Phased Innovation Awards (PIA) Abstracts
Stephen Dewhurst
Overcoming Low HIV-1 Envelope Spike Immunogenicity by High Density Display
The display of proteins in dense, repetitive arrays in known to result in strong humoral immune responses, as exemplified by the virus-like particle (VLP) vaccine for human papillomavirus. This concept has relevance to HIV-1 vaccine development, because recent structural studies of HIV-1 virions have shown that the envelope spikes on the virus surface are sparse and irregularly distributed. This may contribute to low spike immunogenicity, and complicate the generation of broadly neutralizing antibodies. This proposal will test the hypothesis that the immunogenicity of HIV-1 envelope spikes is limited, in part, as a result of their sparse and irregular distribution on the virion surface. To do this, HIV-1 envelope spikes will be displayed at high density on a repetitively ordered scaffold (provided by the bacteriophage lambda capsid). Env display will be acheived using a simple in vitro complementation system, to "decorate" phage particles with soluble Env oligomers, produced in mammalian cells. In the R21 (feasibility) phase of this proposal, experiments will be conducted to generate lambda phage particles that display well-characterized, oligomeric envelope spikes on their surface at both high and low density. The immunogenicity of these phage particles will then be assessed in a small animal model, to determine if ordered, high density display of envelope spikes results in a measurable increase in the magnitude or quality of Env-specific antibody responses (including virus-neutralizing antibodies). If quantifiable milestones are met, the project will progress to the R33 (development) phase. This phase focus on the evaluation of methods intended to further improve the quality and magnitude of the humoral immune response elicited by lambda phage particles displaying HIV-1 Env. The focus will be on the evaluation of practical, translationally-relevant strategies that can be readily applied to enhance humoral immune responses to the phage-displayed envelope spikes. The studies will culminate in a proof-of- concept immunogenicity study in non-human primates, in which the immune response to phage-displayed envelope spikes will compared to that elicited by a soluble oligomeric envelope preparation. Collectively, these experiments are expected to provide a comprehensive proof-of-concept evaluation of our hypothesis that ordered, dense display of HIV-1 envelope spikes on the lambda phage scaffold will allow for the generation of improved antibody responses to HIV-1 Env.
Eckhard Podack
Induction of Mucosal SIV Immunity in Non-Human Primates by Secreted Hsp-Gp96
Cellular immunity and memory is required for clearance of viruses and for protection from viral infection. Cellular immunity by CD8 CTL and NK cells is initiated through activation of the innate immune response, maturation of dendritic cells (DC) and antigen cross presentation to CD8 T cells. Anti viral vaccines stimulating cellular immunity have to imitate this process. Adjuvants for the stimulation of antibody responses are used effectively however our knowledge about adjuvants for the stimulation of CTL immunity is limited. We have demonstrated that the endoplasmic reticulum resident heat shock protein gp96, a chaperone for peptides transported to be presented by MHC-I, is a natural adjuvant for the activation of DC, NK and cognate CD8 T cells. DC and macrophages have receptors for gp96-peptide complexes and become activated upon gp96-binding. Gp96 together with its associated peptides is taken up the APC, the peptide moiety is transported to the ER and used to charge MHC-I molecules. Concomitant activation of DC results in more than million fold enhanced cross priming of cognate CD8 T cells when compared to cross priming by intact protein taken up by DC. To take advantage of this unique adjuvant effect and the ability to transport relevant peptides, we have made a secretable form of gp96, gp96-lg, by replacing the KDEL ER-retention signal with the Fc portion of lgG1. We hypothesize that cell-based gp96-lg vaccines, by prolonged in vivo secretion of gp96-lg-peptide, imitate viral replication and provide immune stimuli comparable to attenuated viruses. In model systems in mice we have shown that gp96-lg transfected, antigen expressing tumor cells secrete gp96-lg in vivo and stimulate the innate DC and NK as well as adaptive, cognate cellular CD8 CTL immune response and generate specific CD8 memory independent of CD4 help and in the absence of lymph nodes. Both systemic and strong mucosal immunity in intraepithelial, lamina propria and Peyer's patch CD8 CTL is generated by gp96-lg vaccines. Because of their unique properties we now plan to evaluate the gp96- vaccines in non-human primate models for SIV for their immunogenicity for mucosal and systemic cellular immunity (R21). In addition we will examine the protective power of SIV-gp96-vaccines against subsequent viral challenge (R33). To maximize the chances of success, the team in Miami (Podack/Pahwa) has entered into a collaboration with experts (Franchini lab) at the NIH bringing together basic immunologists with experts in human and non human primate HIV/SIV pathogenesis.
