Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

This protocol describes a novel method to deliver oral combination antiretroviral drugs, or ART, that successfully suppresses HIV RNA replication in humanized mice. The novel oral ART method surpasses previous delivery attempts due to its safety, ease of preparation and administration, and reduced animal stress resulting from a daily injection. The proposed oral delivery method to suppress HIV RNA replication in humanized mice will be highly valuable for preclinical proof-of-concept studies to develop novel cure treatments that closely mimic the drug delivery in chronic HIV-infected individuals.

Demonstrating the procedure will be Valerie Rezak, a lab manager and technician from our laboratory. Begin by weighing individual drugs. Make 10 food cups with cART using sterile cell scrapers to weigh out 250 milligrams of FTC, 375 milligrams of TDF, and 500 milligrams of RAL or ELV into individual, sterile 50-milliliter centrifuge tubes in a bio-safety cabinet.

Add DMSO to the FTC and TDF tube to make up a final concentration of 250 milligrams per milliliter, and into the RAL or ELV tube to a final concentration of 500 milligrams per milliliter. Mix the drug solution by stirring and pipetting until a clear solution is obtained. Sterilize the solutions using a 0.22-micrometer hydrophilic PVDF membrane filter with a sterile syringe.

Individual drug solutions can be stored at minus 20 degrees Celsius for 12 weeks. Freshly thaw one aliquot of each drug solution before use at 37 degrees Celsius until the solution becomes clear. Mix well using a pipette.

Combine the drugs dissolved in DMSO and mix well to make up a master mix containing one milliliter of FTC, 1.5 milliliters of TDF, and one milliliter of ELV or RAL. Add 350 microliters of cART master mix solution into a cup to prepare one diet gel boost cART cup. Add 0.75 milliliters of trimethoprim sulfamethoxasole into the cup.

Stir thoroughly using one-milliliter sterile pipette tips. Using a pipette tip, aliquot the food cup containing cART onto a 60-millimeter Petri dish. Calculate the amount of cART in the food cup by weighing the food for each cage according to the number of mice.

Remove regular chow from the cage and replace it with a food cup containing cART. Refresh cART food three times per week. Weigh used cups to monitor intake.

Then, weigh the mice weekly to confirm consumption. After four weeks of HIV-1 infection, mice were treated for another 7.5 weeks, either with FTC/TDF/RAL regimen through subcutaneous injection or by oral administration of FTC/TDF/RAL or FTC/TDF/ELV. Mouse weight continually decreased during daily cART subcutaneous injection.

In contrast, diet gel restored mouse weights to pre-ART initiation levels after five weeks of oral cART administration. In addition, no significant weight changes were observed between RAL or ELV groups. Within four weeks, the FTC/TDF/ELV and RAL/ART food regimen suppressed the viral replication by 100%and 80%respectively.

70%of the mice receiving SC injection reached undetectable levels after four weeks of treatment. The results confirmed that oral administration suppressed viral replication faster and more efficiently than subcutaneous injections. Furthermore, the cART food regimen prevented the further decline of CD4 to CD8 ratios in the peripheral blood earlier than daily injections.

It is critical to maintain sterility of any material involved in the protocol during the whole process and avoid multiple freeze and thaw of the cART stock solution.