Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

This procedure describes three models of humanized immunodeficient mice for the study of the dynamics of HIV infection. NSG humanized mice recapitulates on features of human immunity. In this case, permitting the study of chronic, acute, and reactivated HIV infection in pre-clinical settings.

Demonstrating the cell preparation procedure will be Sandra Medina-Moreno, a laboratory research supervisor from our group. When performing this procedure, always use disposable personal protective equipment including sterile scrubs, gloves, dedicated shoes, shoe covers, mask, goggles, hair and beard bonnets, and a sterile lab coat. For the chronic model, resuspend one million frozen CD34+human stem cells in 10 milliliters of RPMI 1640 media with 10%FBS under a certified biosafety cabinet and maintain sterile conditions.

Use 10 microliters of the suspension to count and check cell viability by Trypan Blue exclusion staining in a hemocytometer. Then centrifuge the cell suspension at 400 times g for 15 minutes at room temperature. Discard the supernatant and resuspend the cells in cold 1X PBS at the required concentration.

Keep the cells on ice until injection. Rub clean bedding between hands to mask any other foreign odors on the recipient pups. Then place pups into a sterile 100 square millimeter Petri dish along with a small amount of bedding material from the breeder cage.

Put the Petri dish into a clean transport cage and then place the cage in a Carboy transport cage to protect pups from direct light and noise. Cover the transport cage with a cover pad to avoid exposure of animals to the environment while in transit to the irradiation room. Following anesthetization as described in the text protocol, prepare for engraftment via hepatic injection.

To minimize the time of mouse restraint, let one investigator load the syringe with the stem cell suspension and have the second investigator hold the pup and administer the injection. Load 50 microliters of the CD34+human stem cell suspension into the syringe with an attached needle under the certified biosafety cabinet. Restrain the pups with thumb and index fingers and clean the injection site with 70%alcohol.

Use a shallow needle angle to deliver 50 microliters of cells directly into the liver so as not to completely pierce the liver. Prewarm the dish using an infrared warming pad for rodents at 20 degrees Celsius to ensure the pups will not be over warmed. Place the pups on the Petri dish covered with sterile gauze for one to five minutes to allow recovery.

Immediately before returning the pups to their parents, apply a small amount of menthol and eucalyptus-based ointment using the thumb and index fingers to the snout of both parents to avoid cannibalism or rejection of the pups. Check cages everyday, looking for any signs of graft versus host disease in the pups such as dry skin, no feeding, rash, or alopecia. Wean the pups at three weeks of age and house them in different cages.

Verify engraftment in the peripheral blood by flow cytometry at 14 weeks of age. A representative gating strategy for the evaluation of human CD45+cell reconstruction and percentage of CD4+and CD8+T cells is shown. The success rate of engraftment is between 80%to 100%Use human peripheral blood mononuclear cells derived from a healthy donor for the acute model or from an HIV infected patient who was under antiretroviral therapy for the reactivation model.

Layer 15 milliliters of whole blood in five milliliters of sterile density gradient medium into a 50 milliliter conical tube. Centrifuge at 400 times g for 30 minutes at room temperature without breaks to avoid the buffy coat from becoming mixed with the density gradient medium. Carefully collect the fraction of mononuclear cells between the density gradient medium and the supernatant.

Transfer the buffy coat to a 15 milliliter centrifuge tube containing 10 milliliters of 1X PBS. Centrifuge at 300 times g for 10 minutes at room temperature. Discard the supernatant and remove the remaining red blood cells by lysing with five milliliters of ACK lysis buffer added to the pelleted cells.

Incubate for four minutes at room temperature. After centrifuging again as before, discard the supernatant and resuspend in 10 milliliters of 1X PBS or RPMI 1640 medium. Use 10 microliters of the cell suspension to count the cells and check viability by Trypan Blue exclusion staining in a hemocytometer.

Typically, one to two million cells are obtained for each one milliliter of blood with more than 95%viability. Following centrifugation as before, discard the supernatant and adjust the cell number to the required concentration. Load 3.5 million peripheral blood mononuclear cells in 200 microliters of 1X PBS into a syringe with an attached needle under a certified biosafety cabinet.

Remove the mouse from the cage and hold it by the tail so that it can grip the mesh applying gentle traction backward. Then place the index finger and thumb on the shoulders of the animal grabbing the loose skin of the neck and using the middle finger to stabilize its back. Slide the mouse head backward so that its back is above its head.

This allows the viscera in the abdominal cavity to be displaced backwards and reduces the risk of puncturing internal organs during the injection. Clean the injection site with 70%alcohol. Penetrate the prepared syringe through the abdominal wall and aspirate before injecting the cells.

If any material is aspirated, remove the syringe and discard it. Otherwise, inject the cells slowly in the intraperitoneal cavity before removing the syringe and discarding it. Return the animal to its cage and verify engraftment in peripheral blood by flow cytometry at three weeks post-injection.

Identify mice by ear tagging. Observe the mice used in these experiments closely, twice per day after each procedure for clinical signs of distress. Then proceed to HIV injection as described in the text protocol.

Following HIV injection, there is a rapid increase in plasma viral load usually being detectable after two to three weeks post-infection in both the chronic and acute models with similar kinetics in the reactivation model. The increase in viral load coincide with a decrease in the CD4:CD8 ratio. These changes are not observed in control mice.

Of note, in the humanized PBMC NSG adult mouse model, an initial inversion of the CD4:CD8 ratio is observed and reconstituted along monitoring time. When antiretroviral therapy is administered to HIV infected mice, a suppression of the viral load as well as recovery in the CD4:CD8 ratio occurred as expected reaching similar levels to those in uninfected controls. Typically, after two to three weeks of treatment, a decrease in viral load and increase in the CD4:CD8 ratio is observed in the chronic, acute, and reactivation models.

Use the recommended irradiation dose to avoid lethal overdose or rejection of transplanted cells. Do not completely pierce the liver during injection and use bedding to mask odors that the dam will perceive as foreign. These three models permit testing of antiviral compounds, anti-HIV antibodies or other biological substances that affect viral replication.

Additionally, they allow adaptive transfer experiments for HIV immunotherapy. NSG humanized mice can be reconstituted partially or in full with human cells to respond to pathogens or drugs, mimicking the complexity of the human immune system. All basic biological measures need to be strictly followed since NSG humanized mice can be infected with both murine and human pathogens, particularly in this case which uses HIV virus manipulation.