Haynes Sheppard
Whole Inactivated HIV-1; Stabilization and Immunogenicity Display
A traditionally successful approach to viral vaccines, whole inactivated virus (WIV), has received relatively little attention in the overall effort to develop an effective HIV/AIDS vaccine. Only one inactivated virus product (the Salk/IRC Immunogen, "Remune") has been tested in humans but this product is envelope depleted and tested only as a therapeutic rather than a prophylactic immunogen. Since the primary target epitopes for antibody neutralization are in the envelope glycoprotein, it is generally considered an indespensible component of a prophylactic immunogen. Thus far, however, immunization of humans with monomeric recombinant envelope glycoprotein (rgp120) has resulted in relatively type-specific neutralization, very little neutralization of primary isolates, and no efficacy. The few monoclonal antibodies that demonstrate broad primary-isolate neutralization generally recognize conformational epitopes. The primary hypothesis driving this proposal is that such epitopes can be retained, and presented in an immunogenic form, on WIV. Our prior work focused on 1) developing methods for scalable propagation of diverse primary isolates of HIV-1, 2) assessing the retention and structure/function of gp120 during various methods for propagation, processing, and inactivation, 3) evaluating the combination of two orthogonal inactivation procedures, and 4) assessing the animal immunogenicity of WIV, using one set of possible production protocols and vaccine formulation. Preliminary studies were promising, showing relatively broad heterologous neutralization of primary isolates, despite rather small doses and low purity of the WIV. The objective of this proposal is to evaluate promising but unproven methods for virus stabilization, to achieve dramatic improvements in the yield, purity, and immunogenicity of WIV without sacrificing the structural integrity of the native gp120 trimers. These novel methods for virus stabilization are based on studies of cryptobiotic species that are capable of surviving drastic but transient environmental conditions (e.g. high salt, high heat, freezing, and desiccation) due to high concentrations of solutes, which are variably known as osmolytes, kosmotropes, or compatible solutes. The best studied of these is the disaccharide trehalose, which has been used to stabilize viral vaccines during lyophylization. We hypothesize that the innovative use of such solutes will yield dramatically improved WIV immunogenicity as well as the scalability that will be needed if this candidate were to proceed to human testing. In addition to the benefits of this approach for the WIV vaccine concept, methods for improving the purity and yield of intact viruses during processing and purification could contribute to many other HIV vaccine concepts (e.g. those using recombinant viral vectors such as vaccinia, avipox, VSV, VEE, etc.) and could also impact the gene therapy field where retroviruses are commonly used as the delivery vehicle and product yield is a major limiting factor. If this effort is successful at improving WIV yield and immunogenicity (R21 phase), we will conduct a proof-of-principle efficacy study in macaques using subtype-C WIV as the vaccine, followed by challenge with a heterologous subtype C infectious and pathogenic SHIV (R33 phase). Therefore, this proposal represents a critical step in qualifying this HIV vaccine concept for transition from pre-clinical to clinical studies.
David Curiel
Capsid-Incorporation of HIV Antigens as a Novel Adenovirus HIV Vaccine Approach
Despite the many potential advantages of Ad vectors for vaccine application, full utility of current Ad vaccines may be limited by the host anti-vector immune response. Specifically, the anti-Ad humoral immunity abrogates the effectiveness of subsequent administrations of the Ad vector, confounding expression of the encoded transgene, and thus practically restricting the gains that might be accrued via booster effect. In order to exploit the inherent antigenicity of the Ad vector we have developed a vaccination approach based on incorporation of the immunizing antigen epitope directly into the Ad capsid. This novel paradigm is based upon Ad presenting the antigen as a component of the capsid rather than an encoded transgene. Incorporation of immunogenic peptides into the Ad capsid offers potential advantages. Most noteworthy, the processing of the capsid incorporated antigen via the exogenous pathway should result in a strong humoral response akin to the response provoked by native Ad capsid proteins. In addition, since anti-Ad capsid responses are augmented by repeated vector administration, immune responses against antigenic epitopes that are part of the Ad capsid should be augmented by repeated administration as well, thus allowing boosting. These considerations suggest that this novel capsid-incorporated antigen approach may offer exciting potentials to realize Ad-based vaccine strategies that circumvent the major limitations associated with Ad vectors. Critical to the realization of this approach is to define the optimal configuration of antigen in the adenoviral capsid context. To this end, we have established several key technologies that will enable us to reach our goal. In particular, we have developed the means to incorporate heterologous peptide epitopes within the surface-exposed domains of the major Ad capsid protein hexon. We have begun to determine the size and structural factors that predicate functional utility of these domains in the hexon. In addition, we have developed the means to apply cryoelectron microscopy (cryoEM) single particle reconstruction methods to allow us to explore the capsid-incorporated peptide localization with unprecedented, subnanometer resolution. Based on these technologies, we will be able to establish the critical correlates between antigen locale/accessibility within the capsid context and vaccine efficacy. On the basis of these established feasibilities, we hypothesize that Ad vectors can be created with novel capsid-incorporated antigens that can serve as vaccine agents against HIV in animal models. CryoEM-guided capsid design will be applied to develop an optimized vector with optimal anti-HIV immunization. We envision that our proposed structural studies will provide complementary information to in vitro assays and biological readouts and thereby will enable us to understand the functional determinants of incorporated HIV epitopes. This project will design new and innovative methodologies to create HIV vaccines, in hopes of preventing the spread of HIV disease.
