Alexander K. Andrianov

Microneedle-based vaccines

The threat of pandemic influenza and other public health needs motivate the development of better vaccine delivery systems. To address this need, microneedles have been developed as micron-scale needles fabricated using low-cost manufacturing methods that administer vaccine into the skin using a simple device that may be suitable for self-administration. Delivery using solid or hollow microneedles can be accomplished by (1) piercing the skin and then applying a vaccine formulation or patch onto the permeabilized skin, (2) coating or encapsulating vaccine onto or within microneedles for rapid, or delayed, dissolution and release in the skin, and (3) injection into the skin using a modified syringe or pump. Extensive clinical experience with smallpox, TB, and other vaccines has shown that vaccine delivery into the skin using conventional intradermal injection is generally safe and effective and often elicits the same immune responses at lower doses compared to intramuscular injection. Animal experiments using microneedles have shown similar benefits. Microneedles have been used to deliver whole, inactivated virus; trivalent split antigen vaccines; and DNA plasmids encoding the influenza hemagglutinin to rodents, and strong antibody responses were elicited. In addition, ChimeriVax-JE against yellow fever was administered to nonhuman primates by microneedles and generated protective levels of neutralizing antibodies that were more than seven times greater than those obtained with subcutaneous delivery; DNA plasmids encoding hepatitis B surface antigen were administered to mice and antibody and T cell responses at least as strong as hypodermic injections were generated; recombinant protective antigen of Bacillus anthracis was administered to rabbits and provided complete protection from lethal aerosol anthrax spore challenge at a lower dose than intramuscular injection; and DNA plasmids encoding four vaccinia virus genes administered to mice in combination with electroporation generated neutralizing antibodies that apparently included both Th1 and Th2 responses. Dose sparing with microneedles was specifically studied in mice with the model vaccine ovalbumin. At low dose (1 microg), specific antibody titers from microneedles were one order of magnitude greater than subcutaneous injection and two orders of magnitude greater than intramuscular injection. At higher doses, antibody responses increased for all delivery methods. At the highest levels (20-80 microg), the route of administration had no significant effect on the immune response. Concerning safety, no infections or other serious adverse events have been observed in well over 1,000 microneedle insertions in human and animal subjects. Bleeding generally does not occur for short microneedles (<1 mm). Highly localized, mild, and transient erythema is often observed. Microneedle pain has been reported as nonexistent to mild, and always much less than a hypodermic needle control. Overall, these studies suggest that microneedles may provide a safe and effective method of delivering vaccines with the possible added attributes of requiring lower vaccine doses, permitting low-cost manufacturing, and enabling simple distribution and administration.

Protein release from polyphosphazene matrices

Polyphosphazenes have been exploited as carriers for protein delivery due to versatility of molecular structures and sophisticated spectrum of chemical and physical properties. Ease of structural manipulations for this class of organometallic polymers allows efficient control over physico-chemical parameters of polyphosphazenes including their biodegradability and matrix permeability. Some polyphosphazenes offer additional advantages as protein delivery vehicles since microencapsulation of substrates in these systems can be achieved under remarkably mild physiological conditions. Because of these properties polyphosphazenes have tremendous potential as matrices for protein release as shown by studies both in vitro and in vivo.

Polymeric carriers for oral uptake of microparticulates.

Delivery of vaccine antigens by the oral route is plagued with challenges. Much of the research has focused on the development of microparticles as antigen carriers to the gastrointestinal (GIT) mucosa. Polymers, either natural or synthetic, have been the class of compounds most often investigated for their ability to form microparticles containing antigen. A great deal of research has been performed using model microparticles composed of polystyrene. From this work it has become clear that microparticles are taken up and translocated across the GIT epithelium. Antigen carrying microparticles generated from both hydrophobic and hydrophilic polymers are able to induce significant immune responses after oral immunization. Although very little systematic work on the effects of the physicochemical properties of the polymer composing the microparticles has been done, enough is known to conclude that the surface of the polymeric microparticle can be decisive in determining the overall uptake of the microparticles. Charge and the hydrophobic/hydrophilic balance of the polymer are important physicochemical characteristics that determine the value of the polymer as a microparticulate carrier. This review examines the properties of polymeric matrices that make them viable candidates as oral vaccine delivery vehicles.

