Richard M. Gilley

Controlled vaccine release in the gut-associated lymphoid tissues. I. Orally administered biodegradable microspheres target the peyer's patches

Abstract Microspheres prepared from various polymers were evaluated for their usefulness as carriers for the targeted delivery of vaccine antigens to the gut-associated lymphoid tissues. Following oral administration to mice, microspheres consisting of polystyrene, poly(methyl methacrylate), poly(hydroxybutyrate), poly( dl -lactide), poly( l -lactide), and of poly( dl -lactide-co-glycolide) with various ratios of lactide to glycolide were absorbed into the Peyers patches of the small intestine. In contrast, no or very little uptake was observed with microspheres consisting of ethyl cellulose, cellulose acetate hydrogen phthalate or cellulose triacetate. Tissue penetration was specific to the Peyers patches and was restricted to microspheres ⩽ 10 μm in diameter. Time-course studies on the fate of the poly( dl -lactide-co-glycolide) microspheres within the gut-associated lymphoid tissue showed that the majority of the microspheres dl -lactide-co-glycolide) microspheres containing a toxoid vaccine of staphylococcal enterotoxin B were prepared and characterized for their size distribution, surface morphology and toxoid release kinetics in an aqueous environment. Oral immunization with these microspheres effectively delivered and released the vaccine in the gutassociated lymphoid tissue as determined by their ability to induce a disseminated mucosal IgA anti-toxin antibody response.

Biodegradable microspheres as a vaccine delivery system

The utility of biodegradable and biocompatible microspheres as a vaccine delivery system for the induction of systemic and disseminated mucosal antibody responses was investigated. Intraperitoneal (ip) injection into mice of 1-10 microns microspheres, constructed of the copolymer poly(DL-lactide-coglycolide) (DL-PLG) which contained approximately 1% by weight a formalinized toxoid vaccine of staphylococcal enterotoxin B (SEB), dramatically potentiated the circulating IgG anti-toxin antibody response as compared to the free toxoid. The initiation of vaccine release was delayed in larger microspheres, and a mixture of 1-10 and 20-50 microns microspheres stimulated both a primary and an anamnestic secondary anti-toxin response following a single injection. However, neither free nor microencapsulated SEB toxoid induced a detectable mucosal IgA anti-toxin response following systemic injection. In contrast, three peroral immunizations with toxoid-microspheres stimulated circulating IgM, IgG and IgA anti-toxin antibodies and a concurrent mucosal IgA response in saliva, gut washings and lung washings. Systemic immunization with microencapsulated toxoid primed for the induction of disseminated mucosal IgA responses by subsequent oral or intratracheal (it) boosting in microspheres, while soluble toxoid was ineffective at boosting. These results indicate that biodegradable and biocompatible microspheres represent an adjuvant system with potentially widespread application in the induction of both circulating and mucosal immunity.

Release of Human Serum Albumin from Poly(lactide-co-glycolide) Microspheres

Human serum albumin (HSA) was encapsulated in a 50:50 copolymer of DL-lactide/glycolide in the form of microspheres. These microspheres were used as a model formulation to study the feasibility of controlling the release of large proteins over a 20- to 30-day period. We show that HSA can be successfully incorporated into microspheres and released intact from these microspheres into various buffer systems at 37°C. A continuous release of the protein could be achieved in physiological buffers at 37°C over a 20- to 30-day period from microspheres with high protein loadings (11.6%). These results demonstrate the potential of poly(DL-lactide-co-glycolide) microspheres for continuous delivery of large proteins.

Biodegradable Microspheres: Vaccine Delivery System for Oral Immunization

The potential of biocompatible and biodegradable microspheres as a controlled release oral vaccine delivery system has been examined. Orally-administered 1-10 micron microspheres composed of poly (DL-lactide-co-glycolide) were specifically taken up into the Peyers patch lymphoid tissue of the gut, where those greater than or equal to 5 micron remained for up to 35 days. Microspheres less than 5 micron disseminated within macrophages to the mesenteric lymph nodes and spleen. In contrast to soluble staphylococcal enterotoxin B toxoid, oral immunization with enterotoxoid in microspheres induced circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin response in saliva and gut fluid.

Preparation of injectable controlled-release microcapsules by a solvent-evaporation process☆

Abstract The solvent-evaporation microencapsulation process can be used to prepare high-quality, injectable, controlled-release microcapsules containing as little as 1 wt.% drug to as much as 90 wt.% drug. This process is useful for the microencapsulation of water-soluble and water-insoluble drugs. When the process conditions are optimized, the process affords good yields of a free-flowing powder comprising smooth spherical particles. Using this process, a batch of microcapsules can be made to have a wide or narrow size distribution. The micro-capsules can have diameters from less than 1 μm to as large as 250 μm. During the early stages of a microcapsule development project, problems can often arise, and high-quality microcapsules are difficult to fabricate. Some of these problems and how to remedy them with respect to the solvent-evaporation process are discussed.

[8] Poly(lactide-co-glycolide) microcapsules for controlled release of steroids

Publisher Summary This chapter addresses long-acting, drug-delivery systems comprising steroidal drugs microencapsulated in aliphatic polyester resins. Because the aliphatic polyesters slowly degrade, owing to hydrolysis of ester linkages, when they are exposed to water they disappear from the tissues soon after (or during) drug release. Many drug and biologic substances, other than the steroids, can be microencapsulated in the biodegradable polyesters to produce injectable controlled-release doses. The specific copolymer resins and the appropriate microencapsulation processes must be chosen carefully, however, to achieve the rates and durations of drug release desired. When steroid hormones are microencapsulated in the biodegradable copolyesters in which the drug is homogeneously dispersed within the resin matrix to form a monolithic microcapsule and the microcapsules are injected either intramuscularly or subcutaneously, the drugs are slowly released into the tissues principally by a diffusion mechanism. The microcapsules are designed to release norethisterone at a nearly constant rate for 3 months. The 85:15 poly(DL-lactide-co-glycolide) resin is selected as the biodegradable excipient because the onset of resin fragmentation occurs at about 50 days postinjection. Total resorption of the resin by the tissues is complete in about 180 days.

