Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Summary

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Abstract

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%–50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

Introduction

Human immunodeficiency virus (HIV) is a chronic infection with more than 37 million infected individuals worldwide¹. Combination antiviral therapy (cART) is a life-saving therapy, but a cure is still warranted. Thus, there is a need for animal models that mirror the human immune system and its responses in order to facilitate continued research in HIV. Multiple types of humanized mice that are capable of supporting cell and tissue engraftment have been developed by transplanting human cells into severely immunodeficient mice². Such humanized mice are susceptible to HIV infection and provide an important alternative to no....

Protocol

All cord blood samples were obtained in strict accordance with locally approved protocols, including informed consent of anonymous donation by the parents. All animal experiments were approved and performed in strict accordance with Danish national regulations under the license 2017-15-0201-01312.

CAUTION: Handle HIV exposed mice and blood with extreme caution. Decontaminate all surfaces and liquids that have been in contact with HIV with a confirmed HIV disinfectant (Table of Material.......

Discussion

The severely immunocompromised mouse strain NOD.Cg-Prkdc^scid Il2rg^tm1Sug/JicTac (NOG) is extremely well suited for transplantation of human cells and tissues. Both innate and adaptive immune pathways in these mice are compromised. NOG and NSG mice harbor a Prkdc^scid mutation that results in defective T and B cell function. Furthermore, these mice lack a functional interleukin-2 receptor γ-chain (common gamma chain, IL2rg) which is indispensable in .......

Materials

Name	Company	Catalog Number	Comments
Blue pad	VWR	56616-031	Should be sterilized prior to use
Bovine serum albumin (BSA)	Sigma	A8022
CD19 (clone sj25c1) PE-Cy7	BD Bioscience	557835
CD3 (clone OKT3) FITC	Biolegend	317306
CD3 (clone SK7) BUV395	BD Bioscience	564001
CD34 (clone AC136) FITC	Miltenyi	130-113-740
CD4 (clone SK3) BUV 496	BD Bioscience	564652/51
CD45 (clone 2D1) APC	Biolegend	368511/12
CD8 (clone RPA-T8) BV421	BD Bioscience	562428
ddPCR Supermix for probes (no dUTP)	Bio-Rad	1863025
DMSO	Merck	10,02,95,21,000
DNAse	Sigma	D4263	For suspension buffer
dNTP mix	Life Technologies	R0192
Dulbeccos phosphate-buffered saline (PBS)	Biowest	L0615-500
EasySep Human Cord Blood CD34 Positive Selection Kit II	Stemcell	17896
EDTA	Invitrogen	15575-038
FACS Lysing solution 10X	BD	349202	Dilute 1:10 in dH20 immediately before use
FACS tubes (Falcon 5 mL round-botton)	Falcon	352052
Fc Receptor blocking solution (Human Trustain FcX)	Biolegend	422302
Fetal bovine serum	Sigma	F8192-500
Ficoll-Paque PLUS	GE Healthcare	17144002
Flowjo v.10
Gauze	Mesoft	157300	Should be sterilized prior to use
Heating lamp			Custom made
Hemacytometer (Bürker-Türk)	VWR	DOWC1597418
Isoflurane gas	Orion Pharma	9658
LSR Fortessa X20 flow cytometer	BD
Microcentrifuge tubes, PCR-PT approved	Sarstedt	72692405
Mouse cART food	ssniff Spezialdiäten GmbH	Custom made product
Mouse restrainer		Custom made product
Needle, Microlance 3, 30G ½"	BD	304000
NOG mice NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac	Taconic	NOG-F
Nuclease-free water	VWR chemicals	436912C
Nucleospin 96 Virus DNA and RNA isolation kit	Macherey-Nagel	740691
PCR-approved microcentrifuge tubes	Sarstedt	72.692.405
Penicillin-Streptomycin solution 100X	Biowest	L0022-100
Phusion Hot Start II DNA polymerase	Life Technologies	F549S
Pipette tips, sterile, ART 20P Barrier	ThermoScientific	2149P
Proteinase K	NEB	100005398
QuantaSoft software	Bio-Rad
QX100 Droplet Generator	Bio-Rad	1886-3008
QX100 Droplet Reader	Bio-Rad	186-3003
RBC lysis solution	Biolegend	420301
RNase-free DNAse size F + reaction buffer	Macherey-Nagel	740963
RNAseOUT Recombinant Ribonuclease inhibitor	ThermoScientific	10777-019
RPMI	Biowest	L0501-500	Dissolve in H20
Softject 1 mL syringe	Henke Sass Wolf	5010-200V0
Superscript III Reverse Transcriptase	ThermoFisher Scientific	18080044
Thermoshaker	VWR	89370-910
Trypane blue	Sigma	T8154
Ultrapure 0.5 EDTA, pH 8.0	ThermoFisher Scientific	15575-020
Virkon S (virus disinfectant)	Dupont	7511