Comparative Medicine And Translational Research Training Program
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
Grant 1T32RR024394-01A1 from National Center For Research Resources, IRG: RIRG
Abstract: The National Research Council has documented a dire national need for veterinary specialists trained in biomedical research. Furthermore, veterinary researchers play a key role in comparative and translational research activities since they naturally bridge basic and clinical research. To address this training need, faculty in the CCMTR (CCMTR) at the College of Veterinary Medicine, NC State have established the Comparative Medicine and Translational Research Training Program (CMTRTP). This training program specifically target individuals with the DVM degree who have completed specialty training. Trainees will complete requirements leading to the PhD degree in laboratories of well-funded faculty that have a strong training track record and diverse research expertise. Research projects will emphasize comparative and translational themes fostered by the CCMTR and trainees will participate in multidisciplinary research efforts. It is envisioned that trainees will bridge research programs and serve as a nidus for new faculty collaborations. The training program was initiated in Fall 2007 with funds for 2 slots committed by North Carolina State University. The program will grow by 2 slots each year, reaching a total of 8 slots in year 4. Six trainee slots are requested and 2 will be supported from university resources. Program requirements include (1) a capstone course on comparative medicine and translational research; (2) a professional development course that will culminate in submission of a K award proposal; (3) a seminar series on translational research; (4) a course in research ethics; and (5) an annual research symposium. These requirements are in addition to those associated with a student´s particular graduate program. This novel training program will build upon the strong commitment and track record of the NC State CVM to train veterinary specialists in research
Project start date: 2008-08-01
Project end date: 2013-05-31
Sponsored Links Lab Supply Mall http://www.labsupplymall.com
Grants awarded to Gregg A Dean
In Vivo Significance Of T-reg Cells During FIV Infection
Gregg A Dean, Professor
North Carolina State University Raleigh Sponsored Programs And Regulatory Compliance Raleigh, Nc 27695
Grant 5R21AI065223-02 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1
Abstract: We have described CD4+CD25+ Treg cells in the cat and demonstrated that they are chronically activated in feline immunodeficiency virus (FIV) infected animals. Treg cells from FIV-infected cats significantly inhibited proliferation of CD4+CD25- T cells, and are apparently activated in vivo as a result of the chronic FIV infection. As activated Treg cells are non-antigen specific in their suppressive function, it is possible that these cells could in turn suppress or anergize CD4+ T helper responses to a variety of antigens including FIV antigen and thereby contribute to the acquired immunodeficiency syndrome (AIDS) characteristic of this infection. The same observation has recently been reported in HIV-1 infected people and interestingly, increased levels of Treg function were associated with a favorable clinical status or respose to HAART. Normally, Treg function is critical to limit and down-regulate immune responses, preventing excessive inflammation and autoimmune disease. Whether Treg cells in HIV infection are beneficial or detrimental is unclear and the answer may depend on the stage of disease. It is possible that HIV-induced Treg activity during acute infection may limit the scope and efficacy of the anti-viral response, whereas Treg activity during asymptomatic infection may serve to control chronic immune activation and limit availability of activated target cells. The most definitive way to address the significance of Treg function during HIV infection is to deplete Treg cells in an animal model. In these studies we will employ the FIV/cat model of HIV to 1.) Determine whether depletion of Treg cells before or immediately after FIV infection results in a more effective anti-viral immune response, and 2.) Determine whether depletion of Treg cells during the asymptomatic phase of FIV infection results in the expansion of antiviral T-cell immunity and/or change in plasma viremia. Results from these studies will provide a clinical understanding of Treg function in HIV infection and will guide future approaches to modulate Treg function.
