新型疫苗佐剂的研究进展

与传统的灭活或活体疫苗相比,由基因工程重组抗原或化学合成多肽组成的现代疫苗往往存在免疫原性弱等问题,需要新型的免疫佐剂来增强其作用。尽管传统的铝盐佐剂是目前唯一全球公认的人用佐剂,但存在激发细胞免疫应答能力差等不足,因此,需要研发更为安全有效的人用新型佐剂,尤其是安全无毒、能够刺激较强细胞免疫应答的佐剂,以及适合粘膜疫苗、DNA疫苗和癌症疫苗的免疫佐剂。分析阐述了新型佐剂研究状况和佐剂发展方向,并进一步对新型佐剂的临床前和临床试验研究以及已批准上市的新型疫苗佐剂进行了综述。     

Biotechnology approaches to produce potent,self-adjuvanting antigen-adjuvant fusion protein subunit vaccines

Traditional vaccination approaches (e.g. live attenuated or killed microorganisms) are among the most effective means to prevent the spread of infectious diseases. These approaches, nevertheless, have failed to yield successful vaccines against many important pathogens. To overcome this problem, methods have been developed to identify microbial components, against which protective immune responses can be elicited. Subunit antigens identified by these approaches enable the production of defined vaccines, with improved safety profiles. However, they are generally poorly immunogenic, necessitating their administration with potent immunostimulatory adjuvants. Since few safe and effective adjuvants are currently used in vaccines approved for human use, with those available displaying poor potency, or an inability to stimulate the types of immune responses required for vaccines against specific diseases (e.g. cytotoxic lymphocytes (CTLs) to treat cancers), the development of new vaccines will be aided by the availability of characterized platforms of new adjuvants, improving our capacity to rationally select adjuvants for different applications. One such approach, involves the addition of microbial components (pathogen-associated molecular patterns; PAMPs), that can stimulate strong immune responses, into subunit vaccine formulations. The conjugation of PAMPs to subunit antigens provides a means to greatly increase vaccine potency, by targeting immunostimulation and antigen to the same antigen presenting cell. Thus, methods that enable the efficient, and inexpensive production of antigen-adjuvant fusions represent an exciting mean to improve immunity towards subunit antigens. Herein we review four protein-based adjuvants (flagellin, bacterial lipoproteins, the extra domain A of fibronectin (EDA), and heat shock proteins (Hsps)), which can be genetically fused to antigens to enable recombinant production of antigen-adjuvant fusion proteins, with a focus on their mechanisms of action, structural or sequence requirements for activity, sequence modifications to enhance their activity or simplify production, adverse effects, and examples of vaccines in preclinical or human clinical trials.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

Elicitation of predictable immune responses by using live bacterial vectors

There is an increasing need for novel vaccines able to stimulate efficient and long-lasting responses, which have also low production costs. To confer protective immunity following vaccination, the adequate type of response should be elicited. Vaccines based on attenuated bacterial carriers have contained production and delivery costs, and are able to stimulate more potent immune responses than non-replicating formulations. The improved knowledge on carrier physiology and host response, the availability of different mutants and highly sophisticated expression tools, and the possibility of co-administering modulators enable to trigger predictable responses according to the specific needs. Recent studies support the use of attenuated bacteria not only as conventional carriers, but also as a delivery system for DNA vaccines against infectious agents and tumors. In this review we discuss the most widely used bacterial carrier systems for either antigens or nucleic acid vaccines, and the strategies which have been successfully exploited to modulate the immune responses elicited.     

Innate endogenous adjuvants prime to desirable immune responses via mucosal routes

Vaccination is an effective strategy to prevent infectious or immune related diseases, which has made remarkable contribution in human history. Recently increasing attentions have been paid to mucosal vaccination due to its multiple advantages over conventional ways. Subunit or peptide antigens are more reasonable immunogens for mucosal vaccination than live or attenuated pathogens, however adjuvants are required to augment the immune responses. Many mucosal adjuvants have been developed to prime desirable immune responses to different etiologies. Compared with pathogen derived adjuvants, innate endogenous molecules incorporated into mucosal vaccines demonstrate prominent adjuvanticity and safety. Nowadays, cytokines are broadly used as mucosal adjuvants for participation of signal transduction of immune responses, activation of innate immunity and polarization of adaptive immunity. Desired immune responses are promptly and efficaciously primed on basis of specific interactions between cytokines and corresponding receptors. In addition, some other innate molecules are also identified as potent mucosal adjuvants. This review focuses on innate endogenous mucosal adjuvants, hoping to shed light on the development of mucosal vaccines.     

