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.
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.
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 nonhuman primate simian immunodeficiency virus models, as they are cheaper and simpler to use than nonhuman primates. Humanized mice have facilitated research in HIV viral transmission, pathogenesis, prevention, and treatment3,4,5,6,7,8,9,10,11.
We present a flexible humanized model system for HIV research developed by transplanting cord blood derived human stem cells into mice of the NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) background. Besides being of non-fetal origin, the practical bioengineering of these mice is less technically demanding compared to the microsurgical procedures involved in the transplant of the blood-liver-thymus (BLT) construct.
We show how to establish HIV infection through intravaginal transmission and how to monitor the plasma viral load with a sensitive droplet digital PCR (ddPCR)-based setup. Subsequently, we describe the establishment of standard cART given as part of the daily mouse diet. The aim of these combined methods is to reduce stress to the animals and facilitate large-scale experiments where time spent handling each animal is limited12.
In humans, a CCR5Δ32/wt or CCR5Δ32/ Δ32 genotype causes reduced susceptibility to HIV infection with transmitter/founder viruses13, and some precautions must be taken when bioengineering humanized mice with stem cells for the purpose of HIV studies. This is especially true in our region because naturally occurring variants in the CCR5 gene, particularly Δ32 deletions, are more prevalent in Scandinavian and Baltic native populations compared to rest of the world14,15. Thus, our protocol includes an easy, high-throughput assay for screening donor hematopoietic stem cells for CCR5 variants prior to transplantation.
For the intravaginal exposure we chose the transmitter/founder R5 virus RHPA4259, isolated from a woman in an early stage of infection who was infected intravaginally16. We exposed the mice to a viral dose that was sufficient to yield successful transmission in the majority of mice, but below a 100% transmission rate. Choosing such a dose enables a sufficient dynamic range in transmission rate such that antiviral effects of a drug candidate can result in protected animals in HIV prevention experiments and decreased viral load for treatment studies.
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 Materials).
1. Isolation of human CD34+ stem cells
2. Assessing CD34+ stem cell purity via flow cytometry
3. Genetic screening for CCR5Δ32 variants in cord blood
4. Intravenous stem cell transplant
NOTE: Having one person prepare the cells in the laboratory and another person prepare the mice and workspace for transplants is an efficient approach.
5. Blood collection and processing for analysis
NOTE: Human cell engraftment in the peripheral blood can be evaluated via flow cytometry 3–5 months after human stem cell transplantation.
6. Evaluation of human engraftment via flow cytometry
7. Intravaginal HIV exposure
NOTE: The virus used for intravaginal exposure of the mice can be produced using previously published protocols17. The virus is kept at -80 °C and transported between locations while stored on dry ice following locally approved protocols. The virus is stored on dry ice until immediately before the exposure of the mice. The virus can be diluted into plain RPMI (avoid RPMI that has antibiotics or serum additives) to achieve the appropriate concentration immediately prior to exposure (21,400 IUs were used for this IVAG exposure). Once generated, keep the diluted stock on wet ice throughout the procedure to avoiding freeze-thaw cycles that would occur if the diluted virus was placed back on dry ice once thawed.
8. Processing of blood samples prior to viral load analysis
9. DNA extraction using a proteinase K extraction method
10. RNA extraction, cDNA synthesis, and ddPCR quantification of viral RNA
11. Treatment with cART-containing chow
The gating strategy for the analysis of stem cell purity is depicted in Figure 1. Figure 1A–C shows the purified CD34+ population and Figure 1D–F the CD34- flow-through used to illustrate that the minimal amount of the CD34+ population is lost in the isolation process. The purity of isolated CD34+ stem cells was between 85%–95% with less than 1% T-cell contamination. Figure 1G depicts CCR5 bands from one adult human control donor with the CCR5Δ32/wt genotype, followed by bands from two CCR5wt/wt and one CCR5Δ32/wt stem cell donors. The frequency of the genotype CCR5Δ32/wt in a group of 19 donors was 15.8% (Figure 1H). This is in agreement with larger epidemiological studies14,15 reporting the genotype in up to 23.6% of investigated persons in Denmark.