Keywords: Retroviridae disease, T lymphocyte, feline immunodeficiency virus, immunoregulation, pathologic process, HIV infection, apoptosis, cell cell interaction, disease /disorder model, immune response, immunopathology, cat
Project start date: 2005-06-15
Project end date: 2007-11-30
5R21AI065223-02 (2006): $285138
1R21AI065223-01A1 (2005): $292000
Optimizing Immunogenicity Of Lactobacillus As A Mucosal Vaccine Against HIV-1
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
sponsored Programs And Regulatory Compliance
raleigh, Nc 27695
Grant 1R21AI077409-01A1 from National Institute Of Allergy And Infectious Diseases, IRG: VACC
Abstract: Transmission of HIV-1 occurs predominately at mucosal surfaces and therefore a successful vaccine against HIV-1 must induce mucosal humoral and cellular immune responses. Lactobacillus is a very common commensal of the human gastrointestinal tract and is used commercially to process and preserve food, thus it is safe and unlikely to stimulate anti-vector immune responses. Futhermore, Lactobacillus is easy to manufacture, store and is highly manipulatable as the functional genomics are well understood. The well-known probiotic activities are due at least in part to the interaction of Lactobacillus with dendritic cells. Lactobacillus activates DC and stimulates cytokine production that leads to Th1 polarization of T cells. We have performed preliminary immunogenicity studies and demonstrated proof-of-principle for the use of Lactobacillus as a vaccine vector against HIV-1 but the vector must be further optimized to increase immunogenicity. In the studies proposed here, we will investigate the host/vector interaction to determine the kinetics and location of gastrointestinal colonization by recombinant Lactobacillus and whether recombinant protein expression affects the indigenous flora. The optimal subcellular location for HIV-1 antigen expression will be determined and a comprehensive assessment of mucosal and systemic humoral and cellular immune responses will be performed in mice. To further increase immunogenicity, flagellin will be expressed by the Lactobacillus as a method to increase innate immune activation in the gastrointestinal tract via TLR5 stimulation. Together these studies will determine whether Lactobacillus is worth further development as an oral vaccine vector against HIV-1. The purpose of these studies is to develop a novel HIV vaccine strategy using Lactobacillus. A vaccine using lactobacilli would be safe, inexpensive to manufacture, and could be delivered orally. If successful, this strategy could have a significant impact on HIV prevention
Project start date: 2008-02-15
Project end date: 2010-01-31
Recombinant Lactobacillus As An Oral Mucosal Vaccine Against HIV-1
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
sponsored Programs And Regulatory Compliance
raleigh, Nc 27695
Grant 1R01DE019069-01 from National Institute Of Dental & Craniofacial Research, IRG: ZDE1
Abstract: Transmission of HIV-1 occurs predominately at mucosal surfaces and therefore a successful vaccine against HIV-1 must induce mucosal humoral and cellular immune responses. Lactic acid bacteria have been used for centuries to process and preserve food and are well known for their probiotic activities. Recently members of the lactic acid bacteria family have been explored as vaccine vectors. Lactobacillus gasseri is a common commensal of the human oral cavity and gastrointestinal tract and may be particularly useful as an oral mucosal vaccine vector against HIV-1. L. gasseri colonizes the oral cavity and serves as a source for colonization of the intestinal tract. In addition, L. gasseri activates dendritic cells and stimulates cytokine production that leads to Th1 polarization of T cells. In preliminary studies, recombinant L. gasseri expressing HIV-1 Gag induced serum IgG, mucosal IgA and cell mediated responses measured in the colon and reproductive tract after a single oral dose. Here we propose to better determine the dose, distribution and persistence of L. gasseri after oral immunization. We will then refine L. gasseri as an immunogen by co-expressing flagellin as a means to stimulate the innate immune system through TLR5 and determining whether L. gasseri can deliver a DNA vaccine for the eukaryotic expression of Env. The role of regulatory T cells in mucosal immunization will be assessed by depleting Treg concomitant with oral immunization. Finally, a robust challenge study will be performed to determine whether L. gasseri can induce protective immunity. These studies will be performed using the cat/FIV model of HIV-1 since cats have oropharyngeal mucosa associated lymphoid tissue similar to humans, can be economically studied using statistically relevant numbers, and can be vaginally challenged with pathogenic FIV using cell-associated and cell-free virus. Project Narrative Transmission of HIV-1 occurs predominately at mucosal surfaces and therefore a successful vaccine against HIV-1 must induce mucosal humoral and cellular immune responses. Lactobacillus gasseri is a common probiotic bacterium found in the human oral cavity and gastrointestinal tract and as such may be a particularly effective vaccine vector against HIV-1. Studies proposed here will investigate strategies to engineer recombinant L. gasseri that can induce a protective immune response against HIV-1 via the oral mucosa
