John D. Altman - US grants

Studies of the course of HIV-1 infection in humans point to an important role of major histocompatibility complex (MHC)-restricted, HIV-specific, cytotoxic T lymphocytes (CTL) in the control of viral burden. At the same time, rhesus macaques challenged SIV or chimeric HIV/SIV viruses (SHIV) have emerged as the best animal model for pathogenesis and vaccine studies a major deficiency of this model is that characterization of SIV/SHIV CTL epitopes and their class I MHC restriction elements in macaques lags significantly behind CTL studies in humans. The goal of this project is to intensively study the MHC allele found in the macaques used for the DNA vaccination studies (described by Dr. Harriet Robinson in Project 1 of this program), providing data essential for an adequate interpretation of the results of those vaccine trials. The three specific aims of this project are: 1) genotypic analysis of the macaque class I MHC alleles (Mamu) using locus-specific PCR primers to clone the macaque cDNAs, followed by development of allele-specific primers for more rapid genotypic analyses; 2) characterization of the peptide binding motifs of the Mamu Class I proteins found at high frequency our colony at the Yerkes Regional Primate Center, leading to testable predictions of CTL epitopes; and 3) production of novel MHC tetramers based on the alleles studied in Aim 2, leading to studies of the frequency and phenotype of the SIV/SHIV- specific CD8+ T cells induced by the vaccination protocol or by challenge infection. Together with the data from the cellular immune response project of this program (Project 4, led by Drs. Villinger and McNichol) and from the clinical course of the SHIV-challenged animals, these data will allow us to determine the epitope-specificity of CTL responses which correlate with protection from viral challenge.

DESCRIPTION: Immunity to a number of viruses is known to be mediated by T cells, implying that many vaccines should be engineered to elicit strong T cell memory. While the phenomenon of functional T cell memory may be accounted for by increased frequencies of antigen specific cells with possibly heightened sensitivity, many of the mechanistic details of memory T cell development and maintenance remain unresolved. Precise analysis of memory T cells has been hampered by the absence of methods for detecting and purifying low frequency populations of antigen-specific T cells without resort to in vitro culture techniques. Recently described soluble tetramers of peptide-bound MHC complexes that label T cells according to the specificity of their antigen receptor provide a general solution to the problem. This technique will be applied to answer two specific questions on the nature of T cell memory, using well characterized CD8+ T cell responses to LCMV. 1) What is the lifespan of memory T cells? The MHC tetramers will provide definitive answers to this question through analysis of extremely pure populations of memory T cells (possessing several distinct antigen-specificities) generated during the course of physiologically relevant responses. As a corollary to the lifespan studies, the stability of the expression patterns of a large number of cell adhesion molecules, signaling molecules, and homing receptors will be analyzed on multiple antigen-specific populations within the same mouse. 2) Is the development of memory T cells dependent upon a distinct clonal selection step? If so, there will be detectable differences in the distribution of functional clonotypic T cell receptor (TCR) genes expressed by T cells involved in the primary response and those which form the memory compartment. These populations will be sorted using appropriate MHC tetramers. Pools of sorted cells specific for a single antigen will be analyzed by TCR spectratyping; sequences of functional TCR genes from single sorted cells will be analyzed using highly optimized RT-PCR techniques.