Human CD45+ levels in mice peripheral blood was assessed via flow cytometry 3–5 months after transplantation of human CD34+ stem cells. The gating strategy is presented in Figure 2A–E. Figure 3A and Figure 3B illustrate the variability between 10 and 16 individual mice receiving stem cells from two different donors. Transplantation of 75,000 hCD34+ cells yielded 20%–50% human CD45+ in the peripheral blood. All mice developed human B and T cells, including both CD4- and CD8+ T cells.
For atraumatic intravaginal exposures, the setup depicted in Figure 4 was used. Mice were anaesthetized in a closed chamber and kept under anesthesia during the exposure. Mice were held with the vagina facing up for 5 min after exposure to ensure virus solution engagement with mucosal surfaces.
Figure 5A shows the 64% HIV transmission success rate observed using this model. Mice were challenged with 21,400 infectious units (IU) of RHPA4259 intravaginally. This dose resulted in 64% of mice becoming HIV infected following vaginal exposure. For comparison, data from two different cohorts of mice exposed through an intravenous route are included. As expected, 100% of the mice became HIV+ with similar doses of RHPA and an additional strain (YU2) using this route.
Figure 5B depicts representative results from three mice that were infected with HIV and switched to a diet containing standard cART. Mice were switched back to regular mouse chow after 40 days of cART. In this assay setup, the limit for viral load detection was 725 copies/mL. Viral loads were all below the detection limit after 4 weeks of cART. After cessation of cART, the virus rebounded, mirroring clinical data22. Mice on cART tolerated the change in diet well as indicated in Figure 5C.
Figure 1: Representative flow cytometry gating strategy for validation of stem cell purity and CCR5 donor variant status. (A–C) The gating strategy used for the isolated CD34+ cell population. Doublets and debris are excluded in panel A and B respectively (FSC-A vs. FSC-H and FSC-A vs. SSC-A). (C) The frequency of CD34+ stem cells and CD3+ T cell contamination. (D–F) The CD34- flow-through gating strategy. Percentages in gates are calculated as a fraction of the parent population. (G) The results of a CCR5Δ32/wt PCR analysis. Lane 1: DNA from a human CCR5Δ32/wt donor, lanes 2 and 3: two CCR5wt/wt human stem cell donors, lane 4: A CCR5Δ32/wt human stem cell donor. (H) Frequency of the genotype CCR5Δ32/wt in our group of 19 stem cell samples is 15.8%. Please click here to view a larger version of this figure.
Figure 2: Flow cytometry gating strategy for validation of human cell engraftment and differentiation. The total mononuclear cell population from humanized mice was analyzed via flow cytometry. (A) The percentage of human CD45+ cells was determined as a fraction of the total recorded events. (B) Doublets were subsequently excluded based on FSC-A/FSC-H gating. (C) The true lymphocyte population was defined based on size and granularity. (D) Lymphocytes were then characterized as either CD3+ (T cells) or CD19+ (B cells). (E) CD3+ T cells were either CD4+ T cells or CD8+ T cells. Percentages in gates were calculated as a fraction of the parent population. Please click here to view a larger version of this figure.
Figure 3: Representative humanization levels 4–5 months after stem cell transplantation with cell subtype fractions for 10 and 16 mice generated from two different human donors. (A) The mononuclear cell population (MNC) from 10 and (B) 16 humanized mice were analyzed via flow cytometry and gated as presented in Figure 2. The fraction of human CD45+ cells is presented as %hCD45 (of total MNC), and %B and %T cells as a fraction of hCD45. T cells were subsequently divided into %CD4 and %CD8. Each data point represents one mouse. Data is presented as mean ± SD. Please click here to view a larger version of this figure.
