The authors would like to thank the Biomedicine Animal Facility staff at Aarhus University, particularly Ms. Jani K&#230;r 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.

<ol>
	<li>Skelton, J. K., Ortega-Prieto, A. M., Dorner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Hitchhiker's+guide+to+humanized+mice:+new+pathways+to+studying+viral+infections.">A Hitchhiker's guide to humanized mice: new pathways to studying viral infections.</a> Immunology. 154 (1), 50-61 (2018).</li><li>Denton, P. W., Krisko, J. F., Powell, D. A., Mathias, M., Kwak, Y. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+Administration+of+Antiretrovirals+Prior+to+Exposure+Prevents+Rectal+and+Intravenous+HIV-1+Transmission+in+Humanized+BLT+Mice.">Systemic Administration of Antiretrovirals Prior to Exposure Prevents Rectal and Intravenous HIV-1 Transmission in Humanized BLT Mice.</a> PLoS ONE. 5 (1), 8829 (2010).</li><li>Zou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nef+functions+in+BLT+mice+to+enhance+HIV-1+replication+and+deplete+CD4+++CD8+++thymocytes.">Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4 + CD8 + thymocytes.</a> Retrovirology. 9 (1), 44 (2012).</li><li>Berges, B. K., Akkina, S. R., Folkvord, J. M., Connick, E., Akkina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+transmission+of+R5+and+X4+tropic+HIV-1+via+vaginal+and+rectal+routes+in+humanized+Rag2+-/-+&#947;c+-/-+(RAG-hu)+mice.">Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2 -/- &#947;c -/- (RAG-hu) mice.</a> Virology. 373 (2), 342-351 (2008).</li><li>Veselinovic, M., Charlins, P., Akkina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+HIV-1+Mucosal+Transmission+and+Prevention+in+Humanized+Mice.">Modeling HIV-1 Mucosal Transmission and Prevention in Humanized Mice.</a> Methods Mol Biol. , 203-220 (2016).</li><li>Neff, C. P., Kurisu, T., Ndolo, T., Fox, K., Akkina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+topical+microbicide+gel+formulation+of+CCR5+antagonist+maraviroc+prevents+HIV-1+vaginal+transmission+in+humanized+RAG-hu+mice.">A topical microbicide gel formulation of CCR5 antagonist maraviroc prevents HIV-1 vaginal transmission in humanized RAG-hu mice.</a> PLoS ONE. 6 (6), 20209 (2011).</li><li>Neff, P. C., Ndolo, T., Tandon, A., Habu, Y., Akkina, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+pre-exposure+prophylaxis+by+anti-retrovirals+raltegravir+and+maraviroc+protects+against+HIV-1+vaginal+transmission+in+a+humanized+mouse+model.">Oral pre-exposure prophylaxis by anti-retrovirals raltegravir and maraviroc protects against HIV-1 vaginal transmission in a humanized mouse model.</a> PLoS ONE. 5 (12), 15257 (2010).</li><li>Veselinovic, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+pre-exposure+prophylaxis:+Mucosal+tissue+drug+distribution+of+RT+inhibitor+Tenofovir+and+entry+inhibitor+Maraviroc+in+a+humanized+mouse+model.">HIV pre-exposure prophylaxis: Mucosal tissue drug distribution of RT inhibitor Tenofovir and entry inhibitor Maraviroc in a humanized mouse model.</a> Virology. 464-465, 253-263 (2014).</li><li>Akkina, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+Rag1-/-&#947;c-/-+mice+support+multilineage+hematopoiesis+and+are+susceptible+to+HIV-1+infection+via+systemic+and+vaginal+routes.">Humanized Rag1-/-&#947;c-/- mice support multilineage hematopoiesis and are susceptible to HIV-1 infection via systemic and vaginal routes.</a> PLoS ONE. 6 (6), 20169 (2011).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+administration+of+combinatorial+dsiRNAs+via+nanoparticles+efficiently+suppresses+HIV-1+infection+in+humanized+mice.">Systemic administration of combinatorial dsiRNAs via nanoparticles efficiently suppresses HIV-1 infection in humanized mice.</a> Molecular Therapy. 19 (12), 2228-2238 (2011).</li><li>Balcombe, J. P., Barnard, N. D., Sandusky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+routines+cause+animal+stress.">Laboratory routines cause animal stress.</a> Contemporary Topics in Laboratory Animal Science. 43 (6), 42-51 (2004).