Dennis Burton - US grants

neutralizing antibody; human immunodeficiency virus 1; antiAIDS agent; molecular cloning; AIDS; antigen antibody reaction; passive immunization; antiviral antibody; human genetic material tag; antibody neutralization test; human tissue; enzyme linked immunosorbent assay;

The proposed research aims to characterize human antibody responses to the HIV-1 virus at the molecular level, and to generate human monoclonal antibodies for clinical evaluation in combatting HIV infection. The latter includes passive immunization as a potential treatment for AIDS and prophylaxis in pregnant women to prevent transmission of the virus to offspring. Panels of antibodies against HIV-1 envelope proteins will be generated by establishing Fab libraries from appropriate donors on the surface of M13 phage and selection of specific Fabs by antigen panning. Fabs will be characterized in terms of in vitro neutralizing ability, antigen affinity, epitope recognized and nucleic acid sequence. Fabs will be linked to Fc genetically for the production of glycosylated whole IgG antibodies in eukaryotic cells. A cocktail of antibodies for therapeutic application will be assembled based on exceptional neutralizing ability, reactivity with multiple viral strains, recognition of different epitopes on the viral envelope and possible synergistic effects. The research will provide opportunities to explore the mechanism(s) responsible for virus neutralization and some of the rules governing antibody recognition of viral antigens.

To develop improved vaccines to measles that can safely overcome the maternal antibody barrier and induce long-lasting immunity, it is highly desirable to characterize the human humoral response to measles infection at the molecular level. This characterization can then form the basis for understanding the differences between humoral immunity induced by vaccination and by infection and for probing the specificities of maternal antibodies. Classical technologies can yield occasional human monoclonal antibodies but not in sufficient numbers or with sufficient reproducibility to consider characterizing antibody responses. Combinatorial library technology, i.e., antigen selection from antibody libraries generated in/on phage, offers rapid access to large numbers of human monoclonal antibodies (Fab fragments) and has the potential to allow analysis of human responses. The aim of this project is to use library technology to generate panels of recombinant Fabs against measles antigens from donors who have acquired immunity through natural infection. The Fabs will be characterized in terms of antigen recognized, affinity, amino acid sequence and neutralizing ability in vitro. The epitopes recognized will be defined in detail by competition with mouse antibodies and with other recombinant Fabs and by selection of non-neutralizable virus variants by antibody. The recombinant Fabs will then be used in competition studies with serum antibodies to compare the specificities of antibodies elicited by vaccination and infection and to describe the specificities of maternal antibodies.

The recent outbreaks of disease associated with infection by the hantavirus Sin Nombre and the filovirus Ebola are causes for concern. Small changes in these viruses affecting transmissibility or virulence could lead to devastating epidemics. Panels of human antibodies to these viruses can be a very valuable resource, both in terms of current viruses and variants that may arise in the future. We propose to use antibody phage library technology to generate these panels. In this technology, antibody repertoires are rescued from humans and cloned into bacteriophage display vectors to generate vast antibody libraries. Affinity selection on immobilized antigen allows the retrieval of specific antibodies. The unique strengths of the technology for this project are (a) the ability to select, possibly rare, highly potent human antibodies from immune donors and evolve these antibodies in vitro to be still more effective and (b) the ability to exquisitely target selection to yield broad reactivities across a wide variation in virus phenotype. The latter is particularly important to be able to respond to new emerging viral strains. We propose that the antibodies generated in this project will find application as immunoprophylactic and immunotherapeutic reagents, will be valuable in guiding vaccine design and will be useful as analytical reagents. The studies carried out to date on murine antibodies to the hantaviruses, Hantaan and Puumala, provide strong indicators for the strategy to be pursued and the likelihood of success for Sin Nombre virus. Ebola virus is a more difficult problem but the principle of whether or not monoclonal antibodies can be used against filoviruses needs to be established.