Project start date: 2008-03-20
Project end date: 2013-01-31
CHARACTERIZATION/MODULATION OF CYTOKINE RESPONSES IN FIV
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
sponsored Programs And Regulatory Compliance
raleigh, Nc 27695
Grant 5R29AI040407-05 from National Institute Of Allergy And Infectious Diseases, IRG: ARRA
Abstract: The objective of this proposal is to comprehensively characterize cytokine mRNA and protein expression in the lymphoid tissues of FIV-infected cats and attempt to modulate the cytokine response with anti-cytokine antibodies and recombinant Listeria monocytogenes. Hypothesis Inappropriate cytokine production contributes to the ineffective immune response to FIV, the patho- genesis of FIV, and the susceptibility of FIV infected cats to opportunistic infections. Modulation of cytokines may be useful to study the efficacy of therapeutic interventions and vaccine strategies, as well as the pathogenesis of FIV. Specific Aim 1 to determine the effect of FIV infection on cytokine mRNA and protein production in tissues of cats, multiple lymphoid compartments will be evaluated by QC-RT-PCR to quantify tissue production of cytokine mRNA, by in situ hybridization to localize mRNA within the tissue structure, and by immunocytochemistry, ELIspot, and flow cytometry to determine cytokine protein production within specific cell phenotypes. Specific Aim 2 to determine the significance of IL2, IL4, IL10, IL12, TNF-alpha and IFN-gamma in the immunopathogenesis of FIV, anti-IL2, anti-IL4, anti-IL10, anti-IL12p40, anti-TNF-alpha or anti-IFN-gamma monoclonal and polyclonal antibodies will be administered to acutely FIV infected cats. The effects of cytokine inhibition on viral replication, immune response, and disease progression will be determined. Specific Aim 3 To determine if a strong cell-mediated immunity to FIV is protective, SPF cats will be vaccinated with live recombinant biological vaccine vector; FIVenv- and/or FIVgag- expressing Listeria monocytogenes. Cats will then be challenged with homologous and heterologous FIV and the immune response and cytokine profile associated with either protective or non- protective immunity will be evaluated. Significance Lentivirus- associated immunodeficiency is an expanding global problem affecting humans and cats, however the immunopathogenesis is incompletely understood and current therapies are limited. Understanding the role of the cytokines in the pathogenesis, therapy, and vaccination in FIV infection will have direct implications for HIV infection
Keywords: cytokine, feline immunodeficiency virus, protein biosynthesis Listeria, antibody, cellular immunity, interferon gamma, interleukin 10, interleukin 12, interleukin 2, interleukin 4, messenger RNA, tumor necrosis factor alpha, vector vaccine cat, flow cytometry, immunocytochemistry, in situ hybridization, polymerase chain reaction
Project start date: 1997-08-01
Project end date: 2003-02-28
5R29AI040407-05 (2001): $81125
5R29AI040407-04 (2000): $81125
5R29AI040407-02 (1998): $118000
1R29AI040407-01A2 (1997): $132750
Comparative Medicine And Translational Research Training Program
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
Grant 1T32RR024394-01A1 from National Center For Research Resources, IRG: RIRG
Abstract: The National Research Council has documented a dire national need for veterinary specialists trained in biomedical research. Furthermore, veterinary researchers play a key role in comparative and translational research activities since they naturally bridge basic and clinical research. To address this training need, faculty in the CCMTR (CCMTR) at the College of Veterinary Medicine, NC State have established the Comparative Medicine and Translational Research Training Program (CMTRTP). This training program specifically target individuals with the DVM degree who have completed specialty training. Trainees will complete requirements leading to the PhD degree in laboratories of well-funded faculty that have a strong training track record and diverse research expertise. Research projects will emphasize comparative and translational themes fostered by the CCMTR and trainees will participate in multidisciplinary research efforts. It is envisioned that trainees