Starch microparticles as vaccine adjuvant

The demand for new vaccine adjuvants is well documented. New purified antigens from parasites, bacterial or viral pathogens, as well as recombinant subunit antigens and synthetic peptides, are often inherently weak immunogens; therefore, they need some kind of adjuvant to help initiate an immune response. In addition, there are very few adjuvants using the potential of the mucosal immune system, which may play an important role in the defence against air- and food-borne infections. Starch is a natural biocompatible and biodegradable polymer that is suitable for the production of various particulate adjuvant formulations, which can induce mucosal as well as systemic immune responses. This review gives an account of the different starch adjuvants used in immunisation studies. In particular, the properties of polyacryl starch microparticles as an oral vaccine adjuvant that induce protective immune responses in mice challenge experiments are summarised. In addition, a diphtheria booster vaccine has been proposed to be used to proving the concept in man and the possibilities to design an efficient vaccine formulation for human use are discussed.     

Mucosal vaccine delivery

Vaccine development and vaccination is a major growth area of the pharmaceutical industry. As new vaccine products become available, potential will be given to physicians to provide prophylaxis for diseases that were previously not preventable, or to improve immunisation for some diseases that are currently suboptimally covered. Many factors influence vaccine effectiveness but one of the most important is the route of delivery of the product. Mucosal delivery of vaccines allows primary immunisation at the sites of the body where many of mankind’s mortality- and morbidity-causing diseases are initiated. Effective mucosal immunity is best induced by mucosal delivery of vaccines, due to the specialised and interlinked nature of the mucosal lymphoid tissues. As well as the potential for enhanced immunity, mucosal vaccine delivery is expected to increase patient compliance, make vaccines easier to use and reduce the pain, side-effects and fear of parenteral injection. However, mucosal delivery of vaccines is not straightforward and several strategies have been developed to allow for administration by the oral, nasal, rectal, genito-urinary and even pulmonary routes. These strategies include the use of live attenuated micro-organisms, attenuated toxins, bioadhesive polymers and emulsions, liposomes and proteosomes, biodegradable microparticles and immune stimulatory complexes (ISCOMS) as mucosal vaccine delivery systems/adjuvants. Details of some of the recent advances utilising these systems for mucosal antigen delivery are included in the article with a brief discussion on some of the strengths and weaknesses of the various strategies.     

BCG-loaded chitosan microparticles: interaction with macrophages and preliminary in vivo studies

The aim of this study was to develop a novel BCG-loaded chitosan vaccine with high association efficiency which can afford efficient interaction with APC and elicit local and Th1-type-specific immune response after intranasal administration. Chitosan-suspended BCG and BCG-loaded chitosan-alginate microparticles were prepared by ionotropic gelation. Interaction with APC was evaluated by fluorescence microscopy using rBCG-GFP. Specific immune responses were evaluated following intranasal immunisation of mice. Cellular uptake was approximately two-fold higher for chitosan-suspended BCG. A single dose of BCG-loaded microparticles or chitosan-suspended BCG by intranasal route improved Th1-type response compared with subcutaneous BCG. Chitosan-suspended BCG originated the highest mucosal response in the lungs by intranasal route. These positive results indicate that the proposed approach of whole live BCG microencapsulation in chitosan-alginate for intranasal immunisation was successful in allowing efficient interaction with APC, while improving the cellular immune response, which is of interest for local immunisation against tuberculosis.     