DESCRIPTION: (Taken from applicant's abstract) Following infection with human immunodeficiency virus type 1 (HIV-1), a quasi-steady state is achieved where viral replication appears to be balanced by the immune response and by death (and replacement) of target cell populations. Highly active antiretroviral therapy (HAART) effectively perturbs the steady state by reducing viral loads and unmasks the dynamic characteristics of the CD4+ T cell population; the effects of HAART on HIV-specific CD8+ T cell responses have not been characterized. Direct staining techniques, using soluble tetramers of human leukocyte antigen (HLA) class I complexes with peptides from HIV, allow rapid enumeration of HIV-specific CD8+ T cells and qualitative analyses of many of their phenotypic and functional properties. Preliminary studies of a small number of patients (prior to HAART) reveal frequencies of antigen specific cells often exceeding 1.5% of CD8+ cells, yet in most cases, these cells do not appear to be activated, an unexpected finding given the chronic exposure to antigen. In several cases, antigen-specific cells appear to die following in vitro stimulation with antigenic peptides. The investigator will extend these studies to monitor frequencies and phenotypes of HIV-specific CD8+ T cells as a function of time before and following the initiation of HAART, and to include greater numbers of patients. The prevalence and cause of resting phenotypes will be investigated through phenotypic analysis of cell surface markers, staining of molecules directly responsible for effector function, and analysis of potential negative regulation due to changes in signaling molecule expression or T cell antagonism. Antigen availability will be analyzed by sequencing viral epitopes to monitor CTL escape. The functional potential of the antigen-specific populations will be determined by staining of cultures stimulated with antigenic peptides in vitro for short periods of time. These experiments will reveal the fraction of the populations that proliferate, develop effector function, or are primed to die upon contact with antigen. Finally, the repertoire of the antigen-specific populations as a function of time following viral load reduction will be monitored over time by T cell receptor spectratyping analysis of magnetically sorted antigen specific cells.

DESCRIPTION (Adapted from applicant's abstract): The intent of this proposal is to develop a new assay for the functional assessment of HIV-specific cytotoxic T lymphocytes (CTL), and in the process compare the new assay with other methods for measuring these cells. This assay will be based on the recent description of a novel means of identifying and enumerating antigen-specific CTL. This new technique, tetramer binding analysis, uses soluble multimers of the major histocompatibility complex (MHC) and peptide to directly stain antigen-specific T cells. While excellent for quantitation, this technique does not address function of the identified CTL. The investigators propose to use analysis of conjugate formation between the virus-specific CTL and their specific target cells as a means to examine function in the CTL population. The experiments proposed may more clearly define the role of HIV-1-specific CTL in killing virus-infected cells. In addition, directly comparing different methods of measuring CTL may improve the ability to assess this critical arm of the immune response to viral infection. The proposal is intended to ultimately address the functional activity of real-time cellular immune responses. The application has four Specific Aims. (1) To use tetramer-specific flow cytometric techniques to examine the lytic relationship between an HIV-specific CTL clone and its target cell. (2) To determine the effect of MHC tetramer binding to T cell receptors on the function of the CTL. (3) To analyze conjugates between freshly isolated HIV-specific T cells and autologous infected target cells. (4) To compare the conjugate formation functional analysis of antigen-specific CTL with other methods of antigen-specific CTL measurement in both the HIV and murine LCMV systems.

To introduce the new and robust technology of MHC class I tetramers for the quantification of immune responses in melanoma patients involved in a multi-institutional phase III clinical trial. Recent evidence suggests that circulating CD8+ T cells in melanoma patients may be present in relatively large numbers but may be impotent. Therefore, we will assess the functional potential of melanoma-specific CD8+ T cells by examining their response to peptide stimulation using a combination of MHC tetramer staining, together with analysis of intracellular cytokine production.