DESCRIPTION (Adapted from the applicant's abstract) Passive immunotherapy with potent antibodies could find application in a number of aspects of HIV-1 infection, including the prevention of infection after accidental exposure to the virus, the interruption of transmission of virus from mother to child, and the reduction of viral load in combination with anti-retroviral drugs. Three human monoclonal antibodies (b12, 2G12 and 2F5) and an immunoadhesin (CD4-IgG2) show promise as potential immunotherapeutics by their ability to neutralize a wide range of primary isolates of HIV-1. Our aim is to evaluate the anti-viral efficacy of these antibodies, singly and in combination, in vivo in the hu-PBL-SCID mouse model. The specific aims of this proposal are: (1) To determine the in vitro neutralization properties of a number of antibodies, individually and in combination, for a chosen set of primary isolates (2) To determine the ability of antibodies alone and in combination to protect hu-PBL-SCID mice against challenge with primary isolates. (3) To determine the properties of in vitro selected neutralization escape mutants of primary viruses in the mouse model and the ability of antibodies of protect against these viruses (4) To attempt to generate escape mutants in vivo. The emerging results should assist in the design of immunotherapeutic strategies using neutralizing antibodies and illuminate areas of potential concern such as neutralization escape.

The aims of this study are to prepare and characterize human antibodies to human immunodeficiency virus type I (HIV-1), respiratory syncytial virus (RSV), cytomegalovirus (CMV), varicella zoster virus (VZV), measles virus and cryptosporidium parvum. The success of the approach is dependent upon the availability of tissue containing antibody- producing cells from appropriate donors identified by our colleagues in the Division of Infectious Diseases.

DESCRIPTION (Adapted from the applicant's abstract) Passive immunotherapy with potent antibodies could find application in a number of aspects of HIV-1 infection, including the prevention of infection after accidental exposure to the virus, the interruption of transmission of virus from mother to child, and the reduction of viral load in combination with anti-retroviral drugs. Three human monoclonal antibodies (b12, 2G12 and 2F5) and an immunoadhesin (CD4-IgG2) show promise as potential immunotherapeutics by their ability to neutralize a wide range of primary isolates of HIV-1. Our aim is to evaluate the anti-viral efficacy of these antibodies, singly and in combination, in vivo in the hu-PBL-SCID mouse model. The specific aims of this proposal are: (1) To determine the in vitro neutralization properties of a number of antibodies, individually and in combination, for a chosen set of primary isolates (2) To determine the ability of antibodies alone and in combination to protect hu-PBL-SCID mice against challenge with primary isolates. (3) To determine the properties of in vitro selected neutralization escape mutants of primary viruses in the mouse model and the ability of antibodies of protect against these viruses (4) To attempt to generate escape mutants in vivo. The emerging results should assist in the design of immunotherapeutic strategies using neutralizing antibodies and illuminate areas of potential concern such as neutralization escape.

neutralizing antibody; HIV infections; genetic library; recombinant proteins; HIV envelope protein gp120; epitope mapping; B lymphocyte; antibody specificity; protein structure; clinical research; laboratory mouse; monoclonal antibody; human subject;

DESCRIPTION: (Adapted from the Investigator's abstract): this is a new application from Dr. Dennis Burton to study the anti-viral activity of antibodies to a filovirus. The filoviruses Ebola and Marburg cause severe hemorrhagic fever and high mortality in humans and there are no vaccines or effective treatments available. The induction of a neutralizing antibody response is generally viewed as highly desirable in a vaccine but a formidable challenge is faced in the case of filoviruses because even convalescent patients exposed to large quantities of virus appear to mount very poor neutralizing antibody responses. However a potent neutralizing human monoclonal antibody has been generated to Ebola virus showing that such antibodies do exist and furthermore the antibody has been shown to protect against disease in animal models. A major question is whether a vaccine can be designed that elicits such antibodies. The investigator proposes an approach to this question based on further understanding the properties of the neutralizing human antibody, exploring the reasons for the low humoral neutralizing response to Ebola virus and attempting to enhance this response by immunotargeting strategies. In addition the potential role of a secreted form of the virus envelope in subverting the immune response is to be investigated. The specific aims are: (1) to further investigate the in vivo anti-viral activity of the human neutralizing monoclonal antibody KZ52; (2) to study the immunogenicity of the envelope glycoprotein GP by using various preparations of GP to immunize mice, rabbits and macaques and to select from human and monkey antibody libraries followed by analysis of the corresponding antibody responses; (3) to attempt to enhance the immunogenicity of GP using immunotargeting strategies; (4) to investigate the role of secreted GP (sGP) in infection in vivo by using anti-sGP antibodies to neutralize sGP in infected monkeys and monitoring the effects on the course of disease.

