John D. Clements, Ph.D. - US grants

A variety of techniques, including use of live oral vaccines, have been employed to deliver antigens to the mucosal lymphoid tissues in an attempt to initiate production of specific sIgA. One recent approach has been to employ avirulent mutants of Salmonella as carriers for plasmids which code for virulence determinants of heterologous mucosal pathogens. Antigens expressed by these carrier strains are delivered directly to the antibody- forming cells in the mucosal lymphoid tissues. This has been shown to be an effective means of stimulating significant levels of serum and mucosal antibodies directed against the carrier and against cloned antigens. There are a number of unresolved questions that will influence the utility of this approach to vaccine development. We propose here to address a number of these unresolved questions, using the cholera related enteropathogen, enterotoxigenic E. coli, as a model. We have chosen this system to explore because it is the one with which we are most familiar and within which we have established a large body of preliminary data upon which to build. It is important to note, however, that the questions we propose to address are such that the answers are broadly applicable to those contemplating the use of this system for the development of vaccines against a variety of other human pathogens. Using a murine model, we will examine 1) the effect of multiple use on carrier efficacy, 2) the effect of varying dose and repeated immunization on antibody response, 3) effect of gene location on stability, expression, cellular location, and immunogenicity of cloned antigens, and 4) the immune response to nonimmunogenic peptides when incorporated into genetic fusion polypeptides and delivered as cloned antigens. As a component of this proposal, we will also develop a challenge model system for use in determining the efficacy of this approach to vaccine delivery. This is a technique for vaccine delivery with significant potential to influence the management of infectious diseases on a large scale, not only for vaccines against enteric bacterial pathogens, but also for vaccines against a variety of other bacterial, viral, and parasitic diseases. This proposal examines many of the parameters of the model and will provide valuable, necessary information applicable to the use of these vaccines in humans.

This proposal addresses several related aspects of a research problem with significant potential to influence the management of infectious diseases on a large scale, not only for vaccines against enteric bacterial pathogens, but also for vaccines against a variety of other bacteria, viruses, and parasites. The central theme of this project is for the use of attenuated mutants of Salmonella as immunologic vehicles to deliver heterologous antigens to the mucosal lymphoid tissues. This has been shown to be an effective means of stimulating significant levels of serum and mucosal antibodies as well as cell mediated immunity against the carrier and against foreign antigens delivered by these organisms. During the current funding cycle, we have addressed several broad issues relative to the use of this technique of vaccine delivery. There are, however, a number of fundamental questions remaining to be answered regarding the use of Salmonella as a vaccine carrier. When attenuated Salmonella sp. are utilized as carriers of foreign antigens, an undefined series of events occurs which can result in production of antigen specific mucosal sIgA, serum IgG, cell mediated immunity, or combinations of these immunologic events. We propose to examine a number of specific parameters that will enable us to begin to understand the mechanisms underlying these outcomes. For this competitive renewal, we propose to resolve the underlying mechanisms controlling the immunologic outcome as a function of bacterial attenuation, strain viability, bacterial species, pre-existing immunity, and qualitative and quantitative nature of the target antigen. For these experiments, animals will be inoculated orally and examined for antigen specific humoral and cellular immune responses. Isotype analysis of antigen specific antibodies from sera and mucosa will be performed by ELISA. Cellular responses will be determined by l) PCR to amplify reverse transcribed cytokine mRNAs expressed by leukocytes in vivo and 2) ELISA to determine cytokine secretion from antigen specific lymphocytes in vitro. These experiments should permit the design of specifically tailored Salmonella vaccine delivery systems according to the type and character of immune response appropriate for the target pathogen (i.e., CMI for intracellular bacteria vs. sIgA for mucosal response). In addition, we will more fully examine the issue of bacterial persistence in the tissues as a determinant of the cellular and humoral immune response and complete our studies on development of a mouse model for ETEC diarrheal disease.