will bridge research programs and serve as a nidus for new faculty collaborations. The training program was initiated in Fall 2007 with funds for 2 slots committed by North Carolina State University. The program will grow by 2 slots each year, reaching a total of 8 slots in year 4. Six trainee slots are requested and 2 will be supported from university resources. Program requirements include (1) a capstone course on comparative medicine and translational research; (2) a professional development course that will culminate in submission of a K award proposal; (3) a seminar series on translational research; (4) a course in research ethics; and (5) an annual research symposium. These requirements are in addition to those associated with a student´s particular graduate program. This novel training program will build upon the strong commitment and track record of the NC State CVM to train veterinary specialists in research
Project start date: 2008-08-01
Project end date: 2013-05-31
Interacton Of HIV With Toll-like Receptors
Gregg A Dean, Professor
North Carolina State University Raleigh Sponsored Programs And Regulatory Compliance Raleigh, Nc 27695
Grant 5R03AI058855-02 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1
Abstract: The innate immune system is responsible for the elimination of most pathogens encountered by a healthy individual. Several defects in innate immunity have been associated with HIV-1 infection resulting in increased susceptibility to opportunistic and secondary infections. The underlying mechanisms for innate immune dysfunction during HIV-1 infection are largely unexplored. A family of receptors that recognize pathogen-associated molecular patterns (PAMP) have been identified in humans and are called toll-like receptors (TLR). It is now clear that TLR provide some specificity to the innate immune response as a result of the PAMP recognized, the cellular distribution of different TLR, and the unique down-stream effects of TLR ligation and signal transduction. TLR are at the initial interface between a potential pathogen and the immune system, and in this role, TLR initiate the inflammatory response and induce the adaptive immune response. Because of the pivotal role of TLR in immune responses, it is reasonable to consider whether HIV-1 affects TLR function as has been observed with several other viruses. Here we propose to address three questions Firstly, does HIV induce signal transduction through TLR that could lead to chronic immune stimulation or target cell recruitment? Secondly, does HIV-1 affect TLR expression? Thirdly, does HIV-1 impair TLR function. Through these studies we will learn if HIV/TLR interaction contribute to the immunodeficiency caused by HIV-1.
Keywords: human immunodeficiency virus 1, immune response, intermolecular interaction, toll like receptor, biological signal transduction, messenger RNA, protein structure function, receptor expression, cell line, enzyme linked immunosorbent assay, polymerase chain reaction
Project start date: 2003-12-15
Project end date: 2005-11-30
5R03AI058855-02 (2005): $73000
1R03AI058855-01 (2004): $73000
CYTOKINE RESPONSE IN FIV AND SIV IMMUNODEFICIENCY
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
sponsored Programs And Regulatory Compliance
raleigh, Nc 27695
Grant 5K02AI001529-04 from National Institute Of Allergy And Infectious Diseases, IRG: AIDS
Abstract: The application proposes independent investigator support for a candidate who has been involved in retrovirus research since 1986 and has participated in a number of original and significant studies on the identification of lentivirus target cells, effects of opportunistic infections on immunodeficiency virus pathogenesis, and the role of cytokines in immunodeficiency virus pathogenesis. The objective of this application is to provide salary support, over five years, to reduce the clinical component of the candidates current tenure-track appointment, thus allowing development of his research program and career. The increased time dedicated to research would also allow development of new collaborations. The candidate is in a supportive environment with several central equipment laboratories and service centers. The candidates research focus concerns the role of cytokines in the immunopathogenesis of lentivirus infections and immunomodulatory therapeutic and vaccination strategies to treat or prevent lentivirus infection. a. Candidate Dr. Gregg Dean received his D.V.M. degree in 1988 and his Ph.D. degree in 1991 both from Colorado State University, Ft. Collins. He then trained as a Post Doctoral Fellow at the University of California, Davis, until 1994, where he then served as Faculty Research Virologist, until 1996. Later that year, he was appointed to his current position of Assistant Professor at the College of