Oral Vaccine Delivery Research Articles

Oral vaccine delivery

Oral vaccination of animals and man, to provide effective mucosal and/or systemic immunity, is largely ineffective. This is due mainly to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal wall. Over the past decade or so, a number of proteins have been identified that are effective at eliciting mucosal and systemic immune responses following oral administration. Uptake of these molecules by the gastro-intestinal tract (GIT) epithelium is dependent upon specific binding to the GIT epithelial cells. The identity of these molecules is discussed, as well as their possible application as ‘carriers’ for co-transporting haptens, proteins and nanoparticles across the GIT epithelium.

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A number of antimicrobial agents have been developed since penicillin was discovered. In spite of the strong antimicrobial activity, some of them are difficult to be used clinically due to their various side effects including the organ toxicity. On the other hand, some of them are not suitable for oral administration because of their poor absorbability. To overcome these problems, DDS technology has been applied. Some poorly absorbable drugs became to be used as the oral dosage form by converting to the well-absorbable prodrugs. Lipid-dispersed formulations, such as liposomes and lipid-nanospheres, of amphotericin B could make the antifungal drug less toxic and more effective. Recently, extensive studies are conducted to treat or protect against the virus infections. Oral vaccine delivery is one of the most attractive objectives. The use of the particulate dosage form has been investigated to deliver the vaccine to M cells. However, the most suitable properties of the carrier particles for the extensive binding to M cells and the sequential uptake remain to be clarified. To protect against influenza virus infection, the nasal applications of influenza vaccine were examined. The high protection against the viral challenge by the administration of influenza vaccine with sodium polystyrene sulfonate resin or CT 112 K, a mutant of cholera toxin, as the adjuvant was shown by the experiments in mice. The plasmid DNA could also protect against in influenza virus infection in mice while the method of effective introduction of the plasmid DNA to mucosal cells should be developed. The optimal DDS technology will lead the success of these novel approaches.

Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco

Plant seeds offer unique opportunities for the production and delivery of oral subunit vaccines. We have used the immunodominant glycoprotein B complex of human cytomegalovirus (HCMV), introduced into tobacco plants, as a model system for studying the merit of this promising approach. Given the advantages of expressing proteins in seeds, a novel expression vector was developed incorporating regulatory sequences of glutelin, the major rice seed storage protein, to direct synthesis of recombinant glycoprotein B. Analysis of genomic DNA of 28 selected tobacco transformants by PCR amplification showed that 71% harboured the gB cDNA, a finding further documented by Southern blotting. Specific immunoassays of protein extracts from seeds of positive plants showed that all were producing antigenic glycoprotein B at levels ranging from 70–146 ng/mg extracted protein. In addition, similarity with native glycoprotein B produced in HCMV-infected cells was also demonstrated by inhibition of immunofluorescence on HCMV-infected human fibroblasts. These data are the first to report the expression of an immunodominant antigen of HCMV in plant tissues, indicating the fidelity with which this very large heterologous viral glycoprotein can be synthesized in this model system.

Preparation and characterization of protein-loaded poly(ε-caprolactone) microparticles for oral vaccine delivery

This paper describes the conditions of preparation of poly(ε-caprolactone) (PCL) microparticles with a mean size between 5 and 10 μm, obtained by a double emulsion-solvent evaporation technique, suitable for oral vaccine delivery. Bovine serum albumin (BSA) was used as water-soluble model antigen for encapsulation. Different parameters influencing the microparticle size, the BSA loading and entrapment efficiency were investigated. Spherical, smooth and homogeneously distributed microparticles were produced with a BSA loading and entrapment efficiency reaching, respectively, 5% (w/w) and 30%. Polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) analyses of BSA released from these particles confirmed that the entrapped protein seemed to remain unaltered by the protein encapsulation process.

Cell surface display of human immunodeficiency virus type 1 gp120 on Escherichia coli by using ice nucleation protein.

A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface of Escherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.