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.     

Immunotherapeutic potential of whole tumour cells

Despite the identification of tumour antigens and their subsequent generation in subunit form for use as cancer vaccines, whole tumour cells remain a potent vehicle for generating anti-tumour immunity. This is because tumour cells express an array of target antigens for the immune system to react against, avoiding problems associated with major histocompatibility complex (MHC)-restricted epitope identification for individual patients. Furthermore, whole cells are relatively simple to propagate and are potentially efficient at contributing to the process of T cell priming. However, whole cells can also possess properties that allow for immune evasion, and so the question remains of how to enhance the immune response against tumour cells so that they are rejected. Scenarios where whole tumour cells may be utilised in immunotherapy include autologous tumour cell vaccines generated from resected primary tumour, allogeneic (MHC-disparate) cross-reactive tumour cell line vaccines, and immunotherapy of tumours in situ. Since tumour cells are considered poorly immunogenic, mainly because they express self-antigens in a non-stimulatory context, the environment of the tumour cells may have to be modified to become stimulatory by using immunological adjuvants. Recent studies have re-evaluated the relative roles of direct and cross-priming in generating anti-tumour immunity and have highlighted the need to circumvent immune evasion.     

Vaccine adjuvants: current state and future trends

The problem with pure recombinant or synthetic antigens used in modern day vaccines is that they are generally far less immunogenic than older style live or killed whole organism vaccines. This has created a major need for improved and more powerful adjuvants for use in these vaccines. With few exceptions, alum remains the sole adjuvant approved for human use in the majority of countries worldwide. Although alum is able to induce a good antibody (Th2) response, it has little capacity to stimulate cellular (Th1) immune responses which are so important for protection against many pathogens. In addition, alum has the potential to cause severe local and systemic side-effects including sterile abscesses, eosinophilia and myofascitis, although fortunately most of the more serious side-effects are relatively rare. There is also community concern regarding the possible role of aluminium in neurodegenerative diseases such as Alzheimer's disease. Consequently, there is a major unmet need for safer and more effective adjuvants suitable for human use. In particular, there is demand for safe and non-toxic adjuvants able to stimulate cellular (Th1) immunity. Other needs in light of new vaccine technologies are adjuvants suitable for use with mucosally-delivered vaccines, DNA vaccines, cancer and autoimmunity vaccines. Each of these areas are highly specialized with their own unique needs in respect of suitable adjuvant technology. This paper reviews the state of the art in the adjuvant field, explores future directions of adjuvant development and finally examines some of the impediments and barriers to development and registration of new human adjuvants.     

Development of mucosal influenza inactivated vaccines

In this review recent data on the development of mucosal influenza inactivated vaccines with the use of mucosoadhesive adjuvants are presented. After the intranasal administration of such vaccines the formation of not only systemic, but also local immunity (IgA antibodies), as well as cross protection against the variants of influenza virus within its serotype, can be observed. Some mucosal influenza vaccines have passed clinical tests. Certain drawbacks of Escherichia thermolabile enterotoxin, used as mucosoadhesive adjuvant, and difficulties which may arise in mass immunization with mucosal vaccines due to the necessity of introducing them in two or three intranasal administrations are indicated.     

