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Greenberg Progress Report Abstracts continued

Submitted July 1, 2010

The goal of this project is to develop and utilize mouse models to evaluate the immunogenicity and immune mechanisms engaged by HIV vaccines. These models employ transfer of trackable HIV-specific T and B cells expressing an HIV-specific T cell receptor (TCR) or B cell receptor (BCR).

TCR model: Development of transgenic mice expressing TCR specific for dominant and subdominant CD4 or CD8 epitopes required generating prototype vaccines expressing consensus clade B HIV genes, identifying epitopes recognized in vaccinated C57BL/6 mice, generating CD4 and CD8 T cell clones specific for each epitope, isolating and validating TCR genes from high and low avidity T cell clones, and constructing regulated transgenic expression vectors.  We have generated several transgenic mouse strains expressing high and low avidity TCRs specific for dominant and subdominant CD4 and CD8 gag epitopes, validated the use of TCR-transgenic CD8 T cells recognizing the immunodominant gag epitope, 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 generation of CD4 (and CD8) TCR-transgenic mice recognizing epitopes from env.

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, 4E10, or 2F5 light chains. To permit class-switching and affinity maturation during B cell responses, IgH “knock-in” constructs have been generated and electroporated into embryonic stem (ES) cells. 447 IgH knock-in ES cells are being selected for injection into blastocysts that will be implanted into pseudopregnant female mice, 4E10 IgH knock-in chimeric founders are being crossed to B6, and 2F5 heavy chain knock-in mice are being obtained through CAVD collaborators. To improve the efficiency for generating more Ab-producing knock-in mice, we are introducing targeted recombination exchange sequences into ES cells for rapid introduction of Ig genes. We anticipate having these ES cells developed by Dec 2010.

We have also pursued numerous collaborations with CAVD partners and other vaccine developers to analyze immune responses elicited by novel vaccines and/or adjuvants.

 
Submitted January 15, 2010 (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 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.

 
Submitted July 1, 2009

The overall goal of this project is to develop and utilize genetically-modified mouse models that better model human immune responses to evaluate the immunogenicity and immune mechanisms engaged by candidate HIV vaccines and to provide insights into and test possible improvements in the design of candidate HIV vaccines. Establishment of an informative mouse model to evaluate T cell responses to candidate HIV vaccines has required in part the development of prototype vaccines expressing HIV proteins, identification of epitopes derived from these HIV proteins recognized by immunodominant and subdominant CD4 and CD8 T cell responses, cloning and expansion of T cells recognizing these epitopes, isolation and validation of the clonotypic TCR genes cloned from these T cells for the subsequent generation of TCR-transgenic mice, and administration of the trackable HIV-specific CD4 or CD8 T cells from the transgenic donors into recipient mice prior to vaccination. Using DNA, MVA, and Ad5 prototype vaccines expressing consensus sequence HIV clade B gag, env, pol, & rev genes, we 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, and the TCR genes from selected high-avidity clones isolated and validated by demonstrating that introduction of the TCR genes into a transformed T cell line imparts recognition of the specific epitope. The V(D)J regions from these TCR genes have been inserted into a transgene construct that includes intronic sequences necessary for optimal expression and has previously been successfully used to generate other TCR transgenic mice, and we have now shown that mRNA from these constructs are spliced in-frame. Pronuclear injections with these plasmids encoding TCR  and  genes specific for the immunodominant gag epitope resulted in 8 founder mice that had integrated both TCR chains. These mice are being bred and will serve as T cell donors- first, to validate this model system using the prototype vaccines that have been already generated, and second, for subsequent evaluation of candidate HIV vaccines. The remaining TCR genes, specific for clade B gag, env, pol, & rev epitopes are now being cloned into the transgene constructs, and are scheduled for pronuclear injections to generate the TCR-transgenic mice. 

