Richard Markham - US grants

We have previously shown that T cells from mice immunized with a high molecular weight polysaccharide (PS) isolated from Pseudomonas aeruginosa can inhibit bacterial growth in vitro and adoptively protect non-immune mice in vivo. The current proposal seeks to analyze how T cells are activated by PS and to clone and analyze the activity of regulatory T cells that control this response. We propose a model of T cell activation by PS that involves cross-linking of Fc receptors (FcR) on T cells that have been "pre-armed" with P. aeruginosa-specific antibody produced in undetectable amounts in the spleen. Cross-linking of the FcR results in secretion of an antibacterial lymphokine. This model is supported by several findings: 1. T cell immunity is demonstrable only when there is evidence of B cell activation, although in some cases, this evidence is indirect since specific antibody cannot be detected. 2. PS does not bind to gene products of the major histocompatibility complex. Activation of T cells cannot occur via the T cell receptor. 3. Removal of, or enrichment for, immune T cells bearing FcR removes or enriches antibacterial T cell activity, respectively. The planned studies will (a) evaluate the effect of immunization of FcR expression by T cells, (b) examine the ability of FcR for different immunoglobulin classes to generate antibacterial activity, (c) attempt to elicit T cell antibacterial activity by cross-linking FcR without bacterial products, and (d) seek to directly demonstrate the presence of P. aeruginosa-specific antibody on the surface of T cells. The effect of cloned suppressor and contrasuppressor T cell lines on FcR expression will also be studied.

SCID mouse; human immunodeficiency virus 1; disease /disorder model; AIDS; AIDS therapy; model design /development; virus envelope; CD4 molecule; cellular immunity; biotechnology; bone marrow transplantation; natural killer cells; antiviral agents; humoral immunity; virus antigen; HIV infections; virus DNA; zidovudine; nucleic acid probes; human tissue; flow cytometry; polymerase chain reaction; nucleic acid hybridization;

This proposal will use a cohort of injection drug users to evaluate the hypothesis that progression of HIV-1 infection is attributable to 1) the ability of the virus to spread to new cells and 2) the rate of viral replication in cells that are already infected. Both of these processes require some form of broadly reactive T-cell activator (PHA, anti-CD3, IL-2) to proceed efficiently in vitro, and it is our hypothesis that similar activation is required in vivo, as well. The absence of such activation in the naive host may explain the restricted viral genetic heterogeneity observed in newly infected individuals. We hypothesize that the source of the stimulation in ongoing viral infection is the virus itself and that differences in the course of infection, as measured by CD4 T cell decline and time to AIDS, can be attributed to differences in the ability of different viral variants to induce cellular activation, which in turn permits viral replication and spread. Dividing the AIDS Linked to the Intravenous Experience cohort of injection drug users into groups of rapid progressors, moderate progressors, and slow progressors, we will prospectively follow and compare four parameters in 60 individuals from the ALIVE cohort: 1) the development of genetic variation over time in the third hypervariable domain of the viral envelope 2) the ability of viral variants to elicit tumor necrosis factor-a alpha from host cells and 3) the ability of virus to stimulate proliferation of host cells. We hypothesize that genetic variation can serve as a measure of viral spread since such variation occurs predominantly during the reverse transcription that occurs when new cells are infected. Our preliminary data suggest that such variation increases as CD4 T cell levels decline in the majority of HIV-1 infected subjects and in this proposal we seek to extend and confirm the relevance of this observation to groups progressing at different rates. We seek to show a correlation between CD4 decline and variation and a correlation between rate of CD4 decline and rate of appearance of variation. Our preliminary data also show that low passage viral variants differ in their ability to elicit TNF-alpha secretion, which directly activates viral replication, and to stimulate T cell proliferation, which enhances viral reverse transcription and integration in the host cell genome. We will therefore analyze differences in these capabilities among low passage viruses obtained from the study cohort. By establishing the critical influence of the activating properties of different viral variants on the course of HIV-1 progression, these studies will provide important insights into HIV-1 pathogenesis and provide a new target for therapeutic strategies directed to slowing the progression of this disease.