Oral immunogens have the potential to be some of the most versatile, cost effective and potent vaccines for stimulating mucosal immune responses. However fundamental principles for the design and use of effective oral, multivalent, subunit vaccines are developing. For this proposal, a multidisciplinary team will investigate in depth the immune response against two different subunit vaccine candidates administered orally in a murine model. Vaccine candidates to be used in this proposal are derived from microbes which cause significant diarrheal disease in humans; i.e. the heat labile toxin of E. coli (LT-B) and the capsid protein of Norwalk virus. The form of antigen used as well as the scheme for oral immunization will be varied between immunogens given 1) as recombinant protein, 2) associated with liposomes, 3) as Salmonella constructs, or 4) expressed in plants. Both the inductive and effector phases of the mucosal immune response will be evaluated after primary immunizations and after subsequent secondary immunizations. To evaluate the immune response, advanced technologies will be employed. Specifically, sensitive quantitative competitive-reverse transcribed-polymerase chain reactions (QC-RT-PCR) will be used to define the primary and secondary inductive phases of the cytokine response in the Peyer's patches, intestines, mesenteric lymph nodes and spleen of orally immunized animals. Furthermore the primary and secondary effector phases of the immune response in the same tissues will be detailed using l) QC-RT-PCR for cytokine mRNAs, 2) ELISpot analyses for cytokine secretion, 3) ELISAs to define antigen specific antibody secretion, 4) antibody neutralization assays for LT-B and Norwalk virus, 5) ELISPot analyses for antigen specific antibody secretion, and 6) in vitro antigen-induced T helper lymphocyte activation. Initially, the same antigen preparation (i.e. homologous immunizations) will be given orally to stimulate secondary immune responses. These studies will not only establish a standard level of responsiveness for each immunization scheme, but will permit comparison to identify the "optimal" methods for stimulating the appropriate response for each antigen. Once this has been accomplished, it will be possible to investigate strategies for combinatorial oral immunizations using both antigens. These studies will utilize the "best" immunization strategies for both antigens given to a single animal, and the immune response will be evaluated as described above. Using a similar strategy, the memory immune response will be investigated to determine the potential effects of immunological interference, antigenic competition, or as yet other undefined problems or benefits associated with giving multiple oral immunizations. Ultimately, these studies will address fundamental questions relative to the combinations and immunization schemes which are optimal for the development of multivalent oral vaccines.

A number of vaccine strategies for prevention of AIDS have been proposed, including use of attenuated bacterial and viral vectors expressing various epitopes from HIV, hybrid hepatitis particles expressing a VC loop peptide, DNA vaccines expressing gp120, and synthetic peptides containing B- and T-cell epitopes of HIV as immunogens. HIV subunits (gp120, gp160) and whole killed HIV have been tested in humans and non-human primates. None of these has been effective. A major problem limiting the development of an effective HIV vaccine is that the immune correlates of protection against HIV are not known. In this proposal, the applicants address a new strategy for prevention of AIDS by vaccination. They have developed a novel mucosal adjuvant which has been shown in numerous animal studies to induce protective immunity when coadministered with whole inactivated bacteria or viruses, or subunits or relevant virulence determinants from these pathogens. This adjuvant promotes the development of both antigen- specific humoral(antibody) and cell-mediated immune responses against the pathogen in both the systematic and mucosal compartments. In addition, the adjuvant has recently undergone two Phase I clinical studies in humans and has been shown to be safe and nontoxic at adjuvant-effective doses. The proposed studies will focus on the use of this adjuvant for production of humoral and cell-mediated immune responses against a model of HIV antigen - gp120. The main goal of this study is to characterize the humoral and cellular response against HIV gp120 when administered with the adjuvant, and to determine whether the nature of the humoral or cellular immune responses to this antigen when delivered in the presence or absence of this adjuvant will be influence by the route of immunization, or the number of doses administered. Since the immune protective correlates to HIV infection are unknown, the type of immune response induced by vaccination will be characterized in depth. Serum and mucosal anti-gp120 antibodies will be determined and characterized with respect to antigen-specific Ig isotypes and distribution in serum and mucosal secretions by ELISA and Western blot, and the ability to neutralize HIV infectivity in vitro. Cellular studies will be applied to determine the type of T helper cell response induced and the cytokine profile during the effector phases of the immune response, with special regard to development of TH1 and TH1 type response, as well as CTL activity.