Poly[di(carboxylatophenoxy)phosphazene] is a potent adjuvant for intradermal immunization

Intradermal immunization using microfabricated needles represents a potentially powerful technology, which can enhance immune responses and provide antigen sparing. Solid vaccine formulations, which can be coated onto microneedle patches suitable for simple administration, can also potentially offer improved shelf-life. However the approach is not fully compatible with many vaccine adjuvants including alum, the most common adjuvant used in the vaccine market globally. Here, we introduce a polyphosphazene immuno adjuvant as a biologically potent and synergistic constituent of microneedle-based intradermal immunization technology. Poly[di(carboxylatophenoxy)phosphazene], PCPP, functions both as a vaccine adjuvant and as a key microfabrication material. When used as part of an intradermal delivery system for hepatitis B surface antigen, PCPP demonstrates superior activity in pigs compared to intramascular administration and significant antigen sparing potential. It also accelerates the microneedle fabrication process and reduces its dependence on the use of surfactants. In this way, PCPP-coated microneedles may enable effective intradermal vaccination from an adjuvanted patch delivery system.

Preparation of hydrogel microspheres by coacervation of aqueous polyphosphazene solutions

A new method of preparing ionically cross-linked polyphosphazene hydrogel microspheres which enables effective control over microsphere size distribution has been developed. The synthesized microspheres can be used in protein delivery and, especially, as vaccine delivery vehicles. A new technique utilizes the ability of aqueous solutions of poly[di(carboxylatophenoxy)phosphazene] (PCPP) to form coacervate microdroplets upon addition of sodium chloride. These microdroplets are then stabilized via polymer cross-linking with calcium ions. It was demonstrated that the method enables efficient microencapsulation of proteins. It was also shown that microsphere size can be controlled through the manipulation of microencapsulation conditions. The process is simple, highly reproducible and generates microspheres with a narrower microsphere size distribution, compared to the previous technologies.

Poly[di(carboxylatophenoxy)phosphazene] (PCPP) is a potent immunoadjuvant for an influenza vaccine.

The adjuvant activity of poly[di(carboxylatophenoxy)phosphazene] (PCPP) on the immunogenicity of formalin-inactivated influenza virions and commercial trivalent influenza vaccine was studied. Regardless of which antigen preparation is used, the addition of 100 micrograms PCPP enhances the HAI antibody response 10-fold over the levels elicited by the vaccine alone. Similarly, PCPP enhanced the IgM, IgG, and IgG1 ELISA antibody titers to influenza antigens at least 10-fold higher than the vaccine alone. In contrast, the IgG2a isotype titers were only enhanced about 2-fold. Immunization of aged mice (22 months old) with trivalent influenza vaccine alone did not sero-convert these mice as measured by HAI or ELISA whereas significant sero-conversion was achieved when mice were immunized with PCPP-formulated trivalent vaccine. The adjuvant activity of PCPP was shown to not be due to a site of injection depot effect. PCPP adjuvanticity was positively correlated to the molecular weight of the polymer.

Water-Soluble Phosphazene Polymers for Parenteral and Mucosal Vaccine Delivery

PCPP can be used in two different ways to potentiate an immune response. The soluble form of the polymer has been found to have immunoadjuvant activity. A single subcutaneous injection of polymer/influenza dramatically increases the ELISA, neutralizing, and HI antibodies to influenza virus compared to CFA. The polymer has also succeeded in dramatically increasing the amount of ELISA antibodies to TT. The antibody response elicited was predominantly of the IgG1 isotype. PCPP has also been used to generate micron-sized hydrogel microspheres through a process of divalent ion cross-linking of the polymer strands. These microspheres can induce significantly higher anti-TT serum IgG titers after a single intranasal immunization than TT alone.