Vaccine instability in the cold chain: Mechanisms,analysis and formulation strategies

Instability of vaccines often emerges as a key challenge during clinical development (lab to clinic) as well as commercial distribution (factory to patient). To yield stable, efficacious vaccine dosage forms for human use, successful formulation strategies must address a combination of interrelated topics including stabilization of antigens, selection of appropriate adjuvants, and development of stability-indicating analytical methods. This review covers key concepts in understanding the causes and mechanisms of vaccine instability including (1) the complex and delicate nature of antigen structures (e.g., viruses, proteins, carbohydrates, protein-carbohydrate conjugates, etc.), (2) use of adjuvants to further enhance immune responses, (3) development of physicochemical and biological assays to assess vaccine integrity and potency, and (4) stabilization strategies to protect vaccine antigens and adjuvants (and their interactions) during storage. Despite these challenges, vaccines can usually be sufficiently stabilized for use as medicines through a combination of formulation approaches combined with maintenance of an efficient cold chain (manufacturing, distribution, storage and administration). Several illustrative case studies are described regarding mechanisms of vaccine instability along with formulation approaches for stabilization within the vaccine cold chain. These include live, attenuated (measles, polio) and inactivated (influenza, polio) viral vaccines as well as recombinant protein (hepatitis B) vaccines.     

Liposomes as adjuvant for anti-mycobacterial vaccine development

Mycobacteria are intracellular pathogens that invade and reside inside the macrophages. Recent advances in controlled delivery systems for vaccines such as liposomes have sparked a renewed interest in their potential application for the prevention of mycobacterial infections. The versatility of liposomes in the incorporation of hydrophilic/hydrophobic components, their non-toxic nature, biodegradability, biocompatibility, adjuvanticity, induction of cellular immunity, property of sustained release and prompt uptake by macrophages, makes them attractive candidates for the delivery of antigens. This review focuses on liposome research in the area of mycobacterial diseases and highlights how the various mycobacterial components may be exploited as powerful antigens with liposomes as adjuvants.     

人类口服病毒疫苗的研究进展

研制能同时诱导有效黏膜免疫和系统免疫的疫苗是预防黏膜感染病原体的理想目标。消化道具有许多产生黏膜免疫的位点,包括口腔、胃和小肠等。理想的口服病毒疫苗不仅能诱导较好的局部及远端黏膜免疫,也能产生较好的系统免疫,而且还因为具有无痛接种、可自行服用等优势而备受关注。由于人消化道环境及黏膜免疫的复杂性,目前成功上市的人口服病毒疫苗仅限于3种减毒活疫苗。本文将从消化道黏膜免疫的特点、当前口服病毒疫苗种类及研究现状、口服病毒疫苗所面临的挑战等方面进行综述,期望对我国人口服病毒疫苗的研究和开发提供参考和借鉴。     

New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development.

This review attempts to synthesize the new knowledge of pathogenesis of bacterial enteric infections and relate this information to vaccine development. Discussion focuses on human infections and to those in which significant strides have been made. As a general theme in the pathogenesis of bacterial enteric infections, pathogens can be characterized into 5 groups on the basis of their degree of ultimate invasiveness after ingestion by a susceptible hose: mucosal adherence and enterotoxin production; mucosal adherence and brush border dissolution -- enteropathogenic E. coli (EPEC) of "classical" serotypes; mucosal invasion and intraepithelial cell proliferation; mucosal translocation followed by bacterial proliferation in the lamina propria and mesenteric lymph nodes; and mucosal translocation followed by generalized infection. The review covers cholera (motility and chemotaxis, mucosal adhesion, flagellar sheath protein, hemagglutinins, outer membrane proteins, enterotoxin production, quality and duration of infection derived immunity, immune response in humans, LPS, flagellar sheath protein, cholera lectin, other cholera hemagglutinins, outer membrane protein, previous cholera vaccines, killer whole cell vaccines, toxoids, combination vaccines, attenuated versus cholerae vaccines): enterotoxigenic Escherichia coli (ETEC) (entertoxins, O:H serotypes and enterotoxin phenotypes, colonization factors, immune response in humans, vaccines against ETEC, and toxiods); EPEC (vaccines against EPEC); Shigella (smooth LPS O antigen, epithelial cell invasiveness, Shigella toxin, and Shigella vaccines); and typhoid fever (caccines against typhoid fever). The major attraction of a nonliving oral cholera vaccine is its safety. A review of available information leads to the conclusion that an oral vaccine consisting of a combination of antigens, intending to stimulate both antibacterial and antitoxic immunity, would be most likely to succeed. Current approaches to immunoprophylaxis of ETEC infection involve vaccines that stimulate antitoxic or antiadhesion immunity or both by means of killed antigens or attenuated strains. It is likely that the most effective vaccines will contain appropriate antigens intended to simultaneously stimulate both antibacterial and antitoxic immunity, thereby leading to a synergistic protective effect. Now that the speical enteroadhesive properties of EPEC have been characterized and shown to be associated with a plasmid, it should be possible to identify the phenotypic gene products responsible for this phenomenon. It is likely that fimbriae or outer membrane proteins will prove to be the organelle of adhesion. When such information becomes available, it should be possible to prepare oral vaccines consisting of the purified antigen. Efficacy has been shown for attenuated Shigella strains utilized as oral vaccines. The major thrust in the development of new immunization agensts against typhoid fever is to identify immunizing agents at least equal in efficacy to the parenteral acetone killed vaccine but which cause no adverse reactions.     

