Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice
Özet
This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.
Abstract
The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.
Introduction
The life expectancy of chronic human immunodeficiency virus (HIV)-infected individuals has significantly improved with combinational antiretroviral treatment (cART)1,2. cART successfully reduces the HIV-1 replication and increases CD4+ T cell counts to normalcy in the majority of HIV-1 chronically infected participants3, resulting in improved overall health and dramatically reduced disease progression4. However, the latent HIV-1 reservoir is established even when ART is initiated during acute infection5,6,7. Reservoirs persist over years during ART and rapid viral rebound after ART interruption is well-documented8,9. People living with HIV on ART are also predisposed to a higher risk of comorbidities such as cardiovascular disease, cancer, and neuro disorders10,11,12. Therefore, a functional cure for HIV is needed. Animal models for HIV-1 infection offer obvious advantages in developing and validating novel HIV cure strategies13,14,15. Humanized mice, as a small animal model, can provide multilineage human immune cell reconstitution in different tissues, which allows for the close study of HIV infection16,17,18,19. Among humanized models, the humanized bone marrow-liver-thymus (BLT) model successfully recapitulates chronic HIV-1 infection as well as functional human immune responses to HIV-1 infection20,21,22,23,24. Therefore, the humanized BLT mouse model has been widely used to investigate various aspects in the HIV research field. Humanized BLT mice are not only well-established models for the recapitulation of persistent HIV-1 infection and pathogenesis, but also consequential tools for the evaluation of cell therapy-based intervention strategies. The current authors and others have demonstrated that the humanized BLT mice model recapitulates persistent HIV-1 infection and pathogenesis25,26,27 and provides tools to evaluate cell therapy-based intervention strategies 28,29,30,31,32,33.
cART regimens consisting of combinations of antiretroviral drugs that are taken daily suppress HIV-1 replication to the point that the viral load in successfully treated individuals remains undetectable over long term34. The outcomes of treating HIV-infected humanized mice with clinically relevant cART regimens resemble those observed in HIV-1 infected ART-treated individuals22: HIV-1 levels are suppressed below the limits of detection and interruption of cART results in a rebound of HIV replication from the latent reservoir35. Subcutaneous (SC)27,36,37 or intraperitoneal (IP)37,38,39 injection is the route commonly used for cART treatment in humanized mice. However, intensive daily injection induces stress to animals by physical restraint40. It is also labor-intensive and potentially unsafe for researchers due to increased exposure to HIV while using sharps. Oral administration is ideal to mimic the absorption, distribution, and excretion of cART drugs that are taken by HIV-1-infected individuals. Oral administration typically involves customized and often laborious procedures to put the antiretroviral drugs in sterilized (necessary due to the immunodeficiency of the mice) food24,37,41 or water42,43,44,45,46, which may or may not be chemically compatible with many antiretroviral drugs, or result in something the mice would not readily eat or drink (which would affect dose and drug levels in the body). The novel peroral cART administration method proposed here surpasses previous delivery attempts due to its compatibility with different types of antiretroviral drugs, safety and ease of preparation and administration, and reduction of animal stress and anxiety resulting from the daily injection.
Tenofovir disoproxil fumarate (TDF), Elvitegravir (ELV), and Raltegravir (RAL) are poorly water-soluble drugs. Interestingly, increased bioavailability of TDF is observed with fatty foods, suggesting that competitive inhibition of lipases by fatty food may provide certain protection for TDF47. Therefore, DietGel Boost cups were selected to replace regular rodent chow as the method of delivery based on their modest fat content (20.3 g per 100 g) as compared to regular rodent chow (10 g per 100 g) and a typical mouse high-fat diet (40-60 g per 100 g)48. The total weight of one cup is 75 g; thus, each cup will contain the amount of food, and therefore drug, sufficient for five mice over 3 days.