Starch microparticles as oral vaccine adjuvant: antigen-dependent uptake in mouse intestinal mucosa

An oral vaccine formulation comprised of starch microparticles with conjugated antigens is being developed. In this report we have examined the uptake of such microparticles by the intestinal mucosa and examined whether the conjugated antigen can influence the uptake. Two model antigens were used: recombinant cholera toxin B subunit (rCTB), which is known to bind to the ubiquitous GM1-receptor, and human serum albumin (HSA) which is not known to have any specific binding properties. The uptake was studied in mouse ligated intestinal loops into which the microparticles were injected. The intestinal loops were excised, fixed in ice-cold 95% ethanol. Entire specimens were mounted, exposed to fluorescence-labeled reagents staining the cytoskeleton, the particles and/or M cells and examined in a confocal laser-scanning microscope. A qualitative difference in the uptake of the rCTB- and HSA-conjugated microparticles was seen. The rCTB-conjugated microparticles were found both in villi and in the follicles of the Peyer's patches. HSA-conjugated microparticles could only be detected in the follicles of the Peyer's patches and not in villi. The rCTB conjugated to the microparticles did not lose its ability to bind the GM1-receptor, as shown with a GM1-ELISA, and the uptake of rCTB-conjugated microparticles in villi is most probably facilitated by the rCTB binding to the GM1-receptor. The qualitative difference in uptake could be of importance for the development of an immune response as the cytokine and chemokine microenvironment during antigen presentation will decide the differentiation of the immune response induced.     

Needle-free vaccine delivery

The need for minimally invasive delivery methods is urgent. As the number of registered vaccines increases, so does the number of injections. The use of sharps can be unsafe and needle immunisation is less suitable for mass immunisations during emergencies such as pandemics or bioterrorist attacks. The approach of combining vaccines has limitations due to high development costs, risk of pharmaceutical or immunological interference and economic risks. Advancements in the development of alternatives to injection with syringes and needles are discussed in this paper, and include: mucosal vaccination, injection without needles and vaccine delivery via the skin.     

以壳聚糖为佐剂的幽门螺杆菌疫苗免疫小鼠的TH免疫应答

目的探讨壳聚糖在幽门螺杆菌疫苗中的免疫佐剂效应。方法将20只Balb/c小鼠分为4组,分别以磷酸盐缓冲液、单纯幽门螺杆菌(Hp)抗原、Hp抗原+壳聚糖酸溶液、Hp抗原+壳聚糖颗粒进行口服免疫,用ELISA法检测胃黏膜中白细胞介素(IL)-2I、L-4,IL-10。结果胃黏膜内IL-10和IL-4的含量在以壳聚糖为佐剂组(255.25,237.05 pg/mg和14.70,14.48 pg/mg)显著高于无佐剂组(104.33和6.49 pg/mg)和对照组(67.13,4.19 pg/mg)(P<0.05~0.01)。结论以壳聚糖为佐剂的Hp疫苗可明显促进TH2细胞因子IL-4I、L-10的分泌,这可能在其免疫防御中起作用。     

Microparticles for oral delivery of vaccines

Nonreplicating antigens are poorly immunogenic when given orally, either due to their degradation in the gastrointestinal tract or because they are not efficiently taken up in the gut. Studies in laboratory animals have clearly demonstrated that microparticles can significantly improve the immunogenicity of orally administered antigens. However, the oral delivery of vaccines using microparticles has not been explored extensively in humans and large animals. In this article the progress in oral microparticle antigen delivery will be reviewed and, where possible, studies in humans and large animals will be highlighted. In addition, possible approaches that have the potential to significantly improve microparticle delivery of oral vaccines will be suggested.     

Oral vaccination: where we are?

As early as 900 years ago, the Bedouins of the Negev desert were reported to kill a rabid dog, roast its liver and feed it to a dog-bitten person for three to five days according to the size and number of bites . In sixteenth century China, physicians routinely prescribed pills made from the fleas collected from sick cows, which purportedly prevented smallpox. One may dismiss the wisdom of the Bedouins or Chinese but the Nobel laureate, Charles Richet, demonstrated in 1900 that feeding raw meat can cure tuberculous dogs – an approach he termed zomotherapy. Despite historical clues indicating the feasibility of oral vaccination, this particular field is notoriously infamous for the abundance of dead-end leads. Today, most commercial vaccines are delivered by injection, which has the principal limitation that recipients do not like needles. In the last few years, there has been a sharp increase in interest in needle-free vaccine delivery; new data emerges almost daily in the literature. So far, there are very few licensed oral vaccines, but many more vaccine candidates are in development. Vaccines delivered orally have the potential to take immunization to a fundamentally new level. In this review, the authors summarize the recent progress in the area of oral vaccines.     