DESCRIPTION (Adapted from the applicant's abstract) Passive immunotherapy with potent antibodies could find application in a number of aspects of HIV-1 infection, including the prevention of infection after accidental exposure to the virus, the interruption of transmission of virus from mother to child, and the reduction of viral load in combination with anti-retroviral drugs. Three human monoclonal antibodies (b12, 2G12 and 2F5) and an immunoadhesin (CD4-IgG2) show promise as potential immunotherapeutics by their ability to neutralize a wide range of primary isolates of HIV-1. Our aim is to evaluate the anti-viral efficacy of these antibodies, singly and in combination, in vivo in the hu-PBL-SCID mouse model. The specific aims of this proposal are: (1) To determine the in vitro neutralization properties of a number of antibodies, individually and in combination, for a chosen set of primary isolates (2) To determine the ability of antibodies alone and in combination to protect hu-PBL-SCID mice against challenge with primary isolates. (3) To determine the properties of in vitro selected neutralization escape mutants of primary viruses in the mouse model and the ability of antibodies of protect against these viruses (4) To attempt to generate escape mutants in vivo. The emerging results should assist in the design of immunotherapeutic strategies using neutralizing antibodies and illuminate areas of potential concern such as neutralization escape.

There is concern that variola virus, the causative agent of smallpox which was eradicated as a human pathogen more than two decades ago, forms a threat to humans once again, this time as an agent of bioterrorism. The use of variola virus in a bioterrorist attack would be met by the use of the licensed live vaccinia virus vaccine. This vaccine may cause serious side effects which can be successfully treated with vaccinia immune globulin (VIG) derived from hyperimmune individuals. VIG however is in short supply and future availability is uncertain, and in addition suffers from the general concerns of using human blood products for therapeutic applications. This proposal aims to prepare and characterize human monoclonal antibodies against vaccinia virus which, likely formulated as an antibody cocktail, will constitute a replacement for VIG. We will isolate neutralizing antibodies against both infectious forms of vaccinia virus, i.e. intracellular mature virus (IMV) and extracellular enveloped virus (EEV). We will place particular emphasis on isolating antibodies against EEV, as EEV mediates dissemination of infection and is the viral form against which protective immune responses are directed. Inactivation of vaccinia virus will be studied in vitro and in vivo, and will be aimed at designing an antibody cocktail that provides protection against vaccinia virus infection in pre-exposure and post- exposure immunopropylaxis. The antibody cocktail designed may provide a treatment for smallpox itself. To examine the impact of passive immunization in immunoprophylaxis and immunotherapy of a smallpox-like disease in a non-human primate model, we will use an experimental model of monkeypox virus infection

[unreadable] DESCRIPTION (provided by applicant): Neutralizing antibodies have been shown to protect against HIV challenge in some of the best available animal models. Antibodies given intravenously can protect macaques against intravenous or mucosal SHIV challenge and topically applied antibodies can protect macaques against vaginal SHIV challenge. The mechanisms of this protection are not understood. It is often assumed that neutralization of free virus particles, as measured in classical in vitro assays, is solely responsible because protection is afforded by neutralizing antibodies. However neutralizing antibodies can also mediate a number of other anti-viral activities, notably against infected cells. These include effector functions such as complement activation and antibody dependent cellular cytotoxicity mediated by the Fc part of the antibody molecule. The critical importance of effector functions for antibody-mediated protection against a number of viruses has been well documented in the literature. The purpose of this application is to explore the importance of effector function in antibody-mediated protection against HIV. It is proposed to investigate the ability of a panel of antibody molecules, derived from the human neutralizing antibody IgG1b12, to protect against vaginal challenge with an R5 SHIV in macaques. The conditions for intravenously or topically administered IgG1b12 to protect against vaginal challenge have been established previously. The current application makes use of a panel of IgG1 b12variants in which effector functions have been specifically disabled to better understand the role of effector functions in systemic and mucosal antibody-mediated protection. In addition, a secretory IgA version of b12 (SIgA2b12) will be used to compare the ability of mucosal IgA and IgG to protect and to reveal the importance of functions associated with SIgA in protection. The results will help clarify how antibodies protect against HIV and may have implications for vaccine design and microbicide development. [unreadable] [unreadable] [unreadable]

