不同佐剂与鼠疫F1-V融合重组蛋白抗原滴鼻免疫效果的研究

目的 研究不同佐剂与鼠疫F1-V融合重组蛋白抗原滴鼻免疫Balb/c小鼠,观察机体产生体液免疫和局部粘膜免疫反应的效果,为发展黏膜疫苗提供理论基础.方法 鼠疫F1-V融合重组蛋白抗原按比例分别与PorB(2类外膜蛋白)重组蛋白、蛋白体佐剂制备黏膜疫苗,滴鼻免疫Balb/c小鼠3次,取尾静脉血,采用ELISA检测血清IgG及抗体亚型分类,并检测鼻咽、肺、小肠及阴道灌洗液sIsA;采用FAC检测脾淋巴细胞表型的变化.结果 PorB重组蛋白佐剂疫苗组和蛋白体佐剂疫苗组较无佐剂组体液免疫抗体水平高、蛋白体佐剂疫苗组好于PorB重组蛋白佐剂疫苗组,但无显著性差异.结论 PorB重组蛋白佐剂疫苗和蛋白体佐剂疫苗均能诱导较强的系统免疫和黏膜免疫应答,且PorB重组蛋白佐剂疫苗免疫效果可与蛋白体佐剂疫苗相媲美,可进一步论证是否可用PorB重组蛋白佐剂替代蛋白体佐剂,这为鼠疫粘膜疫苗的研制奠定了基础.     

重组IL-1β蛋白表达纯化及佐剂效应的评价

目的应用原核表达系统制备IL-1β重组蛋白,并作为流感喷鼻疫苗的佐剂,研究其作为喷鼻疫苗佐剂的有效性。方法从Genbank获得IL-1β的基因cDNA序列,在大肠杆菌中表达。分离纯化表达产物,与甲型H1N1流感喷鼻疫苗均匀混合,免疫Balb/c小鼠,通过对体液免疫、黏膜免疫和细胞免疫水平的检测,证明rIL-1β蛋白佐剂的有效性。结果 rIL-1β蛋白在大肠杆菌中获得了高效表达,表达量约是30%,纯度是94.5%。制备的rIL-1β蛋白具有无毒、稳定和良好的生物活性。通过对小鼠的免疫实验证明,rIL-1β作为喷鼻疫苗佐剂对体液、黏膜和细胞免疫应答均有良好的增强效果。结论 rIL-1β蛋白具有黏膜佐剂的作用,为深入研究佐剂效应提供了实验数据。     

壳聚糖在黏膜免疫中的应用

目的黏膜免疫可以诱导有效的黏膜和系统免疫反应,从而保护黏膜表面,是防止病原体在黏膜表面克隆并入侵的关键。但黏膜接种疫苗吸收效率低,黏膜免疫效果往往不理想,因此需要添加有效的黏膜免疫佐剂和载体系统来提高疫苗的效果。壳聚糖是一种被广泛应用的天然聚合物,无毒,具有生物粘附性和免疫调节作用,在黏膜免疫中作为佐剂和载体得到大量应用。     

黏膜传递系统和黏膜佐剂研究进展

黏膜传递系统和佐剂被认为是疫苗的重要组成部分。多年来，这一领域的研究不断深化，包括作为传递系统的重组减毒活细菌或病毒、质粒DNA、脂质体，以及霍乱毒素和大肠杆菌不耐热肠毒素等传统的黏膜佐剂。但由于这些物质存在着毒副作用等局限性，新的更加有效和安全、经济的黏膜传递系统和佐剂一直是人们探索的目标。本文拟对一些近年有关菌影、芽孢等新的黏膜传递系统以及ADP核糖基化肠毒素类佐剂及单磷酰脂质A、CpG-ODN等佐剂新的进展简介如下。     