脊髓灰质炎灭活疫苗和减毒活疫苗联合接种效果分析

目的：探讨脊髓灰质炎灭活疫苗（IPV）和减毒活疫苗（OPV）不同联合接种方式的临床效果。方法本研究为队列研究,将2009~2013年内收集的在管辖区域具备进行计划免疫4个部门的2400例新生儿进行随机分组,其中一组进行1剂IPV和2剂OPV序贯（I-O-O）（A组）,另一组2剂IPV和1剂OPV序贯（I-I-O）（B组）,并观察两组新生儿进行接种后的反应。结果这两种方式对于新生儿计划免疫无明显的差异。结论这两种方式对于新生儿进行脊髓灰质炎的计划免疫无不良反应。     

A review of multiple approaches towards an improved hepatitis B vaccine

Background: Hepatitis B is a DNA virus that can cause liver inflammation, cirrhosis, and cancer in chronically infected and symptomatic carriers. Antiviral treatments are usually limited in their effectiveness in treating the disease states. Vaccination against hepatitis B in pediatric and adolescent populations has proven to be a generally effective means for preventing diseases that could be potentially caused by this virus. Some 5 – 10% of the vaccinees do not develop protective immunity against the virus. Therefore, a significant amount of effort has been made in many research laboratories across the world to increase the potency of the vaccine by various innovative means, e.g., increasing the immunogenicity of the antigen or through introduction of novel adjuvants that elicit strong humoral and cell-mediated immune responses. Objectives/methods: The objective of this review is to highlight publications of significant developments that have been made over the past decade and efforts that are continuing towards producing an improved vaccine. A number of patents that protect novel hepatitis B vaccine formulations, including those claiming novel hepatitis B core antigen formulations and combinations of a vaccine with small molecule therapeutics, are discussed. Conclusion: There have been promising developments in the area of new adjuvants and delivery systems. The practical need for reducing the total number of childhood vaccinations has driven development of, and patent filings on, multivalent and combination vaccine formulations in which the hepatitis B vaccine is included as one component. Efforts and some advances have also been made in the critical area of therapeutic application of the vaccine. The existence of a large population of already infected patients and the inadequacy of most of the current antiviral drugs against hepatitis B diseases have also inspired efforts to produce a vaccine that would be efficacious in clearing an exiting infection.     

Alginate microparticles for Polymyxin B Peyer's patches uptake: microparticles for antibiotic oral administration

Microparticles with size less than 3?µm, able to be taken up by M cell of Peyer's patches for the drug delivery to the Gut Associated Limphoid Tissue (GALT), were developed in order to improve oral bioavailability of Polymyxin B (PMB). Less than 3?µm alginate microparticles resistant to gastro-intestinal media were prepared by spray-drying technique and cross-linking by calcium ions and chitosan. The cross-linked microparticles were evaluated for PMB content by spectrophotometric method, alginate/PMB interaction by rheological study, cross-linking degree by EDS analysis and PMB activity by microbiological assay. By modulating the polymer cross-linking degree, cationic PMB interacted on alginate chains leading to a proper PMB loading as well as antibiotic retention in gastric environment and sustained delivery in intestinal fluid. Moreover, the procedure resulted suitable for PMB biological activity preservation.     

Liposomes as delivery systems for nasal vaccination: strategies and outcomes

Importance of the field: Among the particulate systems that have been envisaged in vaccine delivery, liposomes are very attractive. These phospholipid vesicles can indeed deliver a wide range of molecules. They have been shown to enhance considerably the immunogenicity of weak protein antigens or synthetic peptides. Also, they offer a wide range of pharmaceutical options for the design of vaccines. In the past decade, the nasal mucosa has emerged as an effective route for vaccine delivery, together with the opportunity to develop non-invasive approaches in vaccination.

