Batista: Mouse Genomic Manipulation Platform for Accelerated Vaccine Design
This grant will support a multi-pronged approach employing knock-in (KI) mice to evaluate HIV-1 immunogens and to increase the speed and efficiency of reiterative immunogen refinement, with the goal of developing a broadly effective and globally economical vaccine for HIV-1. Dr. Batista and team have developed a rapid, one-step
in vivo CRISPR/Cas9 nuclease-mediated strategy to generate KI mice. They have used this to produce KI mice bearing pre-arranged human B cell receptor heavy chain into the native murine immunoglobulin locus. The methodology relies on Cas9 nuclease-induced double-stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double-stranded breaks are subsequently repaired via homology-directed repair by a plasmid-borne template containing the pre-arranged human immunoglobulin heavy chain. These KI mice can thus be used as vaccine models to evaluate immune responses against specific infectious disease, allowing the acceleration of HIV vaccine design.
Knock-in mouse models expressing prearranged germline sequences of broadly neutralizing antibodies (bNAbs) will be used to test the efficacy of different HIV-1 immunogens. Responses will be monitored by FACS analysis of germinal center development and epitope-specific binding to immunogens; by isolation of responsive cells and sequencing of their antibodies; and by
in vitro analysis of antibody affinity. In parallel, a series of KI mice based on the VRC01 bNAb will be used. Both protein immunogens and mRNA immunizations with Env epitope sequences will be tested. This funding will also support the generation of mice with enhanced expansion of HLA-restricted CD8+ T cell responses. These KI models will be used to investigate another angle of attack on HIV infection: a CD8+ T cell epitope vaccine for both prevention and therapy.
1. To accelerate the identification of promising immunogen candidates.
2. To extend the array of mouse models to include additional Env epitopes.
3. To increase the power and flexibility of sequence analyses to reveal paths of antibody evolution.
4. To establish and manage a central repository of mouse lines for immunogen evaluation:
a) by maintaining active colonies of germline bNAb KI mice
b) by providing a standardized source of modified mice for the purpose of collaboration