Whole genome screen for antigen processing and presentation: Improving vaccine efficacy

T-cells play an important role in protection against diseases such as tuberculosis, malaria, HIV, or cancer. The capacity to develop more efficacious T-cell based-vaccines that stimulate the immune system and confer protective immunity greatly relies on antigen-presenting cells’ (APC) ability to successfully process and present immunogenic peptides to T-cells. APCs, such as macrophages and dendritic cells, process and present epitopes via Major Histocompatibility Complex (MHC) class-I and II to CD8+ and CD4+ T-cells, respectively. In addition, co-stimulatory ligands, such as CD86, and CD80 will bind to CD28 on the T-cells to activate them.
Moreover, factors such as age or immunodepression can interfere with antigen processing and presentation process, resulting in poor antigen presentation and low vaccine efficacy. In order to improve T-cell activation, we propose to screen all genes involved in antigen processing and presentation.
To achieve this goal, we will use a CRISPR library composed of whole genome-specific single guide RNAs (sgRNA) to knock down gene expression 1 (available from Addgene). We will propagate this library and clone the pooled sgRNAs into a lentiviral vector similarly to what we have performed in the past for screening a sub-library of protease-encoding genes in the context of tuberculosis 2. We will test this strategy by performing a high-content loss-of-function screen in human-monocyte-derived macrophages 3. Fluorescence-activated cell sorting (FACS) will be used to select the populations that show the highest changes in surface expression of a panel of receptors, such as MHC-I, MHC-II, and others. Next, the sgRNA sequences present in the sorted population will be identified by next-generation sequencing. Genes with the most enriched sgRNA sequences will be shortlisted for future phenotypic characterization.
At the end of this project, we expect to have implemented a high-content whole genome screening technology and a preliminary list of the most important genes involved in antigen processing and presentation, to inform better vaccine design. The objective will be to use this technology to further test these genes in primary samples of human origin beyond the limited short span of this project.