Recombination is a fundamental property of genetic material, which allows patterns of evolutionary change that would be virtually impossible without it. Although normally associated with sexual reproduction, recombination may also play a critical role in allowing non-sexually reproducing organisms to adapt to a changing environment. In the case of HIV-1, the recombigenic potential of this retrovirus has been noted in settings in which individuals were dually infected with two divergent viral variants. However, the high mutation rate of this virus, along with its rapid replication to high level and long persistence within the infected host, permit enough diversity to develop to allow the detection, by recently developed techniques, of recombination events within individuals infected with monophyletic viruses. The potential impact of such recombination on disease course, especially in the face of multi-drug therapy which can only be resisted through the acquisition of numerous mutations, is the focus of the proposed studies. The specific aims are to 1) Sequence regions of the env and pol gene from 24 individuals infected by a single source of HIV-1 and followed longitudinally in order to monitor intrahost evolution. 2) Simultaneously estimate the evolutionary trees and recombinational history from the sequence data using newly developed analytical procedures. 3) Use the longitudinal data available on each individual to examine the evolutionary fate of recombinant versus non-recombinant haplotypes in order to test for interactions between recombination, selection, and adaptive evolution of HIV-1. The analysis will separate the effects of intragenic versus intergenic recombination. 4) Test for associations between the evolutionary patterns observed for recombinants and the course of disease progression and treatment regimes. These studies should provide critical insights into the potential rate of emergence of multi-drug resistant variants of HIV-1.

Resistance to antiretroviral therapy always results from selection for resistant viral variants. The resistance genotype can either be pre-existing, frequently as a minority clone, before the initiation of therapy or it can emerge due to ongoing viral replication that occurs despite therapy. In either case the development of a drug-resistance mutation arising during the HIV-1 life cycle is likely a random event, dependent on the rate at which new mutations arise generally in the HIV-1 population existing within an infected individual. The appearance of new mutations, in turn, will reflect intrinsic replicative properties of a given viral variant, as well as host factors, such as T cell activation state or co-receptor expression, that facilitate replication of specific viral variants. We have recently determined that one host factor not frequently considered as a source of increased viral replication, frequency of injection drug use, is positively and highly significantly associated with diversity in the env gene of HIV-1 infected injection drug users. This increase is not due to infection with a second virus and may be attributable to opiate-induced enhancement of viral replication, which has been observed in tissue culture systems. It is likely that this increased diversity will extend to viral genes other than env, including pol. Based on these findings we hypothesize that multi-drug resistant mutants will emerge more readily among injection drug users and those on methadone maintenance programs than in drug-free control subjects. To address this hypothesis we will evaluate specimens collected from over 40 women by the Women's Interagency HIV Study to answer the following questions: 1) Is the higher rate of viral genetic diversity observed in the env gene in frequent drug injectors also observed in the pol gene? 2) Is the higher rate of genetic diversity observed in frequent injection drug users also observed in individuals taking methadone? 3) Does a history of injection drug use or methadone use result in a higher incidence of resistance to HAART? The findings from this study should have important implications for the clinical approach to control of opiate addiction and antiretroviral therapy in the HIV-1- infected, drug-infected population.

DESCRIPTION (provided by applicant): Given the poor near-term prospects for an HIV-1 transmission-blocking vaccine and the failure of condoms as a practical mechanism for controlling sexual transmission of HIV-1, there is urgent need for an effective and acceptable anti-HIV-1 microbicide. Two features, which should dramatically enhance microbicide acceptability would be complete transparency to the user and the absence of a requirement for immediate pre-coital application. Although the relative roles of cell-free and cell-associated virus in human sexual transmission are unknown, we have found using in vivo SCID mouse and in vitro transwell models of HIV-1 sexual transmission that cell-associated transmission of HIV-1 is highly efficient and cell-free virus is poorly transmitted. These results differ from those reported in the macaque model system, which is likely accounted for by the fact that all of the reported macaque studies make no effort to adjust the environment to the neutral pH at which transmission occurs in the human setting. In our model systems, antibody to the cell adhesion molecule ICAM-1 is remarkably effective in blocking cell-associated transmission. Preliminary data suggest that the antibody is acting by a mechanism that involves signal transduction rather than by simply blocking the docking function of ICAM-1. We are now hypothesizing that lactobacilli expressing single chain antibody fragments (scFv or scAb) can be used as a delivery mechanism for providing a sustained, totally transparent,and, in the case of heterosexual transmission, woman-controlled method for blocking cell-associated virus transmission. To evaluate this hypothesis we have established a collaboration with the leading laboratory in the world studying the expression of antibody fragments by lactobacilli. In collaboration with this laboratory, located in the Netherlands, we seek to 1) Examine differential effects of engagement of ICAM-1 in a cross-linking or a non-cross-linking manner on resistance of endothelial monolayers to transmigration of infected cells 2)Engineer lactobacilli to express cell-wall bound or secreted monovalent ordivalent scFv or scAb and evaluate the in vitro efficacy of the secreted products in the transwell assay 3) Evaluate in the SCID mouse model the colonization characteristics and protective efficacy of engineered lactobacilli used to colonize the mouse vagina. It is anticipated that these studies will provide the proof of principle necessary to initiate clinical studies using this approach.