The hu-PBL-SCID mouse model can be a useful tool for assessing the efficiency of certain anti-HIV approaches and for looking at patterns of viral evolution in the face of selection pressure. For instance we have used the model to study viral escape from treatment with cocktails of neutralizing human monoclonal antibodies. Here we propose to use the model for in vivo evaluation of selected entry-blocking strategies and potential subsequent viral evasion. We propose three specific aims: (1) to evaluate in vivo the anti-viral efficiency of a potent human polyclonal neutralizing antibody response by passive transfer of this response to hu-PBL-SCID mice. We will generate in vivo serum escape mutants in order to better understand the specificities of the Abs responsible for the strong serum neutralizing activity. This should help in the design of vaccines capable of inducing an efficient humoral response. (2) to evaluate the role of cell-to-cell transmission in viral replication in vivo. Our results show that in the hu-PBL-SCID mouse, as has been suggested in HIV-1 infected individuals, viral cell-to-cell spread occurring in lymphoid tissue is of primary importance. We will study cell-to-cell spread in the mouse model in order to understand whether cell-to-cell transmission could impair anti-viral strategies based on blocking viral entry. (3) to evaluate the anti-viral efficiency of blocking of HIV-1 coreceptors in vivo. Our preliminary results indicate that Ab-blocking of CCR5 in the hu-PBL-SCID mouse temporarily decreases HIV-1 replication in vivo but may induce the virus to escape coreceptor blocking. We will continue this study and investigate whether blocking coreceptor with Ab cocktails could prevent viral escape. The studies will reveal how HIV-1 can evade entry-blocking strategies, and may provide important information for vaccine design.

DESCRIPTION (provided by applicant): Transmissible spongiform encephalopathies (TSEs) or prion diseases, are neurodegenerative diseases of conformation in which the cellular prion protein (PrPC) misassembles into an abnormal conformer (PrPSc). PrPSc is thought to be the sole constituent of infectious prion particles. Prion propagation proceeds via a highly-specific process of conformational rearrangement in which PrPSc imposes its own structure upon PrPC bound to it. The transmission of bovine spongiform encephalopathy prions to humans, manifesting clinically as variant Creutzfeldt-Jacob disease, and ongoing spread of chronic wasting disease in cervids in North America, have demonstrated the pressing need for effective prion therapeutics. In this proposal we have developed experimental strategies designed to yield small molecule inhibitors of prion propagation suitable for use in the prevention and/or therapy of prion infection and disease. Two distinct approaches have been taken. In the first, drawing upon our considerable experience using PrP-specific antibodies, we seek to identify small compounds binding specifically to key regions of either PrPC or PrPSc. We hypothesize that molecules preventing PrPC-PrPSc interactions will effectively inhibit assembly of the prion replicative complex, preventing generation of nascent prion infectivity. In a second approach, small molecules binding specifically to PrPC, increasing the intrinsic thermodynamic and kinetic stability of this molecule and thereby precluding the possibility of unwanted conformational changes that are intimately associated with prion pathogenesis and PrPSc formation will be identified. Molecules recovered via both of these strategies will be evaluated for their capacity to resolve prion infection in vitro and in vivo.