Submitted January 15, 2009 (Interim Report)

The overall goal of this project is to develop and utilize a novel mouse model to evaluate the immunogenicity and immune mechanisms engaged by candidate HIV vaccines and to provide insight into and test possible improvements in the design of the evaluated candidate HIV vaccines.  Establishment of this mouse model, which employs the administration of trackable HIV-specific T cell receptor (TCR)-transgenic CD4 or CD8 T cells into mice prior to vaccination, has 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, we 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, and the TCR genes from selected high avidity clones isolated.  We continue to validate these TCR by demonstrating that introduction of the genes into transformed T cell lines imparts recognition of the specific epitope.  Pronuclear injections with constructs containing the first pair of these TCR genes resulted in production of 6 founder TCR transgenic mice.  Unfortunately, none of these founder mice expressed significant levels of the TCR.  We have now revised/improved our strategy for production of these TCR transgenic mice, and the next round of pronuclear injections is scheduled for Feb 2009.

Our initial studies and model development has been focused on T cell responses to vaccines, but, in response to the results from the STEP-trial and in order to engage members of the Vaccine Development Consortia (VDC) working on HIV vaccines being designed to generate neutralizing antibody B cell responses, we have begun generation of a second type of mouse model which will employ the transfer of small numbers of trackable B cells from mice expressing the variable region of known neutralizing monoclonal antibodies (mAb), with the initial mAb selected being 4E10 and 447.  This model will allow us to test the ability of candidate HIV vaccines to engage B cells capable of producing a neutralizing antibody specific for a known HIV structure and to generate B cell memory and antibody-producing plasma cells.  As B cells from these mice retain the ability to undergo class switching and affinity maturation, this model will further allow us to evaluate the quality of the B cell response generated and could potentially aid in the identification of new neutralizing antibodies.  We will also be developing fertilized eggs containing an acceptor” site in the Ig locus that will facilitate the future development of antibody “knock-in” mice for additional/new antibodies that appear relevant to the development of a neutralizing antbody HIV vaccine.

We have begun using the model systems we are developing to evaluate candidate HIV vaccines obtained from 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 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 a strong mucosal T cell response, which was not evident from sampling systemic sites such as blood or lymph nodes, indicating that responses in human vaccinees, 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, memory T cell levels were similar between all AAV and Ad5 immunized mice, despite differing initial effector responses. These results have helped our CAVD to prioritize vectors for advancement to preclinical studies in non-human primates. 

Submitted July 1, 2008

The overall goal of this project is to develop and utilize a mouse model to determine the immunogenicity and elucidate the immune mechanisms engaged by individual candidate HIV vaccines, and to then use the lessons learned from such studies to provide insights into possible improvements in the design of candidate HIV vaccines.  Establishment of this mouse model, which employs the administration of readily trackable HIV-specific T cell receptor (TCR)-transgenic CD4 or CD8 T cells into mice prior to vaccination, has required the development of prototype vaccines expressing each HIV protein, induction of immune responses to these vaccines followed by identification of the epitopes within each of these HIV proteins in the context of mouse MHC molecules that are recognized by the immunodominant and subdominant CD4 and CD8 T cell responses, cloning and expansion of T cells recognizing these epitopes that represent dominant and subdominant responses, and isolation and validation of the clonotypic TCR genes encoding the receptors in these T cells for the subsequent generation of TCR-transgenic mice.  Using DNA, MVA, and Ad5 prototype vaccines expressing consensus sequence HIV clade B gag, env, pol, & rev genes, we have identified immunodominant and subdominant HIV-specific CD8 and CD4 T cell responses elicited following immunization. T cell clones specific for these epitopes have been generated, and the TCR genes from these clones isolated and validated by demonstrating that introduction of the genes into transformed T cell lines imparts recognition of the specific epitope.  We have also generated constructs containing these isolated TCR genes for pronuclear injection into fertilized eggs for the production of TCR transgenic mice and expect the first transgenic strain to be available in the next several months.  In addition to the progress made in developing this model to specifically study responses to HIV vaccines, we have also begun testing model vaccine vectors expressing non-HIV encoded antigens that are similar to vectors being developed at individual Vaccine Development Centers (VDC) to define the principles and possible limits of the model.  Using previously developed transgenic mice expressing a TCR specific for the LCMVgp33 epitope, we have evaluated peak, memory, and recall T cell responses to DNAgp33 and Ad5gp33 vaccines that are being studied in the McElrath VDC.  We have also begun analysis of Adeno-associated virus vaccines, being developed and studied by the Zamb VDC, that express the SIVmac239 gag gene or a gag-flagellin fusion protein.  Consistent with the goals of the CAVD and HIV Vaccine Enterprise, we are currently finalizing analysis of this data and the results will be posted on the VISC data portal for sharing with the individual VDC as well as the larger CAVD community.