DESCRIPTION: (Adapted from Applicant's Abstract) The intent of this proposal is to ascertain the effect of in vivo anti-4-1BB treatment on rhesus macaque cellular immune responses to SIV vaccination and/or infection. In so doing, we will explore new ways to stimulate SIV-specific cellular immunity and at the same time, determine the effect of this treatment on the course of disease in SIV-infected animals. Monoclonal antibodies to the 4-1BB receptor (CDw137), a member of the TNF receptor superfamily expressed on activated T cells and NK cells, preferentially stimulate CD8+ T cells in vitro and in vivo. Recent data suggests that ligation of the 4-1BB receptor on CD8+ T cells not only provides necessary co-stimulation and thus activation but may also prolong their survival. The latter effect is intriguing given the importance of CD8+ T cells in controlling viremia in both HIV and SIV infections. This proposal therefore addresses the areas of emphasis of the program announcement in that we are potentially identifying a co-stimulator that may optimize the CD8+ T cell response and ultimately be used as part of a vaccine against HIV. The specific aims are: 1) To test the in vitro effect of anti-4-1BB monoclonals on macaque lymphocytes in terms of activation, proliferation and cytokine secretion. 2) To determine the effect of anti-4-1BB monoclonals on CD8+ T cell responses induced by vaccination of Rhesus macaques with a DNA prime followed by a modified vaccinia Ankara (MVA) boost, both encoding SIVmac239 genes. 3) To administer anti-4-1BB during the course of an acute SIVmac239 infection and thus determine the effect of the treatment on viral loads, CD4 counts, anti-SIV CD8 activity and ultimately disease course.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The National Institute of Allergy and Infectious Diseases (NIAID) supports reagent programs and repositories that provide centralized resources for the research community. These facilities support research by providing reliable sources of quality assured materials, specimens, or experimental animals at reasonable cost to qualified investigators. The NIAID Tetramer Facility, established at Emory University in 1998 under the direction of John Altman, Ph.D., serves the scientific community as a centralized source of quality-controlled tetramer reagents for basic, preclinical, and clinical research. The NIAID Division of AIDS oversees the Facility, which is contracted by the AIDS Research and Reference Reagent Program with subcontracts to McKesson BioServices and Virginia Mason. The Facility has provided tetramer reagents, comprising human, murine, and non-human primate MHC alleles to researchers at non-profit organizations both in the U.S. and abroad, including investigators supported by ten NIH Institutes, the Food and Drug Administration, the Department of Defense, the Centers for Disease Control and Prevention, and private non-profit U.S. foundations, as well as to researchers in Europe, Australia, Israel, and Canada;has produced tetramers applicable to a wide range of T cell studies, including research on AIDS and other infectious diseases, cancer, organ transplantation, autoimmunity, clinical vaccine evaluation, and basic studies on T cells and immune responses;and has developed standardized methodology for tetramer usage and evaluated new advances in tetramer design and production to assure that the Facility's reagents reflect state-of-the-art capabilities.

immune response; virus antigen; cellular immunity; Primates; animal colony; T lymphocyte; microorganism immunology;

enzyme linked immunosorbent assay; serology /serodiagnosis

human immunodeficiency virus; AIDS vaccines; leukocyte activation /transformation; cytotoxic T lymphocyte; Primates; animal colony;

Macaca; animal colony;