Protocol
Anonymized human fetal tissue was acquired commercially. Animal research was carried out according to protocols approved by the University of California, Los Angeles, and (UCLA) Animal Research Committee (ARC) in accordance with all federal, state, and local guidelines. Specifically, all the experiments were carried out in accordance with the recommendations and guidelines for housing and care of laboratory animals of the National Institutes of Health (NIH) and the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International under UCLA ARC Protocol Number 2010-038-02B. All surgeries were performed under ketamine (100 mg/kg)/xylazine (5 mg/kg) and isoflurane anesthesia (2-3 vol%) and all efforts were made to minimize animal pain and discomfort.
1. Humanized mice infected with HIV-1
NOTE: Humanized mice were constructed as previously described in30,31,49. The protocol is briefly described below.
- Purify CD34+ hematopoietic progenitor cells from the human fetal liver via anti-CD34 microbeads according to the manufacturer's protocol.
- Anesthetize 6-8-week-old NOD/SCID/IL2Rγ−/− (NSG) male and female mice and sub-lethally irradiate (2.7 Gy) before the surgery.
- Implant thymus, derived from the same donor as the fetal liver, under the kidney capsule along with the liver.
- Following implantation, inject mice with 0.5 million to one million CD34+ cells, intravenously.
- After 8-10 weeks, collect 100 µL of mouse blood via retro-orbital bleed50 into microcentrifuge tubes containing 5 µL of EDTA and centrifuge at 350 x g for 3 min.
- Store the plasma at -80 °C to monitor viral load after the mouse has been infected with HIV-1. Add 2 mL of 83% NH4C solution and incubate for 5 min at room temperature to lyse red blood cells.
- Add 10 mL of RPMI with 10% Fetal Bovine Serum (FBS) to stop lysis. Spin at 300 x g for 5 min.
- Aspirate supernatant. Stain cells with antibody panel (see Table of Materials) and analyze by flow cytometry to check human immune cell engraftment.
- Infect mice exhibiting more than 50% of circulating CD45+ cells by retro-orbital vein injection51,52 with at least 200 ng of p24 of an HIV-1 strain (i.e., NFNSXSL930,53,54) using an insulin syringe. Collect blood biweekly for flow cytometry analysis and to measure the viral load.
2. Preparation of ART drugs
- Weigh individual drugs; for example, to make 10 food cups with cART, use sterile cell scrapers to weigh out 250 mg of FTC (Emtricitabine), 375 mg of TDF, and 500 mg of RAL or ELV into individual sterile 15 mL centrifuge tubes in a biosafety cabinet.
- Add 1 mL of DMSO into 250 mg FTC tube (final concentration of 250 mg/mL), add 1.5 mL of DMSO into 375 mg TDF tube (final concentration of 250 mg/mL), and add 1mL of DMSO into 500 mg RAL or ELV tube (final concentration of 500 mg/mL). Stir or pipet the drug mixture until fully dissolved and a clear solution is obtained.
- Use a 0.22 µM pore size hydrophilic PVDF membrane filter to sterilize solutions with a sterile syringe. Individual drug solutions can be stored at -20 °C for 12 weeks.
- When ready to use, freshly thaw one aliquot of each drug solution at 37 °C until the solution becomes clear. Mix well using a pipet.
- Combine drugs and mix well to make up master mix: 1 mL of FTC in DMSO, 1.5 mL of TDF in DMSO, and 1 mL of ELV or RAL in DMSO.
NOTE: This amount will make 10 food cups. - Add 350 µL of cART master mix solution into one cup to make one DietGel Boost cART cup.
- Add 0.75 mL of Trimethoprim-Sulfamethoxazole (0.48 mg/mL final concentration) into the cup.
- Stir thoroughly using 1 mL sterile pipette tips.
- Aliquot the food cup containing cART from the original cup with a micro spatula onto a 60 mm Petri dish as needed. Weigh the food on a scale to calculate the amount of food cup containing cART for each cage according to the number of mice.
3. Administration of ART drugs to HIV-1 infected mice
- Remove regular chow from the cage and replace it with a food cup containing cART.