Veterinary Medicine, Department of Microbiology, Parasitology, and Pathology at North Carolina State University (NCSU). b. Career Development Plan Plans are presented to extend the candidate´s research program by expanding his technical staff and number of graduate students, to maintain a well funded research program, to train research scientists, and to pursue funding for remodeling of the laboratory animal facilities allowing the establishment of a specific-pathogen-free cat colony at NCSU. c. Research Plan The core research project (a 5-year R29 funded 7/11/97) consists of three specific aims. First, to comprehensively evaluate cytokine mRNA and protein production within multiple lymphoid compartments of Fly-infected cats, determine phenotype of cytokine expressing cells, and localize cytokine production defects. Second, to determine the significance of IL4, IL10, IL12, TNF-a and IFN-gamma in the immunopathogenesis of FIV. Third, to evaluate L. monocytogenes as a biological vaccination vector and determine the correlates of successful or unsuccessful immunity. The candidate is addressing three additional specific aims through collaborative studies. Fourth, to determine the effect of simian immunodeficiency virus (SIV) infection on cytokine mRNA and protein production in tissues of infant macaques treated and not treated with PMPA. Fifth, to determine cytokine mRNA and protein expression in the thymus of pathogen-free and Fly-infected cats. Sixth, to determine the effect of treatment with insulin-like growth factor-1 on cytokine expression in the thymus and other lymphoid organs in pathogen-free and Fly-infected cats. d. Environment and Institutional Commitment NCSU has provided the candidate with a private office, 300 sq. ft. of laboratory space, startup funds, access to the central equipment laboratory, and three years of support for a technical assistant
Keywords: cytokine, feline immunodeficiency virus, immunodeficiency, protein biosynthesis, simian immunodeficiency virus AIDS vaccine, HIV infection, Listeria, disease /disorder model, insulinlike growth factor, interferon alpha, interferon gamma, interleukin 10, interleukin 12, interleukin 4, vaccine development, vector vaccine Macaca, cat, in situ hybridization, polymerase chain reaction
Project start date: 1998-08-01
Project end date: 2003-07-31
5K02AI001529-04 (2001): $96080
5K02AI001529-03 (2000): $96080
5K02AI001529-02 (1999): $67068
1K02AI001529-01 (1998): $67068
CYTOKINE-MEDIATED PATHOGENESIS OF HIV INFECTION
Gregg A Dean, Professor
Medical Pathologyuniversity Of California Davis
office Of Research - Sponsored Programs
davis, Ca 95618
Grant 1K11AI001262-01 from National Institute Of Allergy And Infectious Diseases, IRG: SRC
Abstract: Proposed here is a Phase II research program designed to provide the candidate the opportunity to obtain research experience, initiate a research program, and develop into an independent investigator able to successfully compete for NIH research support. Feline immunodeficiency virus (FIV) induces a moderate to severe acute disease followed by a long clinical latency that, in nature, culminates in terminal acquired immunodeficiency syndrome (AIDS). While some experimentally infected cats do progress to AIDS, many do not, providing the opportunity to compare a successful to an unsuccessful host immune response against an immunodeficiency-inducing lentivirus. Two hypotheses are proposed to explain this dichotomy. Firstly, a primarily cellular immune response may predominate in FIV-infected cats that remain in a clinically latent phase, while animals progressing to AIDS may have a diminished cellular immune response. Whether an immune response is primarily cellular (T- helper lymphocyte type 1(Th1) predominates) or humoral (Th2 predominates) is reflected in the cytokine profile and may determine whether the immune system can contain viral replication and disease progression or not. Therefore, by quantitating viral DNA and RNA, as well as, mRNA levels of IL2, IL4, IL10, and INF-g in peripheral blood mononuclear cells and lymphoid tissues of experimentally FIV-infected cats at various time post inoculation, the correlation of particular cytokine profiles with status and degree of viral replication can be determined. By similarly evaluating naturally infected cats with AIDS to identify cytokine profiles characteristic of end-stage disease, changes in cytokine levels that may be indicative of a shift from Th1 to Th2 predominance. A second hypothesis is that maintenance of normal TNF-a levels may explain why cats remain in the clinically latent phase of disease. TNF-a is elevated in HIV-infected people with AIDS and approximately 50% of patients with ARC as well as in some individuals with lymphadenopathy. However, while serum TNF-a levels of FIV-infected cats are transiently elevated during the acute phase of infection, levels return to normal. These data suggest TNF-a may be play an important role in the progression of immune- deficiency. This hypothesis will be tested by administering TNF-a continuously to FIV-infected cats in clinical latency using subcutaneous osmotic pumps, and measuring viral DNA, viral RNA and cytokine mRNA production as well as monitoring the clinicopathologic condition of cats to see if TNF-a induces end-stage disease (AIDS)