DESCRIPTION (provided by applicant): Failure of antiretroviral therapy (ART) has been a particular problem among HIV-1 infected illicit drug users. This failure has primarily been attributed to poor compliance with antiretroviral drug regimens. Studies from this laboratory have indicated that a higher viral mutation frequency that we have documented in injection drug users (IDU) and the resulting higher frequency of primary resistance mutations in pol might contribute to the poor response in this group. Genotypic resistance analysis using population sequencing has proved useful in guiding selection of antiretroviral therapy in individuals who have developed viral relapse after initial treatment. Our preliminary data analyzing up to 10 viral clones from protease inhibitor (PI) and non-nucleoside reverse transcriptase inhibitor (NNRTI) naive IDU have indicated that a strikingly and significantly high proportion of IDU subject-visits (28%) carry PI resistance mutations compared to the much lower proportion (8%) found in non-IDU. Almost none of these resistance mutations were detected using standard population genotyping techniques. Similar high levels of primary resistance have been found in a cohort of non-injection illicit drug users followed by investigators at Vanderbilt Medical School. This proposal hypothesizes that use of sensitive sequencing techniques prior to initiation of HAART will be predictive of rapid development of resistance, therefore enabling the development of personalized and more effective initial HAART regimens. Specifically in cohorts from the Johns Hopkins and Vanderbilt Schools of Medicine of 150 HAART-naive drug users who rapidly failed HAART and 150 comparable subjects for whom therapy was successful we will 1) Determine whether identification of resistance mutations by standard clonal analysis using the Sanger sequencing method to study the pol region from 20 HIV-1 clones from a single visit predicts risk of subsequent therapy failure better than population genotyping from that same visit 2) evaluate whether identification of resistance mutations by high throughput clonal analysis of relatively short sequences from the RT and protease regions (454 sequencing) predicts risk of subsequent therapy failure better than: i) standard population genotyping and ii) analysis of 20 clones (sequenced using the Sanger method) in which both NRTI and NNRTI or PI resistance mutations can be identified on the same viral clone and 3) Evaluate, using 454 sequencing technology, the frequency of clonal resistance needed for clones with PI or NNRTI resistance to predict increased risk of rapid development of therapy failure. The results of this study could provide a new standard of care for initiation of HAART and could greatly reduce the financial and social impact of HAART failure. This study is designed to evaluate new technologies that will render anti-HIV-1 drug therapy more effective. This new technology will permit better characterization of the viral strains that are infecting an individual so that the therapy can be specifically targeted to those viruses.