Areas covered in this review: This review focuses on the recent strategies and outcomes that have been developed around the use of liposomes in nasal vaccination.

What the reader will gain: The various formulation parameters, including lipid composition, size, charge and mucoadhesiveness, that have been investigated in the design of liposomal vaccine candidates dedicated to nasal vaccination are outlined. Also, an overview of the immunological and protective responses obtained with the developed formulations is presented.

Take home message: This review illustrates the high potential of liposomes as nasal vaccine delivery systems.     

Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast

Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.     

新的疫苗技术——鼻腔疫苗

综述了鼻腔疫苗给药的研究进展及原理,重点介绍了鼻腔免疫佐剂及给药装置的研究进展.     

Starch Microparticles as Oral Vaccine Adjuvant: Antigen-dependent Uptake in Mouse Intestinal Mucosa

An oral vaccine formulation comprised of starch microparticles with conjugated antigens is being developed. In this report we have examined the uptake of such microparticles by the intestinal mucosa and examined whether the conjugated antigen can influence the uptake. Two model antigens were used: recombinant cholera toxin B subunit (rCTB), which is known to bind to the ubiquitous G_M1-receptor, and human serum albumin (HSA) which is not known to have any specific binding properties. The uptake was studied in mouse ligated intestinal loops into which the microparticles were injected. The intestinal loops were excised, fixed in ice-cold 95% ethanol. Entire specimens were mounted, exposed to fluorescence-labeled reagents staining the cytoskeleton, the particles and/or M cells and examined in a confocal laser-scanning microscope. A qualitative difference in the uptake of the rCTB- and HSA-conjugated microparticles was seen. The rCTB-conjugated microparticles were found both in villi and in the follicles of the Peyer's patches. HSA-conjugated microparticles could only be detected in the follicles of the Peyer's patches and not in villi. The rCTB conjugated to the microparticles did not lose its ability to bind the G_M1-receptor, as shown with a G_M1-ELISA, and the uptake of rCTB-conjugated microparticles in villi is most probably facilitated by the rCTB binding to the G_M1-receptor. The qualitative difference in uptake could be of importance for the development of an immune response as the cytokine and chemokine microenvironment during antigen presentation will decide the differentiation of the immune response induced.     

Uptake of Ovalbumin-conjugated Starch Microparticles by Pig Respiratory Nasal Mucosa In Vitro

The uptake of ovalbumin-conjugated starch microparticles (OVA-MP) was studied after application to porcine respiratory nasal mucosa in vitro. Nasal mucosa from freshly slaughtered pigs was mounted in horizontal Ussing chambers, which permit monitoring of the viability of the tissue exposed to microparticles and ensure that the microparticles are deposited on the mucosa. The antigen-conjugated starch microparticles have previously been shown to produce strong mucosal, cellular and systemic immune responses to conjugated model antigens following oral administration. Intranasal administration of vaccines for mucosal immunisation is an interesting alternative to oral administration, since nasal delivery systems generally require lower doses of antigen and the site of application is better suited for protection against air-borne antigens. Most of a nasally administered dose is deposited on the surface of the respiratory area of the nasal mucosa. It is therefore important to examine whether the microparticles are taken up in this area and, if so, by which cell type.

Confocal laser scanning microscopy and transmission electron microscopy (TEM) of the nasal tissue both showed intracellular OVA-MP in non-ciliated epithelial cells after 45 min' incubation. The morphology of the cells in the TEM preparations did not support the presence of either M cells (specialised antigen sampling cells) or adjacent lymphocytes. Anticytokeratin-18 (Ac18) was used as a potential M cell marker. However, there was no indication of Ac18 binding to M cells, but it did bind to mucus-producing cells in the respiratory nasal mucosa. In conclusion, OVA-MP were taken up intracellularly by non-ciliated epithelial cells in the nasal respiratory mucosa of pigs, in vitro.     