[unreadable] DESCRIPTION (provided by applicant): A major goal of HIV vaccine candidates is to elicit broadly neutralizing antibodies. Many features of HIV envelope glycoproteins (Env), the target of neutralizing antibodies, make this a difficult goal. These features include poor exposure of conserved regions of Env, better exposure of variable regions and a dense carbohydrate covering. Nevertheless, a small number of broadly neutralizing monoclonal antibodies (mAbs) directed against conserved epitopes have been isolated and they represent potential tools for vaccine design. This application concentrates on one of these, mAb b12, and, in particular, on attempts to design molecules that focus antibody responses to the 1012 epitope, which overlaps the conserved CD4 binding site on gp120. We have solved the structure of b12 and characterized its interaction with Env gp120 in some detail by modeling, mutagenesis and other studies. We have shown how gp120 can be engineered, notably by the introduction of extra carbohydrate groups, to preferentially bind b12 relative to a panel of non- and weakly neutralizing mAbs. Here we propose to investigate the immunogenicity of engineered gp120s in animal models and to use the resulting data to improve immunogen design. We also propose to investigate the ability of the engineering strategy to immunofocus responses to the b12 epitope on recombinant trimeric Env and virus-like particles (VLPs). Finally, in a conceptually different approach, we will chemically modify gp120 bound to b12 and then attach immuno-inert large moieties, including carbohydrates, to mask the regions not contacted by b12. Immunization with this molecule should, in principle, be akin to immunizing with the footprint of b12. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The design of an immunogen able to elicit broadly neutralizing antibodies is a major, but so far elusive, goal of HIV vaccine research. The existence of such antibodies is indicated by the description of rare broadly neutralizing HIV sera and a handful of broadly neutralizing human monoclonal antibodies (bnMAbs) isolated from HIV infected individuals. To understand how to better induce broadly neutralizing HIV antibodies it is important to identify the appropriate B cell precursors and to stimulate them with effective antigens. For this reason we propose to develop mouse strains carrying B cells with germline versions of bnMAbs and to follow their responses to test immunogens. This approach renders visible the responses of the very B cells that we wish to recruit into the HIV antibody response and should allow rapid screening of vaccine candidates for the ability to elicit neutralizing antibodies in a context in which the appropriate precursors are present in high frequency. Failure of response in this context would not be ascribable to a deficiency in appropriate B cells unless such B cells are eliminated by immune tolerance, an outcome that can be experimentally excluded. The specific aims of the proposal are (1) to generate transgenic (knockin) mice carrying B cells expressing germline versions of the bnMAbs b12, 2G12 and 2F5 and (2) To investigate antibody responses in the transgenic mice to a wide variety of immunogens using a number of immunization strategies. The project is a close collaboration between a laboratory (Nemazee) with expertise in B cells, including knockin mice, and one (Burton) with expertise in antibodies and HIV. It is expected to generate valuable reagents and animals for the proposed UO1 consortium.

[unreadable] DESCRIPTION (provided by applicant): Human metapneumovirus (HMPV), although only recently discovered, has rapidly become recognized globally as a major respiratory pathogen in young children, the elderly and immunocompromised individuals. It is closely related to human respiratory syncytial virus (HRSV), and produces an analogous clinical syndrome in infected individuals. In this highly focused application, we propose to thoroughly characterize the human antibody response against HMPV by recovering an extensive panel of over 50 monoclonal antibodies elicited in different individuals as a result of natural infection with this pathogen. Studies will be directed to antibodies recognizing the fusion (F) protein of the virus, which is largely invariant between heterologous virus strains, is highly immunogenic and is the target of neutralizing antibody responses. Using this human monoclonal antibody panel, we will map antigenically the HMPV F protein and identify antibodies with exceptional virus neutralizing properties that are able to protect rodents from virus challenge and are well positioned for immediate clinical development as passive prophylactic agents for the prevention of HMPV disease in vulnerable patient cohorts. The neutralization potency of selected antibodies will be further improved in vitro with the aim of enhancing their potential clinical efficacy in protecting from HMPV infections. [unreadable] [unreadable] The proposed grant as four aims. (1) To recover an expansive panel of human monoclonal antibodies against the HMPV fusion (F) protein, (2) To determine the neutralization potency of the monoclonal antibodies against HMPV in vitro and in vivo, (3) To evolve ultra-potent HMPV neutralizing antibodies from the naturally occurring antibodies we first recover. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Human metapneumovirus (HMPV), although only recently discovered, has rapidly become recognized globally as a major respiratory pathogen in young children, the elderly and immunocompromised individuals. In this program, we propose to thoroughly characterize the human antibody response against the fusion (F) protein of the virus. The F protein is largely invariant between heterologous virus strains, is highly immunogenic, and is the target of neutralizing antibody responses. Human antibodies with exceptional HMPV neutralizing properties and that protect rodents from virus challenge will be identified and evolved. Such antibodies will be positioned for immediate clinical development as passive prophylactic agents for the prevention of HMPV disease in vulnerable patient cohorts. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Approximately 170 million people are infected by hepatitis C virus (HCV) worldwide and many of these will go on to develop disease, primarily cirrhosis of the liver and hepatoma. A vaccine is required to help limit spread of the disease. Most effective vaccines developed to date elicit neutralizing antibodies (NAbs). There have been at least two major roadblocks en route to developing a component that elicits NAbs to HCV. The first is the lack of a conventional neutralization assay because of an inability to culture HCV in vitro. This roadblock has recently been at least partly dismantled by the development of systems for culturing HCV, albeit currently with certain limitations. The second roadblock is the great sequence variation of HCV found in infected donors that suggests a vaccine should elicit NAbs effective against a wide variety of different viruses, i.e. the vaccine should elicit broadly neutralizing antibodies. We propose a program to investigate the necessary conditions to induce a strong neutralizing antibody response against multiple HCV isolates and use the knowledge gained to design a NAb-inducing component of an HCV vaccine. We will systematically dissect NAb responses to HCV in natural infection, and isolate mAbs that are most potent and broad against neutralizing diverse isolates of HCV (Aim 1). These prototype mAbs will help us to identify neutralizing epitopes on HCV and we will explore the interaction between these epitopes and NAbs at the molecular level (Aim 2). We will apply knowledge gained from these molecular studies to the design of immunogens in order to produce NAb responses in animals (Aim 3). The prototype broadly neutralizing mAbs and Abs from animals immunized with the novel immunogens will be tested in a small animal model (Aim 4).