[unreadable] DESCRIPTION (provided by applicant): Previous studies from this laboratory have demonstrated significantly higher viral genetic diversity and a higher frequency of primary resistance to protease inhibitors (PI) among illicit drug users compared to HIV-1 infected non-drug users. To explore the potential link between drug use and greater viral replication, diversity and antiretroviral resistance, we propose to exploit the findings that 1) cocaine abuse stimulates high levels of the AP-1 transcription factor associated immediate early genes and 2) only about 40% of Clade B infected individuals carry an HIV-1 LTR length polymorphism (MFNLP) containing an AP-1 binding site. Using a large cohort of cocaine using HIV-1 infected subjects followed at the Vanderbilt Clinical Care Center, we will address the hypothesis that cocaine users infected with HIV-1 carrying the Clade B HIV-1 LTR polymorphism with an AP-1 binding site will have higher viral loads, greater genetic diversity, and greater primary resistance to antiretrovirals than either cocaine users whose virus does not carry the LTR polymorphism or non-cocaine users. To explore this hypothesis we will 1) Identify among a cohort of HIV-1 positive, antiretroviral therapy naive individuals those cocaine-using and non-cocaine using individuals with virus carrying the MFNLP within the LTR. We will thereby have identified four groups for additional analyses: HIV-1 infected cocaine users with and without the MFNLP and non-cocaine users with and without the MFNLP. 2) Within the four groups quantify the expression levels within peripheral blood CD4 T lymphocytes and monocytes of c-fos and JunB, as representative immediate early gene components of AP-1. Similarly, using gel-shift assays or a new high throughput kit, we will quantify levels of AP-1 within those cell populations from the four groups. 3) Using 454 high throughput sequencing technology we will sequence the protease region to define within host genetic diversity and primary resistance mutations to PI. Using diversity, resistance mutations to PI and viral load as outcomes, we will examine using a multivariate analysis, the effect of cocaine use, immediate early gene expression levels, AP-1 quantities, and the presence of the MFNLP. PUBLIC HEALTH RELEVANCE: Certain variants of HIV-1 carry genetic alterations that might enable them to replicate more rapidly in cells exposed to cocaine. This study will evaluate whether the presence of those variants in cocaine users alters various parameters of disease progression. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Although DNA vaccines offer many advantages, their inability to elicit sustained protective immune responses in non-human primates and humans has been disappointing. Based on substantial evidence derived from tumor model systems and preliminary studies with a candidate Plasmodium yoelii vaccine, this proposal hypothesizes that an effective DNA-based vaccine against malaria can be developed by expressing malarial protein antigens as fusion proteins linked to chemokine ligands for receptors on immature dendritic cells (iDC). This vaccine model differs from related approaches in that it attracts the appropriate antigen-presenting cell to the inoculation site and by fusing the vaccine epitopes of interest to the chemokine, ensures the efficient uptake of the antigen for processing. This hypothesis will be tested in a murine model of Plasmodium yoelii infection and immunogenicity. Specifically, we will 1. Evaluate ex vivo the responses elicited in BALB/c mice by a candidate multi-epitope DNA vaccine administered by in vivo electroporation that targets the pre-erythrocytic stages of P. yoelii. The candidate vaccine to be evaluated will consist of a plasmid that expresses macrophage inflammatory protein 31 (MIP- 31) fused to (a) The SYVPSAEQI immunodominant P. yoelii Class I-restricted T cell epitope in BALB/c mice (b) The (QGPGAP)4 immunodominant P. yoelii B cell epitope in BALB/c mice and (c) The YNRNIVNRLLGDALNGKPEEKA immunodominant Class II-restricted T cell epitope in BALB/c mice. A similar DNA construct will be created that lacks the MIP-31 component and several additional positive and negative control immunization groups will also be established. Outcomes of immunization will be measured by interferon gamma (IFN-3) Elispot assays, antibody concentrations measured in ELISA and immunofluorescence assays, and by flow cytometry evaluation the development of central memory T cells. 