QS-21: a water-soluble triterpene glycoside adjuvant

QS-21 is a purified immunological adjuvant derived from a natural source, the bark of the tree Quillaja saponaria. It is a water soluble triterpene glycoside with amphiphilic character that can be mixed with a soluble antigen in a fully soluble vaccine formulation or combined with emulsion or mineral salt adjuvants. QS-21 has been shown to enhance antibody and cell-mediated immune responses to subunit antigens, as well as DNA vaccines in animal models. It acts as an immunostimulatory adjuvant, eliciting production of immunomodulatory cytokines, and not as an antigen depot. QS-21 is currently under clinical evaluation with various vaccines. This includes a Phase II evaluation of a QS-21 adjuvanted pneumococcal polysaccharide vaccine and a Phase III evaluation of a QS-21 adjuvanted GM2-KLH (ganglioside GM2 vaccine) immunotherapeutic product for melanoma. At present, more than 1600 individuals have received vaccines containing QS-21 adjuvant. In most studies, QS-21-containing vaccines have been well-tolerated. No serious adverse events have occurred.     

Recent developments in leishmaniasis vaccine delivery systems

Background: The observation that recovery from infection with Leishmania confers immunity to reinfection suggests that control of leishmaniasis by vaccination may be possible. New generation vaccines, particularly those based on recombinant proteins and DNA, are found to be less immunogenic. Objective: There is an urgent need for the development of new and improved vaccine adjuvants. Methods: Based on their principal mechanisms of action, adjuvants can be broadly separated into two classes: immunostimulatory adjuvants and vaccine delivery systems. Vaccine delivery systems can carry both antigen and adjuvant for effective delivery to the antigen-presenting cells (APCs). In this article, we review the adjuvants, the delivery systems and their combinations used in the search of an effective vaccine against leishmaniasis. Conclusion: Based on current knowledge, cationic liposomes appear to have better prospects as effective delivery systems for developing a vaccine for leishmaniasis.     

Delivery strategies of melanoma vaccines: an overview

For many years, various cancer vaccines have been widely evaluated, however clinical responses remain rare. In this review, we attempt to address the question of which delivery strategies and platforms are feasible to produce clinical response and define the characteristics of the strategy that will induce long-lasting antitumor response. We limit our analysis and discussion to microparticles/nanoparticles, liposomes, heat-shock proteins, viral vectors and different types of adjuvants. This review aims to provide an overview of the specific characteristics, strengths and limitations of these delivery systems, focusing on their impacts on the development of melanoma vaccine. To datze, only adoptive T-cell transfer has shown promising clinical outcomes compared to other treatments.     

HPLC法测定人用皮卡狂犬病疫苗和皮卡佐剂中硫酸卡那霉素的含量

目的:建立测定人用皮卡狂犬病疫苗和皮卡佐剂中硫酸卡那霉素含量的HPLC方法。方法:色谱柱为CAPCELL PAKC8 DD(4.6 mm×250 mm,5μm);流动相为磷酸二氢钾缓冲液(0.02 mol.L-1,pH 7.5)-甲醇-乙腈(23∶7∶40);流速为1.0mL.min-1;柱温为45℃;采用柱前衍生化法,衍生化试剂为2,4,6-三硝基苯磺酸(TNBS);检测波长为350 nm。结果:在0.01～1.0 mg.mL-1范围内,硫酸卡那霉素的浓度与峰面积呈现良好的线性关系(A=3×106C-3313.9,r=1.000,n=7);在80%,100%,120%3个浓度下的平均回收率(n=3)分别为82.5%(RSD=1.6%),90.3%(RSD=2.0%),81.6%(RSD=1.8%);方法的最低检测限为0.41μg.mL-1;最低定量限为1.39μg.mL-1;供试品溶液在8 h内稳定(RSD=1.1%)。结论:本方法准确、快速,通用性良好。