Research Overview
 

1. Transgenic Plant based oral vaccines for Hepatitis B

A highly effective and safe hepatitis B vaccine was licensed in the U.S. in 1986. It has been a great commercial success, with sales in the industrialized world exceeding $1 billion/year. With this outstanding product on the market, it is valid to ask why we have pursued an alternative vaccine to prevent hepatitis B virus (HBV) infections. The answer lies in finding an equivalent or improved vaccine that will serve poorer countries of the world, where the HBV disease burden remains at epidemic levels and vaccine costs preclude the use of the currently available product. The existing vaccine to prevent HBV infection falls in the category of ‘‘subunit vaccines.’’ The gene encoding the hepatitis B surface antigen (HBsAg) is expressed in yeast cells grown by fermentation; the cells are broken, the protein is collected, and the HBsAg is caused to refold by chemical treatment to yield virus-like particles that can be formulated for injection. The resulting vaccine is the epitome of modern macromolecular pharmaceuticals derived from recombinant DNA technology; it is a superior product that contains only a ‘‘subunit’’ of the disease-causing agent. However, it is technology-intensive and therefore comparatively expensive as a health-care product for developing countries.

Although the licensed HBV vaccines developed to date have been for parenteral delivery, there is no a priori reason to exclude oral delivery. Oral vaccines are highly desirable from several standpoints including simplicity of use, increase in compliance (as a result of increased ease/comfort of delivery), enhanced immune responses at mucosal sites, and stimulation of humoral immunity. For pathogens that cause enteric, respiratory, or sexually transmitted diseases, mucosal immune responses will provide an essential first line of defense. However to date, subunit vaccines have not been licensed for oral delivery, in part because of the lack of cost-effective production technology.

We have been exploring a strategy of producing and delivering oral subunit vaccines as constituents of transgenic, edible plants such as potatoes, tomatoes, or bananas. Indigenous technology in developing countries could be harnessed for plant-derived vaccine production to overcome current constraints. Plants are a potential source of HBsAg that is not dependent upon process technology to ensure protein folding and particle assembly. In addition, a plant based HBsAg expression system makes possible the testing of an oral immunization strategy by simply feeding the plant samples.

Fig. 1. TEM of nontransformed and HBsAg-expressing potato cells. A) Thin-section TEM image of a nontransformed FL1607 potato cell. (Bar 5 315 nm.) (B) Thin-section TEM image of a transgenic HB114–16 transgenic potato cell. Arrows indicate membrane-bound vesicles containing 17-nm putative HBsAg particles. (Bar 5 200 nm.) (C) Enlarged image of membrane-bound vesicles containing 17 nm putative HBsAg particulate structures indicated by arrows. (Bar 5 96 nm.) Cp, chloroplast; E.R., endoplasmic reticulum; Mito., mitochondrion; Vac., vacuole.

We have previously shown the expression of HBsAg in tobacco plants and have demonstrated that the plant derived antigen was immunogenic when administered parenterally to mice (PNAS 92; 1995, Nature Biotech. 18; Nov. 2000, Vaccine 23; 2005). Further we have demonstrated that plant-based rHBsAg is an oral immunogen in mice (PNAS 98; Sept. 2001) and stimulated serum antibodies specific for HBsAg at levels that very significantly exceed the protective level (in humans) of 10 mIU/ml. The unique features of bioencapsulation of the antigen within plant cells (Fig. 1) may be the actual reason why plant-based HBsAg is effective.

These combined experiments demonstrated that plants can express, fold, assemble, and process foreign antigens and can provide both a simple vaccine-manufacturing process as well as a matrix suitable for oral immunization (Expert Reviews in Vaccines 5:, 2006).

The question that we next asked was: Will a plant-derived oral subunit vaccine comprised of an antigen from a nonenteric pathogen function at induction sites in the human gut and generate a systemic immune response that is predictive of disease prevention? A randomized, placebo-controlled, double-blind trial, evaluated the safety and immunogenicity of orally delivered HBsAg expressed in transgenic potatoes. Health care workers with a history of previous parenteral immunization with the licensed hepatitis B vaccine volunteered to consume multiple doses of transgenic or control potatoes. We found that ingestion of transgenic potatoes expressing HBsAg by previously vaccinated volunteers provoked increases in the titers of serum antibody specific for HBsAg in 19 of 33 subjects, which is remarkable in view of the facts that (a) the vaccine was delivered without any adjuvant, (b) the HBsAg, an antigen derived from a nonenteric pathogen, was delivered orally, and (c) the recombinant subunit HBsAg was a nonreplicating vaccine. The anti-HBs titers were boosted up to 56-fold after three doses or up to 33-fold after only two doses of transgenic potato. We are greatly encouraged that this prototype study of human immunization against HBsAg gave a strong and sustained systemic antibody response in nearly 60% of the volunteers who ate transgenic potatoes (PNAS 102; 2005). Based on the success of this trial we are now designing a second clinical trial that will involve testing the safety and immunogenicity of orally delivered HBsAg expressed in transgenic potatoes co-delivered with a mucosal adjuvant.

