OA-37-733-24 An in vitro intracellular Mycobacterium tuberculosis and HIV-1 co-infection model suitable for imaging-based platforms

Background: Mycobacterium tuberculosis (Mtb) is an obligate, intracellular bacillus, causing the human dis- ease Tuberculosis (TB). The Human Immunodeficiency Virus (HIV-1) is a lentivirus that causes AIDS (Acquired Immune Deficiency Syndrome) in humans. Both diseases are among the leading causes of mortality worldwide. Mtb and HIV-1 are classed as hazard group 3 (HG3) pathogens by the UK Advisory Committee for Danger- ous Pathogens, as such they are worked with and stored in containment level 3 (CL3) laboratories. The aim of this work was to develop a safe methodology to co-in- fect macrophage cells with Mtb and HIV-1 suitable for UK CL3 laboratories to monitor pathogen interactions and for drug screening purposes. Design/Methods: We utilised multiple fluorescence- based methodologies to quantify the co-infection. By employing genetically modified Mtb and HIV-1, both carrying fluorescent molecules, we used flow cytometry and microscopy to study levels of infection in the pres- ence or absence of drugs. Much optimisation was required in the development of these methodologies, from cell number, MOI and viral load.

Results: Co-infections were quantified and monitored using different approaches: fluorometry, luciferase, confocal microscopy and flow cytometry. Co-infections were performed in presence or absence of antiretrovi- ral (Efavirenz [EFV]) and/or a first-line TB antibiotic (Rifampicin [RIF]). EFV completely inhibited HIV-1 infection in a single and co-infection setting, and inter- estingly RIF also reduced the HIV-1 infection rate by 12%. RIF reduced Mtb viability by more than 50% in a single infection and by 57% in a co-infection setting. The combination of drugs and presence of both virus and bacteria significantly affected infection rates and pathogen viability.

Conclusions: We have successfully developed a method- ology to co-infect THP-1 cells with HIV-1 pseudo vi- rus particles and Mtb, ensuring host cell viability. Our method can be easily applied to address biological ques- tions concerning the HIV-1/Mtb co-infection process as well as be used for drug screening.