AIDS; AIDS Virus; AIDS, International; AIDS/HIV; AIDS/HIV problem; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immune Deficiency Syndrome Virus; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Acquired Immunodeficiency Syndrome Virus; Address; Adoption; Advertising; Analysis, Data; Antibodies; Arts; Assay; Bioassay; Biologic Assays; Biological Assay; Boxing; Categories; Cell Isolation; Cell Mediated Immunology; Cell Segregation; Cell Separation; Cell Separation Technology; Cell-Mediated Immunity; Cells; Cellular Assay; Cellular Immunity; Charge; Chimera Protein; Chimeric Proteins; Clinical Trials; Clinical Trials, Unspecified; Color; Communities; Computer Analysis; Computer Programs; Computer software; Custom; Cytofluorometry, Flow; Cytokines, Chemotactic; Data; Data Analyses; Development; Environment; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Fusion Protein; Grant; HIV; HIV/AIDS; HIV/AIDS problem; HTLV-III; Hearing; Homologous Chemotactic Cytokines; Human; Human Immunodeficiency Viruses; Human T-Cell Leukemia Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Lymphotropic Virus Type III; Human, General; Immune Function, Cellular; Immunity, Cellular; Immunodeficiency Disorder; Immunodeficiency Syndrome; Immunologic Deficiency Syndrome, Acquired; Immunologic Deficiency Syndromes; Immunologic Techniques; Immunological Deficiency Syndromes; Immunological Technics; Immunological Techniques; Immunology; Immunology (Including BRMP); Immunology (NCI Program); In Vitro; Individual; Instrumentation, Other; Intercrines; International; International AIDS; Investigators; LAV-HTLV-III; Label; Laboratories; Libraries; Lymphadenopathy-Associated Virus; Mammals, Primates; Man (Taxonomy); Man, Modern; Mediating; Microfluorometry, Flow; Mission; Moab, Clinical Treatment; Monkeys; Monoclonal Antibodies; NIH Program Announcements; PBMC; Pathogenesis; Performance; Peripheral Blood Mononuclear Cell; Prevention; Price; Primates; Production; Program Announcement; Purpose; Reagent; Research; Research Personnel; Research Resources; Researchers; Resource Sharing; Resources; SIS cytokines; Sampling; Scientist; Services; Software; Sorting - Cell Movement; Staining method; Stainings; Stains; Standardization; Technics, Immunologic; Training; Training and Education; Vaccinated; Validation; Viral Diseases; Virus Diseases; Virus-HIV; Visit; Work; assay development; cell mediated immune response; cell sorting; chemoattractant cytokine; chemokine; clinical investigation; computational analysis; computer program/software; cytokine; data acquisition; design; designing; experiment; experimental research; experimental study; flow cytophotometry; hearing perception; hypoimmunity; immune deficiency disorder; immune function; immunodeficiency; in vivo; innovate; innovation; innovative; instrument; instrumentation; member; method development; microbial; multidisciplinary; non-human primate; nonhuman primate; novel; pathogen; pre-clinical; preclinical; pricing; repository; research study; sorting; sound perception; therapeutic vaccine; vaccine development; vaccine efficacy; viral infection; virus infection

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of this project has been to identify strategies by which the immunogenicity of AIDS vaccines that are derived from Modified Vaccinia Ankara (MVA)-based viral vectors can be significantly augmented. In this reporting period, we determined that combinatorial deletion of several endogenous poxvirus immune-evasion genes from the backbone of MVA-based AIDS vaccine vectors confers an enhanced ability upon such vectors to elicit HIV-specific CD8 and CD4 T cell and antibody responses in MHC-disparate populations of rhesus macaques. The capacity of engineered MVA vectors that express SIV angtigens to elicit systemic and mucosal cellular immunity and to protect against pathogenic SIV challenge has also been investigated in Mamu-A*01-positive rhesus macaques. The development of highly immunogenic AIDS vaccine vectors from platforms other than adenovirus serotype-5 remains a high priority for the field and for future clinical assessments of the T cell hypothesis of AIDS vaccines.