The WHO report of Infectious Disease deaths for 1995 indicated that there were more than 13 million deaths world-wide during that year. The majority of those deaths were caused by organisms that first make contact with and then either colonize or cross mucosal surfaces to infect the host. A number of strategies have been developed to facilitate mucosal immunization to prevent these diseases, including addition of bacterial products with known adjuvant properties. The two bacterial products with the greatest potential to function as mucosal adjuvants are cholera toxin (T), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli. A number of mutants of CT and LT have been developed in an attempt to dissociate the desirable adjuvant properties of these molecules from their toxic effects. Both active-site and protease-site mutants have been constructed and evaluated in a variety of animal models with different antigens. Important questions regarding the adjuvanticity of CT and CT and mutants of these toxins remain to be answered. Some of these questions are practical and the answers will impact the immediate and short term use of these molecules in human vaccines. Other questions address the underlying mechanisms associated with adjuvanticity and the answers will have their greatest impact in the design of future adjuvants and vaccine strategies and in the development of a better understanding of vaccine induced immunity. The proposal includes a series of Specific Aims designed to directly address these issues. One of the most important aspects of the proposed study is a side-by-side comparison of CT, LT, active-site mutants, protease-site mutants, and recombinant B-subunits for the ability to induce specific, targeted immunologic outcomes as a function of route of immunization and nature of the co-administered antigen. With the information obtained in the proposed studies, future vaccine strategies can be designed employing the optimum adjuvant/antigen formulation and route of administration for a variety of bacterial and viral pathogens. This proposal also examines the underlying cellular and intracellular signaling pathways activated by these different molecules to better understand the mechanisms of adjuvanticity at the cellular level.

DESCRIPTION (provided by applicant): An ideal vaccine against potential agents of biological warfare/bioterrorism should be safe, easy to deliver, provide long-lasting protection, require only one or a few doses, and provide protective immunity against different agents. Moreover, since mucosal and respiratory surfaces represent the first productive points of contact with the human host for many aerosolized biological agents, the role of mucosal immunity in protection needs to be examined. Nonparenteral delivery of vaccines (i.e., by the mucosal or transcutaneous route) has been shown to be effective at inducing both humoral and cellular antigen-specific immune responses in both the systemic and mucosal compartments of immunized animals and humans. Such "needle free" immunizations offer many potential advantages over parenteral immunization and are being evaluated by a number of groups for use in biodefense vaccines. Induction of immune responses by these nonparenteral routes is dependent upon the co-administration of appropriate adjuvants that can initiate and support the transition from innate to adaptive immunity. In this application we are proposing to investigate three novel adjuvants (LTR192G), CpG ODN, CTA1-DD) for their ability to induce high titer, long lived, protective antibody responses against relevant vaccine antigens from B. anthracis (rPA), Y. pestis (F1-V), botulinum toxin (Hc), and staphylococcal enterotoxin B (SEBv). The findings from these studies should be broadly applicable to other antigens from these and other biological agents. The primary objective of these studies is the optimization and preclinical testing of these novel adjuvants, each of which has previously shown promise in other infectious disease vaccines delivered mucosally or transcutaneously. It is expected that one or more of these adjuvants will be considered as candidates for future Phase I-II-III testing in clinical trial programs. Another objective of this application is to determine if nonparenteral boosting can induce a protective secondary immune response in animals that have been parenterally primed and if that response can be redirected to include a mucosal immune component. Challenge studies will allow us to correlate immune responses with protective efficacy and determine the contribution of mucosal immune responses to protection. Finally, we will determine the effectiveness of a combined vaccine consisting of relevant antigens from the four different pathogens with the specific objective of determining synergy or interference between the vaccine components and the role of adjuvants and route of delivery in overcoming interference.

DESCRIPTION (provided by applicant): Until recently, anthrax was primarily a concern of individuals working in animal husbandry and military planners concerned about the potential use of anthrax spores as an agent of biological warfare. The use of anthrax as an agent of bioterrorism on civilian populations was a theoretical risk, heightened by the discovery that certain rogue nations (notably Iraq, Syria, and China) either had developed or were attempting to develop anthrax or other biologic agents as a weapon of mass destruction, The post- September 11 release of anthrax spores resulted in five civilian deaths, eighteen infections, and required that more than 30,000 individuals to undergo prophylactic antibiotic therapy. This event also highlighted the need for improved vaccines that would be appropriate for pre- or post- exposure immunization of civilian and military populations against potential bioterrorism agents, including anthrax and plague, Vaccines combining protective antigens from different microorganisms with potential for use against civilian or military populations as biological warfare/biological terrorism agents would be advantageous because they would both broaden the coverage of such vaccines and reduce the overall number of immunizations. The first logical combination to examine would be rPA from B. anthracis and F1-V from Y. pestis since they have individually been shown to induce protective responses. Combining vaccines to decrease the number of immunizations and to increase vaccine coverage is not a new concept in vaccine development and combination vaccines such as DTP and MMR have been used for many years. However, several examples of immunologic interference between individual components of combination vaccines have been observed both in clinical trials and in laboratory tests. We are therefore proposing to examine the potential of a combined vaccine consisting of rPA and F1-V with the specific objective of determining synergy or interference between the vaccine components. For this application, we propose to address a number of interrelated questions regarding immunization with a combined vaccine containing rPA and F1-V as immunogens to protect against anthrax and plague.

