ABSTRACT
Cancer vaccines, though attractive, are not very effective in inducing levels of immunity that can protect the individual from tumor growth. This is due to their inability to trigger the appropriate cells that initiate the primary immune response. Oral vaccines are currently being investigated for their efficacy in stimulating the mucosal as well as systemic immunity. The mucosal route of entry and initiation of primary immune response is well established where pathogens and other invasive microbes enter the host system via regions in the small intestine. Bernadette et al. proposed a study wherein they formulated a novel prophylactic oral microparticulate vaccine for melanoma composed of whole cell lysate (WCL) for melanoma using the spray drying technique. They used an M-cell targeting ligand called AAL (aleuria aurantia lectin), which helped in better uptake into Peyer’s patches of the
small intestine [12]. Lectins, which are naturally occurring proteins with an affinity for sugar residues, have been incorporated to bind specifically the surface receptors on microfold cells (M-cells) in the intestine [13]. The M-cells are specialized cells found in areas of the small intestine called Peyer’s patches that effectively bind, transport and deliver macromolecules and microorganisms to the cells of the underlying antigen-presenting cells (APCs) of the mucosal immune system [14]. The major challenge in designing a successful vaccine is the delivery of antigens to the part of the immune system where maximum stimulation and proliferation of the professional immunopotent cells can be achieved. The purpose of this project was the formulation and evaluation of albumin microparticles for cancer (melanoma) treatment using whole cell antigens as the immunogenic drug. A novel approach was proposed wherein the drug was incorporated into a biodegradable and sustained release polymer matrix containing albumin and cellulose polymers. This is due to their inability to trigger the appropriate cells that initiate the primary immune response. To surmount this shortcoming, an oral vaccine formulation was devised that would target specific regions in the small intestine that are generally responsible for pathogenic entry and initiation of immune responses.The mucosal route of entry and initiation of primary immune response is well established where pathogens and other invasive microbes enter the host system via regions in the small intestine. These regions are called Peyer’s patches and have pockets containing specialized lymphoid follicles that recognize and orchestrate the stimulation of the immune system. Peyer’s patches usually consist of M-cells, which can be used as a target site for antigenic entry. A ligand that will bind selectively to M-cells in Peyer’s patches of the small intestine may be added to the formulation where the antigen of interest can be made available for processing and presentation to the immune cells of the mucosal associated lymphoid tissue (MALT). It is present in most pathogenic microorganisms and helps to enhance their entry into the M-cells. The microparticle formulation containing the antigen when associated with the ligand specifically targets the M-cell. Thereafter, it is taken up
preferentially and transported across the cell where it encounters the underlying APCs and other immunopotent cells [16]. These antigens are processed and presented either through the endogenous (MHC Class I) or exogenous (MHC Class II) pathway.In order to design a successful vaccine, not only does the physical form and foreignness of the antigen play a role but also the manner in which the antigen gets processed and presented to the immune cells. Whole cell based vaccines can overcome the complications by providing a wide coverage of potential tumor antigens [17]. Thus, a microparticulate whole cell antigen vaccine was developed that would provide a robust immune response towards all potential melanoma tumor antigens. 8.2 Formulation of the Vaccine in Albumin
MicroparticlesMicroparticles of the melanoma vaccine were prepared by a spray drying technique (U.S. patent, filed October 2009). The WCL antigen-loaded microparticles were prepared using an albumin, Ethylcellulose (EC) and HPMCAS in an appropriate ratio. The use of a protein biodegradable adjuvant such as albumin in this antigen formulation helps to increase uptake into phagocytic macrophages and other APCs that are available underlying the follicle-associated epithelium of Peyer’s patches in the small intestine. Chitosan was used as a cationic polymer which imparted a positive surface charge to the microparticles which was used to our advantage [18]. The particles remained in contact with the negative mucosal lining of the intestine for a prolonged period of time. Various excipients were incroporated, such as ethylcellulose and HPMCAS as enteric coating polymer to protect the particles from the acidic environment of the stomach.The ligand AAL (Aleuria Aurantia Lectin) was added to the aqueous polymer solution and finally the WCL was added to the polymer solution before spray drying using the Buchi Mini Spray dryer. AAL targets these particles containing antigen to the M-cells [19]. Further, the antigen is transported within the particle to the macrophages and dendritic cells [20] underlying Peyer’s patches to produce antigen specific serum IgG response and to protect the
animals after injection with tumor cells for the post vaccination challenge study. 8.3 Characterization of the Particulate Vaccine
8.3.1 Scanning Electron Microscopy of Antigen Microparticles The surface morphology, size, and uniformity of the microparticles were determined by the JEOL scanning electron microscope. The formulations were evenly spread on metal stubs and coated with gold and dried. These gold-coated microparticles were vacuum
dried and then imaged under a nitrogen airflow stream. Earlier studies have indicated that microspheres between 1 and 5 μm are phagocytosed efficiently by APCs, such as macrophages [21] and dendritic cells [22]. Majority of the microparticles had a uniformly spherical surface morphology, with a small fraction exhibiting doughnut shape (Fig. 8.1).