The overall mission of this CHAVI-ID application is to define immunogens and immunization regimens that induce sustained HIV cross-protective B cell and CD4+ T cell responses in preclinical models and, thereby, guide product development strategies for a preventive human AIDS vaccine. Focus #1 will concentrate on B cell and antibody research to facilitate the development of immunogens and immunization protocols that elicit protective broadly neutralizing antibody responses. Our overall strategy in this focus can be briefly summarized in the following goals: 1) To fully define a comprehensive map of the broadly neutralizing (bn) epitopes of the HIV Env spike. Breakthroughs in generating broadly neutralizing human monoclonal antibodies (bnMAbs) in the last two years are bringing this goal within reach; 2) To determine which of the HIV bnMAbs provide the most effective protection against viral challenge in the non-human primate (NHP) model. At the same time, we shall closely monitor developments in the field, including suggestions that some non-neutralizing antibodies may have protective qualifies that might be exploited in vaccine discovery; 3) To determine which HIV broadly neutralizing Abs (bnAbs) are most readily elicited through natural infection and how and when they are elicited using several large cohorts to which we have unique access; 4) To determine which immunogens and immunization strategies best stimulate HIV bnAb generation in the knock-in mouse and NHP models, drawing upon data collected on bnAbs in goals 1-3 and emerging from Focus #2. As we gather together information on optimal immunogens and immunization strategies, we will move forward with small-scale human trials with the advice and close involvement of our Vaccine Discovery Scientific Research Support Component.

The CHAVI-ID will be established as an integrated, priority-setting consortium with major components at multiple participating institutions and substantial NIH programmatic involvement in a collaborative partnership role with the Awardee to advance the science. Therefore, the Operations and Management Scientific Research Support Component (SRSC) is designed to function across institutional and geographic boundaries to provide the administrative activities that allow successful scientific collaboration and flow of resources. For efficiency of operations, the overall administrative and scientific management will be centralized, with the Operations and Management SRSC based at The Scripps Research Institute (TSRI), the Sponsoring Institution. The SRSC will be overseen by the CHAVI-ID Director and an on-site experienced Chief Operating Officer will dedicate 100% effort to managing CHAVI-ID administrative activities. An Operations Team will function on the premise of flexibility, as evolving scientific priorities and collaborative demands are to be expected during the extended period of the award. However, the flexibility will not be at the expense of efficiency or responsiveness to the needs of the collaborating scientific institutions. This management structure will effectively leverage the associated resources and program offices offered by TSRI. The Specific Aims of the SRSC are: Aim 1: To establish a highly efficient operations and management structure that will provide essential support services to all CHAVI-ID investigators and leverage available resources at the sponsoring and collaborating institutions Aim 2: To assist the flow of information and scientific collaboration between CHAVI-ID Focus Teams by supporting meeting, teleconference, and information technology demands Aim 3: To implement the decisions of the CHAVI-ID Director and Leadership regarding direction of the Strategic Plan, allocation of future funding support, and changing composition of the group of collaborating CHAVI-ID scientists.