Work in the lab has also tested viral expression system to get high yield rapid production of hepaptitis B in plants and we have shown that the antigen is able to generate immune responses in mice (Plant Biotechnology Journal In Press, 2007). We have also tested different strategies to achieve targeting of HBsAg to the gut mucosal system (Manuscript under review).

2. Nanoparticle vaccine delivery systems

Nanomedicine is emerging as an important field by which drugs, therapeutic and imaging agents and vaccines can be delivered effectively, since their bioavailability and pharmacokinetics can be improved by encapsulation within nanoparticles. A significant advantage of using biodegradable polymers are their long safety history, proven biocompatibility and the ability to control the time and rate of polymer degradation and encapsulated cargo release; these qualities make them attractive for the formulation of nanoparticle based vaccine delivery systems Current vaccines face ongoing challenges in terms of both efficacy and ease of delivery. Cancers and many chronic diseases have yet to be prevented by vaccination and several vaccines require multiple injections. We have begun to test the hypothesis that: vaccine antigens that are conjugated to or loaded within the nanoparticles are protected from rapid degradation in vivo, allowing slow and sustained stimulation of immune responses thereby reducing the need for multiple doses; that judiciously chosen biodegradable nanoparticle formulations will allow oral vaccine delivery; and that following uptake by DCs the antigen is released intracellularly and stimulates robust innate and protective adaptive immune responses.

In recent years, significant effort has been devoted to develop nanoparticle/microparticle mediated vaccine delivery systems prepared from biodegradable and biocompatible polymers. A wide variety of polymeric materials have been explored in order to induce systemic and local immune response after administration by various routes. Other than poly (DL-lactide-co-glycolide acid (PLGA), limited studies have been reported on any other nano/microparticulate formulation for hepatitis B vaccination. By using a combination of different polymers, tailor-made vaccines with variable release kinetics can be developed. Entrapment efficiency, release kinetics and other physical characteristics, such as morphology, porosity and size distribution, which influence the efficacy of the formulations, can be controlled by using appropriate combination of different polymers. Thus we anticipate that systematic evaluation of appropriate combinations of various biodegradable and biocompatible polymers in nanoparticulate formulations may lead to a successful vaccine formulation for single dose injectable or a multiple dose oral/intranasal vaccine delivery system for hepatitis B (Adv Exp Med Biol. 601; 2007).

Fig. 2 FRET Images (63x magnification) of HBsAg encapsulated nanoparticle vaccine uptake by BMDC. Excitation by single wavelength of 543 nm.

We have formulated a variety of nanoparticles by double emulsion/solvent evaporation method and found that we can encapsulate recombinant human hepatitis B antigen (HBsAg) with high efficiency. We have studied the size distribution of the nanoparticles encapsulating hepatitis B surface antigen (HBsAg) by dynamic light scattering (DLS) and the morphology by transmission electron microscopy (TEM) and evaluated the stability and release kinetics of the antigen in vitro. We are utilizing a combination of confocal microscopy, image stream analysis and FRET to visualize nanoparticle vaccine uptake by bone marrow derived dendritic cells (Fig. 2).

To demonstrate the ability of the encapsulated antigen to provoke an in vivo immune response, the nanoparticle based vaccines are being used to immunize mice and the antibody response elicited is compared with that from mice immunized with non-encapsulated recombinant HBsAg (Manuscript submitted).

3. Activation of innate immune cells in the human lung and potential contribution to tumorigenesis.

Inflammation is recognized as an important factor in cancer development. Cancer is generally regarded as a disease that occurs in a series of well defined steps that are orchestrated by both activating (oncogenes) and deactivating (tumor suppressor genes) mutations, however a single mutation does not result in tumor development. Additional genetic and epigenetic events are needed to facilitate tumor development. Thus there is a phase of initiation, promotion and finally malignant conversion. An initiated cell must acquire the capacity of infinite self renewal, resistance to apoptosis and insensitivity to growth inhibitory signals.

Lung epithelial cells are consistently exposed to many irritants (cigarette smoke, asbestos, diesel) and pathogens (bacteria and viruses). Chronic injury and repeated cycles of tissue repair in the presence of an ongoing inflammatory reaction can provide a milieu that is conducive for the selection of cells with enhanced proliferative capacity or reduced sensitivity to growth arrest.

Figure 3

Carcinogens in cigarettes cause mutations and initiate the oncogenic process (Fig 3 left side). Progression of tumorigenesis is supported by intrinsic (enhanced activity of ongogenic gene products or loss of tumor suppressor genes) and extrinsic factors (tissue milieu and growth factors). Inflammation plays a vital role in tumorigenesis through the generation of reactive oxygen species (ROS) leading to mutations, and through the induction of cytokines that have growth regulatory action, enhancing the proliferation of transformed epithelium (Fig 3). Tumor promotion is greatly enhanced by the milieu created by lung diseases, which have a greatly enhanced inflammatory component. Thus, a person suffering from COPD has 4-6 times greater risk of developing lung cancer.