2. Evaluate the protective efficacy of this candidate vaccine in mouse malaria challenge models. Outcomes will be followed using real-time PCR to quantitate malaria gene expression in liver samples from the mice and levels of blood parasitemia attained in immune compared to nonimmune mice. PUBLIC HEALTH RELEVANCE: This project is designed to develop a vaccine for malaria. Should this approach be successful, this vaccine could save the more than 1 million deaths that occur annually as a result of this infection. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The major hypothesis of this proposal is that bacteria capable of sustained vaginal colonization of a broad cross-section of women can be engineered to produce alpaca- derived beta integrin-specific variable region antibodies (VHH) that will inhibit infection with HIV-1. This approach to preventing HIV-1 transmission offers the unique advantage of targeting receptor-ligand interactions that are completely independent of mutable viral proteins. A similar delivery system targeting Glycoprotein D of herpes simplex type 2 will also prevent infection with that virus, which enhances transmission of HIV-1 infection. This hypothesis will be pursued by demonstration of the efficacy of bacterially-delivered VHH using three different mouse model systems and two different bacterial strains, one of which, Streptococcus gordonii, is selected specifically for the purpose of demonstrating proof of principle in mouse model systems that require pre-treatment of mice with progesterone. Lactobacillus rhamnosus GR-1 will be used in a mouse model that does not require progesterone pre-treatment, as this strain of lactobacillus has been demonstrated to be effective in colonizing a broad cross-section of women. Lactobacilli are appealing as a microbicide vehicle because they are "generally regarded as safe" organisms by the FDA, constitute the primary bacterial component of the normal flora of the female genitourinary tract, intrinsically inhibit the growth of more virulent bacteria, would be transparent to users, and would allow dissociation of microbicide application from coitus. Alpaca-derived antibody binding domains (VHH) are developed from a class of camelid antibodies consisting solely of heavy chains and the binding determinants of which are single protein domains that are much smaller than any which might be generated from classical antibodies. Using this technology we will: 1) Raise antibodies in alpacas against the ectodomain of CD18, the beta-chain component of the CD11a/CD18 heterodimer that is LFA-1. From circulating PBMC of the immunized alpacas we will generate a library of VHH screened by phage display for binding affinity. Those VHH will then be expressed from plasmids and/or the bacterial chromosome of Lactobacillus rhamnosus GR-1 and Streptococcus gordonii. 2) Using the methods described in Aim 1 generate VHH targeting HSV-2 glycoprotein D. 3) Evaluate the potential toxicity of anti-CD18 and anti-GpD by measuring transepithelial resistance in two different in vitro model systems. 4) Test the ability of the CD18 and GpD-specific VHH to block transmission or neutralize infection in transwell or in vitro neutralization assays. 5) Evaluate colonization of the mouse vagina and secretion of CD18-specific VHH by transformed S. gordonii in progesterone-treated Hu-PBL-SCID mice and by L. rhamnosus in non-progesterone-treated humanized bone marrow liver thymus NOD- SCID (BLT) mice. Evaluate the ability of colonized mice to resist infection with cell- associated and cell-free HIV-1. 6) Evaluate colonization of the mouse vagina and production of Glycoprotein D-specific VHH by transformed S. gordonii progesterone- treated BALB/c mice. Evaluate in these mice the appearance of anti-Glycoprotein D and anti-CD18 VHH antibodies in vaginal lavage fluid and serum and examine vaginal lavage fluid for the appearance of inflammatory cells induced by the VHH. The passive protective ability of in situ-produced HSV-2 specific VHH will also be assayed in this mouse vaginal challenge model. and 7) If dictated by results of the previous studies, develop a plasmid-based expression system that does not depend on antibiotic selection. At the completion of these studies, we will have proved the potential efficacy of this approach using in vivo model systems and will have developed the VHH constructs that could be used in the clinical setting as well as established their production from a lactobacillus species that also may be applicable to the clinical setting.