</li><li>Trecarichi, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+protective+effect+of+CCR5-Delta+32+heterozygosity+in+a+cohort+of+heterosexual+Italian+HIV-1+exposed+uninfected+individuals.">Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals.</a> AIDS Research and Therapy. 3 (1), (2006).</li><li>Novembre, J., Galvani, A. P., Slatkin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geographic+spread+of+the+CCR5+&#916;32+HIV-resistance+allele.">The geographic spread of the CCR5 &#916;32 HIV-resistance allele.</a> PLoS Biology. 3 (11), 1954-1962 (2005).</li><li>Solloch, U. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequencies+of+gene+variant+CCR5-&#916;32+in+87+countries+based+on+next-generation+sequencing+of+1.3+million+individuals+sampled+from+3+national+DKMS+donor+centers.">Frequencies of gene variant CCR5-&#916;32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national DKMS donor centers.</a> Human Immunology. 78 (11-12), 710-717 (2017).</li><li>Ochsenbauer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Transmitted/Founder+HIV-1+Infectious+Molecular+Clones+and+Characterization+of+Their+Replication+Capacity+in+CD4+T+Lymphocytes+and+Monocyte-Derived+Macrophages.">Generation of Transmitted/Founder HIV-1 Infectious Molecular Clones and Characterization of Their Replication Capacity in CD4 T Lymphocytes and Monocyte-Derived Macrophages.</a> Journal of Virology. 86 (5), 2715-2728 (2012).</li><li>Andersen, A. H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Acting,+Potent+Delivery+of+Combination+Antiretroviral+Therapy.">Long-Acting, Potent Delivery of Combination Antiretroviral Therapy.</a> ACS Macro Letters. 7 (5), 587-591 (2018).</li><li>Caro, A. C., Hankenson, F. C., Marx, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+thermoregulatory+devices+used+during+anesthesia+of+C57BL/6+mice+and+correlations+between+body+temperature+and+physiologic+parameters.">Comparison of thermoregulatory devices used during anesthesia of C57BL/6 mice and correlations between body temperature and physiologic parameters.</a> Journal of the American Association for Laboratory Animal Science JAALAS. 52 (5), 577-583 (2013).</li><li>Gatlin, J., Padgett, A., Melkus, M. W., Kelly, P. F., Garcia, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+engraftment+of+nonobese+diabetic/severe+combined+immunodeficient+mice+with+human+CD34++cells+transduced+by+a+self-inactivating+human+immunodeficiency+virus+type+1+vector.">Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector.</a> Human Gene Therapy. 12 (9), 1079-1089 (2001).</li><li>Leth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+transcriptional+activity+during+frequent+longitudinal+sampling+in+aviremic+patients+on+antiretroviral+therapy.">HIV-1 transcriptional activity during frequent longitudinal sampling in aviremic patients on antiretroviral therapy.</a> AIDS. 30 (5), 713-721 (2016).</li><li>Halper-Stromberg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broadly+neutralizing+antibodies+and+viral+inducers+decrease+rebound+from+HIV-1+latent+reservoirs+in+humanized+mice.">Broadly neutralizing antibodies and viral inducers decrease rebound from HIV-1 latent reservoirs in humanized mice.</a> Cell. 158 (5), 989-999 (2014).</li><li>Rothenberger, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+number+of+rebounding/founder+HIV+variants+emerge+from+multifocal+infection+in+lymphatic+tissues+after+treatment+interruption.">Large number of rebounding/founder HIV variants emerge from multifocal infection in lymphatic tissues after treatment interruption.</a> Proceedings of the National Academy of Sciences. 112 (10), 1126-1134 (2015).</li><li>Rongvaux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Hemato-Lymphoid+System+Mice:+Current+Use+and+Future+Potential+for+Medicine.">Human Hemato-Lymphoid System Mice: Current Use and Future Potential for Medicine.</a> Annual Review of Immunology. 31 (1), 635-674 (2013).</li><li>Walsh, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+Mouse+Models+of+Clinical+Disease.">Humanized Mouse Models of Clinical Disease.</a> Annual Review of Pathology: Mechanisms of Disease. 