Bacteria are always present in the upper airways, however in the absence of lung disease the tracheobronchial tree is maintained sterile. Tobacco smoking causes impairment of mucociliary clearance and this allows bacterial pathogens to gain access to the lower respiratory tract. The bacteria then persist (colonize) by further impairing mucociliary clearance as a result of their products (Fig 9, right side). Inflammatory responses to infection must be precisely regulated to facilitate microbial killing while limiting host tissue damage.

The question we ask is how do inflammatory cells get co-opted into the tumorigenic process? There is now excellent evidence that many tumors types including cervical, gastric, hepatocellular and colon are initiated by infections. Persistent infections induce chronic inflammation. Leukocytes and phagocytes can induce damage in proliferating cells via ROS and RNS that these cells produce to combat pathogens. Repeated tissue damage, with concomitant tissue repair in the presence of ROS/RNS that interact with DNA in proliferating epithelium can result in point mutations, deletions or rearrangements.

The recognition by specific TLRs of microbes or their products results in the initiation of an acute inflammatory response, induction of genes of inflammatory chemokines and cytokines, the recruitment of neutrophils and activation of macrophages. Additionally, in the past few years there is a body of evidence that has emerged indicating that TLRs contribute to the activation of adaptive immunity and that this “cross talk” helps shape the quality of the adaptive immune response. Since innate immune cells produce a vast array of growth factors, cytokines and chemokines and a variety of proteinases they can thereby potentiate tumor development. Our goal is to evaluate how ‘irritants’ produced by gram negative bacteria that colonize the airways (of COPD patients during periods of exacerbations) can activate cells of the innate immune system. Since macrophages are one of the key cells perpetuating chronic inflammation (in the lung and elsewhere) we focus on the interaction of an endotoxin (made by gram negative bacteria that colonize the lungs of chronic smokers and COPD patients) via interactions with TLR receptors on the macrophage. We also study how this interaction results in the production of cytokines and small molecular inflammatory mediators and thereby creates an environment conducive to progression of the initiated epithelial cell to malignancy.

4. Cellular immune responses in COPD patients with correlation to clinical outcome----a role for T regulatory cells?

In early studies supported by a program project grant had noted that P6, an outer membrane protein of the gram negative bacterium NTHI, was able to provoke T cell dependent antibody responses even when mice were immunized in the absence of any adjuvant (Vaccine 18; 2000;Vaccine 23; 2005). Being cognizant of the fact that OMP P6 is a lipoprotein known to possess a cys-pam3 motif, that is likely to interact with the toll-like receptors on cells of the immune system, we have begun test the hypothesis that the lipoprotein P6 by activating, via its lipid moiety, cells of the innate immune system (DC signaling via TLR2) potentiates/enhances antigen presentation to P6 specific T cells. In other words – can lipoprotein P6 of NTHI bridge innate and adaptive immune responses?

To investigate the role of this motif in influencing dendritic cell activation and function we utilize recombinant P6 proteins either retaining or lacking the lipid component and test them for their ability to interact with bone marrow derived dendritic cells from TLR2 +/+ and TLR2 -/- mice and promote their maturation, alter their endocytic capacity, cytokine secretion and their ability to stimulate antigen specific T cells. Our results clearly point to a key role for the lipid component of P6 in the maturation and functional activation of DCs and have important implications in the design of a potential vaccine for NTHI based on the OMP P6.

Also in earlier published studies we had reported (Am. J. Respir. Crit. Care Med. 65, 2002) that some COPD patients with exacerbations due to NTHI in the prior year had significantly lower lymphocyte proliferative responses to P6 compared with COPD patients with no exacerbations due to NTHI in the prior year and compared with age- and sex-matched healthy controls. Our results supported the working hypothesis that patients with COPD who experience exacerbations due to NTHI do not respond adequately to P6 and suggest that the ability to make a proliferative response to P6 may be associated with relative protection from exacerbations due to NTHI. To determine if the non responsiveness to P6 observed in a subset of patients could be due to the action of regulatory cells (Tregs), we have begun to identify these cells in human peripheral blood mononuclear cell populations (PBMCs). Human immunophenotyping of T reg cells has been complex and typically the most suppressive T cells are the CD4+ T cells that show CD25 high expression. Another marker that has been used to define T reg cells is the transcription factor FoxP3. FoxP3 is intracellular and therefore cells need to be permeabilized, and this limits their use in the functional studies. We perform cell sorting experiments based on the protocol established by Bluestone and colleagues that the IL7 receptor is an excellent biomarker for human T regs and results in a highly purified population of Tregs and importantly these cells are highly suppressive in functional assay.