Acid Sensitive Polymer Microparticles For Subunit Vaccine Delivery

Vaccines are one of the most significant contributions to modern medicine, as they are credited with such feats as the near or complete eradication of devastating diseases (e.g., polio, smallpox). Despite these successes, more conventional formulations (e.g., live-attenuated pathogens) have critical drawbacks that limit their use to healthy patients. Subunit vaccines are an appealing alternative because they contain safe antigens and can be universally administered. The Food and Drug Administration’s (FDA’s) mid-1980s approval of the first subunit vaccine containing a recombinant protein antigen has fostered our ability to develop new protein-based formulations. However, a common issue with protein antigens continues to be their lack of immunogenicity and structural sensitivity. Compounds known as adjuvants are used to boost the efficacy of protein-based subunit vaccines. Aluminum salts (alum) were the only FDA-approved adjuvants for many years, but they have a poorly-defined mechanism of action and, more importantly, have limited efficacy against many pathogens. A more recent generation of adjuvants known as pathogen-associated molecular patterns (PAMPs) analogs can induce broader immune responses against many pathogens. In 2009, the FDA approved an adjuvant formulation containing alum along with a PAMP analog (monophosphoryl lipid A). This has catalyzed the use of countless other PAMP analog adjuvants, but their widespread application is still limited by factors such as the intracellular location of many of their target receptors. Vehicles such as polymer particles can aid in enhancing antigenicity and adjuvanticity by offering sustained and passive targeted delivery to phagocytic antigen-presenting cells (APCs). However, many traditional polymer particle formulation methods are denaturing to structurally sensitive protein antigens. Furthermore, many of these processes encapsulate proteins and adjuvants inefficiently. In this work, acid-sensitive polymer particles that exploit the acidic endocytic pathway of APCs are developed and used to deliver protein antigens and/or PAMP analog adjuvants to boost the efficacy of subunit vaccines. Particular attention is paid to developing (i) dual-functioning immunostimulatory polymer vehicles that can deliver protein antigens, (ii) mild processes that can efficiently encapsulate both protein antigens and adjuvants, and (iii) polymer particles that can be conjugated with protein antigens post-fabrication.

The prevention and treatment of cancer and infectious diseases requires vaccines that can mediate cytotoxic T lymphocyte-based immunity. A promising strategy is protein subunit vaccines composed of purified protein antigens and immunostimulatory adjuvants, such as Toll-like receptor (TLR) agonists. In this research, we developed two new biodegradable polymeric delivery vehicles for protein antigens and TLR agonists, as model vaccine delivery systems. This work was guided by the central hypothesis that an effective vaccine delivery system would have stimulus-responsive degradation and release, biodegradability into excretable non-acidic degradation products, and the ability to incorporate various TLR-inducing adjuvants. The first vaccine delivery system is a cross-linked polyion complex micelle which efficiently encapsulates proteins, DNA, and RNA. The micelle-based delivery system consists of a block copolymer of poly(ethylene glycol) (PEG) and poly(L-lysine), cross-linked by dithiopyridyl side groups to provide transport stability and intracellular release. The second delivery system consists of solid biodegradable microparticles encapsulating proteins, nucleic acids, and hydrophobic compounds. The microparticles are composed of pH-sensitive polyketals, which are a new family of hydrophobic, linear polymers containing backbone ketal linkages. Polyketals are synthesized via a new polymerization method based on the acetal exchange reaction and degrade into non-acidic, excretable degradation products. In addition, the technique of hydrophobic ion pairing was utilized to enhance the encapsulation of ovalbumin, DNA, and RNA in polyketal microparticles via a single emulsion method. Using in vitro and in vivo immunological models, we demonstrated that the micelle- and polyketal-based vaccine delivery systems enhanced the cross-priming of cytotoxic T lymphocytes. The model vaccines were composed of ovalbumin antigen and various TLR-inducing adjuvants including CpG-DNA, monophosphoryl lipid A, and dsRNA. The results demonstrate that the cross-linked micelles and polyketal microparticles have considerable potential as delivery systems for protein-based vaccines.

