Generation of an In Vitro Cell Culture Model of Malaria-HIV Co-Infection

Begin with a multi-well plate containing human peripheral blood mononuclear cells, or PBMCs derived from a human immunodeficiency virus, or HIV-infected donor.

PBMCs contain various immune cells, including HIV-infected CD4-positive T cells with integrated proviral DNA.

Seed the wells with a mixture of Plasmodium falciparum parasitized erythrocytes, or PfRBCs — expressing parasite-derived antigens, and uninfected RBCs.

During incubation, the PfRBCs' antigens interact with receptors of immune cells, activating them and releasing cytokines.

These cytokines trigger downstream signaling in CD4 T cells, initiating transcription of HIV proviral DNA and synthesis of new viral RNAs.

The viral RNAs and HIV proteins form virus particles that bud off, infecting new CD4 T cells and spreading HIV infection.

Progressive HIV infection disrupts cytokine secretion, impairing the immune response against PfRBCs and leading to persistent malaria infection.

Over time, infected RBCs release parasites that invade new RBCs, proliferating malarial parasites and establishing the malaria-HIV co-infection cell culture model.

To set up the co-infection cultures, begin by seeding 1 x 10⁶ of freshly-isolated HIV-positive and HIV-negative PBMC in 100 microliters of RPMI-S+ per well into a 24-well plate. Bring the total volume of each well up to 500 microliters with 400 microliters of RPMI-S+.

Then, in triplicate and in a total volume of 500 microliters per well, add 3 x 10⁶ uninfected red blood cells or 3 x 10⁶ parasitized red blood cells to the HIV-infected and HIV-uninfected cells. Place the co-infection plate in a cell culture incubator.

Then, after the appropriate experimental time period, pellet the cells, and collect 700 microliters of culture supernatant from each well. Centrifuge the supernatant to remove any debris. Then, aliquot the samples as appropriate; label the tubes, and freeze them at minus 20 degrees Celsius or below for later analysis.