PLG microparticles stabilised using enteric coating polymers as oral vaccine delivery systems

Novel poly(dl-lactide-co-glycolide) microparticles for oral vaccine delivery were formulated using the enteric polymers Eudragit ® L100-55 and carboxymethylethylcellulose (CMEC) as stabilisers. To serve as a control, microparticles were also produced using the conventional PVA surfactant. In all three cases the antigen, ovalbumin (OVA)-loaded microparticles produced were less than 5 μm in diameter and had a spherical, smooth rounded appearance. The presence of surfactants at the microparticle surface was demonstrated by the surface analysis techniques, XPS and SSIMS. Incubation of microparticles with solutions of pepsin or trypsin led to the removal of a proportion of the antigen associated with all three systems. However, in three CMEC-stabilised microparticle formulations and one of three Eudragit formulations, a high percentage of the associated antigen was protected from removal by a solution of pepsin at pH 1.2 compared with the PVA-stabilised microparticles. In addition, with certain CMEC and Eudragit formulations a degree of protection was also afforded to the associated OVA against removal by trypsin at pH 7.4. Following the incubation of microparticles in simulated gastric fluid a higher percentage of intact antigenic OVA was detected in microparticles stabilised using CMEC than in the PVA - and Eudragit - stabilised formulations. Oral immunisation of mice with OVA-loaded microparticles stabilised using either of the three surfactants led to the induction of specific serum IgG and salivary IgA antibodies. Significantly higher levels of specific salivary IgA antibody to OVA were measured in mice immunised with the CMEC-stabilised microparticles than with the other two formulations. This novel approach in PLG microparticle formulation may have potential in increasing the efficacy of microparticulate systems for the oral administration of vaccines.

Microparticulate drug delivery systems.

Chitosan was proposed as a drug carrier for mucosal administration in ocular, buccal, nasal, gastroenteric and vaginal-uterine therapies based on its bioadhesive properties and biodegradability in vivo under the action of hydrolases. Examples are the delivery of acyclovir via ocular administration, and the delivery of 5-aminosalicylic acid to the colon. Microparticles may need to be cross-linked to retard their degradation in acidic media; yet cross-linking with glutaraldehyde introduces cytotoxic characteristics and depresses bioadhesion. Alternative cross-linking approaches are discussed along with the suitability of chitosan for the oral delivery of vaccines.

Preface

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.

Effect of size, surface charge, and hydrophobicity on the translocation of polystyrene microspheres through gastrointestinal mucin

Microspheres (MS) have been proposed for use as oral vaccine delivery vehicles (VDV); however, due to poor and variable absorption their clinical utility is limited. The effects of size, ζ-potential, and surface hydrophobicity on the translocation (PT) permeabilities of polystyrene (PS) MS with varying surface functional groups (amidine, carboxyl, carboxylate-modified [CML], and sulfate) were determined through gastrointestinal (GI) mucin. PT were determined, under steady-state conditions, using a modified Ussing-type diffusion chamber and a mucin packet developed for use with the Transwell-Snapwell system. PT followed the Stokes-Einstein relationship, demonstrating the limited ability of larger MS (>0.5 μm) to diffuse through the mucin layer. PT also varied according to the surface characteristics. Even though the ζ-potential did not correlate with the transport of MS through mucin, surface ionization appears to be important in MS translocation. The PS-amidine MS were significantly less hydrophobic and had a higher PT than that of the other MS, suggesting that hydrophobicity is also a significant factor in MS transport through mucin. While these results suggest that mucin may be a significant barrier to the oral absorption of vaccines and VDVs in vivo, the rate-limiting barrier for the absorption of MS will be the intestinal mucosa. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 1481–1492, 1997

Evaluation of oral poliovirus vaccine delivery during the 1994 national immunization days in Pakistan.

Pakistan conducted national immunization days (NIDs) for the first time in 1994. To estimate coverage, to evaluate risk factors for failure to be immunized, and to determine the effectiveness of mass media, parents of 1288 children in 714 households in four districts were surveyed after the first NID round. In each district, a high proportion of children (93%-96%) received oral poliovirus vaccine (OPV) during the NID. In three districts, unimmunized or partially immunized children were less likely to receive NID OPV than were fully immunized children (Kohistan, P < .001; Quetta, P < .001; and Sibi, P = .05). Although a high proportion of children in each age cohort received NID OPV, in three districts children 0-11 months of age were less likely to receive NID OPV than were older children. Television and radio reached a high proportion of survey households, but other mass media were less effectively utilized. Risk factor and media effectiveness surveys provide important information that is useful for planning future NIDs.