Donald Forthal
Fcg Receptor Polymorphisms and Risk of HIV Infection
Interactions between antibody and Fcg receptors (FcgRs) mediate many biological activities of antibody. FcgRIIIa, found on cells targeted by HIV-1, is encoded by a gene with a polymorphism affecting IgG binding affinity. This polymorphism results in either a phenylalanine (F) or a valine (V) in a part of the receptor that binds to IgG; there are three possible genotypes: FF, FV, or VV. Participants in a large HIV vaccine trial (Vax004) who had the VV genotype were more likely to become infected if they received vaccine than if they received a placebo. This finding indicated that the vaccine increased the risk of infection, probably due to antibody-dependent enhancement (ADE) of infection. ADE presents a major potential obstacle to the development of vaccines that depend wholly or partly on HIV-specific antibody responses. In the proposed research, assays will be developed to test the mechanisms responsible for the findings in the Vax004 trial. The long-term goals are to understand early events in HIV infection and develop vaccination strategies that take advantage of FcgR biology and avoid future harm. In the R21 phase, the following hypothesis will be tested: the relationship between FcgRIIIa genotype and HIV infection risk observed in Vax004 can be modeled in vitro. The SPECIFIC AIMS are: 1) develop an in vitro model of FcgRIIIa genotype-dependent differences in HIV-1 replication based on direct infection of FcgRIIIa-bearing cells. Natural killer cells (NKs), macrophages (Mfs) and dendritic cells (DCs) expressing the different forms of FcgRIIIa will be used as target cells for antibody-coated HIV and; 2) develop in vitro models of FcgRIIIa genotype-dependent differences in HIV replication based on indirect effects of FcgRIIIa-bearing cells. Assays will measure HIV replication in CD4+ lymphocytes after transfer from FcgRIIIa-bearing cells or after cross-linking FcgRIIIa. If successful in developing such assays, mechanisms that account for the increased infection risk after vaccination in VV individuals will be investigated in the R33 phase. The SPECIFIC AIMS in the R33 phase are: 1) determine the intracellular fate of opsonized virus in Mfs or DCs with high and low affinity FcgRIIIa forms; 2) determine if the efficiency of virus transfer to lymphocytes and if the production of pro-viral and anti-viral cytokines/chemokines are regulated in an FcgRIIIa-dependent manner and; 3) develop reproducible assays to test multiple virus strains and sera for FcgRIIIa genotype-associated enhancement. Information gained from this research will be crucial to understanding early events in HIV infection and provide direction for the development of effective HIV vaccines.
Anna-Lise Williamson
BCG as an HIV Vaccine Vector
In South Africa more than 11% of the population are infected with HIV-1 so there is a clear need for a prophylactic HIV-1 vaccine. The focus of this application is the use of Mycobacterium bovis bacille Calmette-Gu[1]rin (BCG) as an HIV vaccine vector. BCG - which is better known as the tuberculosis vaccine - is widely used to immunize infants, has strong adjuvant potential in man and animals, can be administered orally, is inexpensive to produce, is heat stable, elicits long-lasting cellular immune response and has a very low rate of complications. These characteristics make BCG a very attractive vehicle for recombinant vaccines. This vaccine is likely to induce good cellular immune responses but not neutralising antibodies. Despite these advantages there are also problems expressing viral antigens in BCG, which result in low immunogenicity and genetic instability. The hypothesis being tested in this application is that immunogenicity and stability of recombinant BCG (rBCG) expressing HIV antigens can be improved either through modification of the HIV genes or by regulation of HIV gene expression. The aims of the proposal are: To modify HIV genes to increase the stability of rBCG and increase immunogenicity. To develop systems which allow for down regulation of expression of HIV genes in rBCG and up regulation in animals. To assess the immunogenicity of rBCG expressing HIV genes in combination with matched virus like particles (VLP) and modified vaccinia virus Ankara (MVA) based HIV candidate vaccines initially in mice and then in non-human primates. The vaccine will be designed for South Africa where HIV-1 subtype C is the dominant circulating subtype. Genes were selected from recently transmitted HIV and closest to the South African consensus sequence. As it is likely that a vaccine will be more effective with more genes, this project will initially focus on gag and reverse transcriptase but will then move on to include the gene encoding HIV-1 subtype C gp120.