Figure 4: Experimental lab bench setup for intravaginal exposure of mice. Experimental setup for HIV exposure of humanized mice through the intravaginal route. The procedure is performed in a flow bench where all reagents and surfaces have been sterilized prior to use. Please click here to view a larger version of this figure.
Figure 5: Rate of HIV strain transmission through different exposure routes and efficacy and safety of cART-containing chow in viral suppression. (A) Humanized NOG mice were successfully infected with two different strains of HIV through either the intravaginal or the intravenous route. Mice were exposed with 21,400 IUs of RHPA4259 intravaginally, 5,157 IUs IV with RHPA4259, or 3,000 IUs IV with YU2. Details regarding IV exposure of humanized mice are not included in this protocol. HIV infections were successfully treated with a cART regimen delivered through mouse chow. (B) The viral load decreased to below detection for all three mice on cART and rebound emerged after the cessation of cART. The dotted line indicates limit of quantification at 725 copies/mL. Mice fed with cART chow had similar weight development as mice housed on non-cART chow during the same time period, indicating no taste-preference or side effects of the cART diet. (C) Weights are presented as fold change compared to the start of cART. Each data point represents the mean of three animals ± SD. Please click here to view a larger version of this figure.
Antibody target | Clone | Fluorophore |
CD3 | clone SK7 | BUV395 |
CD34 | clone AC136 | FITC |
CD45 | clone 2D1 | APC |
Table 1: Antibodies used for determination of stem cell purity. Suggested multicolor flow cytometry panel for evaluation of stem cell purity. Listed are the antibody target, the clone, and the fluorophore.
CCR5Δ32 detection | Primers |
Forward primer | 5'CTTCATTACACCTGCAGCT'3 |
Reverse primer | 5'TGAAGATAAGCCTCACAGCC'3 |
Table 2: CCR5Δ32 variant detection PCR primers. Forward and reverse primers used for detection of the 32 bp deletion in the CCR5 gene.
No. of Cycles | 1 | 45 | 1 | ∞ |
Temperature (°C) | 98 | 98/63/72 | 72 | 10 |
Time | 30 s | 10 s/30 s/15 s | 5 min | ∞ |
Table 3: CCR5Δ32 variant detection PCR program. PCR cycling program used for amplification of the CCR5 gene.
Antibody target | Clone | Fluorophore |
CD4 | SK3 | BUV 496 |
CD8 | RPA-T8 | BV421 |
CD3 | OKT3 | FITC |
CD19 | sj25c1 | PE-Cy7 |
CD45 | 2D1 | APC |
Table 4: Antibodies used for determination of mouse humanization. Suggested multicolor flow cytometry panel for humanization. Listed are the antibody target, the clone, and the fluorophore.
No. of Cycles | 1 | 1 | ∞ |
Temperature (°C) | 51 | 80 | 4 |
Time | 45 min | 15 min | ∞ |
Table 5: cDNA amplification program. Program used for amplification of complementary strand DNA to the viral RNA.
HIV quantification | Primers |
Forward primer | 5'AGGGCAGCATAGAGCAAAAA'3 |
Reverse primer | 5'CAAAGGAATGGGGGTTCTTT'3 |
FAM probe | 5'ATCCCCACTTCAACAGATGC'3 |
Table 6: HIV ddPCR primers. Primers and probes used for ddPCR amplification of viral cDNA.
No. of Cycles | 1 | 39 | 1 | ∞ |
Temperature (°C) | 95 | 95/54.5 | 98 | 4 |
Time | 10 min | 30 s/1 min | 10 min | ∞ |
Table 7: HIV ddPCR program. PCR cycling program used for amplification of viral RNA.
Raltegravir (RAL) | 4800 mg/kg |
Tenofovir disoproxil fumarate (TDF) | 720 mg/kg |
Emtricitabine (FTC) | 520 mg/kg |
Table 8: Mouse cART chow diet. Mouse chow diet was formulated as previously published21. The chow diet was made on a base of standard mouse chow, and after production, the food was γ-irradiated with 25 kGy and double-bagged. The chow was stored at -20 °C until use.