This comprehensive volume compiles the concepts essential for the understanding of the pharmaceutical science and technology associated with the delivery of subunit vaccines. Twenty-one chapters are divided into four main parts: (I) Background; (2) Delivery Systems for Subunit Vaccines; (3) Delivery Routes, Devices and Dosage Forms; and (4) Pharmaceutical Analysis and Quality Control of Vaccines. Part one provide a basic background with respect to immunology and general vaccine classification. In part two, it presents representative types of vaccine delivery systems individually with focus on the physicochemical properties of the systems and their significance for the immune response they stimulate. These delivery systems include aluminum adjuvants, emulsions, liposomes, bilosomes, cubosomes/hexosomes, ISCOMs, virus-like particles, polymeric nano- and microparticles, gels, implants and cell-based delivery systems. Following these chapters, part three addresses the challenges associated with vaccine delivery via specific routes of administration—in particular subcutaneous, intramuscular, oral, nasal, pulmonary, transdermal and vaginal administration. Furthermore, the specific administration routes are discussed in combination with device technologies relevant for the respective routes as well as dosage forms appropriate for the device technology. Finally, the fourth part concerns pharmaceutical analysis and quality control of subunit vaccines.

This practical guide offers concise coverage of the scientific and pharmaceutical aspects of protein delivery from controlled release microparticulate systems-emphasizing protein stability during encapsulation and release.

The key aspect of a vaccine delivery platform is to be easily programmed towards different antigens and adjuvants application. Antigens or adjuvants that are derived from various pathogens are diverse in a number of physical chemical properties, such as size, charge, and solubility. A polymer nanoparticle system made of the mixture of pH-insensitive polymer, such as poly(lactic-co-glycolic acid) (PLGA) and pH-sensitive polymer has been developed in our lab for vaccine delivery. By modulating the ratio of pH-responsive copolymers, we were able to control both intracellular and extracellular release of protein antigens with different charge properties. Co-delivering the mixture of optimized antigen and adjuvants particles to mice subcutaneously, the quantity and quality of both primary and memory immunity were successfully improved compared to the positive control which contained antigen mixed with aluminum hydroxide (Alum) adjuvant. Unlike Alum, which induced Th2-dominant humoral responses, blend particles elicited robust Th1-dominant responses. Additionally, the quality of cellular responses was improved by blend particle systems. Higher level of polyfunctional CD4+ T cells was induced by particles compared to Alum control. In combination of a novel mucosal vaccination strategy, this particle system was tested to protect mice from genital Herpes Simplex Virus-2 (HSV-2) infection. More than 500 millions of people are estimated to be infected with HSV-2 worldwide and HSV-2 seropositive persons have a 2 to 4 fold increased risk of acquiring HIV-1 infection. Lacking sufficient immunity, candidate HSV-2 vaccines that were protective in animal model, however, have not been successful in human clinical studies in decades. Our results showed that our strategy recruited sufficient immunity in local tissue and has efficiently protected the mice against HSV-2 infection. This pH-responsive blend particle system represents a simple and promising delivery platform for subunit vaccine development against viral infections.

Biomacromolecules such as nucleic acids and peptides have great potential as therapeutics but must overcome many challenging biological barriers to succeed in the clinic. In this work, we present several investigations of stimulus-responsive polymers developed to navigate both extracellular and intracellular barriers to biologic drug delivery. We begin with a review of past applications of pH-responsive chemistries in nucleic acid delivery (Chapter 1). After screening a panel of lytic peptides in a pH-sensitive polymer conjugate system that mediates endosomal escape of plasmid cargo (Chapter 2), we evaluate the most promising gene delivery vector in a mouse model of traumatic brain injury (Chapter 3). Our efforts to develop engineered stem cells transplants as alternatives to gene delivery are then discussed (Chapter 4). Finally, we adapt our pH-responsive polymer for the delivery of peptide vaccine cargo (Chapter 5) and detail future directions to further improve antigen and adjuvant delivery with polymers (Chapter 6).