Keywords: HIV infection, cytokine, feline immunodeficiency virus, pathologic process cellular immunity, helper T lymphocyte, humoral immunity, interferon gamma, interleukin 2, interleukin 4, tumor necrosis factor alpha biopsy, cat, polymerase chain reaction, radioimmunoassay
Project start date: 1994-09-01
Project end date: 1997-08-31
1K11AI001262-01 (1994): $87480
Basis And Modulation Of The Innate Immune Defect In FIV
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
sponsored Programs And Regulatory Compliance
raleigh, Nc 27695
Grant 5R01AI047707-06 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1
Abstract: Deficiencies in the innate immune response of HIV infected people and FIV infected cats are well documented. The defect often presents clinically as reduced competence in eliminating intracellular opportunistic pathogens. Not only is innate immunity responsible for the control and elimination of the majority of opportunistic infections, it is the innate response that initiates the type and strength of the adaptive immune response. We have employed Listeria monocytogenes (LM) as an immune system probe to investigate the effects of FIV infection on innate immune function. We have found that the innate immune response to LM is impaired in acutely and chronically FIV infected cats. We also determined that locally administered IL15 could improve the innate immune response and that this was associated with increased NK cells in draining lymph nodes. Activated, mature dendritic cells (DC) play a key role in initiating the innate immune response by recruiting and activating NK cells and modulating Treg function. It is now clear that cross-talk between DC, NK cells and Treg is critical for coordination of the innate response. As such, these three cell types create a fundamental regulatory axis for innate responses and subsequent adaptive responses. We will determine the contribution of each of these cell types to the innate immune defect associated with FIV infection. Specific Aim 1 will determine whether incomplete DC maturation could underlie innate immune dysfunction during lentivirus infection and whether dendritic cells in FIV infected animals respond normally to TLR ligands. Specific Aim 2 will determine the nature and mechanism of NK cell dysfunction in vivo. Specifically, this aim will assess NK cell chemotaxis, proliferation and function during an innate immune challenge of FIV+ cats, whether DC-NK cross-talk results in NK cell activation, and whether systemic IL15 treatment restores innate immune function. Specific Aim 3 will determine if regulatory T cells suppress innate immune function in FIV infected cats, whether such an effect is mediated by TGF-a and if Treg are preferentially induced by DC. Together these studies will elucidate the cellular mechanisms that underlie the innate immune defect and investigate potential molecular mechanisms
Keywords: cellular immunity, feline immunodeficiency virus, humoral immunity, microorganism immunology B lymphocyte, Lentivirus, T lymphocyte, gene delivery system, immunologic memory, interferon gamma, interleukin 10, interleukin 15, macrophage, natural killer cell, recombinant protein, tumor necrosis factor alpha, virus protein cat, tissue /cell culture
Project start date: 2001-07-01
Project end date: 2011-06-30
2R01AI047707-05A2 (2007): $334125
1R01AI047707-01A2 (2001): $306375
5R01AI047707-04 (2004): $256375
5R01AI047707-03 (2003): $256375
RECOMBINANT LISTERIA--BIOLOGIC VACCINE VECTOR FOR FIV
Gregg A Dean, Professor
Molecular Biomedical Sciencesnorth Carolina State University Raleigh
sponsored Programs And Regulatory Compliance
raleigh, Nc 27695
Grant 5R21AI047046-02 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1
