.2.4 Facilitates Chromatin Transcription (FACT) Complex
				5.2.5 Mammalian Target of Rapamycin (mTOR)
				5.2.6 Bromodomain-Containing Protein 4 (BRD4)
				5.2.7 Estrogen Receptor 1 (ESR-1)
				5.2.8 Splicing Factor 3B Subunit 1 (SF3B1)
				5.2.9 Inhibitors of Signaling Pathways that Block Reversal of HIV Latency
				5.2.10 Use of Gene Editing Platforms for Transcriptional Repression of  HIV
		6 Conclusion
		References
	Chapter 27: Pathways Toward a Functional HIV-1 Cure: Balancing Promise and Perils of CRISPR Therapy
		1 Introduction
		2 Evolving Toward a Therapy
		3 Inactivating the HIV Genome
		4 Ablation of HIV Cellular Entry
		5 Modulation of Intracellular Signaling Host Antiviral Pathways
		6 Optimizing On- Versus Off-Target Effects
		7 Induction of HIV Escape Mutants
		8 CRISPR Delivery
		9 HIV Elimination Using CRISPR
		10 Overcoming CRISPR Therapeutic Limitations
		References
Index