Annual and Interim Progress Report Summaries
Principal Investigator: Phil Greenberg
Project: Defining and Comparing Immunogenicity and Improving Activity of Candidate HIV Vaccines by Developing and Employing Novel Mouse Models
Submitted January 15, 2010
The overall goal of this project is to develop and utilize mouse models to evaluate immunogenicity and immune mechanisms engaged by candidate HIV vaccines. Establishment of these models, which employ transfer of small numbers of trackable HIV-specific B and T cells, has required identification and isolation of antibodies (Ab) and T cell receptors (TCR) specific for HIV epitopes, and construction of transgenic vectors to express these molecules in developing B cells or T cells.
TCR model:
We have previously generated prototype vaccines expressing consensus clade B HIV genes, identified the epitopes recognized following vaccination of wild-type B6 mice, propagated CD4 and CD8 T cell clones specific for each epitope, isolated TCR genes from high-avidity T cell clones, and made vectors for expression in transgenic mice. We have been generating the TCR-transgenic mice with constructs containing the endogenous V-region promoter, intron, and enhancer elements to obtain expression of the HIV-specific TCR appropriately during T cell development. Our first TCR-transgenic mouse with specificity for the immunodominant gag epitope recognized by CD8 T cells has been validated, and we are currently transferring cells from this mouse strain into recipient mice to comparatively evaluate responses to candidate HIV vaccines. We have also generated TCR-transgenic mice specific for 2 subdominant gag CD8 T cell epitopes, the dominant gag CD4 T cell epitope, and the dominant rev CD8 epitope. These mice will soon be fully validated, making it possible to assess the ability of vaccine reagents/regimens to induce and sustain responses with breadth. Finally, to address specific questions associated with the RV144 HIV vaccine clinical trial, regarding the CD4 response generated by canarypox vectors expressing env followed by boosting with gp140 protein, as described in the RV144 HIV vaccine clinical trial, we have pushed forward generation of CD4 (as well as CD8) TCR-transgenic mice recognizing epitopes from env, with microinjection of TCR genes into pronucleii of fertilized eggs in Jan/Feb 2010. These TCR-transgenic mice will facilitate comparative analysis and mechanistic studies of candidate HIV vaccines.
BCR model:
We have obtained coding sequences for the 447, 4E10, and 2F5 HIV-specific neutralizing antibodies from CAVD collaborators. Transgenic mice have been generated expressing 447 and 4E10 light chains. To permit class-switching and affinity maturation during B cell responses, we have adopted a “knock-in” strategy for the Ig heavy chains. These constructs have been generated and electroporated into embryonic stem cells and ES cells with the 447 IgH have been selected. These ES cells will be injected into blastocysts, implanted into pseudopregnant female mice, and chimeric founders selected. 4E10 knock-in ES cells are currently being selected and 2F5 heavy chain knock-in mice being obtained through CAVD collaborators. To improve the efficiency for generating more Ab-producing knock-in mice, we are introducing targeted recombination sites into ES cells that can be used to rapidly introduce Ig genes. We anticipate having these ES cells developed by June 2010.
In addition, we have pursued numerous collaborations with CAVD partners and other vaccine developers to analyze immune responses elicited by novel vaccines and/or adjuvants.