[unreadable] DESCRIPTION (provided by applicant): A great deal of effort has been directed towards developing nonparenteral (needle-free) alternatives to traditional vaccine delivery. Nonparenteral vaccines offer a number of potential advantages over traditional vaccines including 1) the potential to confer mucosal as well as systemic immunity, 2) increased stability, 3) increased shelf-life, 4) elimination of needles and the need for specially trained healthcare specialists to administer vaccines, and 5) potentially lower costs. One such approach, transcutaneous immunization (TCI), is a non-invasive, safe method of delivering antigens directly onto bare skin. Immunization is achieved by direct topical application of a vaccine antigen. Despite the attractiveness of TCI, the technology is limited by the relative inefficiency of transport of large molecular weight vaccine antigens across intact skin. Recent innovations in transdermal delivery of drugs, including chemical enhancers, electricity, ultrasound, and microneedles, demonstrate the feasibility of large-molecule transport through the skin's permeation barrier, specifically the stratum corneum. This outer layer of the skin is composed of tightly packed lipid molecules and the dense, crystalline arrangement of these lipids creates the essential barrier to prevent water loss and pathogen entry. Recent evidence has shown that this barrier can be overcome by properly structured nano-sized particles (nanocarriers). This proposal will compare three different nanocarriers (temperature-responsive hollow nanospheres, nanohydrogels, and star copolymers) for the ability to incorporate a model vaccine antigen and deliver that antigen through the stratum corneum to immunoresponsive cells in the epidermis. The specialized assembly of each type of nanocarrier gives each unique properties and different interactions within the lipid channels of the stratum corneum. The use of nanocarriers for vaccine delivery is a platform technology, applicable to delivery of a variety of existing and potential vaccines. For the purposes of this proposal, we will utilize two different proteins: 1) Bovine Serum Albumin that has been fluorescently labeled to monitor incorporation and permeation of a macromolecular antigen, and 2) F1-V, a vaccine antigen from Yersinia pestis, the causative agent of plague, which we and others have shown to protect against aerosol challenge with virulent Y. pestis. The proposed studies will address important questions in vaccine delivery by application of nanotechnology through the exploitation of the novel properties of nanocarriers. The findings of these studies will be broadly applicable to a variety of vaccines and therapeutics and will further highlight the important role of nanotechnology in science and medicine. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal by Tulane University is a request for funds to remodel select biomedical research laboratories in the J. Bennett Johnston Health and Environmental Research Building (JBJ building) on our Health Science Center campus. The main objective of this remodeling is to create open collaborative spaces that foster interdisciplinary research. The proposed remodeling encompasses 2,876 m2 (31,915 sq. ft.) on three floor of a building that was built fifteen years ago with very traditional, discipline oriented laboratories and offices. Tulane University is committed to remaining a top-tier research institution known for high quality, cutting-edge, basic and translational research. The proposed changes will provide modern, open laboratories that promote collaborative interactions between investigators, and support areas for each suite that include equipment rooms, tissue culture rooms, and meeting areas (Team rooms). Each suite is located adjacent to other investigators in the same research field to facilitate interaction and use of core equipment. In addition, we will upgrade the fa[unreadable]ade of the building to improve ambient lighting in the laboratory areas and improve the overall comfort in the work environment, thereby increasing productivity while realizing cost savings in electrical lighting. The project team will pursue a rating of LEED Silver for this project. The proposed remodeling reflects a change in the research culture at Tulane University by providing a means for looking beyond traditional discipline boundaries, recognizing opportunities across a broader landscape, and promoting interdisciplinary and translational research in areas where we have historic and current strengths - Infectious Diseases, Cardiovascular Disease - Renal - Hypertension, and Cancer. In each case, those groups will bring together scientists and engineers from across the university, including some of our most productive investigators in each field, as well as junior investigators who will benefit from the mentoring provided by the senior investigators in those units.