Mucosal Immunisation for Enteric Diseases

Oral delivery of vaccines results in these being taken up by specialised microfold epithelial cells covering Peyer’s patches of the gastrointestinal tract, therefore stimulating regulatory T cells and surface IgA positive (sIgA+) B cells. T helper cells can be divided into 2 subsets, type 1 (TH1) and type 2 (TH2), according to their function and the cytokines they secrete. TH1 cytokines such as interleukin (IL)-2, interferon-γ and tumour necrosis factor-β (TNFβ) elicit activation of T cells and macrophages, whereas TH2 cytokines such as IL-4, IL-5, IL-6 and IL-10 favour mucosal and parenteral B cell responses. Therefore, TH2-type T cells are of particular interest for mucosal responses, since they help in the differentiation of sIgA+ B cells into IgA-producing plasma cells. As a result, one can take advantage of the fact that different forms of antigen delivery systems generally influence the outcome of an immune response, and use those that best induce mucosal responses. An example of this would be orally administered live attenuated Salmonella versus the oral administration of cholera toxin. The first induces a dominant TH1-type response, and the second a TH2-type response. Thus these 2 delivery systems can be exploited in order to elicit the desired immune response depending on the protective response required. Alternatively, encapsulating antigens into polyglycolide microspheres or liposomes, or incorporating them into immune-stimulating complexes, has facilitated the delivery of antigens which otherwise do not result in an immune response when given orally. Much progress is being made in the construction of attenuated viral and bacterial vectors for the delivery of antigen to mucosal sites. For example, poliovirus has been used as a vector to deliver both rotavirus and HIV antigens. Bacterial vectors and attenuated mutant bacteria for use in vaccines have also been extensively researched. Examples of these include Salmonella typhi mutants, Vibrio cholerae, Shigella species, Helicobacter pylori and Campylobacter jejuni. In addition, new approaches are being developed to induce responses at mucosal surfaces such as the gastrointestinal, respiratory and genitourinary tracts. These include the use of adjuvants that stimulate mucosal responses such as cholera toxin and Escherichia coli heat labile toxin, as well as the coexpression of cytokine genes with antigenic proteins on live vectors to drive the immune response so that mucosal responses are favoured. Furthermore, nucleic acid vaccines and the potential use of transgenic plants are new technologies that are contributing to our ability to induce responses at mucosal surfaces.

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.

Oral vaccination with alginate microsphere systems

Oral vaccination is a simple, efficient way of inducing immunity at mucosal surfaces. The slow development of oral vaccines has been mainly due to the lack of suitable delivery systems. We have used hydrogel microspheres to deliver various vaccines to several animal species by oral administration. Oral delivery of vaccines using alginate microspheres elicited the production of secretory IgA (sIgA) at the mucosal surfaces in mice, rabbits, and cattle. Oral vaccination of chicken resulted in an increased delayed-type hypersensitivity, a cell-mediated immune response, indicating a positive response to the vaccine. Our studies have clearly shown that alginate microspheres are effective for the oral administration of vaccines.

Update on the Center for Recombinant Gamete Contraceptive Vaccinogens.

The overall goal of the Center for Recombinant Gamete Contraceptive Vaccinogens is to develop a contraceptive vaccine which will induce antibodies in the female reproductive tract at sufficient levels to block fertilization. It is envisioned that antibodies developed by this vaccine will act to agglutinate, immobilize or coat either the egg or the sperm in the oviduct, uterus, cervix or vagina. Thus, the vaccine will exert its effects before the fertilization event, as a "prefertilization contraceptive." The Center has focused its activities over the past 4 years on four areas: 1) sperm antigens: the identification and characterization of novel sperm surface molecules which are attractive candidates for inclusion in a vaccine; 2) zona antigens: the identification of synthetic peptides derived from the zona pellucida which possess contraceptive potential without adverse pathology; 3) oral vaccine delivery: the development of live Salmonella vectors which deliver vaccine antigens through the oral route and which will stimulate secretory immunity in the female reproductive tract; and 4) animal testing: the testing of vaccine formulations in in vitro fertilization models, in small animals, in monkeys and in baboons. In addition, preparations for human testing are underway through preparation of an Investigational New Drug Application to begin Phase I human trials of a LDH-C4 peptide vaccine.