12 (1), 187-215 (2017).</li><li>Denton, P. W., Garc&#237;a, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mouse+models+of+HIV+infection.">Humanized mouse models of HIV infection.</a> AIDS Reviews. 13 (3), 135-148 (2011).</li><li>Denton, P. W., S&#248;gaard, O. S., Tolstrup, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+animal+models+to+overcome+temporal,+spatial+and+combinatorial+challenges+in+HIV+persistence+research.">Using animal models to overcome temporal, spatial and combinatorial challenges in HIV persistence research.</a> Journal of Translational Medicine. 14 (1), (2016).</li><li>Andersen, A. H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAIMP+administration+in+humanized+mice+induces+a+chimerization-level-dependent+STING+response.">cAIMP administration in humanized mice induces a chimerization-level-dependent STING response.</a> Immunology. 157 (2), 163-172 (2019).</li><li>Tanaka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Mature+and+Functional+Human+Myeloid+Subsets+in+Hematopoietic+Stem+Cell-Engrafted+NOD/SCID/IL2r+KO+Mice.">Development of Mature and Functional Human Myeloid Subsets in Hematopoietic Stem Cell-Engrafted NOD/SCID/IL2r KO Mice.</a> The Journal of Immunology. 188 (12), 6145-6155 (2012).</li><li>Quan, P. L., Sauzade, M., Brouzes, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DPCR:+A+technology+review.">DPCR: A technology review.</a> Sensors (Switzerland). 18 (4), (2018).</li><li>Denton, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+HIV+Latency+in+Humanized+BLT+Mice.">Generation of HIV Latency in Humanized BLT Mice.</a> Journal of Virology. 86 (1), 630-634 (2012).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+immune+system+mouse+model+with+robust+lymph+node+development.">A human immune system mouse model with robust lymph node development.</a> Nature Methods. 15 (8), 623-630 (2018).</li><li>Satheesan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+Replication+and+Latency+in+a+Humanized+NSG+Mouse+Model+during+Suppressive+Oral+Combinational+Antiretroviral+Therapy.">HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy.</a> Journal of Virology. 92 (7), 02118 (2018).</li><li>Bachmanov, A. A., Reed, D. R., Beauchamp, G. K., Tordoff, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+intake,+water+intake,+and+drinking+spout+side+preference+of+28+mouse+strains.">Food intake, water intake, and drinking spout side preference of 28 mouse strains.</a> Behavior Genetics. 32 (6), 435-443 (2002).</li><li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+human+T-cell+subsets+with+HLA-restricted+immune+responses+in+HLA+class+I+expressing+NOD/SCID/IL2r+null+humanized+mice.">Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r null humanized mice.</a> Proceedings of the National Academy of Sciences. 107 (29), 13022-13027 (2010).</li><li>Willinger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+IL-3/GM-CSF+knock-in+mice+support+human+alveolar+macrophage+development+and+human+immune+responses+in+the+lung.">Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung.</a> Proceedings of the National Academy of Sciences. 108 (6), 2390-2395 (2011).</li><li>Hanazawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+immunosuppressive+myeloid+cell+populations+in+human+interleukin-6+transgenic+NOG+mice.">Generation of human immunosuppressive myeloid cell populations in human interleukin-6 transgenic NOG mice.</a> Frontiers in Immunology. 9, (2018).</li><li>Huntington, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-15+trans-presentation+promotes+human+NK+cell+development+and+differentiation+in+vivo.">IL-15 trans-presentation promotes human NK cell development and differentiation in vivo.</a> The Journal of Experimental Medicine. 206 (1), 25-34 (2009).</li><li>Rongvaux, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+function+of+human+innate+immune+cells+in+a+humanized+mouse+model.">Development and function of human innate immune cells in a humanized mouse model.</a> Nature Biotechnology. 32 (4), 364-372 (2014).</li></ol>

