NSG newborn mouse engraftment with human cord blood CD34+ cells
Newborn animals were transplanted with 1 × 105 human CD34+ hematopoietic stem cells obtained from umbilical cord blood as previously described [14, 18, 23, 32] (Lonza, donor 28753, Cat No. #2C-101). Newborns 3–4 days old were irradiated with 100 cGy and injected intrahepatically with CD34+ cells within 3 h after irradiation. CD34+ cells were thawed and assessed for viability before injections. Animals were housed under pathogen-free conditions at the IHV Animal Facility, School of Medicine, University of Maryland (SOM UM) Baltimore, Maryland. All experimental protocols were in accordance with the NIH guide for the care and use of laboratory animals and approved by the SOM UM IACUC. Twenty weeks after CD34+ HSC transplantation, mice were selected based on the expansion of human CD45+ cells with T>B cells (CD3+ cell numbers higher than CD19+ cell numbers), as judged by flow cytometry. Animals with less than a minimum of 20% human CD3+ T cells were not selected for this study [23, 32, 33]. The 13 mice selected were divided into three experimental groups: 8 to be infected intraperitoneally with 10,000 TCID50% of HIV-1 BaL (200 µL total volume per mouse) at around 20 weeks of age, of which 5 mice were to be treated with oral cART for 17 weeks, and 5 mice to remain uninfected as a control group. HIV-1 BaL, an R5 replication competent virus that contains most of the BaL env gene in an HIV-1 IIIB backbone, titer 1 × 105/mL , was used to infect 8 mice. Viral stocks were provided by the µQuant Core Lab, IHV SOM UM. The NIH regulation  mandating inclusion of both mouse genders in experimental groups was respected; each experimental group had sex ratios of 1:1. To monitor graft versus host disease (GVHD) that could interfere with the experimental outcome, the animals were closely observed for hair and weight loss, as they are the main indications of GVHD. No hair changes or weight reduction higher than 10% were observed. Mice were euthanized at the end of week 20 according to the IACUC protocol regulations.
Treatment with antiretroviral drugs (cART)
Doses for standard cART were chosen based on the published therapeutic efficacy in chronic HIV-1 infection in humans [36, 37] adjusted for mouse body weights. Food pellets (Mod TestDiet) containing cART [a mixture of Tenofovir Disoproxil (TDF), Emtricitabine (FTC) and Dolutegravir (DTG)] were administered to mice daily for 17 weeks. Cylindrical pills were manufactured as single doses of 60 mg TDF, 60 mg FTC, and 48 mg DTG per kg of food. Pellets also contained antibiotic (amoxicillin, 0.12%) and were irradiated before use. Food pellets containing cART mix were orally administered daily to each mouse. Mice were monitored for drug intake and given regular food pellets after finishing the medicated pellets. Mice were weighed twice per week and monitored for fur loss or change of behavior twice daily. Fresh water was changed every 2 days and always available ad libitum.
Staining procedures for confocal microscopic visualization of human lymphocytes and HIV-1 structural protein, p24
Primary PBMCs isolated from donor whole blood were cultured and activated from frozen stocks for 3 days prior to infection, then split into infected and non-infected groups, with the infected group being cultured with HIV BaL (MOI = 0.00075) for 3 h at 37 °C 5% CO2. Afterwards, media was replaced and both groups cultured separately for 1 week. Infected and non-infected primary cells were stained using rabbit α-CD4 (Invitrogen, Cat. MA5-16338) primary antibody at a 1:25 dilution followed by goat α-rabbit AF647 (Abcam, Cat. ab150079) secondary antibody at a 1:250 dilution for 45 min each at 4 °C. Cells were fixed and permeabilized with Foxp3 Fixation and Permeabilization Buffer (Invitrogen, Cat. 00-5523-00) for 45 min at 4 °C, then stained using mouse α-p24 FITC (Beckman Coulter, Cat. 6604665) at 1:25 for 45 min at 4 °C followed by 50 µg/mL DAPI for 1 min. Cells were imaged in 1% PFA diluted in PBS. Controls were stained without rabbit α-CD4 primary and/or with mouse IgG1 Alexa Fluor 488 (Biolegend, Cat. 400134) in place of α-p24 FITC. HIV-1 negative and control cells showed no positive staining (not shown).