NOTE: On average, a mouse will eat up to 5 g of food per day. Approximately one food cup can be administrated to five mice for 2 days. - Refresh cART food three times per week.
- Weigh used cups to monitor intake. Weigh mice weekly to confirm consumption.
4. Monitor viral load by real-time PCR
- Assess human immune cells (CD4 and CD8 T cell levels) and HIV-1 replication in BLT mice every 2 weeks by retro-orbital bleeding. Harvest plasma by following instructions in steps 1.5-1.8.
- Monitor plasma viral loads of mice infected with HIV-1 before and during oral cART administration for 8 weeks. Extract plasma viral RNA from plasma using a viral RNA extraction kit and quantify it by real-time PCR using the primers and probes (see Table of Materials) as previously described27,30,31. Use the following cycling protocol: 48 °C (15 min), 95 °C (10 min), then cycling 95 °C (15 s), 60 °C (1 min) for 45 cycles.
5. Assess CD4/CD8 ratios by flow cytometry
- Prepare single-cell suspensions from peripheral blood of biweekly bleeds following steps 1.5-1.8.
- Stain cells with surface markers and analyze by flow cytometry. Use the following surface marker antibodies27,30,43,49 in flow cytometry: CD45 (clone HI30), CD8 (clone SK1), CD3 (clone OKT3), CD4 (clone RPA-T4)27,30,42,49.
Representative Results
Assuming an average mouse weighing 25 g consumes 4 g of food per day, the daily drug dose through oral intake corresponds to 2.88 mg/kg TFV, 83 mg/kg FTC, and 768 mg/kg RAL. To test whether the optimized food regimen is toxic and influences overall health compared to daily injection of cART, mice weight was monitored weekly before and during cART through oral or subcutaneous injection. There were no significant weight differences before cART administration in each group (Figure 1). However, mouse weight continually decreased during daily cART SC injection. In contrast, FTC/TDF/ELV or FTC/TDF/RAL in DietGel restored mouse weights to pre-ART initiation levels after 5 weeks of oral cART administration. In addition, no significant weight changes were observed between Raltegravir or Elvitegravir groups.
To test if oral cART administration suppresses viral load as effectively as a daily injection, biweekly plasma viral loads were assessed using RT-PCR. Figure 2 shows that FTC/TDF/ELV ART food regimen 100% efficiently suppressed the viral replication to undetectable levels within 4 weeks; FTC/TDF/RAL ART food regimen can suppress 80% of the mice to undetectable levels within 4 weeks, whereas only 70% of mice receiving SC injection reached undetectable levels after 4 weeks of treatment. The results demonstrated that oral administration suppresses viral replication faster and more efficiently than SC injection. Furthermore, the cART food regimen prevented the further decline of CD4/CD8 ratios in the peripheral blood earlier than the SC daily injection (Figure 3). These results suggested that the proposed oral cART regimen can successfully suppress plasma viremia below the detection level, rapidly restore CD4 T cell levels, and improve the overall health of the animals in HIV-1 infected humanized mice.
Figure 1: Mouse body weight changes before and during cART treatment after HIV-1 infection in different groups. Humanized mice were infected with HIVNFNSXSL9 after immune reconstitution. After 4 weeks of HIV-1 infection, mice were either treated for another 7.5 weeks with FTC/TDF/RAL regimen through subcutaneous (SC) injection, or by oral administration of either FTC/TDF/RAL or FTC/TDF/ELV. Mouse body weights were measured starting from 1 week before HIV infection. All statistical comparisons were conducted using the Mann-Whitney test, reporting group mean (± S.E.). Green asterisk stars show statistical differences between FTC/TDF/ELV food oral group and FTC/TDF/RAL SC injection group. *P < 0.05, **P < 0.01, ***P < 0.001. n=6-7 in each group. Please click here to view a larger version of this figure.