The authors declare no conflicts of interests.

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 &#947;-chain (common gamma chain, IL2rg) which is indispensable in ...

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 CCR5&#916;32/wt or CCR5&#916;32/ &#916;32 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 &#916;32 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blue pad</td>
			<td>VWR</td>
			<td>56616-031</td>
			<td>Should be sterilized prior to use</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A8022</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19 (clone sj25c1) PE-Cy7</td>
			<td>BD Bioscience</td>
			<td>557835</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 (clone OKT3) FITC</td>
			<td>Biolegend</td>
			<td>317306</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 (clone SK7) BUV395</td>
			<td>BD Bioscience</td>
			<td>564001</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 (clone AC136) FITC</td>
			<td>Miltenyi</td>
			<td>130-113-740</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 (clone SK3) BUV 496</td>
			<td>BD Bioscience</td>
			<td>564652/51</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 (clone 2D1) APC</td>
			<td>Biolegend</td>
			<td>368511/12</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8 (clone RPA-T8) BV421</td>
			<td>BD Bioscience</td>
			<td>562428</td>
			<td></td>
		</tr>
		<tr>
			<td>ddPCR Supermix for probes (no dUTP)</td>
			<td>Bio-Rad</td>
			<td>1863025</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Merck</td>
			<td>10,02,95,21,000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma</td>
			<td>D4263</td>
			<td>For suspension buffer</td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Life Technologies</td>
			<td>R0192</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbeccos phosphate-buffered saline (PBS)</td>
			<td>Biowest</td>
			<td>L0615-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human Cord Blood CD34 Positive Selection Kit II</td>
			<td>Stemcell</td>
			<td>17896</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Lysing solution 10X</td>
			<td>BD</td>
			<td>349202</td>
			<td>Dilute 1:10 in dH20 immediately before use</td>
		</tr>
		<tr>
			<td>FACS tubes (Falcon 5 mL round-botton)</td>
			<td>Falcon</td>
			<td>352052</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc Receptor blocking solution (Human Trustain FcX)</td>
			<td>Biolegend</td>
			<td>422302</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma</td>
			<td>F8192-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17144002</td>
			<td></td>
		</tr>
		<tr>
			<td>Flowjo v.10</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Mesoft</td>
			<td>157300</td>
			<td>Should be sterilized prior to use</td>
		</tr>
		<tr>
			<td>Heating lamp</td>
			<td></td>
			<td></td>
			<td>Custom made</td>
		</tr>
		<tr>
			<td>Hemacytometer (B&#252;rker-T&#252;rk)</td>
			<td>VWR</td>
			<td>DOWC1597418</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane gas</td>
			<td>Orion Pharma</td>
			<td>9658</td>
			<td></td>
		</tr>
		<tr>
			<td>LSR Fortessa X20 flow cytometer</td>
			<td>BD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes, PCR-PT approved</td>
			<td>Sarstedt</td>
			<td>72692405</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse cART food</td>
			<td>ssniff Spezialdi&#228;ten GmbH</td>
			<td>Custom made product</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td></td>
			<td>Custom made product</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, Microlance 3, 30G &#189;&#34;</td>
			<td>BD</td>
			<td>304000</td>
			<td></td>
		</tr>
		<tr>
			<td>NOG mice NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac</td>
			<td>Taconic</td>
			<td>NOG-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>VWR chemicals</td>
			<td>436912C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleospin 96 Virus DNA and RNA isolation kit</td>
			<td>Macherey-Nagel</td>
			<td>740691</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR-approved microcentrifuge tubes</td>
			<td>Sarstedt</td>
			<td>72.692.405</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution 100X</td>
			<td>Biowest</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion Hot Start II DNA polymerase</td>
			<td>Life Technologies</td>
			<td>F549S</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, sterile, ART 20P Barrier</td>
			<td>ThermoScientific</td>
			<td>2149P</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>NEB</td>
			<td>100005398</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaSoft software</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QX100 Droplet Generator</td>
			<td>Bio-Rad</td>
			<td>1886-3008</td>
			<td></td>
		</tr>
		<tr>
			<td>QX100 Droplet Reader</td>
			<td>Bio-Rad</td>
			<td>186-3003</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC lysis solution</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free DNAse size F + reaction buffer</td>
			<td>Macherey-Nagel</td>
			<td>740963</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAseOUT Recombinant Ribonuclease inhibitor</td>
			<td>ThermoScientific</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Biowest</td>
			<td>L0501-500</td>
			<td>Dissolve in H20</td>
		</tr>
		<tr>
			<td>Softject 1 mL syringe</td>
			<td>Henke Sass Wolf</td>
			<td>5010-200V0</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>VWR</td>
			<td>89370-910</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypane blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 0.5 EDTA, pH 8.0</td>
			<td>ThermoFisher Scientific</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon S (virus disinfectant)</td>
			<td>Dupont</td>
			<td>7511</td>
			<td></td>
		</tr>
	</tbody>
</table>

humanized nog mice for intravaginal hiv exposure and treatment of hiv infection

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%&#8211;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.

The gating strategy for the analysis of stem cell purity is depicted in Figure 1. Figure 1A&#8211;C shows the purified CD34+ population and Figure 1D&#8211;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%&#8211;95% with less than 1% T-cell contamination. <strong class=...

Watch this Scientific Journal Video about Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection at JoVE.com

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

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 ...

humanized-nog-mice-for-intravaginal-hiv-exposure-treatment-hiv

Research

JoVE Journal

Immunology and Infection

8.4K Views.  Aarhus University. 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.

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

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...