DESCRIPTION (provided by applicant): This application requests a Roche Genome Sequencer FLX (GS FLX), developed by 454 Sequencing for placement in the Johns Hopkins Bloomberg School of Public Health. This instrument generates over one million unique high quality sequencing reads with an average of 350 to 400 bases pairs in length within a ten hour instrument run time. The total average yield per instrument run is 400 to 500 megabases. The instrument will be used by a diverse group of investigators addressing questions related to the major disease epidemics confronting the world today. Representative studies include those of primary resistance to antiretroviral therapy for HIV among drug users and among infants born to mothers receiving prophylactic antiretroviral therapy. These studies will evaluate the usefulness of this technology for guiding initial antiretroviral therapy in those distinct settings. The genetic basis for resistance to anti-tuberculosis agents will be studied among tubercle bacilli lacking the traditional resistance mechanisms. Differences among malaria parasites that can or cannot complete the transmission cycle in mosquitoes will be defined using this instrument, as will the mosquito?s innate immune response to the presence of malaria. The biology of cancer may be elucidated by studies identifying compensatory mutations in yeast knockout strains, with many secondary mutations yielding phenotypes observed in cancer cell lines. Similarly studies will characterize distinct genetic features of those hepatitis B virus variants identified in individuals with hepatocellular carcinoma. All of these studies have in common the need to detect either rare mutations in a large and diverse gene pool or mutations in a large genome that account for a distinct phenotype. In all cases that need is addressed by the high throughput sequencing capability of the Genome Sequencer FLX system. Analytical tools provided with the instrument will aid in the proposed analyses, but this project also takes advantage of a bioinformatics core established specifically for purposes of genome analysis at Johns Hopkins University. Finally the instrument will be housed in a pre-existing core facility originally established for array analysis but with the instrumentation and available expertise to exploit the capabilities of high throughput sequencing.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): Human cytidine deaminases APOBEC3 (A3) proteins are potent host defenses against HIV. These antiviral proteins induce lethal modification of cytosines to uracils in newly synthesized minus-strand viral DNA, resulting in abortive viral infection. HIV must overcome these host cellular defenses for successful viral replication. HIV-1 encodes a protein, Vif, which suppresses the antiviral effects of A3 proteins by targeting them for degradation through the 26S proteasome. Vif hijacks cellular Cullin5 (Cul5), ElonginB, and ElonginC to form a viral E3 ubiquitin ligase that targets A3G for polyubiquitination and degradation. Thus, identification of novel strategies to preserve the antiviral functions of A3 is n exciting new target for antiretroviral therapy. In this application, we propose to capitalize our expertise in HIV-1 Vif/A3 system and our new understanding of the viral evasion mechanism to develop a rapid cell-based assay for the identification of small molecule inhibitors of Vif and to further optimize and adapt the system for application to high throughput molecular screening of large compound libraries to identify molecules that inhibit HIV-1 replication. The proposed research is based on our recent discovery that CBF? is a key and unique regulator of HIV-1 Vif function. This study is expected to provide us with critical information regarding the design and development of effective intervention strategies against HIV.

Abstract The current proposal employs a vaccine platform, originally developed for malaria, to elicit protective immunity to Zika virus infection. The novel vaccine platform employs fusion of the vaccine antigen to a series of virus-derived chemokine-like molecules to direct the vaccine antigen to the immature dendritic cells that initiate the adaptive immune response. This vaccine construct will be used in combination with the MF59 adjuvant, which has been approved for clinical use in pregnant women for other vaccines by the US FDA. In its initial iteration including the human-derived chemokine MIP3? (CCL20), this vaccine platform has proven to be remarkably effective at eliciting high concentrations of antibody and protective immunity in a malaria mouse model challenge system. To avoid use of human-derived products in the vaccine construct MIP3? will be replaced in the currently proposed studies with the viral chemokines, for which similar immune enhancing capabilities have been observed. Domain III of the Zika virus E protein, which lacks N-linked glycosylation sites, will be targeted because of the ease of its expression in bacterial expression systems. Initial studies will optimize the vaccine construct, ensuring proper conformation and evaluating the affinity and magnitude of antibody responses elicited in wild-type C57BL/6 mice receiving different vaccine dosing regimens. Subsequent studies will evaluate the maintenance of protective antibody concentrations over a six month period, using both in vitro PRNT assays and in vivo challenge systems. For longevity of protection studies, in vivo challenge will be performed evaluating the protective efficacy of passively transferred antibody in Irf-3, 5 and 7 triple knockout mice available at this institution, These mice, under 11 weeks of age, have been demonstrated to be susceptible to Zika virus infection.