Figure 2: Food oral cART administration shows faster viral suppression. As described in Figure 1, mice were either untreated or treated with FTC/TDF/RAL regimen through subcutaneous injection, and through oral administration of food cup mock, FTC/TDF/RAL, or FTC/TDF/ELV food regimens for another 7.5 weeks. (A) Plasma viral load over time after HIV-1 infection in different groups. (B) Summary of viral load over time after HIV-1 infection in different groups, reporting group geometric mean with 95% confidence interval (CI). Black arrows indicate the time of cART initiation for cART treated groups. (C) Survival analysis of time post cART treatment to undetectable viral load for each group. n= 6-7 in each group. Please click here to view a larger version of this figure.
Figure 3: ART food oral administrations show faster restoration of CD4/CD8 ratio. CD4/CD8 ratio in peripheral blood over time after HIV-1 infection for each group. All statistical comparisons were done using the Mann-Whitney test, reporting group mean (± S.E.). Red asterisk stars show statistical differences between FTC/TDF/RAL food oral group and FTC/TDF/RAL SC injection group. *P < 0.05. n=6-7 in each group. Please click here to view a larger version of this figure.
Discussion
An oral cART administration method is developed here for HIV-1 infected humanized mice by combining three antiretroviral drugs within high nutrient food. Compared to administration by daily injections, oral delivery is easier to use, limits administration frequency, reduces animal handling, minimizes stress, and improves safety55. Up to this point, only a few studies in humanized mice24,37,41 have used food pellets containing crushed ART drugs to treat mice. However, this method is challenging to apply widely due to limited access to manufacturing special food pellets. Other studies42,43,44,45,46 have used drinking water as a cART delivery system. However, compounding a medication into drinking water might alter the stability, purity, or even potency of the active ingredients. In addition, many antiretroviral drugs, including TDF, RAL, and ELV, are poorly water-soluble. Studies have shown that oral bioavailability of TDF was increased 40% after a high fatty meal56, suggesting that competitive inhibition of lipases by food may provide certain protection for TDF57. DietGel Boost is a food supplement that provides hydration, nutrition, and enrichment products that improve the overall welfare of research animals58. The nutrient-fortified gel consists of 25%-30% pure water with added carbohydrates, proteins, fats, minerals, and electrolytes, and is certified free of phytoestrogens and nitrosamines58. It provides an economical, effective, and labor-efficient alternative to mash diets58. Since 20.3% of total fat was included in the Boost cup, we propose that high nutrient levels can dissolve the TDF better, and hence increase its oral bioavailability. Therefore, a high nutrient food suspension was utilized to deliver the cART drugs to mimic the peroral delivery of cART drugs that HIV-1-infected individuals currently use.
Mice have a higher metabolism than humans, and thus, the dose of the distinct compounds was converted and used by a formula as described in reference59. Human doses of 0.4 mg (total dose) of RAL, 0.1 mg (total dose) of FTC, and 2.14 mg (total dose) of TDF were converted based on correction factor (Km, estimated by dividing the average body weight (kg) of species by its body surface area (m2)) to estimate the mouse equivalent dose values of 37 (Km) and 3 (Km) for humans and mice59, respectively. Considering the relatively low solubility of TDF, RAL, and ELV in water, DMSO was used here as a solvent for cART drugs. The final concentration of DMSO contained in the oral cART food is 0.0059% (v/v). The DMSO concentration is very low and relatively safe as a drug solvent60,61,62,63. Importantly, no fur loss or any changes of behavior in mice were observed in these studies.
The procedure described above is a highly robust and repeatable cART delivery method for treating HIV-1 infected humanized mice. This protocol can be followed easily. The critical steps in the protocol are 1) to keep sterile the whole process of any material involved in the protocol related to the DietGel food considering immunodeficiency of the humanized mice, and 2) to avoid multiple thaw/freeze cART stock solutions, and aliquot cART drugs appropriately according to mouse numbers and groups. The data suggest that oral administration of three-drug cART (TDF, FTC, and RAL or ELV) pre-mixed within the food cup efficiently suppresses HIV-1 replication and reduces viral load in plasma to undetectable levels within 4 weeks of treatment. Oral cART food administration not only prevented a further decline in CD4 T cells, but also resulted in an increased CD4 T cell percentage in the peripheral blood. In addition, the oral cART administration method restored mouse weight faster than daily injection and improved overall health.
