HIV REV DEPENDENT REPORTER CELL FOR NEUTRALIZING ANTIBODY DISCOVERY

The development of an effective vaccine is the most plausible approach to eliminate the HIV AIDS pandemic The detection of broadly neutralizing antibody bNABs responses is critical for vaccine development In this grant application we propose to develop a novel sensitive method for bNAB detection and quantification An HIV Rev dependent A R derived reporter cell line Rev A R NanoLuc HaloTag A R is a T lymphoblast that expresses physiologically relevant levels of the HIV receptors CD and CXCR CCR and is highly susceptible to X and R viruses A R has also been shown to be the optimal cell line currently available for bNAB detection We plan to engineer A R to carry two highly sensitive reporters NanoLuc the most sensitive luciferase currently available and HaloTag the most flexible fluorescent reporter Both reporters are engineered to exclusively depend on HIV and Rev for expression eliminating non specific noises from environmental stimuli This novel system will provide unmatched specificity and sensitivity providing a new standard for NABs detection and quantification We plan to carry out two specific aims in the phase I stage Aim will be to create a high sensitivity A R derived Rev dependent reporter cell line Rev A R for bNAB detection We will first construct the Rev dependent vector pNL NanoLuc HaloTag RRE for assembly into lentiviral viral particles which will be used to transduce A R cells Subclones will be selected for optimal performance in NAB detection Aim will be to characterize and validate Rev A R We will test the specificity of the reporter cells Additionally Rev A R andapos s susceptibility to primary viral isolates will be tested We will also perform bNAB screen using a panel of antibodies and patient sera to optimize the system for Phase II development We aim to develop the system into a standard assay product for academic and industry laboratories for bNAB screening and validation The detection of HIV broadly neutralizing antibodies bNAB is critical for vaccine development In this grant application we propose to develop a novel sensitive reporter cell line for bNAB detection and quantification This novel system will offer unmatched specificity and sensitivity providing a new standard for academic and industry laboratories for bNAB screening and validation