51 chromium release assay; ATGN; Alleles; Allelomorphs; Antigenic Determinants; Antigens; Assay; Binding Determinants; Bioassay; Biologic Assays; Biological Assay; CD8; CD8B; CD8B1; CD8B1 gene; CML test; CTL; Cell Communication and Signaling; Cell Signaling; Cell-Mediated Lympholytic Cells; Cells; Class; Clinical Trials; Clinical Trials, Unspecified; Collaborations; Color; Coloring Agents; Complement; Complement Proteins; Complex; Cytofluorometry, Flow; Cytolytic T-Cell; Cytotoxic T Cell; Cytotoxic T-Lymphocytes; Detection; Development; Dyes; Epitope Mapping; Epitopes; Epitopes, T-Lymphocyte; Flow Cytofluorometries; Flow Cytometry; Flow Microfluorimetry; Frequencies (time pattern); Frequency; Genes; Human; Human, General; Immune; Immune response; In complete remission; Individual; Instrumentation, Other; Intracellular Communication and Signaling; LYT3; Lytic; Man (Taxonomy); Man, Modern; Manufacturer; Manufacturer Name; Maps; Memory; Methods; Methods and Techniques; Methods, Other; Microfluorometry, Flow; ORFs; Open Reading Frames; Peptide Library; Peptides; Phenotype; Population; Poxvirus officinale; Preparation; Production; Property; Property, LOINC Axis 2; Protein Coding Region; Proteins; Q-Dot; Quantum Dots; Reagent; Recombinants; Research Resources; Resolution; Resources; Signal Transduction; Signal Transduction Systems; Signaling; Smallpox; Smallpox Vaccine; Specificity; Staining method; Stainings; Stains; Standards; Standards of Weights and Measures; T Cell Specificity; T-Cell Epitopes; T-Cell Immunologic Specificity; T-Cells; T-Lymphocyte; T-Lymphocyte Epitopes; T-Lymphocytes, Cytotoxic; Techniques; Technology; Thymus-Dependent Lymphocytes; VSV; Vaccines; Vaccinia; Vaccinia virus; Variola; Vesicular Stomatitis Virus; Vesicular stomatitis Indiana virus; Work; Yellow fever virus; base; biological signal transduction; cell mediated lymphocytolysis test; chromium release assay; clinical investigation; complete response; cytokine; flow cytophotometry; gene product; host response; immunogen; immunoresponse; improved; instrument; instrumentation; novel; pathogen; recombinant vaccinia virus; response; small pox; small pox vaccine; technique development; thymus derived lymphocyte; tool; variola major; vector

In the period immediately following infection, many viruses cause a transient, type I interferon-dependent lymphopenia. The reason that mammalian hosts adopt such a strategy has been the subject of considerable speculation, but few of the proposals have been completely satisfactory. Recently, we made the unexpected discovery that a strain of lymphocytic choriomeningitis virus (LCMV) that is normally rapidly cleared by immmunocompetent mice[unreadable]the Armstrong strain[unreadable]induces a profound lymphopenia, but the clone 13 strain, which establishes a high level chronic infection, does not. In order to test the hypothesis that the failure of clone 13 to induce lymphopenia was associated with the failure of mice to clear clone 13, we induced transient lymphopenia during the acute phase of infection by treatment with the drug FTY720, a sphingosine analog that sequesters lymphocytes in lymphoid organs by blocking signals required for their exit. The results were stunning: a transient, three day course of FTY720 at days 0, 1, and 2 of the infection promoted complete clearance of clone 13, including from organs such as the kidneys where the virus normally persists for months. We then obtained a result that is potentially even more important: a transient course of FTY720 given at 30 days post clone 13 infection also induced complete clearance of the virus. In both experiments, clearance was completely dependent upon CD4 cells, demonstrating that the drug is not acting directly on the virus. These discoveries raise a number of important questions that we will address in this grant. In Aim 1, we will explore the mechanisms through which FTY720 is promoting clearance of LCMV. In Aim 2, we will ask whether FTY720 also induces reversal of LCMV-induced generalized immunosuppression. Finally, in Aim 3, we will ask whether FTY720 also improves immune responses to other viral infections. For these experiments we will turn to four well established mouse models of viral infection: lethal intranasal infection with vaccinia virus, lethal intranasal infection with influenza virus, infection of newborn tumor-susceptible mice with polyoma virus, and establishment of latent infection with gammaherpesvirus 68. Treatment with FTY720, which acts on hostimmune cells and has no direct specific antiviral effects, might prove useful in treating chronic viral infections in humans, such as HIV, HBV, or HCV.