Vaccine-Containing Biodegradable Microspheres Specifically Enter the Gut-Associated Lymphoid Tissue Following Oral Administration and Induce a Disseminated Mucosal Immune Response

Biodegradable and biocompatible microspheres have been investigated for their usefulness as a vaccine delivery system for both parenteral and enteral immunization. Microspheres composed of poly(DL-lactide-co-glycolide) which contained a toxoid vaccine of Staphylococcal enterotoxin B were found to strongly potentiate the circulating anti-toxin antibody response following intraperitoneal injection. Following oral administration, microspheres less than 10 microns in diameter were specifically taken up into the Peyers patches of the gut-associated lymphoid tissue, where those greater than or equal to 5 microns remained fixed for an extended period. Microspheres less than 5 microns were disseminated within macrophages to the mesenteric lymph nodes, blood circulation and spleen. Oral immunization with enterotoxoid-containing microspheres induced circulating toxin-specific antibodies and a concurrent secretory IgA anti-toxin response in saliva, gut wash fluids and bronchial-alveolar wash (BAW) fluids. In contrast, soluble enterotoxoid was completely ineffective as an oral immunogen.

Biodegradable microspheres for the delivery of oral vaccines

Mucosal membranes are the most frequent portals of entry of almost all infectious agents. The most important protective humoral factors on mucosal surfaces are locally produced antibodies predominantly of the IgA isotype. Therefore, the induction of specific protective immune responses at mucosal surfaces is a highly desirable goal in the prevention of many infectious diseases. This can be achieved by novel vaccination strategies using effective antigen delivery systems. The ingestion or inhalation of antigens results in the induction of immune responses on mucosal surfaces due to the dissemination of antigen-specific and IgA-committed precursors of plasma cells from intestinal and respiratory lymphoid tissues to other mucosal surfaces. However, only minute quantities of antigens are absorbed from mucosal surfaces due to their degradation by enzymes and hydrochloric acid and inefficient uptake. The protection and enhanced uptake of antigens provided by particles with slow biodegradation could facilitate oral immunization. This possibility has been explored in several recent studies which indicate that simple proteins or complex antigens of viral and bacterial origin incorporated into biodegradable microspheres induce, after ingestion, both mucosal and systemic immune responses. Experimental animals orally immunized with an influenza virus vaccine in biodegradable microspheres displayed virus-specific antibodies in saliva and in serum, and were protected against challenge with the live viruses. Many theoretical and practical advantages of oral over systemic immunization should stimulate further studies of applications of oral vaccines.

Induction of protective immune responses against Venezuelan equine encephalitis (VEE) virus aerosol challenge with microencapsulated VEE virus vaccine.

Venezuelan equine encephalomyelitis (VEE) virus, a member of the family Togaviridae, genus Alphavirus, causes disease in humans and equids. The virus is normally transmitted by the bite of an infected mosquito however, it can also be highly infectious by aerosol. The purpose of the present study was to determine the effectiveness of formalin-fixed, 60Co-irradiated VEE virus microencapsulated in poly DL-lactide-co-glycolide in inducing immune responses protective against aerosol challenge with virulent VEE virus. Balb/c mice were primed by subcutaneous injection of microencapsulated VEE virus vaccine, followed 30 days later by a single immunization with the same vaccine given via the oral, intratracheal (i.t.) or subcutaneous (s.c.) route. Mice boosted by the i.t. or s.c. route had higher plasma IgG anti-VEE virus levels than orally immunized animals. The responses in the former groups were similar in magnitude to those seen in mice primed and boosted by the i.t. route. Antibody activity was detected in bronchial-alveolar and intestinal washes, fecal extracts and saliva from immunized animals. The levels of IgG and IgA antibody activity in bronchial-alveolar wash fluids from mice boosted by the i.t. route were higher than those seen in animals immunized by the oral or s.c. route with the microsphere vaccine. Mice immunized with the microencapsulated VEE virus vaccine were protected from lethal VEE virus infection following aerosol challenge at approximately three months after the initial immunization. Mucosal immunization via the i.t. route appeared to be the most effective regimen, since 100% of the mice resisted aerosol challenge.

Immune Responses to Influenza Virus in Orally and Systemically Immunized Mice

In our studies on the induction of an immune response by oral immunization, we have explored the potential of a novel approach for antigen delivery by microencapsulation. This procedure preserved the immunogenicity of the influenza virus introduced by either systemic or oral routes. Furthermore, the levels of specific antibodies in serum and in saliva were enhanced and lasted longer (up to 4 months) in animals immunized with of antigens in microencapsulated form than in animals immunized with equal doses of free suspension. Preliminary challenge experiments showed a correlation between levels of antibodies and protection. All mice systemically immunized were protected against the virus, while mice orally immunized with lower doses of microencapsulated antigen had better survival rates than those immunized with higher doses. Additional experiments suggested that low doses of immunogen were able to generate better protective immunity than high doses, which may instead be tolerogenic. Further experiments with a well characterized microencapsulated antigen (size of microcapsules, time of release of antigen, as well as its dose and form) will be necessary to establish conditions for optimal immunization protocols applicable for the oral or systemic routes.