Transgenic Plants Created for Oral Immunization Against Diarrheal Diseases.

We have created transgenic plants that express subunit antigens of infectious bacteria and viruses. These proteins have been isolated and characterized to demonstrate that they retain immunogenic properties. When raw potatoes containing the recombinant immunogens were fed to mice, the animals were orally immunized. Research completed to date has demonstrated the feasibility of using a genetically-engineered food as an inexpensive oral vaccine production and delivery system for diarrheal disease. The system could be a convenient means to deliver vaccines to travelers. It also has great potential as an appropriate technology for producing vaccines "in country" in the developing world. This review focuses on work from our group on recombinant subunit vaccines in potato tubers against enterotoxigenic Escherichia coli and Norwalk virus.

Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract.

Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine. Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions. A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium. Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions. Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella. Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine.

Recent Development in Oral Vaccine Delivery Systems.

Oral delivery of vaccines is the least effective method of vaccinating fish. However, anal intubation of antigens is very effective in inducing high serum antibody levels and protection against vibriosis and enteric redmouth. It is generally considered that degradation of antigen in the stomach and anterior intestine prevents immunostimulation taking place and that if antigens could be protected during passage through the foregut effective immunization could be achieved.Recent attempts to protect antigens have included the concomitant administration of antacids and antiproteases. Protective coating procedures have included encapsulation of particulated antigen with methacrylic acrylic acid polymers which resist acid but dissolve in the high pH of the intestine.Incorporation of antigen into microparticles of poly lactide co-glycolide polymers protects against enzyme degradation and allows increased uptake of intact antigen.Certain substances can enhance uptake of antigen by the intestine of fish and have adjuvant activity, such as cholera toxin β-subunit and Quil-A saponin.Overcoming problems of palatability of oral vaccines delivered to fish fry has been achieved by incorporating vaccine into live food such as Artemia. However, delivery of antigen to very young fish, before they are fully immunocompetent can induce immune suppression.Many of these methods have improved the level of antigen uptake by the intestine and the antibody response in fish and the prospects for improving the efficacy of oral vaccination appear optimistic.

A distinctive electrophysiological signature from the Peyer's patches of rabbit intestine.

1. Rabbit small intestinal segments containing Peyer's patches (PP) were examined in Ussing chambers using short-circuit current (Isc) recording. By comparison with control small intestinal mucosal segments, rabbit PP-containing epithelia exhibited decreased basal Isc, increased transepithelial resistance (TER) and unchanged potential difference (PD). 2. Carbachol caused a decrease in Isc in rabbit PP epithelia. Forskolin, dibutyryl cyclic GMP, histamine and the calcium ionophore, A23187, were without effect. In contrast, control epithelial segments of rabbit intestine responded to carbachol and forskolin with an increased Isc, indicative of electrogenic chloride secretion. The EC50 for carbachol was approximately 2 microM in both types of epithelia. Methacholine also caused an outward current in rabbit PP epithelia which had similar properties to that of carbachol. The effect of the cholinomimetics on rabbit PP was basolateral-sided, reversible, and sensitive to low concentrations of the general muscarinic cholinoceptor blockers, atropine, scopolamine and also to the M1 cholinoceptor blocker, pirenzepine. 3. The Isc response to cholinomimetics in rabbit PP was insensitive to bumetanide, amiloride, TEA, barium, acetazolamide, piroxicam and omeprazole, but was attenuated in the presence of ouabain. Using bilaterally-substituted solutions, the carbachol effect on rabbit PP Isc was abolished in chloride/bicarbonate-free, but not in chloride-free solutions, suggestive of stimulation of electrogenic bicarbonate absorption by the agent. Substitution for sodium abolished both the basal current and the Isc response to carbachol. Part of the effect of carbachol on PP Isc appeared to be mediated by submucosal neurones because addition of tetrodotoxin reduced the effect by 60%. 4 As microfold (M) epithelial cells predominate in the PP of the rabbit, the unusual phenotype of cholinomimetic-induced outward current may be used as an electrophysiological marker for these potential sites of oral vaccine delivery, and in particular it may also be of use as a marker for rabbit M cells.

P16 Tomato lectin as an adjuvant for oral vaccine delivery

P16 Tomato lectin as an adjuvant for oral vaccine delivery

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.