David Watkins
Vaccine Regimens to Induce CD4+ and CD8+ T cells against SIV Epitopes
The only solution to the HIV epidemic in the developing world is a vaccine that either prevents infection or reduces transmission. Recent data from our laboratory suggest that CD4+ T cell responses along with CD8+ T cell responses against subdominant epitopes can lead to control of replication of SIVmac239. The goal of this proposal, therefore, is twofold. First, we would like to investigate the importance of CD4 help in an HIV vaccine. Second, we want to use innovative new technologies to vaccinate for specific peptides rather than whole proteins. This would eliminate the use of large, commonly seen vectors, thereby circumventing immunodominant responses against vector-derived epitopes. Our new vaccination regimen should induce both CD8+ and CD4+ T cells that efficiently control viral replication. Using newly developed, novel vaccine regimens, we hypothesize that vaccinating macaques with CD4 and subdominant CD8 epitopes will generate immune responses that should control SIV replication. In Specific Aim I of the R21, we will induce CTL specific for subdominant Mamu A*01-restricted SIV epitopes. In Specific Aim II we will engender multiple CD4+ T cell responses against epitopes that are commonly seen in SIV-infected elite controller rhesus macaques. In the R33, we will use the most effective vaccine regimen(s) from the R21 phase to engender CD4+ T cell responses and subdominant CD8+ T cell responses. Animals will be vaccinated with subdominant CD8+ T cell epitopes, CD4+ T cell epitopes, or a mixture of the subdominant CD8+ T cell and CD4+ T cell epitopes. These experiments will allow us to elucidate the role of CD8+ T cell responses against subdominant CD8+ T cell epitopes and CD4 help in an effective HIV vaccine. Project Narrative: Recent vaccine trials have failed to protect individuals from HIV infection or reduce transmission of the virus. Using newly developed vaccine modalities, we now have the tools to circumvent problems characteristically seen in common vaccine vectors as well as address the contribution of individual immune responses against HIV.
Michael Cho
Targeting gp41 to elicit neutralizing antibodies against HIV-1
There is a global urgency to develop a protective vaccine against HIV-1. Neutralizing antibodies (Nabs) can provide effective prophylaxis against HIV-1 infections. However, eliciting Nabs that are broadly reactive against many antigenically diverse HIV-1 isolates has been a major challenge and it remains a critical roadblock to HIV-1 vaccine development. The primary objective of the studies being proposed is to generate novel antigens that are able to elicit broadly reactive Nabs on the mucosal surface, with a long-term goal of developing a vaccine against the virus. Specifically, we propose to target the membrane-proximal external region (MPER) of gp41 by generating recombinant lactic acid bacteria (LAB) that express antigenically intact gp41 protein fragments on the cell surface using a novel technology. The scope of this proposal is limited to the following specific aims: (1) to generate recombinant Lactobacillus casei that display HIV-1 gp41 MPER on the cell surface, (2) to devise an optimal vaccine strategy and to evaluate immunogenic properties of recombinant L. casei in mice, and (3) to elicit broadly reactive Nabs against HIV-1 gp41 MPER in XenoMouse. Successful completion of this study will generate novel vaccine candidates and strategies for eliciting protective humoral immune responses, which would represent a major step forward in AIDS vaccine development efforts. PUBLIC HEALTH RELEVANCE: The major goals of this proposal are to design, to generate and to evaluate vaccine candidates that can elicit potent immune responses against HIV-1, the virus that causes AIDS. Towards these goals, we are taking a novel approach of generating antigens that can elicit virus-neutralizing antibodies that are broadly reactive against many different variants at the site of virus entry. Successful completion of proposed studies will overcome a critical roadblock to AIDS vaccine development.