Importantly, this method removed the risk of the researcher's exposure to sharps during daily injection of cART drugs into HIV-1 infected humanized mice. The proposed method that successfully suppresses HIV-1 RNA replication in humanized mice will be highly valuable for pre-clinical proof-of-concept studies for developing novel cure treatments that closely mimic the drug delivery in cART-treated chronic HIV-1 infected individuals.
Açıklamalar
The authors have nothing to disclose.
Acknowledgements
We would like to thank Drs. Romas Geleziunas and Jeff Murry and the people at Gilead for providing the antiretroviral drugs used in this study. This work was funded by NCI 1R01CA239261-01 (to Kitchen), NIH Grants P30AI28697 (the UCLA CFAR Virology Core, Gene and Cell Therapy Core, and Humanized Mouse Core), U19AI149504 (PIs: Kitchen & Chen), CIRM DISC2-10748, NIDA R01DA-52841 (to Zhen), NIAID R2120200174 (PIs: Xie & Zhen), IRACDA K12 GM106996 (Carrillo). This work was also supported by the UCLA AIDS Institute, the James B. Pendleton Charitable Trust, and the McCarthy Family Foundation.
Materials
| 60 mm petri dish | Thermo Scientific Nunc | 150288 | For aliquoting ART food |
| APC anti-human CD8 Antibody | Biolegend | 344722 | For flow cytometry |
| BD LSRFortessa | BD biosciences | For flow data collection | |
| CD34 microbeads | Miltenyi Biotec | 130-046-702 | For NSG-BLT mice generation |
| Centrifuge tubes | Falcon | 14-432-22 | For dissolving ART |
| DietGel Boost | ClearH2O | 72-04-5022 | For making ART food |
| Elvitegravir | Gilead | Gifted from Gilead | |
| Emtricitabine | Gilead | Gifted from Gilead | |
| FITC anti-human CD3 Antibody | Biolegend | 317306 | For flow cytometry |
| Flowjo software | FlowJo | For flow cytometry data analysis | |
| HIV-1 forward primer: 5′-CAATGGCAGCAATTTCACCA-3′; | IDT | Customized | For viral load RT-PCR |
| HIV-1 probe: 5′-[6-FAM]CCCACCAACAGGCGGCCT TAACTG [Tamra-Q]-3′; |
IDT | Customized | For viral load RT-PCR |
| HIV-1 reverse primer: 5′-GAATGCCAAATTCCTGCTTGA-3′; | IDT | Customized | For viral load RT-PCR |
| Human fetal tissue | Advanced Bioscience Resources, Inc | ||
| Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ | The Jackson Laboratory | 5557 | For constructing the humanized mice |
| Pacific Blue anti-human CD45 | Biolegend | 304022 | For flow cytometry |
| PerCP anti-human CD4 Antibody | Biolegend | 300528 | For flow cytometry |
| QIAamp Viral RNA Kits | Qiagen | 52904 | For measuring viral load |
| Raltegravir | Merck | Gifted from Merck | |
| Sterile cell scrapers | Thermo Scientific | 179693 | For aliquoting ART food |
| TaqMan RNA-To-Ct 1-Step Kit | Applied Biosystems | 4392653 | For plasma viral load detection |
| Tenofovir disoproxil fumarate | Gilead | Gifted from Gilead | |
| Trimethoprim-Sulfamethoxazole | Pharmaceutical Associates | NDC 0121-0854-16 | For keeping ART food sterile. Each 5mL teaspoon contains 200 mg Sulfamethoxazole, USP 40 mg Trimethoprim, USP NMT 0.5% Alcohol |
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