Greenberg Mouse Immunology Laboratory

OVERVIEW:

HIV remains a global health threat, and no HIV vaccine developed has yet achieved prolonged or significant protection in humans. Designing more effective HIV vaccines would be greatly facilitated by a more comprehensive understanding of how vaccines stimulate immune responses. Mouse models that would allow researchers to monitor immune responses to vaccines could provide insights that would facilitate designing and refining preventative HIV vaccines.

Researchers in the Mouse Immunology Laboratory led by Phil Greenberg and Joe Blattman are developing sensitive mouse models to evaluate the immunogenicity of candidate HIV vaccines. Such models will allow researchers to evaluate the abilities of vaccine candidates to elicit effector, long-term memory and mucosal T cell responses and/or functional an neutralizing B cell antibody responses, to determine the innate and adaptive immunologic mechanisms required to achieve these responses, to provide a comparative means for predicting which candidate vaccines will exhibit the greatest activity in humans, and to identify principles that can be incorporated into HIV vaccine vectors or formulations to augment the generation of potentially protective human immune responses.

The development of mouse models that allows researchers to monitor CD4 and CD8 T cell responses has been the first step, and  creating mouse models that allows the evaluation of broadly neutralizing antibody responses is now proceeding. Creation of these mice has required researchers to genetically modify mice to make components of the murine immune system more closely resemble the human system. These models being developed should provide an accurate screen for determining what vaccines and formulations should be selected for testing in non-human primates and humans, and insights into how candidate vaccines might be improved to better achieve the desired immune responses.

RESEARCH OBJECTIVES:

1. Identify immunodominant and subdominant CD4 and CD8 T cell responses to HIV proteins in vaccinated mice; create transgenic mice expressing TCR genes specific for the HIV epitopes recognized; and develop a mouse model utilizing transfer of T cells from TCR-transgenic mice to facilitate quantitation and in vivo tracking of responses to HIV vaccines.
2. Employ this sensitive mouse model for examining HIV-specific T cell responses to evaluate and compare the immunogenicity of candidate HIV vaccines, as well as the ability of these vaccines to induce long-lasting memory responses and the generation of T cell responses that home to mucosal sites.
3. Adapt this mouse model for use with gene-deficient or transgenic mice to determine which cellular and molecular components of the innate and adaptive immune system, as well as which negative regulators of responses, are engaged during responses to candidate HIV vaccines.
4. Improve HIV vaccine immunogenicity by addition/augmentation of identified positive innate and adaptive immune signals, or by disruption/removal of negative signals, to enhance the magnitude of responses and the homing of responding cells to mucosal sites induced by individual candidate HIV vaccines.
5. Create knock-in and transgenic mice that express immunoglobulin (Ig) genes in naïve trackable B cells that encode broadly neutralizing antibodies; determine if immunogens are capable of eliciting such antibody responses and/or have the potential to induce even higher affinity and more strongly neutralizing antibodies.    

PROGRESS:

Establishment of the mouse model, for studying T cell responses, which employs the administration of trackable HIV-specific T cell receptor (TCR)-transgenic CD4 or CD8 T cells into mice prior to vaccination, required the development of prototype vaccines expressing HIV proteins as a representative set of immunogens, identification of the epitopes derived from these HIV proteins recognized by host immunodominant and subdominant CD4 and CD8 T cell responses, cloning and expansion of T cells specific for these epitopes, and isolation and validation of the clonotypic TCR genes from these T cells for the subsequent generation of TCR-transgenic mice.  

• Using DNA, MVA, and Ad5 prototype vaccines expressing consensus HIV clade B gag, env, pol, & rev genes, the researchers have identified immunodominant and subdominant HIV-specific CD8 and CD4 T cell responses elicited following immunization.  T cell clones specific for each of these epitopes have been generated, the TCR genes from selected high avidity clones isolated, and the cloned TCR genes validated for reactivity and affinity for the HIV protein. 
• The researchers are now producing TCR-transgenic mice that will generate T cells expressing these receptors.  Constructs for efficient expression of TCR genes were developed, and pronuclear injections with constructs containing the first pair of these TCR genes, which encodes a receptor for the immunodominant CD8 T cell response to gag has been completed, and multiple founder strains were generated. These mice are now being bred for use in the mouse model.
• Pronuclear injections are proceeding for TCR-transgenic that will encode subdominant CD8 responses to gag, dominant and subdominant CD4 responses to gag, and CD4 responses to env. After these mice have been validated, the remaining TCR transgenic mice will be made from TCR genes isolated from already produced CD4 and CD8 T cell clones.

The researchers have begun generation of a second type of mouse model that will allow testing of the ability of candidate HIV vaccines to engage B cells capable of producing a neutralizing antibody specific for a known HIV epitope and to generate B cell memory and antibody-producing plasma cells.

• The researchers are generating mice that will express the broadly neutralizing 4E10 antibody, which recognizes an epitope within the conserved membrane proximal external region (MPER) of gp41.  Mouse embryonic stem cell clones have been isolated in which the 4E10 antigen-binding domain has been inserted into the immunoglobulin heavy chain genomic locus.  These clones have been identified by PCR assay and confirmed by southern blot.  These stem cells will be used to generate 4E10 heavy chain knock-in mice. 
• The researchers have generated transgenic mice expressing the 4E10 kappa light chain, in which cells expressing the transgene are marked by a cell-surface Thy1.1-PDGFR fusion protein.  Three transgenic founder mice have been identified which express the Thy1.1 marker on a subset of B cells.  Crossing these mice with the 4E10 heavy chain knock-In mice will allow tracking of 4E10 antibody-expressing cells in vivo after adoptive transfer and immunization
• Additionally, the researchers are also generating mice expressing the V3 loop-specific HIV-neutralizing 447D antibody.  DNA constructs for the generation of these mice are complete, and expression is being verified in vitro before the generation of heavy chain knock-in and light chain transgenic mice
• Embryonic cells are being developed that will serve as efficient universal recipients of knock-in heavy chain and light chain genes, so that mice can be rapidly made to evaluate new antibodies of interest.

The model systems under development are already being used to evaluate candidate HIV vaccines obtained from the VDC expressing model antigens as a means to test vector immunogenicity and the mechanisms engaged by such vectors.  This includes DNA, MVA, Ad5, and LM expressing the glycoprotein (GP) from LCMV as well as Adenoviral and Adeno-associated virus  (AAV) vectors expressing the gag protein from SIVmac239 or a gag-flagellin fusion protein designed to engage TLR5 and thereby increase immunogenicity.

• DNA vaccination surprisingly resulted in previously unrecognized strong mucosal T cell response, which was disproportionate to the weak responses detected from sampling systemic sites such as blood or lymph nodes. These results suggest that responses to human vaccines, which are typically measured in blood, may under-represent the total responses to DNA vaccination.
• Immunization with AAV vectors expressing flagellin (or the minimal TLR5 binding domain of flagellin) fused to the N-terminus of the gag protein resulted in higher initial responses than did AAV expressing gag alone or flagellin fused to the C-terminus of gag. However, evaluation of memory T cell responses at later time points revealed similar magnitudes with all of the mice regardless of the AAV vector used. These memory T cell responses in AAV immunized mice were similar to those detected in Ad5, and higher than those found in DNA immunized mice. These results have helped to prioritize vectors for advancement to preclinical studies in non-human primates.