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines

Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.     

Improving vaccines against tuberculosis

Tuberculosis remains a major cause of mortality and physical and economic deprivation worldwide. There have been significant recent advances in our understanding of the Mycobacterium tuberculosis genome, mycobacterial genetics and the host determinants of protective immunity. Nevertheless, the challenge is to harness this information to develop a more effective vaccine than BCG, the attenuated strain of Mycobacterium bovis derived by Calmette and Guérin nearly 90 years ago. Some of the limitations of BCG include the waning of the protective immunity with time, reduced effectiveness against pulmonary tuberculosis compared to disseminated disease, and the problems of a live vaccine in immuno-compromised subjects. Two broad approaches to vaccine development are being pursued. New live vaccines include either attenuated strains of Mycobacterium tuberculosis produced by random mutagenesis or targeted deletion of putative virulence factors, or by genetic manipulation of BCG to express new antigens or cytokines. The second approach utilizes non-viable subunit vaccines to deliver immunodominant mycobacterial antigens. Both protein and DNA vaccines induce partial protection against experimental tuberculosis infection in mice, however, their efficacy has generally been equivalent to or less than that of BCG. The comparative effects of cytokine adjuvants and vaccines targeting antigen presenting cells on enhancing protection will be discussed. Coimmunization with plasmid interleukin-12 and a DNA vaccine expressing Antigen 85B, a major secreted protein, was as protective as BCG. The combination of priming with DNA-85B and boosting with BCG was superior to BCG alone. Therefore it is possible to achieve a greater level of protection against tuberculosis than with BCG, and this highlights the potential for new tuberculosis vaccines in humans.     

Lipid and carbohydrate based adjuvant/carriers in immunology.

This review discusses various issues regarding vaccines; what are they and how they work, safety aspects, the role of adjuvants and carriers in vaccination, synthetic peptides as immunogens, and new technologies for vaccine development and delivery including the identification of novel adjuvants for mucosal vaccine delivery. There has been a recent increase of interest in the use of lipids and carbohydrates as adjuvants, and so a particular emphasis is placed on adjuvants derived from lipids or carbohydrates, or from both.     

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses

There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell-mediated (Th1) immunity (e.g. Freund's complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non-toxic adjuvant that is able to stimulate both cell-mediated and humoral immunity. Inulin-derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf-life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin-based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin-derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.     

Pathogen-associated molecular patterns on biomaterials: a paradigm for engineering new vaccines

Vaccine development has progressed significantly and has moved from whole microorganisms to subunit vaccines that contain only their antigenic proteins. Subunit vaccines are often less immunogenic than whole pathogens; therefore, adjuvants must amplify the immune response, ideally establishing both innate and adaptive immunity. Incorporation of antigens into biomaterials, such as liposomes and polymers, can achieve a desired vaccine response. The physical properties of these platforms can be easily manipulated, thus allowing for controlled delivery of immunostimulatory factors and presentation of pathogen-associated molecular patterns (PAMPs) that are targeted to specific immune cells. Targeting antigen to immune cells via PAMP-modified biomaterials is a new strategy to control the subsequent development of immunity and, in turn, effective vaccination. Here, we review the recent advances in both immunology and biomaterial engineering that have brought particulate-based vaccines to reality.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.