DESCRIPTION (provided by applicant): The central hypotheses of this CHAVI-ID application are that a successful HIV vaccine should elicit protective antibodies (Abs), and that the combination of B cell and CD4+ T ceil responses is critical for the induction and long-term maintenance of vaccine protection. The overall mission of the application is to define immunogens and immunization regimens that induce sustained HIV cross-protective B cell and CD4+ T cell responses in preclinical models and, thereby, guide product development strategies for a preventive human AIDS vaccine. We propose to develop an HIV vaccine based on a deep understanding of the critical attributes of immune responses that provide protection against AIDS viruses, through two focused and highly integrated efforts. Focus #1 will concentrate on B cell and antibody research to guide the development of immunogens that elicit protective HIV antibody responses in appropriate animal models. Focus #2 will concentrate on CD4+ T cell research, taking advantage of key preliminary data to maximize the T cell help offered to B cell responses through immunization, and to harness the direct antiviral activity of CD4+ T cells. We argue that this combined approach will lead to the discovery of novel immunogens and immunization strategies that will generate measurable cross-protective antibody responses. We will then build on such advances iteratively, by improving the most promising constructs and protocols until vaccine protection is achieved. A critical component of our proposal is the strength of our research team in immunology, virology and structural biology. The Director, Dennis Burton, has made major contributions to understanding broad antibody neutralization of HIV and antibody protection in animal models of HIV infection. The Scientific Leadership Group (SLG) members are Rati Ahmed, Michel Nussenzweig, Bruce Walker and Ian Wilson, who have made crucial advances in describing the structure and function of antibodies against HIV and the induction and maintenance of B and T cell immunity to the virus. The team will be sustained by five Scientific Research Support Components (SRSCs), including strong Operations and Management, that have been assembled to maximally accelerate progress toward the designated goals.

DESCRIPTION (provided by applicant): In this application, we are proposing to create a novel class of anti-HIV-1 immunotherapeutics by conjugating HIV-1 fusion inhibitors to exceptionally broadly neutralizing antibodies (bNABS). The cell surface receptors utilized by HIV-1 (CD4, CCR5, CXCR4) are attractive targets for therapy because they are under no pressure to evolve resistance. Unfortunately, HIV envelope protein (Env) mutations are readily evolved to escape small molecule receptor blockade. Likewise, bNABs have been discovered that neutralize up to 90% of tested viral strains but when HIV is challenged with antibodies, in vivo resistance quickly develops. In both cases, the resistance is due to mutations in the Env, although mutations to escape one molecule can make HIV more sensitive to another. Our hypothesis is that a bNAB modified with anti-HIV fusion inhibitor would be an exceptionally potent compound and that a sufficiently modified bNAB would be multifunctional and act as single molecule cocktail. Such a compound would make evolution of resistance very difficult for HIV because it would require many mutations to escape the binding sites of the bNABs and the sterically blocked cell receptor sites. We propose to synthesize validated small molecule HIV inhibitors that target the co-receptors CCR5 and CXCR4, as well as, small molecules that bind and disrupt the CD4/gp120 fusion. These compounds will be synthesized linked to protein reactive moieties which will allow for specific modification of bNABs. Our lab has recently developed two different approaches for specific targeting of tyrosine residues for this purpose. The well characterized and very broadly neutralizing antibodies PG9 and VRCO1 will serve as the scaffold for modification. In addition to chemical modification, we will also investigate creating genetic fusions to combine the Fv's of PG9 and VRCO1 onto a single molecule, as well as, engineering the Fc domain to increase in vivo half-life. Multi-specific antibodies will be assessed for their ability to neutralize a broad panel of HIV-1 isolates including strains which are resistant to the bNABs and the small molecule fusion inhibitors. Successful candidate antibodies will then be assayed for half-life and toxicity. Finally, multi-specific antibodies will be used in an HIV escape assay to test our hypothesis that these molecules can pose an evolutionary challenge that HIV cannot answer. We anticipate that the products of this research will be exceptionally potent and broadly active HIV-1 immunotherapeutics and prophylactics.