Controlled release using ionotropic polyphosphazene hydrogels

Abstract Poly(bis(carboxylatophenoxy)phosphazene) (PCPP) forms hydrogels when treated in aqueous media with salts of divalent cations, such as calcium chloride. This system has potential as a material for the controlled release of drugs. To study the ability of the PCPP hydrogel matrix to release macromolecular substrates, 24-nm fluorescent polystyrene beads or proteins with varying molecular weights were encapsulated in polyphosphazene microspheres. The influence of polyphosphazene concentration and ionic crosslinker content on the outward diffusion of substrates and on microsphere morphology was investigated. In order to create a poly- L -lysine (PLL) outer membrane coating around the PCPP microspheres, the spheres were treated with PLL solution (single coating) or sequentially with PLL and PCPP solutions (double coating). The formation of these membranes (stable poly electrolyte complexes) strongly influences the permeability of the polyphosphazene microspheres and contributes to their stability in physiological saline solution. This enables liquefaction and swelling of the internal core of the microcapsules in saline solution via ion-exchange reactions with monovalent salts and chelating agents. Increasing the PLL molecular weight in the coating process results in a significant enhancement of the substrate release rate. It is possible that the mechanism of this process includes partial coating rupture without loss of bead coherence. The effect of microsphere coating on release profiles can be controlled by varying the molecular weights, the concentration of PLL and reaction times between PCPP microspheres and PLL.

Microneedles with Intrinsic Immunoadjuvant Properties: Microfabrication, Protein Stability, and Modulated Release

ABSTRACTPurposeIntradermal immunization using microneedles requires compatible immunoadjuvant system. To address this challenge, we investigated microneedles coated with polyphosphazene polyelectrolyte, which served both as microfabrication material and an immunoadjuvant compound.MethodsCoated microneedles were fabricated by depositing formulations containing Poly[di(carboxylatophenoxy)phosphazene], PCPP, on metal shafts, and their physico-chemical characterization was conducted.ResultsMicrofabrication of PCPP-coated microneedles exhibited strong dependence on protein-PCPP interactions in solutions and allowed for high efficiency of protein encapsulation. 70°C thermal inactivation studies demonstrated a remarkable increase in functional stability of protein in coated microneedles compared to solution formulation. A potential for modulation of protein release from coated microneedles has been demonstrated through ionic complexation of PCPP with small ions.ConclusionsMicroneedles containing PCPP coatings provide improved protein stability, modulated release, and protein-friendly microfabrication process.

Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes

Adjuvants improve the adaptive immune response to a vaccine antigen by modulating innate immunity or facilitating transport and presentation. The selection of an appropriate adjuvant has become vital as new vaccines trend toward narrower composition, expanded application, and improved safety. Functionally, adjuvants act directly or indirectly on antigen presenting cells (APCs) including dendritic cells (DCs) and are perceived as having molecular patterns associated either with pathogen invasion or endogenous cell damage (known as pathogen associated molecular patterns [PAMPs] and damage associated molecular patterns [DAMPs]), thereby initiating sensing and response pathways. PAMP-type adjuvants are ligands for toll-like receptors (TLRs) and can directly affect DCs to alter the strength, potency, speed, duration, bias, breadth, and scope of adaptive immunity. DAMP-type adjuvants signal via proinflammatory pathways and promote immune cell infiltration, antigen presentation, and effector cell maturation. This class of adjuvants includes mineral salts, oil emulsions, nanoparticles, and polyelectrolytes and comprises colloids and molecular assemblies exhibiting complex, heterogeneous structures. Today innovation in adjuvant technology is driven by rapidly expanding knowledge in immunology, cross-fertilization from other areas including systems biology and materials sciences, and regulatory requirements for quality, safety, efficacy and understanding as part of the vaccine product. Standardizations will aid efforts to better define and compare the structure, function and safety of adjuvants. This article briefly surveys the genesis of adjuvant technology and then re-examines polyionic macromolecules and polyelectrolyte materials, adjuvants currently not known to employ TLR. Specific updates are provided for aluminum-based formulations and polyelectrolytes as examples of improvements to the oldest and emerging classes of vaccine adjuvants in use.