疫苗蛋白体佐剂的研制

蛋白体（proteosmes）是由B群2b型脑膜炎双球菌外膜蛋白中3个孔道蛋白形成的囊泡样大小不同的毫微粒结构体，具有疫苗投递载体和佐剂的特征。近几年，国外蛋白体佐剂应用在很多疫苗的研制中，并且具有突破性的进展，主要用于鼻内接种疫苗。本研究选用B群2b型脑膜炎奈瑟双球菌29353菌株，以不同培养条件优化蛋白体的提取，从稳定性和安全性的某方面检测蛋白体作为疫苗佐剂的可能性，为研制黏膜疫苗提供安全可靠的佐剂。     

壳聚糖在黏膜免疫中的应用

目的 黏膜免疫可以诱导有效的黏膜和系统免疫反应,从而保护黏膜表面,是防止病原体在黏膜表面克隆并入侵的关键.但黏膜接种疫苗吸收效率低,黏膜免疫效果往往不理想,因此需要添加有效的黏膜免疫佐剂和载体系统来提高疫苗的效果.壳聚糖是一种被广泛应用的天然聚合物,无毒,具有生物粘附性和免疫调节作用,在黏膜免疫中作为佐剂和载体得到大量应用.     

壳聚糖在黏膜免疫中的应用

目的 黏膜免疫可以诱导有效的黏膜和系统免疫反应,从而保护黏膜表面,是防止病原体在黏膜表面克隆并入侵的关键.但黏膜接种疫苗吸收效率低,黏膜免疫效果往往不理想,因此需要添加有效的黏膜免疫佐剂和载体系统来提高疫苗的效果.壳聚糖是一种被广泛应用的天然聚合物,无毒,具有生物粘附性和免疫调节作用,在黏膜免疫中作为佐剂和载体得到大量应用.     

壳聚糖在黏膜免疫中的应用

目的 黏膜免疫可以诱导有效的黏膜和系统免疫反应,从而保护黏膜表面,是防止病原体在黏膜表面克隆并入侵的关键.但黏膜接种疫苗吸收效率低,黏膜免疫效果往往不理想,因此需要添加有效的黏膜免疫佐剂和载体系统来提高疫苗的效果.壳聚糖是一种被广泛应用的天然聚合物,无毒,具有生物粘附性和免疫调节作用,在黏膜免疫中作为佐剂和载体得到大量应用.     

壳聚糖在黏膜免疫中的应用

目的 黏膜免疫可以诱导有效的黏膜和系统免疫反应,从而保护黏膜表面,是防止病原体在黏膜表面克隆并入侵的关键.但黏膜接种疫苗吸收效率低,黏膜免疫效果往往不理想,因此需要添加有效的黏膜免疫佐剂和载体系统来提高疫苗的效果.壳聚糖是一种被广泛应用的天然聚合物,无毒,具有生物粘附性和免疫调节作用,在黏膜免疫中作为佐剂和载体得到大量应用.     

壳聚糖在黏膜免疫中的应用

目的 黏膜免疫可以诱导有效的黏膜和系统免疫反应,从而保护黏膜表面,是防止病原体在黏膜表面克隆并入侵的关键.但黏膜接种疫苗吸收效率低,黏膜免疫效果往往不理想,因此需要添加有效的黏膜免疫佐剂和载体系统来提高疫苗的效果.壳聚糖是一种被广泛应用的天然聚合物,无毒,具有生物粘附性和免疫调节作用,在黏膜免疫中作为佐剂和载体得到大量应用.     

Recent advances in mucosal vaccines and adjuvants

Mucosal vaccines may be used both to prevent mucosal infections through the activation of antimicrobial immunity and to treat systemic inflammatory diseases through the induction of antigen-specific mucosal tolerance. New, efficient mucosal adjuvants for human use have been designed based on, amongst others, bacterial toxins and their derivatives, CpG-containing DNA, and different cytokines and chemokines, with the aim of improving the induction of mucosal Th1 and Th2 responses. Mucosal delivery systems, in particular virus-like particles, have been shown to enhance the binding, uptake and half-life of the antigens, as well as target the vaccine to mucosal surfaces. DNA vaccines are currently being developed for administration at mucosal surfaces. However, there have also been failures, such as the withdrawal of an oral vaccine against rotavirus diarrhea and a nasal vaccine against influenza, because of their potential side effects.     