Vaccination represents one of the most cost-effective methods for the treatment and prevention of disease. Due to safety concerns surrounding the use of live attenuated and killed/inactivated pathogens, there has been significant interest in the development of subunit vaccines, which are composed of discrete antigenic proteins or polysaccharides. Unfortunately, protein-based vaccines are poorly immunogenic and typically require the use of adjuvants for the induction of protective immunity. Because of their tunability and synthetic addressability, polymeric particulate carriers represent a promising approach for enhancing the efficacy of protein-based vaccines, and are the subject of this dissertation. In particular, we report on the development of acid-degradable materials, which are capable of releasing encapsulated protein antigens and immunostimulatory molecules following uptake by cells of the immune system and subsequent trafficking to acidic endosomal vesicles. Specific emphasis is placed on the development, functionalization and immunological evaluation of biodegradable acid-sensitive particle systems. Chapter 1 introduces various delivery strategies for enhancing the potency of subunit vaccines and discusses the basics of vaccine immunology. Additionally, the field of polymeric particulate antigen carriers is reviewed, with a focus on relevant design criteria for materials intended to interact with the immune system. In Chapter 2, the synthesis of acid-sensitive acrylamide-based microparticles containing an immunomodulatory agent is discussed. The optimal conjugation strategy for an immunostimulatory DNA sequence is investigated and particles containing a model protein antigen are studied in several models, including a cancer immunotherapy study, to ascertain in vivo the importance of co-delivering antigen and maturation signals to cells of the adaptive immune system. Chapter 3 discusses the synthesis of a second generation acid-sensitive polyurethane particle system which is designed to degrade entirely into biocompatible small molecule byproducts. The ability of these particles to elicit an immune response to a model antigen is studied and a method to monitor the production of polymer degradation byproducts in cells is presented. Chapters 4 and 5 investigate the synthesis, characterization, and a method for the functionalization of a third generation acid-sensitive particle system. The preparation of these particles from acetal-modified dextran and their pH-dependent degradation behavior is described. Additionally, a facile method for the surface functionalization of these particles using alkoxyamine reagents is presented.

This book provides a comprehensive overview of how use of micro- and nanotechnology (MNT) has allowed major new advance in vaccine development research, and the challenges that immunologists face in making further progress. MNT allows the creation of particles that exploit the inherent ability of the human immune system to recognize small particles such as viruses and toxins. In combination with minimal protective epitope design, this permits the creation of immunogenic particles that stimulate a response against the targeted pathogen. The finely tuned response of the human immune system to small particles makes it unsurprising that many of the lead adjuvants and vaccine delivery systems currently under investigation are based on nanoparticles. Provides a comprehensive and unparalleled overview of the role of micro- and nanotechnology in vaccine development Allows researchers to quickly familiarize themselves with the broad spectrum of vaccines and how micro- and nanotechnologies are applied to their development Includes a combination of overview chapters setting out general principles, and focused content dealing with specific vaccines, making it useful to readers from a variety of disciplines

The authoritative reference on recent developments in vaccinology New technologies, including recombinant protein and DNA, have sparked phenomenal progress in vaccine development and delivery systems. This unique resource brings scientists up to date on recent advances and provides the information they need to select candidate adjuvants. With chapters written by leading experts in their fields, Vaccine Adjuvants and Delivery Systems: * Provides a comprehensive overview of the rapidly evolving field and developing formulation methods * Covers cutting-edge technologies and gives the current status of adjuvants in clinical trials and those still in the pre-clinical stage * Includes detailed information on specific vaccine adjuvants, including MF59, TLR4 agonists, new iscoms, cytokines, polyphosphazenes, and more * Provides a historical perspective on the development of vaccine adjuvants and discusses the mechanisms of adjuvant actions * Covers some novel adjuvants and delivery systems and the safety evaluation of adjuvants A great reference for researchers, scientists, and students in vaccinology, biotechnology, immunology, and molecular biology, this resource is also valuable for researchers and scientists in veterinary medicine who work to prevent diseases in animals.

Nanoscience or the science of the very small offers the pharmaceutical scientist a wealth of opportunities. By fabricating at the nanoscale, it is possible to exert unprecedented control on drug activity. This textbook will showcase a variety of nanosystems working from their design and construction to their application in the field of drug delivery. The book is intended for graduate students in drug delivery, physical and polymer chemistry, and applied pharmaceutical sciences courses that involve fundamental nanoscience. The purpose of the text is to present physicochemical and biomedical properties of synthetic polymers with an emphasis on their application in polymer therapeutics i.e., pharmaceutical nanosystems, drug delivery and biological performance. There are two main objectives of this text. The first is to provide advanced graduate students with knowledge of the principles of nanosystems and polymer science including synthesis, structure, and characterization of solution and solid state properties. The second is to describe the fundamentals of therapeutic applications of polymers in drug delivery, targeting, response modifiers as well as regulatory issues. The courses, often listed as Advanced Drug Delivery and Applied Pharmaceutics; Polymer Therapeutics; or Nanomedicine, are designed as an overview of the field specifically for graduate students in the Department of Pharmaceutical Sciences Graduate Programs. However, the course content may also be of interest for graduate students in related biomedical research programs. These courses generally include a discussion of the major principles of polymer science and fundamental concepts of application of polymers as modern therapeutics. All courses are moving away from the above mentioned course names and going by ‘pharmaceutical nanoscience or nanosystems’. This area of research and technology development has attracted tremendous attention during the last two decades and it is expected that it will continue to grow in importance. However, the area is just emerging and courses are limited but they are offered.