Mycobacterium tuberculosis (Mtb) is a major opportunistic pathogen and the leading infectious disease- related cause of death among people living with HIV worldwide. Shorter TB treatment regimens are urgently needed to minimize drug interactions with antiretroviral drugs, improve medical adherence, and curb the emergence of TB drug resistance. The prolonged duration of TB treatment is believed to reflect the unique ability of a subpopulation of bacteria to remain in a non-replicating, persistent state in the infected host. These ?persister? bacteria evade immune-based clearance mechanisms and become tolerant to first-line anti-TB drugs, which more effectively target actively dividing bacteria. One of the important mechanisms by which persisters acquire this phenotype is through induction of the stringent response. The stringent response enzyme RelMtb is required for bacterial growth restriction and antibiotic tolerance, and is essential for long-term Mtb survival during various in vitro and in vivo stress conditions. Deficiency of relMtb renders Mtb more sensitive to isoniazid in various models. We have generated a therapeutic DNA vaccine targeting Mtb stringent response genes, which induces antigen-specific cellular immunity, and, when combined with the first-line drug isoniazid, exhibits significantly greater activity against Mtb in the lungs of chronically infected mice relative to isoniazid alone. We also have developed a novel vaccination strategy involving fusion of the antigen of interest with the dendritic cell-targeting chemokine MIP3?, which significantly enhances immunogenicity. This vaccine platform is designed to address the fact that in addition to the T-cell deficiencies of HIV infection, there is clear documentation of deficiency of DC function in both the TB and HIV settings. However, it remains to be determined if enhanced immunity to Mtb persistence factors can shorten the duration of curative antibiotic treatment in the immune-competent and T-cell-deficient host. In the current proposal, we will directly address these knowledge gaps by testing the novel hypothesis that enhanced cellular immunity against Mtb stringent response factors potentiates the activity of the first-line anti-TB regimen and accelerates cure in the standard murine model of TB. Since HIV infection is associated with disturbed T-cell homeostasis, including depletion of CD4+ T cells and persistent expansion of CD8+ T cells, we will characterize the contribution of each of these cell types to the therapeutic efficacy of the stringent response vaccine in mice. This proposal represents a unique collaboration between Investigators with significant expertise in microbiology, molecular biology, immunology, DNA vaccines, and animal models. Our findings are expected to inform future studies investigating therapeutic TB vaccines, and lay the groundwork for the development of novel strategies to therapeutically target Mtb persisters, with the goal of shortening treatment for drug-susceptible and drug-resistant TB in HIV-infected and uninfected individuals.

Mycobacterium tuberculosis (Mtb) is the leading infectious disease-related cause of death among people living with HIV worldwide. Shorter tuberculosis (TB) treatment regimens are needed to achieve global TB elimination. The protracted nature of the current 6-month TB treatment course reflects the unique ability of a subpopulation of ?persister? bacteria to remain in a growth-limited, antibiotic-tolerant state through various adaptive strategies, including induction of the stringent response. The key stringent response enzyme RelMtb is essential for long-term Mtb survival under physiologically relevant stresses in vitro and in animal lungs. Recently, we have generated a therapeutic relMtb DNA vaccine, which induces RelMtb-specific cellular immunity, and significantly augments the activity of the first-line drug isoniazid against active TB in mice. We also have developed a novel vaccination strategy involving fusion of the antigen of interest with the immature dendritic cell (iDC)-targeting chemokine MIP3?, which significantly enhances antigen-specific T-cell responses. In the current proposal, we will determine if this iDC-targeting strategy, as well as a promising new adjuvant approach involving the use of cyclic dinucleotides to activate the stimulator of interferon genes (STING) pathway, enhance the immunogenicity of our relMtb DNA vaccine. The ideal vaccine platform will be used to test the novel hypothesis that enhanced cellular immunity against RelMtb potentiates the activity of the first- line anti-TB regimen and accelerates cure in the standard murine model of TB. Since HIV infection is associated with disturbed T-cell homeostasis, including depletion of CD4+ T cells and persistent expansion of CD8+ T cells, we will characterize the contribution of each of these cell types to the therapeutic efficacy of the relMtb DNA vaccine in mice. In order to transition our findings to the clinical setting, we will next test the immunogenicity of this vaccination strategy in rhesus macaques, which develop immune response patterns most analogous to those of humans. Finally, leveraging archived clinical samples available through the RePORT South Africa longitudinal cohort of HIV-infected and uninfected patients with pulmonary TB receiving first-line anti-tubercular treatment, we will measure RelMtb-specific T-cell responses during TB therapy. This proposal represents a unique collaboration between Investigators with significant expertise in microbiology, molecular biology, immunology, DNA vaccines, and animal models. Our findings are expected to have far- reaching implications for the development of novel adjunctive therapies for shortening the duration of treatment for drug-susceptible and drug-resistant TB, as well as novel diagnostic tools for confirming the adequacy of TB treatment in HIV-infected and uninfected individuals.