Mucosal vaccines: a paradigm shift in the development of mucosal adjuvants and delivery vehicles

Mucosal immune responses are the first‐line defensive mechanisms against a variety of infections. Therefore, immunizations of mucosal surfaces from which majority of infectious agents make their entry, helps to protect the body against infections. Hence, vaccinization of mucosal surfaces by using mucosal vaccines provides the basis for generating protective immunity both in the mucosal and systemic immune compartments. Mucosal vaccines offer several advantages over parenteral immunization. For example, (i) ease of administration; (ii) non‐invasiveness; (iii) high‐patient compliance; and (iv) suitability for mass vaccination. Despite these benefits, to date, only very few mucosal vaccines have been developed using whole microorganisms and approved for use in humans. This is due to various challenges associated with the development of an effective mucosal vaccine that can work against a variety of infections, and various problems concerned with the safe delivery of developed vaccine. For instance, protein antigen alone is not just sufficient enough for the optimal delivery of antigen(s) mucosally. Hence, efforts have been made to develop better prophylactic and therapeutic vaccines for improved mucosal Th1 and Th2 immune responses using an efficient and safe immunostimulatory molecule and novel delivery carriers. Therefore, in this review, we have made an attempt to cover the recent advancements in the development of adjuvants and delivery carriers for safe and effective mucosal vaccine production.     

Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA

Mucosal immunisation may be used both to protect the mucosal surfaces against infections and as a means for immunological treatment of peripheral immunopathological disorders through the induction of systemic antigen-specific tolerance ('oral tolerance'). The development of mucosal vaccines, whether for prevention of infectious diseases or for oral tolerance immunotherapy, requires efficient antigen delivery and adjuvant systems that can help to present the appropriate vaccine or immunotherapy antigens to the mucosal immune system. The most potent (but also toxic) mucosal adjuvants are cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT), and much effort and significant progress have been made recently to generate toxicologically acceptable derivatives of these toxins with retained adjuvant activity. Among these are the non-toxic, recombinantly produced cholera toxin B-subunit (CTB). CTB is a specific protective antigen component of a widely registered oral cholera vaccine as well as a promising vector for either giving rise to mucosal anti-infective immunity or for inducing peripheral anti-inflammatory tolerance to chemically or genetically linked foreign antigens administered mucosally. CT and CTB have also recently been used as combined vectors and adjuvants for markedly promoting ex vivo dendritic cell (DC) vaccination with different antigens and also steering the immune response to the in vivo-reinfused DCs towards either broad Th1 + Th2 + CTL immunity (CT) or Th2 or tolerance (CTB). Another type of mucosal adjuvants is represented by bacterial DNA or synthetic oligodeoxynucleotides containing CpG-motifs, which especially when linked to CTB have been found to effectively stimulate both innate and adaptive mucosal immune responses. The properties and clinical potential of these different classes of adjuvants are being discussed.     