Abstract: Current data suggest a successful vaccine against the human immunodeficiency virus (HIV) must l. induce an immune response at mucosal barriers, 2. induce a strong cytotoxic T-lymphocyte (CTL) response, and 3. induce an immune response that will be protective against multiple viral subgroups. Proposed here are studies to explore the use of recombinant Listeria monocytogenes as a biologic vaccine vector against lentivirus infections. The cell mediated immune response induced by Listeria monocytogenes infection is well characterized. L. monocytogenes infects macrophages, the central player in the immune response. After infection the bacterium enters the cytoplasm where it secretes antigens that are presented by the MHC I pathway for the induction of a cell-mediated immune response. L. monocytogenes infection of macrophages also leads to high levels of interleukin (IL) 12 and interferon (IFN)-gamma production (a type 1 cytokine profile). Listeria can be used to deliver lentiviral antigens into the cytoplasm of macrophages either through stable integration of viral sequences into the bacterial genome or by transfecting the bacteria with a plasmid containing viral genes that are driven by a eukaryotic promoter. The latter approach results in posttranslational modification of proteins while the first approach does not. Other key features of Listeria are that it´s natural route of infection is through the gastro-intestinal mucosa and it can be attenuated to eliminate virulence even in immunocompromised individuals. The studies herein will l. determine the viral gag genes that are most efficiently produced by recombinant L. monocytogenes in feline macrophages, 2. compare the immune response induced by FIV protein-expressing recombinant L. monocytogenes when administered by a parenteral or mucosal route and determine the efficacy of each route, 3. determine if FIVgag-expressing L. monocytogenes can deliver an envelope DNA vaccine plasmid that will be immunogenic. These studies will be performed using the feline immunodeficiency virus (FIV) infection of cats as a model for HIV infection of people. The value of the FIV model has been proven and will allow the comprehensive immunologic evaluation of a statistically significant number of animals in a highly controlled system. These studies will help determine whether it is rational to pursue the use of L. monocytogenes as a biologic vaccine vector against HIV in humans
Keywords: AIDS vaccine, Listeria, feline immunodeficiency virus, vaccine development, vector vaccine HIV infection, active immunization, disease /disorder model, drug administration route cat
Project start date: 2000-03-15
Project end date: 2002-08-31
5R21AI047046-02 (2001): $220500
1R21AI047046-01 (2000): $220500
Recombinant Listeria: A Biologic Vaccine Vector For FIV
Gregg A Dean, Professor
North Carolina State University Raleigh Sponsored Programs And Regulatory Compliance Raleigh, Nc 27695
Grant 1R21AI055442-01A2 from National Institute Of Allergy And Infectious Diseases, IRG: ZRG1
Abstract: A successful vaccine against HIV will likely require a strong, broad memory T cell and neutralizing humoral immune response. It is not clear how to stimulate such a broad immune response and strategies to date have not been successful. Thus, it is seems reasonable to pursue novel vaccine strategies in the well characterized feline immunodeficiency virus (FIV)/cat model. Listeria monocytogenes (LM) is a Gram positive, facultative intracellular bacterium that can be used to deliver foreign antigens to antigen presenting cells resulting in induction of a strong cell-mediated immune response. We have generated recombinant LM that express FIV Gag from sequence inserted into the bacterial genome and delivers a DNA vaccine for eukaryotic expression of Env proteins. We have shown a single oral immunization with this vaccine vector induced a strong cell-mediated response and high titer vaginal IgA. Upon vaginal challenge with pathogenic FIV, significant protection was demonstrated. Here we propose to further develop and test a novel prime-boost immunization strategy that would combine the benefits of two novel vaccine strategies. In specific aim 1, we will generate Gag and Pol expressing recombinant LM using two different attenuated Listeria isolates. After in vitro validation, we will test these recombinant LM for safety and immunogenicity in vivo. The optimal dose for oral immunization will also be determined. In the second specific aim, we will develop and test a novel DNA vaccine that will be used as the prime. The prime will consist of a PLG-microsphere encapsulated DNA vaccine containing FIV gag, pol, and env sequences. In addition, synthetic adjuvants will be added to the microspheres to specifically stimulate toll-like receptor (TLR) 4 or TLR 9. The prime will be given either orally or parenterally to compare the two routes. Immunogenicity will be proven in vivo. In the third specific aim, the optimized prime and optimized boost will be tested in a three-arm, challenge study. This strategy will determine if a microencapsulated DNA vaccine with a TLR-specific adjuvant, can effectively prime the immune system for a subsequent oral boost with recombinant LM resulting in a protective immune response at mucosal surfaces. These studies will be performed in the FIV/cat model that allows challenge by vaginal exposure to cell-associated and cell-free virus.