DESCRIPTION: The goal of the proposed research is to develop a potentially curative AIDS treatment. We hypothesize that novel cell-penetrating zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) will allow for direct gene editing in human CD4+ T-cells and CD34+ hematopoietic stem cells (HSCs), thereby bypassing concerns and difficulties posed by contemporary delivery systems. Direct application of engineered nuclease proteins will lead to biallelic disruption of CCR5/CXCR4 genes and allow for HIV-1 resistant cells to repopulate and protect the body, eventually allowing affected individuals to go off drug therapy and on to a life free of disease. Success may require combining our therapy with emerging drugs that activate latent viral reservoirs allowing them to be eliminated by the gene edited immune cells we develop here. Driving our enthusiasm is the report of an HIV-1 positive patient cured by stem cell transplant from a ?32 donor. The Berlin patient had no detectable levels of HIV-1 years after the procedure despite discontinuing retroviral therapy. We aim to recapitulate this success using our innovative approach that is free of nucleic acids and viral delivery vehicles. While clinical trials using adenoviral delivery of ZFN SB-728 are currentl underway, recent data indicates that low efficiency of biallelic modification observed in phase I studies may limit its potential to patients whom are ?32 heterozygous. Significant improvements in ZFN nucleases are needed. In preliminary studies we have shown that cell-penetrating ZFN proteins can knock-out CCR5 after their application to cell lines, T-cells, and HSCs. We found that cells treated with our proteins showed a high rate of biallelic modification. In one experiment, 58% of modified cells showed disruption at both alleles. The efficiency of our approach is similar to DNA vector transfection; however, our approach provides dramatic reductions in off-target cleavage. In new preliminary data, we have developed a novel approach to cell- permeable TALENs and demonstrated gene editing at CCR5. Here we develop a family of very efficient CCR5/CXCR4 targeting nucleases using design and in vitro evolution approaches. ZFNs and TALENs will be optimized with respect to catalytic activity, sequence specificity, cell-permeability, and biallelic gene editing. Coupled with charge engineering of nucleases should allow for dramatic increases in the quantity of transduced protein delivered to cells. Because our approaches have allowed us to identify FokI nuclease variants with >15 fold improvement in activity and novel sequence specificity, we are confident that the nucleases developed herein will edit T cells and HSCs at levels exceeding that reported by viral delivery systems. The nucleases developed here will demonstrate higher specificity and improved safety profiles. We will optimize treatment of human T-cells and HSCs with purified proteins, study their transplant into immunocompromised mice, and their ability to limit HIV-1 infection. The efficiency and safety of ZFN/TALEN protein delivery will be compared to conventional delivery methods. We believe that the development of the permeable nucleases proposed here could prove to be a breakthrough in HIV-1 therapy and many other genetic diseases.

Project Summary/Abstract (30 lines) The overarching goal of this CHAVD is to develop a sequential HIV vaccine regimen that induces sustained protective levels of broadly neutralizing antibodies (bnAbs) in humans. bnAbs provide complete protection against HIV infection in preclinical models. We hypothesize that an effective HIV vaccine will need to consistently induce bnAbs against 2-3 different sites on the HIV Envelope glycoprotein (Env) of the virus in most (>90%) of vaccine recipients. Targeting multiple sites is necessary to provide adequate coverage against the huge diversity of global isolates. We propose a sequential strategy in which a series of designed immunogens guide antibody responses from precursors to bnAbs. We are intensely developing and testing immunogens. We have shown proof-of-principle of the sequential strategy in preclinical models and our first three immunogens are entering manufacturing or clinical trials shortly. The second major goal of this CHAVD is to generate immunogens that induce protective non- neutralizing antibody (nnAb) responses that can either act alone or augment the protective activity of bnAb responses. To accomplish this goal, we propose a tiered approach in which the ability of nnAbs to capture infectious virions and to clear infected cells in vivo in two established mouse models will be explored. The most effective nnAbs, alone and in combination with other nnAbs and bnAbs, will then be assessed for their ability to protect nonhuman primates against virus challenge. Protective nnAbs will be used as templates for immunogen design. To support our translational effort, we have organized state-of the-art Manufacturing, Clinical Trials Sample Analysis and Management and Operations units. In addition, we have established twelve Scientific Research Support Units, headed by leaders in their fields, to underpin the diverse scientific and technical capabilities required to execute our comprehensive and highly integrated vaccine program Finally, this CHAVD proposal is built upon a highly successful, innovative and efficient CHAVI- ID program that has made major scientific contributions to the HIV vaccine field. The CHAVD provides the opportunity to advance and translate these contributions into an HIV vaccine.