New generation of mucosal adjuvants for the induction of protective immunity

Invasion of infectious agents through mucosal surfaces can be prevented by use of the common mucosal immune system (CMIS), which interconnects inductive tissues, including Peyer's patches (PPs) and nasopharyngeal-associated lymphoreticular tissue (NALT), and effector tissues of the intestinal and respiratory tracts. In order for the CMIS to induce maximal protective mucosal immunity, co-administration of mucosal adjuvant has been shown to be essential. When vaccine antigen is administered together with mucosal adjuvant, antigen-specific T-helper (Th) 1 and Th2 cells, cytotoxic T lymphocytes (CTLs) and IgA B cell responses are effectively induced by oral or nasal routes via the CMIS. In the early stages of induction of mucosal immune response, the uptake of orally or nasally administered antigens is achieved through a unique set of antigen-sampling cells, M cells located in follicle-associated epithelium (FAE) of inductive sites. After successful uptake, the antigens are immediately processed and presented by the underlying dendritic cells (DCs). Elucidation of the molecular/cellular characteristics of M cells and mucosal DCs will greatly facilitate the design of a new generation of effective mucosal adjuvants and of a vaccine delivery vehicle that maximises the use of the CMIS. Our recent efforts at mucosal vaccine development have focused on nasal administration of vaccine antigen together with nontoxic mutant-based or cytokine-/chemokine-based adjuvant for the induction of the protective immunity. To this end, a chimeric form of a nontoxic adjuvant combining the merits of mutant cholera toxin A subunit (mCT-A) and heat labile toxin B subunit (LT-B) was created as the second generation of detoxified toxin-based mucosal adjuvant. When a vaccine antigen was coexpressed together with an immune stimulatory/delivery molecule in crop seed, this edible vaccine is not only effective but also extremely practical in that it can be produced in huge quantities and preserved and shipped over long distances at room temperature without altering the quality of the vaccine. Because such qualities would greatly facilitate global vaccination, this new generation edible vaccines with a built-in adjuvant and/or M cell-targeted edible vaccine promises to be a powerful weapon for combating infectious diseases and bioterrorism.     

Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin,cholera toxin B subunit and CpG DNA

The mucosal immune system consists of an integrated network of lymphoid cells that work in concert with innate host factors to promote host defence. Mucosal immunization can be used both to protect the mucosal surfaces against colonization and invasion by microbial pathogens and to provide a means for immunological treatment of selected autoimmune, allergic or infectious-immunopathological disorders through the induction of antigen-specific tolerance. The development of mucosal vaccines, whether for prevention of infectious diseases or for oral tolerance immunotherapy, requires efficient antigen delivery and adjuvant systems. Significant progress has recently been made to generate partly or wholly detoxified derivatives of cholera toxin (including the completely nontoxic cholera toxin B subunit) and the closely related Escherichia coli heat-labile enterotoxin, with retained adjuvant activity. Cholera toxin B subunit is a protective component of a widely registered oral vaccine against cholera, and has proven to be a promising vector for either giving rise to anti-infective immunity or for inducing peripheral anti-inflammatory tolerance to chemically or genetically linked foreign antigens administered mucosally. Promising advances have also recently been made in the design of efficient mucosal adjuvants based on bacterial DNA that contains CpG-motifs and various imidazoquinoline compounds binding to different Toll-like receptors on mucosal antigen-presenting cells.     

Implication of nanoparticles/microparticles in mucosal vaccine delivery

Although polymeric nanoparticles/microparticles are well established for the mucosal administration of conventional drugs, they have not yet been developed commercially for vaccine delivery. The limitation of the mucosal (particularly oral) route of delivery, including low pH, gastric enzymes, rapid transit and poor absorption of large molecules, has made mucosal vaccine delivery challenging. Nevertheless, several polymeric delivery systems for mucosal vaccine delivery are currently being evaluated. The polymer-based approaches are designed to protect the antigen in the gut, to target the antigen to the gut-associated lymphoid tissue or to increase the residence time of the antigen in the gut through bioadhesion. M-cell targeting is a potential approach for mucosal vaccine delivery, which can be achieved using M-cell-specific lectins, microbial adhesins or immunoglobulins. While many hurdles must be overcome before targeted mucosal vaccine delivery becomes a practical reality, this is a potential area of research that has important implications for future vaccine development. This review comprises various aspects that could be decisive in the development of polymer based mucosal vaccine delivery systems.     

Efficient mucosal delivery of the HIV-1 Tat protein using the synthetic lipopeptide MALP-2 as adjuvant