The severely immunocompromised mouse strain NOD.Cg-Prkdcscid Il2rgtm1Sug/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 Prkdcscid 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 the binding complexes of many key cytokines such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Immunodeficient mice, such as the NOG, transplanted with a human immune system are a powerful tool for the study of HIV transmission and immunology. Contributions in these fields made using humanized mice have been extensively reviewed2,23,24,25,26. The use of these mice to study human innate immune responses are also gaining increased attention27,28.
The aim of this manuscript is to supply a comprehensive protocol of mouse and ddPCR procedures to go from a naive mouse to HIV transmission and treatment data. Our system utilized ddPCR for quantification of viral RNA and DNA. In a ddPCR reaction, the reactants are partitioned into up to 20,000 droplets, each containing a single, separate micro PCR reaction. The amplification of a target inside a droplet leads to a positive fluorescent signal for that droplet. Thus, the readout is binary and by applying Poisson statistical analyses, the number of positive reactions can be directly translated to a number of template copies in the original sample. The benefit of ddPCR lies in its ability to directly quantify a target independent of a standard curve. This is particularly attractive when analyzing RNA samples that are challenging to utilize as PCR standard curves due to their labile nature29. Moreover, by analyzing multiple replicas of the same sample and merging the individual data points for the final sample quantification, the binary nature of ddPCR makes it possible to lower the detection limit of template copies per mL of sample29. This is especially important in a humanized mouse setting, where only limited sample material is available and high sensitivity is required.
Administration of cART to humanized mice can be done either by oral gavage or intraperitoneal injections with solutions of cART30,31,32, and as shown recently, by formulation into the diet21. One of our major aims was the implementation of a cART regimen in the mouse diet to reduce potential stress on the animals due to the extra handling steps inherent in other drug delivery methods. The dose of medicine that a mouse will eat can be accurately estimated based on the average daily food intake of the mice33. Oral delivery through the diet serves as the easiest delivery route with both minimal stress for the animal and minimal workload for the handler. We based our combination of antiviral drugs on previous published studies in humanized mice21,30. Furthermore, our cART strategy is clinically relevant given that the drug combination utilized clinically is orally administered by patients around the globe.
Certain limitations are noted regarding the use of NOG mice. Importantly, human T cells in these mice are cultivated in a mouse thymic environment, as opposed to a human environment. The recent focus is on generating xenorecipient strains that have a favorable environment for the development of robust human immune responses. These new strains include immunodeficient mice that are transgenic for human MHC molecules, such as A2. These models enable HLA-restricted antigen T-cell responses that result in better maturation and effector functions of the adaptive immune system34. Another approach is to replace mouse genes with key human cytokines for IL-3/GM-CSF35, IL-636, IL-1537, TPO, M-CSF38, and IL-7/TSLP31. Such models have gained increased attention for their ability to generate better differentiation of innate cell types. Our protocol is easily adaptable for the humanization and HIV infection of mice using any such enhanced genetic background immunodeficient strain.
In summary, the ease and utility of the described approach facilitates research in HIV-related fields in vivo. Humanized mice can be a very powerful tool in guiding research towards generating better research hypotheses. Along with the generation of more "human" humanized mice with human transgenes, we believe our standardized protocol will contribute to the streamlining of experimental procedures across different research environments.
The authors have nothing to disclose.
The authors would like to thank the Biomedicine Animal Facility staff at Aarhus University, particularly Ms. Jani Kær for colony maintenance efforts and for tracking mouse weights. The authors would like to thank Professor Florian Klein for developing standard-of-care cART and for guidance. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pRHPA.c/2635 (cat# 11744) from Dr. John Kappes and Dr. Christina Ochsenbauer.
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 |