DESCRIPTION (provided by applicant): Substantial progress has been made towards the eradication of polio through the use of two different vaccines: the inactivated poliovirus vaccine (IPV) and the attenuated oral poliovirus vaccine (OPV). OPV has been useful in eradicating polio from most of the world, due to its simplicity of administration, development of herd immunity resulting from excretion, ease of production, and low cost. Unfortunately, use of OPV in developing countries often requires up to 10 immunizations to achieve equivalent protective levels of immunity compared to children in developed countries. A number of groups have proposed replacing OPV with IPV as part of the final polio eradication campaign. Unfortunately, IPV is more expensive than OPV and does not induce heard immunity or mucosal protection. There are number of potential approaches for improving the immunogenicity and efficacy of IPV and other vaccines, including alternate delivery routes (e.g., intradermal or sublingual) and the use of nano-scale delivery systems. A number of micro- and nano-carriers have been developed that may be appropriate for vaccine delivery, including liposomes, micro-, nano-, and multiple-emulsions, polymeric nano- particles, dendrimers, and immunostimulatory complexes (ISCOMS). Our own nano- and micro-scale technology vaccine delivery research over the last five years has led us to appreciate the potential of nano- carriers to enhance the immunogenicity and efficacy of multiple vaccines by different routes of immunization. For this project, our primary focus will be on the use of nano-scale carriers to facilitate intradermal and sublingual delivery of IPV, building upon our earlier findings. The proposed studies will address important questions in vaccine delivery by application of nano-technology through the exploitation of the novel properties of nano-carriers. The findings of these studies will be broadl applicable to a variety of vaccines and will further highlight the important role of nano-technology in science and medicine. The specific aims are 1) Optimize the incorporation and stability of IPV within specialized nano-carriers; 2) Evaluate the ability of the nano-carrier formulations to enhance production of serum and mucosal antibodies and neutralizing antibodies against all three serotypes of poliovirus following intradermal or sublingual immunization in a murine model; 3) Evaluate the ability of the nano-carrier formulations to enhance the immunologic responses to fractional doses of IPV following intradermal or sublingual immunization in a murine model; 4) Evaluate the effect of pre-existing antibodies against IPV on the immune responses to IPV formulated in the different nano-carriers following intradermal or sublingual immunization in a murine model, and 5) Evaluate serum and mucosal, humoral and cellular responses following intradermal or sublingual immunization with IPV formulated in the optimum nano-carrier for each route in non-human primates.

DESCRIPTION (provided by applicant): Enterotoxigenic E. coli (ETEC) are a significant cause of morbidity and mortality world-wide, especially in children in developing countries. The World Health Organization estimates that the annual diarrhea burden due to ETEC in children less than five years of age in the developing world is approximately 210 million cases and 380,000 deaths. ETEC cause disease by colonizing the small intestine by means of colonization factors (CFs) and by production of either a heat-labile (LT) or heat-stable (ST) enterotoxin. The majority of clinical isolates produce both enterotoxins and the physiologic or immunologic consequences of simultaneous exposure to these two potent enterotoxins are unknown. Our preliminary data indicate that when LT and ST are both present, they work additively, increasing movement of water into the intestine over and above levels observed with either toxin alone. Our preliminary data also demonstrate that the levels of inflammatory cytokines produced by intestinal epithelial cells (IECs) in response to LT are significantly reduced in animals exposed to both enterotoxins. Based on our preliminary findings, our hypotheses for the proposed studies are 1) LT combined with ST increases fluid movement (diarrhea) when compared to either toxin alone by increasing cGMP, 2) the production of ST by LT/ST ETEC can significantly reduce the ability of the host to mount an effective innate immune response and 3) the production of ST by LT/ST ETEC can significantly reduce the ability of the host to mount an effective adaptive immune response (e.g., serum and mucosal antibody) against the infecting organisms (colonizing factors or LPS) or its heat-labile toxins. The specific aims are 1) Determine the role of LT/ST on accumulation of cyclic nucleotides in T84 cells; 2) Determine the role of LT/ST on innate immune responses in the murine intestine; and 3) Determine the role of LT/ST on adaptive immune responses to enterotoxigenic E. coli. At the conclusion of these studies, we will have a better understanding of the physiologic and immunologic consequences of simultaneous exposure to the two potent enterotoxins made by these organisms (LT and ST) and will have the information necessary to transition into non-human primate studies or human trials.