A major requirement for HIV/AIDS research is the development of a mucosal vaccine that stimulates humoral and cell-mediated immune responses at systemic and mucosal levels, thereby blocking virus replication at the entry port. Thus, a vaccine prototype based on biologically active HIV-1 Tat protein as antigen and the synthetic lipopeptide, macrophage-activating lipopeptide-2 (MALP-2), asa mucosal adjuvant was developed. Intranasal administration to mice stimulated systemic and mucosal anti-Tat antibody responses, and Tat-specific T cell responses, that were more efficient than those observed after i.p. immunization with Tat plus incomplete Freund's adjuvant. Major linear B cell epitopes mapped within aa 1-20 and 46-60, whereas T cell epitopes were identified within aa 36-50 and 56-70. These epitopes have also been described in vaccinated primates and in HIV-1-infected individuals with better prognosis. Analysis of the anti-Tat IgG isotypes in serum, and the cytokine profile of spleen cells indicated that a dominant Th1 helper response was stimulated by Tat plus MALP-2, as opposed to the Th2 response observed with Tat plus incomplete Freund's adjuvant. Tat-specific IFN-gamma-producing cells were significantly increased only in response to Tat plus MALP-2. These data suggest that Malp-2 may represent an optimal mucosal adjuvant for candidate HIV vaccines based on Tat alone or in combination with other HIV antigens.     

Immunological Foundations to the Quest for New Vaccine Adjuvants

Developing efficient adjuvants for human vaccines that elicit broad and sustained immune responses at systemic or mucosal levels remains a formidable challenge for the vaccine industry. Conventional approaches in the past have been largely empirical and--at best--partially successful. Importantly, recent advances in our understanding of the immune system, most particularly with respect to early proinflammatory signals, are leading to the identification of new biological targets for vaccine adjuvants. This review covers both the current status of adjuvant testing in humans, the residual needs for vaccines in development, and the emerging immunological foundations for adjuvant design. A better understanding of the biology of toll-like receptors, non-conventional T cell subpopulations, T and B cell memory, regulatory T cells, and mucosal immunity has profound implications for a modern approach to adjuvant screening and development. The future lies in the high throughput screening of synthetic chemical entities targeting well-characterized biological molecules. Used alone or in combination, such synthetic adjuvants will allow stimulation or modulation in a safe and efficient manner of strong effector, regulatory and memory immune mechanisms.     

Translational Mini‐Review Series on Vaccines for HIV: T lymphocyte trafficking and vaccine‐elicited mucosal immunity

Many pathogens use mucosal surfaces to enter and propagate within the host, making particularly desirable vaccines that target immune responses specifically to mucosal compartments. The majority of mucosal vaccine design strategies to date have been empirical in nature. However, an emerging body of basic immunological knowledge is providing new insights into the regulation of tissue‐specific lymphocyte trafficking and differentiation. These insights afford the opportunity for the rational design of vaccines that focus immune responses at mucosal surfaces. Mucosal cellular immunity may prove critical for protection in the context of HIV infection, and thus there has been considerable interest in developing vaccines that target HIV‐specific cellular immune responses to the gastrointestinal and vaginal mucosa. However, the optimal strategies for eliciting mucosal cellular immune responses through vaccination remain to be determined. Here, we review both recent vaccine studies and emerging paradigms from the basic immunological literature that are relevant to the elicitation of potent and protective mucosal cellular immune memory. Increasing the synergy between these avenues of research may afford new opportunities for mucosal vaccine design.     

Development of adenoviral vector-based mucosal vaccine against influenza

The recent pandemic threat of the influenza virus makes the increased safety and efficiency of vaccination against the pathogen a most important issue. It has been well established that for maximum protective effect, the vaccination should mimic natural infection. Therefore, recent efforts to develop a new influenza vaccine have focused on intranasal immunization strategies. Intranasal immunization is capable of inducing secretory IgA and serum IgG responses to provide a double defense against mucosal pathogens. On the other hand, it is desirable that a live pathogen is not present in the vaccine. In addition, for optimal induction of the immune responses via the nasal route, efficient and safe mucosal adjuvants are also required. This is possible to attain using an adenoviral vector for vaccine development. Adenoviral vectors are capable of delivering and protecting the antigen encoding sequence. They also possess a natural mechanism for penetrating into the nasal mucous membrane and are capable of activating the innate immune response. This review describes the basic prerequisites for the involvement of recombinant adenoviruses for mucosal (nasal) vaccine development against the influenza virus.