For tissue imaging, slides were warmed (45 °C) for at least 10 min to remove paraffin, then submerged in xylene. Containers with slides were placed into dehydration buckets containing 100% ethanol and incubated for a few minutes. The procedure was repeated several times with lower concentrations of ethanol, and the slides were then incubated at 95 °C in Dako Target Retrieval Solution (Dako, Cat No. S2368) for 20 min and cooled per the manufacturer’s instruction. Blocking was overnight at 4 °C with 20% BSA in 1× PBS. Surface marker staining used 1:25 α-CD4 Alexa Fluor 488 (Stemcell Technologies, Cat. 60016AD), 1:25 primary antibody α-CD45 (Leica BioSystems, Cat. NCL-L-LCA), or 1:25 primary antibody α-CD68 (Dako, Cat. M0814) for 1 h at room temperature (RT). Slides stained against CD45 or CD68 were then incubated with 1:250 secondary antibody α-mouse IgG (H + L) DyLight 488 (Vector, Cat. DI-2488) for 1 h at RT. Slides were permeabilized with 0.1% Triton X-100 in 1× PBS at RT for 10 min, blocked overnight with 20% BSA in 1× PBS at 4 °C, and labeled with 1:25 primary antibody α-p24 (Sino, Cat No. 40243-RP01) for 1 h at RT, followed by secondary antibody labeling with 1:250 α-rabbit Alexa Fluor 647 (abcam, Cat No. ab150079) for 1 h at RT. Slides were DAPI stained using 50 µg/mL DAPI for 2 min and treated with 1× True Black Blocker (Biotium, Cat No. 23007) for 30 s. Slides were cover slipped with Vectashield Mounting Media (Vector, Cat No. H-1000) prior to imaging. Control samples were stained with the same conditions described above, and for additional controls, HIV-1 negative cells/tissues were stained with DAPI and secondary antibody only (not shown).
Confocal images of cell-associated fluorescence were acquired using the Zeiss LSM 800 confocal system (Carl Zeiss Microscopy, Germany) via the Airyscan super resolution mode. Three laser lines, 405 nm (blue, for nuclei), 488 nm (green, for leukocyte surface antigens [CD45 or CD4 in liver, LN, and spleen; CD68 in brain] and 647 nm (red, for HIV-1 protein, p24) were used. Blue, green, and red signals were separated by a quad DAPI/FITC/TRITC/Cy5 dichroic beam splitter and further acquired using a Gasp detector. A Plan-Apochromat 63x/1.4 Oil DIC objective was used to visualize multi-color labelled cell/tissue samples. ZEN Blue 2.3 software (Carl Zeiss Microscopy, Germany) was used to generate original images. All images were acquired under the same instrument settings. Signal to noise ratio was accounted for by averaging data all images acquired using the same gain offset, detector, and laser excitation power. Saturated signal was avoided by using the software-controlled range for a minimum pixel saturation.
Peripheral blood viral load
Mice were periodically bled retro-orbitally. RNA was extracted from plasma using a QIAamp Viral RNA Mini Kit (QIAGEN, Cat. 52904) as per the manufacturer's instructions. HIV-1 RNA was converted to cDNA using a SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen 18080-400) as per the manufacturer's instructions. HIV-1 cDNA was amplified and quantified using qPCR with the following protocol: a single cycle at 50 °C for 2 min, a single cycle at 95 °C for 15 min, 40 cycles at 94 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s, followed by a single cycle at 72 °C for 30 s using a BioRad LightCycler and BioRad iQ5 software. HIV-1 RNA data were graphed using GraphPad Prism 9 for each mouse (n = 3 per treatment; randomly chosen), along with an average for each treatment.
DNA standard curve
To generate a standard to quantify HIV-1 DNA copies via qPCR, HIV-1 gag amplicons were cloned using a TOPO™ TA Cloning™ Kit (Invitrogen, Cat. 450640), transfected into One Shot® TOP10 E. coli (Invitrogen, Cat. C404004), grown on an LB agar plate containing 50 µg/mL kanamycin, and DNA extracted from overnight cultures using a QIAprep® Spin Miniprep Kit (QIAGEN, Cat. 27104). Plasmid DNA was sequenced to determine HIV-1 gag integrity and serially diluted for standards .
Standards were qPCR amplified alongside mouse tissue DNA samples using primers specific to either HIV-1 gag (at 30,000, 3000, 300, 30, and 3 standard copies) or human β-globin (at 100,000, 10,000, 1000, 100, and 10 standard copies). Cycle thresholds were plotted against initial template quantity on a log scale to create standard curves . DNA sample thresholds were plotted against the standard curves to quantify DNA copy number.
HIV-1 gag DNA quantification in tissue.
Viral DNA loads in mouse tissue were quantified by a qPCR assay measuring total viral DNA (integrated and unintegrated, linear, and circular forms). DNA was extracted from mouse tissue (brain, liver, spleen, and lymph node) using a QIAGEN DNeasy® Blood and Tissue Kit (QIAGEN, Cat. 69506) and analyzed using the QuantStudio 3 PCR system (Applied Biosystems) with 10 µL of genomic DNA in each sample. Samples were analyzed along with the set of standards for both HIV-1 gag DNA and human β-globin DNA in 96-well plates in triplicate. DNA copy numbers were quantified with the QuantStudio 3 PCR system using the standard TaqMan protocol. This consisted of a single cycle at 50 °C for 5 min, a single cycle at 95 °C for 10 min, and 45 cycles at 95 °C for 15 s and 60 °C for one minute. All qPCR experiments were repeated three times for reproducibility. Unknown HIV-1 gag or human β-globin DNA copy numbers were determined by extrapolation from the known standard DNA copy numbers for HIV-1 gag DNA or human β-globin. We expressed HIV-1 gag copies as copies per 106 cells. All statistics were calculated using GraphPad Prism 9.