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Building upon surprising results obtained in our laboratory in the mouse/LCMV model, this project was designed to test the ability of FTY720[unreadable]a drug that blocks exit of lymphocytes from lymph nodes and that has been through phase II clinical trials for treating multiple sclerosis and preventing kidney transplant rejection[unreadable]to enhance the immunological control of SIV in chronically infected rhesus macaques. We had discovered a completely novel and surprising immunotherapy that leads to complete clearance of an otherwise chronic infection of LCMV in the mouse. Our work is focusing on one of two treatment modes in the SIV/NHP model: (1) immediate post-exposure treatment, or (2) treatment of an established of chronic infection. The latter was chosen because of (1) its greater potential impact (in the context of treating HIV), and (2) its likelihood to produce data that is more interpretable because it allowed us to determine drug-associated changes in viral load relative to known set points (allowing us to use fewer animals). There are three possible results for these experiments: (1) no change in viral load;(2) a drug associated increase in viral load, possibly due to concentrating target cells for infection (CD4+) in lymph nodes;or (3) a drug associated decrease in viral load, as in the LCMV model. If the latter is observed, human clinical trials may be warranted, and it helps that FTY720 has been extensively tested in human clinical trials for treatment of MS and solid organ transplantation. We are still evaluating the data.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles;class II reagents for mice and human alleles;mouse and human CD1d tetramers;and human CD1a tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles;class II reagents for mice and human alleles;mouse and human CD1d tetramers;and human CD1a tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In the period immediately following infection, many viruses cause a transient, type I interferon-dependent lymphopenia. The reason that mammalian hosts adopt such a strategy has been the subject of considerable speculation, but few proposals have been completely satisfactory. In our past work, we made the unexpected discovery that a strain of lymphocytic choriomeningitis virus (LCMV) that is normally rapidly cleared by immmunocompetent mice[unreadable]the Armstrong strain[unreadable]induces a profound lymphopenia, but the clone 13 strain, which establishes a high level chronic infection, does not. In order to test the hypothesis that failure of clone 13 to induce lymphopenia was associated with failure of mice to clear clone 13, we induced transient lymphopenia during the acute phase of infection by treatment with drug FTY720, a sphingosine analog that sequesters lymphocytes in lymphoid organs by blocking signals required for exit. The initial results were impressive: a transient, three day course of FTY720 at days 0, 1, and 2 of infection promoted complete clearance of clone 13, including from organs such as kidneys where virus normally persists for months. We then obtained a result that is potentially even more important: a transient course of FTY720 given at 30 days post clone 13 infection also induced complete clearance of virus. In both experiments, clearance was completely dependent upon CD4 cells, demonstrating that the drug is not acting directly on virus. Our most recent work has sought to validate these results.

