Humaniserede NOG-mus til intravaginal hiv-eksponering og behandling af hiv-infektion
Summary
Vi har udviklet en protokol til generering og evaluering af en humaniseret og human immundefekt virus-inficeret NOG musemodel baseret på stamcelletransplantation, intravaginal human immundefekt virus eksponering, og dråbe digital PCR RNA Kvantificering.
Abstract
Humaniserede mus udgør en sofistikeret platform til at studere human immundefekt virus (HIV) virologi og til at teste antivirale lægemidler. Denne protokol beskriver etableringen af et menneskeligt immunsystem i voksne NOG-mus. Her forklarer vi alle de praktiske skridt fra isolering af navlestrengsblod afledt menneskelige CD34 + celler og deres efterfølgende intravenøse transplantation i mus, til manipulation af modellen gennem hiv-infektion, kombination antiretroviral behandling ( cART) og blodprøvetagning. Ca. 75.000 hCD34+ celler injiceres intravenøst i musene og niveauet af human kimærisme, også kendt som humanisering, i det perifere blod anslås langsgående i flere måneder ved flow cytometri. I alt 75.000 hCD34 + celler giver 20% -50% menneskelige CD45 + celler i perifere blod. Musene er modtagelige for intravaginal infektion med HIV og blod kan udtages en gang om ugen til analyse, og to gange om måneden i længere perioder. Denne protokol beskriver en analyse til kvantificering af plasmaviral belastning ved hjælp af dråbe digital PCR (ddPCR). Vi viser, hvordan musene effektivt kan behandles med en standard-of-care cART regime i kosten. Levering af cART i form af regelmæssig mus chow er en betydelig raffinement af den eksperimentelle model. Denne model kan bruges til præklinisk analyse af både systemiske og topiske præeksponeringprofylakseforbindelser samt til test af nye behandlinger og hiv-helbrede strategier.
Introduction
Human immundefekt virus (HIV) er en kronisk infektion med mere end 37 millioner smittede personer på verdensplan1. Kombination antiviral behandling (cART) er en livreddende behandling, men en kur er stadig berettiget. Der er således behov for dyremodeller, der afspejler det menneskelige immunsystem og dets reaktioner for at lette fortsat forskning i hiv. Flere typer humaniserede mus, der er i stand til at støtte celle- og vævsengraftment, er udviklet ved at transplantere humane celler til alvorligt immundefekte mus2. Sådanne humaniserede mus er modtagelige for hiv-infektion og udgør et vigtigt alternativ til ikke-menneskelige primat simian immundefekt virus modeller, da de er billigere og enklere at bruge end ikke-menneskelige primater. Humaniserede mus har faciliteret forskning i hiv-virusoverførsel, patogenese, forebyggelse og behandling3,4,5,6,7,8,9,10,11.
Vi præsenterer et fleksibelt humaniseret modelsystem til hiv-forskning udviklet ved at transplantere navlestrengsblod afledt menneskelige stamceller i mus af NOD. Cg-PrkdcscidIl2rgtm1Sug/ JicTac (NOG) baggrund. Ud over at være af ikke-føtal oprindelse, den praktiske bioengineering af disse mus er mindre teknisk krævende i forhold til de mikrokirurgiske procedurer, der er involveret i transplantation af blod-lever-thymus (BLT) konstruktion.
Vi viser, hvordan man etablerer hiv-infektion gennem intravaginal transmission, og hvordan man kan overvåge plasma viral belastning med en følsom dråbe digital PCR (ddPCR)-baserede setup. Efterfølgende beskriver vi etableringen af standard cART givet som en del af den daglige musekost. Formålet med disse kombinerede metoder er at reducere stress en dyr og lette storstilede forsøg, hvor den tid, der bruges på at håndtere hvert dyr, er begrænset12.
Hos mennesker forårsager en CCR5Δ32/wt eller CCR5Δ32/ Δ32 genotype nedsat modtagelighed for hiv-infektion med sender/stiftervirus13,og der skal træffes visse forholdsregler ved bioengineering humaniserede mus med stamceller med henblik på hiv-studier. Dette gælder især i vores region, fordi naturligt forekommende varianter i CCR5-genet, især Δ32-sletninger, er mere udbredte i skandinaviske og baltiske indfødte befolkninger sammenlignet med resten af verden14,15. Således omfatter vores protokol en nem, høj gennemløbsanalyse til screening af donorhæmatopoietiske stamceller til CCR5 varianter før transplantation.
For intravaginal eksponering valgte vi senderen / grundlægger R5 virus RHPA4259, isoleret fra en kvinde i en tidlig fase af infektion, der var smittet intravaginalt16. Vi udsatte musene for en viral dosis, der var tilstrækkelig til at give vellykket overførsel i de fleste mus, men under en 100% transmissionshastighed. Valg af en sådan dosis muliggør et tilstrækkeligt dynamisk interval i transmissionshastigheden, således at antivirale virkninger af en lægemiddelkandidat kan resultere i beskyttede dyr i hiv-forebyggelsesforsøg og nedsat virusbelastning til behandlingsundersøgelser.
Protocol
Representative Results
Discussion
Den alvorligt immunkompromitterede musestamme NOD. Cg-Prkdcskafil2rgtm1Sug/JicTac (NOG) er særdeles velegnet til transplantation af humane celler og væv. Både medfødte og adaptive immunveje i disse mus er kompromitteret. NOG og NSG mus har en Prkdcskaftmutation, der resulterer i defekt T- og B-cellefunktion. Desuden mangler disse mus en funktionel interleukin-2 receptor γ-kæde (fælles gammakæde, IL2rg), som er uundværlig i de bindende komplekse…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Biomedicine Animal Facility personale på Aarhus Universitet, især Jani Kær for koloni vedligeholdelse indsats og for sporing mus vægte. Forfatterne vil gerne takke professor Florian Klein for at udvikle standard-of-care cART og for vejledning. Følgende reagens blev opnået gennem NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: pRHPA.c/2635 (kat # 11744) fra Dr. John Kappes og Dr. Christina Ochsenbauer.
Materials
| 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 |
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