Geoffrey Stone
Immunostimulatory single cycle SIV Vaccines incorporating TRAF-mediated molecular signaling
Two components are essential for a vaccine: an "antigen," which is typically a protein from a pathogenic microbe; and an adjuvant, which is a molecular signal to the immune system that the antigen is important and should be defended against. Typically, live attenuated viral vaccines for diseases such as influenza utilize the immunostimulatory nature of the virus itself to adjuvant the immune response. However, HIV virions typically do not induce dendritic cell activation following infection. In addition, the virus can actively suppress the immune response with accessory proteins such as Nef. Therefore, a challenge for live attenuated HIV vaccine development is to find an adjuvant capable of inducing strong immune stimulation that can also be encoded within the HIV genome. A potential source of adjuvant is the human immune system itself. Both the human body and viral pathogens are known to produce adjuvant molecules that can initiate and amplify human immune responses. These include CD40, GITR, OX40 and various genes that utilize the TRAF intracellular signaling pathway to activate immune cells. However, it was not previously known that these TRAF-mediated receptor molecules must be clustered. Such clustering can be artificially induced via a "multimeric" ligand where many trimeric ligand molecules are linked together. The main goal of this proposal is to identify TRAF-mediated intracellular signaling systems such as CD40L/CD40 that have the potential to induce dendritic cell immune activation. The project will then encode these genes within a single cycle SIV vaccine to enhance CD40 and other TRAF-mediated intracellular signaling pathways. The hypothesis is that this immunostimulatory single cycle SIV vaccine will result in increased dendritic cell licensing and maturation in vitro, and enhanced vaccine efficacy in animal models. PUBLIC HEALTH RELEVANCE: This project will develop new forms of immune-stimulating attenuated SIV that could significantly improve the strength of experimental AIDS vaccines. The result of these studies will establish whether attenuated HIV vaccines can be boosted using key components of the mammalian immune system or viral mimics of these molecules.
Amy Sims
Human Coronaviruses as Multigene Mucosal Vaccine Vectors for HIV
An effective HIV-1 vaccine approach not only will need to induce broadly reactive neutralizing antibody and cellular immune responses at systemic and mucosal sites, but the vaccine also will have to be feasible for distribution to parts of the world that need it most in terms of safety, manufacturing cost, number of inoculations, and ease of administration. The common cold human coronavirus (HCoV) OC43 is attractive as a vaccine vector for HIV, and recent breakthroughs in coronavirus reverse genetics have now made it feasible for their exploitation as vaccine vectors. Coronavirus genomes are the largest RNA genomes in nature (~30kb), and contain multiple genes that are not essential for viral replication, theoretically allowing the insertion of multiple heterologous genes into a single virus with a packaging capacity much larger than most other vector systems. Because HCoV OC43 causes only a mild upper respiratory tract infection, it is anticipated to be relatively safe as a vaccine vector, allowing for replication competent viruses to be used to present heterologous antigen at mucosal surfaces for multiple rounds of vector replication, much like a live attenuated vaccine. Coronavirus genomes also can be engineered such that recombination with naturally circulating strains yields a dead virus. We recently completed the development of a reverse genetics system for HCoV OC43, which now makes it technically feasible to develop a HCoV OC43-based vaccine vector. In this phased innovation R21/R33 project we propose to engineer the HCoV OC43 genome as a multiple heterologous gene expression vector for SIV, which we will assess using the SIVsmE660/macaque model. In the R21 proof-of-concept phase we will: (i) engineer HCoV OC43 to express the SIVsmH4 matrix/capsid (MA/CA) gene from different regions of the vector genome, and (ii) evaluate the systemic, mucosal, and cellular immunogenicity of MA/CA-expressing HCoV OC43 vectors in mice. In the R33 phase we will: (i) characterize infectivity of HCoV OC43 in macaques, (ii) engineer and validate a recombination-resistant HCoV OC43 construct to express up to four SIVsmH4 proteins-Gag, Env, Nef and Vif, and (iii) vaccinate macaques with the multigene HCoV OC43 vector and assess vaccine efficacy by mucosal challenge with SIVsm E660. This project will provide the first critical evaluation of the potential use of common cold human coronaviruses as live mucosal vaccine vectors for HIV. PUBLIC HEALTH RELEVANCE: In the proposed study we will develop human coronaviruses, which cause about one- third of all common colds, as vaccine vectors to deliver HIV antigens. There are several potential attractive features of coronavirus vectors as vaccines for HIV, such as safety, simplicity in manufacturing, the ability to induce immune responses in sites of HIV transmission, and the ability to induce immune responses with a limited number of inoculations. We will evaluate the potential use of coronaviruses as HIV vaccine vectors using the SIV/macaque model.
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