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to develop a safe and efficacious AIDS vaccine. A number of candidate AIDS vaccines derived from the attenuated poxvirus Modified Vaccinia virus Ankara (MVA) have been (or currently are being) evaluated in clinical trials. However, a number of factors limit the immunogenicity and utility of these vaccine candidates. These include priming HIV-specific T cell responses that are of limited breadth, due to unfavorable antigenic competition with poxvirus proteins encoded by the vector, as well as the development of potent anti-vector immunity, following immunization with recombinant MVA vaccines, that progressively diminishes the effectiveness of homologous booster immunizations to boost HIV-specific T and B cell responses. As a result, and in light of the STEP trial results, there is an urgent need to discover new viral vector modifications and immunization strategies that maximize the magnitude, quality, and breadth of HIV-specific T cell responses that are elicited by vaccine vectors. In addition, it is quite likely that an AIDS vaccine will need to engender broadly neutralizing antibody responses, in addition to CTLs, to be maximally effective. As such, it is imperative to identify the best vectors and methodologies for presenting relevant envelope antigens (once developed) in order to elicit high-titer neutralizing antibody responses that are durable. Toward achieving these goals, we propose to evaluate a number of novel vector modifications that are hypothesized to enhance the cellular and humoral immunogenicity of MVA-based AIDS vaccines, and to mitigate the negative effects of pre-existing, or immunization-induced, vector-specific immunity. We will pursue the following specific aims: 1) We will test the hypothesis that the immunogenicity of recombinant MVA-based AIDS vaccines, particularly in hosts exhibiting vector-specific immunity, can be significantly enhanced through vector modifications that delete relevant determinants of MVA-specific humoral immunity and that attenuate the ability of complement to neutralize virion infectivity or to facilitate the clearance of MVA-transduced cells in vivo;2) We will test the hypothesis that the breadth and magnitude of HIV-specific T cell and antibody responses that are elicited by recombinant MVA-based AIDS vaccines can be significantly enhanced through vector modifications that specifically target antigenic stimulation of TLR-5-mediated innate immune signaling pathways. PUBLIC HEALTH RELEVANCE: The world desperately needs an AIDS vaccine. We propose to develop novel Modified Vaccinia Ankara (MVA)-based AIDS vaccines that are more immunogenic than vectors derived from the parental strain of MVA. Rationally improving MVA vectors to elicit higher levels of more highly diverse HIV-specific T cell and antibody responses and to mitigate their induction of, and sensitivity to, vector-specific neutralizing antibodies, should result in new candidate AIDS vaccines that exhibit greater efficacy than current alternatives.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protection against many intracellular pathogens is provided by CD8 T cells, and pathogen-specific CD8 T cells are thought to need CD4 T cell help to develop into effective memory CD8 T cells. Since murine CD8 T cells do not transcribe MHC class II genes, several models have proposed antigen presenting cells (APCs) as intermediaries required for CD4 T cells to deliver their help to CD8 T cells. We have demonstrates the presence of MHC class II molecules on activated murine CD8 T cells in vitro as well as in vivo. These CD8 T cells acquire MHC class II from their activating APCs, particularly CD11c positive dendritic cells (DCs), via a process called trogocytosis. Transferred MHC class II molecules on activated murine CD8 T cells were functionally competent and could directly stimulate specific indicator CD4 T cells. CD8 T cells that were "helped" in vitro and subsequently allowed to rest in vivo showed enhanced recall responses upon challenge compared to "helpless" CD8 T cells;in contrast, no differences were seen upon immediate challenge. These data indicate that direct CD8:CD4 T cell interactions may significantly contribute to help for CD8 T cells. Furthermore, this mechanism may enable CD8 T cells to communicate with different subsets of interacting CD4 T cells that could modulate immune responses. These studies could have implications for HIV/AIDS research.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. For assessment of human T cell responses to influenza A and B viruses, we have constructed a large panel of replication-restricted, recombinant vesicular stomatitis viruses, each expressing a single protein from a variety of influenza strains. The complete panel covers the HA and NA proteins from influenza vaccine strains used from 2000-2011, as well as the internal components of the A/California/04/09 strain from the recent pandemic, the PR8 strain that is used for production of the conventional vaccine, and the A/Ann Arbor/06/60 strain that is used for the production of the live-attenuated FluMist vaccine. We are using these to measure T cell responses in 60 donors who were vaccinated during the 2010-2011 season. In general, CD8 T cell responses to vaccination have been low-to-undetectable, a result that was not unexpected, especially for the conventional inactivated vaccine. In contrast, CD4 T cell responses are more frequent, with a response range of 0-631 per million CD4 cells.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles;class II reagents for mice and human alleles;mouse and human CD1d tetramers;and human CD1a tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles; class II reagents for mice and human alleles; mouse and human CD1d tetramers; and human CD1a tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles; class II reagents for mouse, non-human primate and human alleles; mouse and human CD1d tetramers; and human CD1a-c tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles; class II reagents for mouse, non-human primate and human alleles; mouse and human CD1d tetramers; and human CD1a-c tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.

The NIH Tetramer Facility provides custom synthesis and distribution of soluble major histocompatibility complex (MHC)-peptide tetramer reagents that can be used to detect antigen-specific T cells. These reagents include custom class I tetramers for mouse, non-human primate, and human alleles; class II reagents for mouse, non-human primate and human alleles; mouse and human CD1d tetramers; and human CD1a-c tetramers. The NIH Tetramer Facility also is developing novel technologies to improve production and expand the range of available MHC and CD1 tetramers. The tetramer reagents can be applied to studies ranging from basic immunology and protection against microbial pathogens to control of immune-mediated diseases and tumor metastases.