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 16, 2011 (Interim Report)
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 or targeting vectors to express these molecules in developing B cells or T cells.
We have developed transgenic mice expressing high and low avidity TCR specific for dominant and subdominant CD4 or CD8 epitopes derived from HIV consensus clade B gag and env proteins. validated the use of TCR-transgenic CD8 T cells recognizing immunodominant and subdominat gag epitopes, and are using these transgenic cells to comparatively evaluate responses to HIV vaccines. The remaining TCR transgenic mice, currently being validated, will make it possible to assess the ability of vaccines to induce responses with breadth. To address questions regarding CD4 responses generated by canarypox vectors expressing env followed by boosting with gp140 protein, as described in the RV144 HIV vaccine clinical trial, we have prioritized validation of CD4 (and CD8) TCR-transgenic mice recognizing epitopes from env. We have now used these mice, as well as wild type B6 mice or P14 mice recognizing the gp33-41 model epitope, to evaluate responses to numerous vaccines and adjuvants contributed by collaborating VDC.
We are developing mouse models to study human HIV-specific B cell responses with the goal of eliciting protective B cell responses, including neutralizing antibody responses, by vaccination. By expressing human HIV-specific immunoglobulin genes in mice, we have developed in vivo systems that can quantify specific B cell responses to immunization in terms of both magnitude and quality. The B cell models that we are developing fall into 3 groups: immunoglobulin (Ig) retrogenic mice, IgH knock-in/Igk transgenic mice, or IgH/Igk knock-in mice.
Retrogenic mice can be generated in a 2-3 month time frame by expressing the human variable region of any antibody with mouse membrane-bound IgM constant domain in mouse bone marrow cells, utilizing a replication-deficient retrovirus. Adoptively transferred retrovirally-transduced bone marrow generates naïve B cells expressing the immunoglobulin of interest in the recipient mouse, along with a marker that labels these specific B cells. Thus a mouse bearing small numbers of these trackable HIV-specific B cells can be immunized with a candidate vaccine, and the extent to which the vaccine engages those specific cells can be quantified for comparison of different vaccines. While these mouse models can be generated quickly, retroviral expression of Ig genes does not permit Ig isotype switching, affinity maturation, or production of secreted antibody. Retrogenic mice are therefo
re most useful in evaluating the activation of naïve B cells bearing specific receptors by priming immunization.
To make IgH knock-in/Igk transgenic mice, we have utilized gene targeting in mouse embryonic stem (ES) cells to express the immunoglobulin heavy chain (IgH) from the mouse genomic locus, and a non-targeted transgene construct to express the light chain (Igk). We have generated 4E10 IgH knock-in/Ig transgenic mice, and KL25 (LCMV-specific) IgH knock-in/Igk transgenic mice. Additionally, 447-52D and VRC01 IgH knock-in/Igk transgenic mice are being developed. B cells from mice generated in this way are capable of isotype switching and production of secreted immunoglobulin, making them useful models for optimization of vaccination regimens and comparison of boosting immunogens.