Keywords: AIDS vaccine, Listeria, feline immunodeficiency virus, immune response, recombinant virus, transfection /expression vector, vaccine development, vector vaccine, viral vaccine, virus antigen, HIV envelope protein, biological model, gag protein, immunomodulator, microcapsule, nonhuman therapy evaluation, oral administration, toll like receptor, vaccine evaluation, biotechnology, cat, female, laboratory mouse
Project start date: 2004-09-30
Project end date: 2006-09-30
1R21AI055442-01A2 (2004): $607668
CYTOKINE-MEDIATED PATHOGENESIS OF HIV INFECTION
Gregg A Dean, Professor
North Carolina State University Raleigh Sponsored Programs And Regulatory Compliance Raleigh, Nc 27695
Grant 7K11AI001262-03 from National Institute Of Allergy And Infectious Diseases, IRG: SRC
Project start date: 1994-09-01
Project end date: 1997-08-31
7K11AI001262-03 (1996): $88668
Related Publications
Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection. Immunology. 2008 Sep 2. [Epub ahead of print] PMID: 18775027 Failure of TLR4-driven NF-kappa B activation to stimulate virus replication in models of HIV type 1 activation. AIDS Res Hum Retroviruses. 2007 Nov; 23( 11): 1387-95. PMID: 18184082 Cloning of feline FOXP3 and detection of expression in CD4+CD25+ regulatory T cells. Vet Immunol Immunopathol. 2008 Mar 15; 122( 1-2): 159-66. Epub 2007 Nov 21. PMID: 18180044 In vivo depletion of CD4+CD25+ regulatory T cells in cats. J Immunol Methods. 2008 Jan 1; 329( 1-2): 81-91. Epub 2007 Oct 22. PMID: 17980384 Phagocytic plasmacytoma in a dog. Vet Clin Pathol. 2007 Sep; 36( 3): 293-6. PMID: 17806081 
Vaccination of cats with attenuated feline immunodeficiency virus proviral DNA vaccine expressing gamma interferon. J Virol. 2007 Jan; 81( 2): 465-73. Epub 2006 Nov 1. PMID: 17079309 Cytokine modulation of the innate immune response in feline immunodeficiency virus-infected cats. J Infect Dis. 2006 Jun 1; 193( 11): 1520-7. Epub 2006 Apr 26. PMID: 16652280 Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus. Vet Immunol Immunopathol. 2006 Apr 15; 110( 3-4): 245-55. Epub 2005 Nov 15. PMID: 16293316 Methodology for isolation and phenotypic characterization of feline small intestinal leukocytes. J Immunol Methods. 2005 Jul; 302( 1-2): 36-53. PMID: 16023664 Toll-like receptor expression in feline lymphoid tissues. Vet Immunol Immunopathol. 2005 Jul 15; 106( 3-4): 229-37. PMID: 15963821 Feline cytokine ELISPOT: issues in assay development. Methods Mol Biol. 2005; 302: 167-78. PMID: 15937351 Pre-existing immunity to pathogenic Listeria monocytogenes does not prevent induction of immune responses to feline immunodeficiency virus by a novel recombinant Listeria monocytogenes vaccine. Vaccine. 2005 Feb 10; 23( 12): 1479-90. PMID: 15670884 Cloning and expression of feline interleukin 15. Cytokine. 2005 Jan 21; 29( 2): 77-83. PMID: 15598442 Assessment of CD4+ and CD8+ IFN-gamma producing cells by ELISPOT in naïve and FIV-infected cats. Vet Immunol Immunopathol. 2004 Nov; 102( 1-2): 77-84. PMID: 15451617 Oral immunization with recombinant listeria monocytogenes controls virus load after vaginal challenge with feline immunodeficiency virus. J Virol. 2004 Aug; 78( 15): 8210-8. PMID: 15254192 Peptide mapping of feline immunodeficiency virus by IFN-gamma ELISPOT. Vet Immunol Immunopathol. 2004 Jul; 100( 1-2): 49-59. PMID: 15182995 Transmission and immunopathogenesis of FIV in cats as a model for HIV. Curr HIV Res. 2003 Jan; 1( 1): 15-29. Review. PMID: 15043209 In vivo cytokine response to experimental feline infectious peritonitis virus infection. Vet Microbiol. 2003 Dec 2; 97( 1-2): 1-12. PMID: 14637034 A randomized controlled trial of misoprostol monotherapy for canine atopic dermatitis: effects on dermal cellularity and cutaneous tumour necrosis factor-alpha. Vet Dermatol. 2003 Feb; 14( 1): 37-46. PMID: 12603684 Evaluation of FIV protein-expressing VEE-replicon vaccine vectors in cats. Vaccine. 2002 Dec 13; 21( 3-4): 258-68. PMID: 12450701