Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Summary

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Abstract

Humanized NOD/SCID/IL-2 receptor γ-chain^null mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34⁺ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Introduction

The humanized NOD/SCID/interleukin (IL)-2 receptor γ-chain^null (hereafter referred to as huNS γ-chain^null) mouse model has been widely used for studying the pathogenesis of infections, autoimmunity, and cancer, as well as for pre-clinical studies of drugs and human cell-based therapies^1,2. These mice are based on a non-obese diabetic (NOD) background, with the scid mutation and targeted mutation at the IL-2 receptor γ-chain locus (common γ-chain for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21), which induce a severe impairment in the development of mouse T-, B-, ....

Protocol

In this work, all animal care and procedures were performed according to protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine (protocol numbers 1018017, 1018018, and 0318009).

1. Human CD34⁺ HSC engraftment of newborn mice

1. Always use disposable personal protection equipment (PPE), including sterile scrubs, gloves, dedicated shoes, shoe covers, mask, goggles, hair/beard bonnet, and sterile lab coats.
2. Re-suspend 1 x 10⁶ of frozen CD34⁺ HSCs (see Table of Materials) in 10 mL of RPMI 1640....

Results

As described above, at 14 weeks post-HSC injection (chronic model) or at 3 weeks post-PBMC injection (acute and reactivation models), the mice are bled for screening the level of human cells engraftment by flow cytometry. A representative gating strategy for the evaluation of 1) human CD45⁺ cells reconstitution and 2) percentage of CD4⁺ and CD8⁺ T-cells is shown in Figure 1A. Typically, the level of engraftment (percentage of human CD45^+.......

Discussion

Important advances have been achieved in the development of immunodeficient mouse strains for humanization, with a number of different options that can be used according to the research interest¹. Provided here is a general protocol for the humanization of NS γ-chain^null mice and genetically similar strains to be employed in three different models for studying HIV infection. In the first experimental approach, irradiated newborn mice are injected with human CD34⁺ HSCs, w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0. 5 ml Microcentrifuge tubes	Neptune	3735.S.X
1. 5 ml Microcentrifuge tubes	Neptune	3745.S.X
10 ml Serologial pipetes	stellar sceintific	VL-4090-0010
15 ml conical tubes	Stellar scientific	T15-600
25 ml Serologial pipetes	stellar sceintific	VL-4090-0025
5 ml Serologial pipetes	stellar sceintific	VL-4090-0005
50 ml conical tubes	Stellar scientific	T50-600
ACK lysis buffer	Quality biological	118-156-101
Alcohol prep pads	Fisher scientific	06-669-62	Sterile
Anti-Human CD3 clone UCHT1	Biolegend	300439	APC conjugated
Anti-Human CD4 clone OKT4	Biolegend	317420	AF488 conjugated
Anti-Human CD45 clone 2D1	Biolegend	368522	BV421 conjugated
Anti-Human CD8 clone SK1	Biolegend	344710	PerCP-Cy5.5 conjugated
Biosafaty cabinet level 2			If posible connected to an exauste chimeny when handling Isoflurane
Bonnet	Fisher scientific	17-100-900	Single use cap for basic protection
Cavicide	Metrex	13-1000	Surface desinfectant
CD34+ cells	Lonza	2C-101	As many vials available from a single donor
Centrifuge	Beckman	65-6KR
Clear jar	Amazon	77977
Cotton gauze pad	Fisher scientific	22-415-468	Sterile
Disposable lab coats	Fisher scientific	19-472-422
EDTA micro tubes	Greiner bio-one	450480
Face Mask	Fisher scientific	17-100-897
FACS lysing solution	BD	340202
FBS premium HI	Atlanta biologicals	S1115OH
Ficoll	GE health one	17-1440-02
Flow cytometer			We used FACS Aria II
Flow cytometry tubes	Falcon	352054	5 ml polystyrene and round bottom
HIV BaL			Prepared in our uQUANT core facility
Human PBMCs			HIV positive and negative volunteers
Infrared warming pad	Venet scientific	DCT-25	Temporary therapeutic warming pad for small animals
Isentress (Raltegravir)	Merck	NSC 0006-0227061	Antiretroviral medication to treat human immunodeficiency virus (HIV)-Integrase inhibitor
Isoflurane	Henry Schein	NDC 11695-6776-2
Mark I irradiator			Equipment belonging to university of Maryland
Micro pipettes
Microcentrifuge	Eppendorf
Mouse ear tags	National Band & Tag company	1005-1L1
Natelson blood collection tubes	Fisher scientific	02-668-10
NOG-EXL	Taconic	HSCFTL-13395-F
NSG mice	Jackson	5557	Time pregnant females for CD34 engraftment and Juveniles for PBMCs engraftment
NSG-SGM3	Jackson	13062
Paraformaldehyde 16%	Electron microscopy sciences	15710
PBS 1X pH 7.4	Gibco	100-10-023
Petri dishes	Fisher scientific	08-757-28
Quantistudio qPCR machine	Thermo	QS3
Reagent reservoirs	Costar	4870
RPMI media 1640 1X	Gibco	11875-093
Shoe covers	Fisher scientific	17-100-911
Sterile disposable Gloves	Microflex	SUF-524
SuperScript II First-Strand Synthesis SuperMix	Invitrogen	10080-400	cDNA synthesis
Syringes 28-G x 1/2	BD	329-461
Syringes 29-G x 1/2	BD	324-702
Truvada (Emtricitabine and Tenofovir	Gilead	NDC 61958-0701-1	Antiretroviral medication to treat human immunodeficiency virus (HIV)-Nicleoside analog-transcriptase inhibitor
Trypan blue	Sigma	T8154	Cell count and viability
Vick Vaporub	School health	43214	Ointment based on menthol and eucalyptus
Water molecular biology grade	Quality biological	351-029-131