Submitted January 15, 2008 (Interim Report)

This project is designed to facilitate development of a unique mouse model in which selected human genes are expressed in mice to make the mice more representative of how humans will respond to immunogens, and which can be used not only to test and compare candidate HIV vaccines but also to identify strategies to improve such vaccines. The model also utilizes a method previously validated in our lab that employs the transfer of small numbers of readily trackable TCR-transgenic T cells specific for a protein of interest into intact wild-type mice that can then be vaccinated and examined in detail to determine the nature and magnitude of T cell responses, and which is being adapted for these studies by including the generation of TCR-transgenic T cells specific for individual HIV proteins. By using gene-deficient mice as the recipients in selected studies, the immune pathways engaged by individual vaccines can be determined and the lessons learned from these studies then used to suggest potential improvements to candidate vaccines. Generation of the necessary TCR-transgenic mice requires the identification of CD4 and CD8 T cell responses in wild-type mice immunized with vaccines expressing HIV proteins, propagation of T cell clones specific for epitopes derived from these proteins, and isolation of TCR genes from these HIV-specific T cell clones. We have identified and defined one immunodominant CD8 T cell response and two CD4 T cell responses in each of the HIVgag, HIVenv, and HIVrev proteins and have generated several T cell clones characteristic of each of these responses. We have also determined the avidity of individual clones and selected representative clones for TCR isolation. Using RACE PCR, we have isolated the rearranged TCR genes from the selected clones and are confirming that these TCR confer specificity by re-expressing these genes in an immortalized TCR-negative murine T cell line. Selected and validated TCR will be inserted in a transgene targeting vector with endogenous mouse TCR promoters and enhancer elements that will be used to generate the TCR-transgenic mice required for this project. The micro-injection of blastocysts for generation of TCR-transgenic mice will be performed by Xenogen Biosciences/Caliper Life Sciences which has the capability of concurrently handling the generation and initial breeding of the large number of transgenic mice required for this project. Although we are still awaiting the development of the TCR-transgenic mice for this project, we have concluded a “proof-of-principal” study of this mouse model utilizing TCR-transgenic CD8 T cells specific for a model antigen and prototype vaccines expressing the model antigen, and have demonstrated that divergent vaccines given by different routes, that engage distinct innate immune pathways and result in different magnitude of T cell responses, engage the transferred T cells proportionally equivalent to the endogenous host response, thereby affirming that the transferred TCR-transgenic response adequately predicts vaccine outcomes. We anticipate that the mouse models will be developed and validated on time for use in evaluation of HIV vaccines obtained from individual VDCs.

Submitted July 1, 2007

The overall goal of this project is to develop and utilize a mouse model to elucidate the immunogenicity and immune mechanisms engaged by individual candidate HIV vaccines and to use the lessons learned from such studies to provide insight into possible improvements in HIV vaccine design.  Establishment of this mouse model, which employs the adoptive transfer of HIV-specific T cell receptor (TCR)-transgenic CD4 or CD8 T cells into vaccinated mice, requires the identification of epitopes derived from HIV proteins and presented in the context of mouse MHC molecules that are recognized by CD4 and CD8 T cells, cloning and expansion of the T cells recognizing these epitopes, and isolation of the clonotypic TCR genes from these T cell clones for the subsequent generation of TCR-transgenic mice.  Using DNA, MVA, and Ad5 prototype vaccines expressing the consensus gag protein from HIV clade B, we have identified one novel CD8 response (Dbgag268-276) and one novel CD4 response (IAbgag301-309) as well as confirmed previously identified T cell responses.  We have now generated T cell clones specific for these epitopes and isolated TCR genes from these clones.  We are currently confirming that these TCR impart recognition of the specific epitope by introduction into transformed T cell lines and are generating targeting constructs for injection into blastocysts for producing TCR transgenic mice.  In addition, we have now developed DNA, MVA, and Ad5 prototype vaccines expressing other consensus clade B HIV proteins (env, pol, nef, tat, & rev) and will be identifying T cell responses to these.

 
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