Outer membrane vesicles as platform vaccine technology

Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications.     

Delivery of a Chlamydial Adhesin N-PmpC Subunit Vaccine to the Ocular Mucosa Using Particulate Carriers

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), remains the world’s leading preventable infectious cause of blindness. Recent attempts to develop effective vaccines rely on modified chlamydial antigen delivery platforms. As the mechanisms engaged in the pathology of the disease are not fully understood, designing a subunit vaccine specific to chlamydial antigens could improve safety for human use. We propose the delivery of chlamydia-specific antigens to the ocular mucosa using particulate carriers, bacterial ghosts (BGs). We therefore characterized humoral and cellular immune responses after conjunctival and subcutaneous immunization with a N-terminal portion (amino acid 1–893) of the chlamydial polymorphic membrane protein C (PmpC) of Ct serovar B, expressed in probiotic Escherichia coli Nissle 1917 bacterial ghosts (EcN BGs) in BALB/c mice. Three immunizations were performed at two-week intervals, and the immune responses were evaluated two weeks after the final immunization in mice. In a guinea pig model of ocular infection animals were immunized in the same manner as the mice, and protection against challenge was assessed two weeks after the last immunization. N-PmpC was successfully expressed within BGs and delivery to the ocular mucosa was well tolerated without signs of inflammation. N-PmpC-specific mucosal IgA levels in tears yielded significantly increased levels in the group immunized via the conjunctiva compared with the subcutaneously immunized mice. Immunization with N-PmpC EcN BGs via both immunization routes prompted the establishment of an N-PmpC-specific IFNγ immune response. Immunization via the conjunctiva resulted in a decrease in intensity of the transitional inflammatory reaction in conjunctiva of challenged guinea pigs compared with subcutaneously and non-immunized animals. The delivery of the chlamydial subunit vaccine to the ocular mucosa using a particulate carrier, such as BGs, induced both humoral and cellular immune responses. Further investigations are needed to improve the immunization scheme and dosage.     

Mucosal immunization using recombinant plant-based oral vaccines

The induction of mucosal immunity is very important in conferring protection against pathogens that typically invade via mucosal surfaces. Delivery of a vaccine to a mucosal surface optimizes the induction of mucosal immunity. The apparent linked nature of the mucosal immune system allows delivery to any mucosal surface to potentially induce immunity at others. Oral administration is a very straightforward and inexpensive approach to deliver a vaccine to the mucosal lining of the gut. However, vaccines administered by this route are subject to proteolysis in the gastrointestinal tract. Thus, dose levels for protein subunit vaccines are likely to be very high and the antigen may need to be protected from proteolysis for oral delivery to be efficacious. Expression of candidate vaccine antigens in edible recombinant plant material offers an inexpensive means to deliver large doses of vaccines in encapsulated forms. Certain plant tissues can also stably store antigens for extensive periods of time at ambient temperatures, obviating the need for a cold-chain during vaccine storage and distribution, and so further limiting costs. Antigens can be expressed from transgenes stably incorporated into a host plant's nuclear or plastid genome, or from engineered plant viruses infected into plant tissues. Molecular approaches can serve to boost expression levels and target the expressed protein for appropriate post-translational modification. There is a wide range of options for processing plant tissues to allow for oral delivery of a palatable product. Alternatively, the expressed antigen can be enriched or purified prior to formulation in a tablet or capsule for oral delivery. Fusions to carrier molecules can stabilize the expressed antigen, aid in antigen enrichment or purification strategies, and facilitate delivery to effector sites in the gastrointestinal tract. Many antigens have been expressed in plants. In a few cases, vaccine candidates have entered into early phase clinical trials, and in the case of farmed animal vaccines into relevant animal trials.     

细菌菌影作为新型疫苗和递送载体的研究进展

细菌菌影（bacterial ghost,BG）是革兰阴性菌在噬菌体PhiX174的裂解基因E的作用下形成不含核酸、核糖体等胞质内容物的细菌空壳。这种细菌空壳保留了与天然细菌一样的完整外膜结构,且不具有活菌样的致病作用,可作为疫苗无需佐剂就能诱导机体产生体液免疫应答和细胞免疫应答。菌影内部及外膜上可装载DNA、抗原和药物等异源物质,易被机体免疫细胞识别捕获,使其成为一种新型的生物递送载体。另外,菌影具有制备简单,易于保存等优点。细菌菌影在疾病预防和治疗方面具有广阔的应用前景。     

Cell-penetrating peptides: Application in vaccine delivery

Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.     

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.     

Plant-based,adjuvant-free,potent multivalent vaccines for avian influenza virus via Lactococcus surface display

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHA^{H5N6 + H9N2} BLP or a combination of tHA^H5N6 BLP and tHA^H9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.     

Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells

The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer.     

Advances in the development of Salmonella-based vaccine strategies for protection against Salmonellosis in humans

Salmonella spp. are important human pathogens globally causing millions of cases of typhoid fever and non-typhoidal salmonellosis annually. There are only a few vaccines licensed for use in humans which all target Salmonella enterica serovar Typhi. Vaccine development is hampered by antigenic diversity between the thousands of serovars capable of causing infection in humans. However, a number of attenuated candidate vaccine strains are currently being developed. As facultative intracellular pathogens with multiple systems for transporting effector proteins to host cells, attenuated Salmonella strains can also serve as ideal tools for the delivery of foreign antigens to create multivalent live carrier vaccines for simultaneous immunization against several unrelated pathogens. Further, the ease with which Salmonella can be genetically modified and the extensive knowledge of the virulence mechanisms of this pathogen means that this bacterium has often served as a model organism to test new approaches. In this review we focus on (1) recent advances in live attenuated Salmonella vaccine development, (2) improvements in expression of foreign antigens in carrier vaccines and (3) adaptation of attenuated strains as sources of purified antigens and vesicles that can be used for subunit and conjugate vaccines or together with attenuated vaccine strains in heterologous prime-boosting immunization strategies. These advances have led to the development of new vaccines against Salmonella which have or will soon be tested in clinical trials.     

Vaccine process technology

The evolution of vaccines (e.g., live attenuated, recombinant) and vaccine production methods (e.g., in ovo, cell culture) are intimately tied to each other. As vaccine technology has advanced, the methods to produce the vaccine have advanced and new vaccine opportunities have been created. These technologies will continue to evolve as we strive for safer and more immunogenic vaccines and as our understanding of biology improves. The evolution of vaccine process technology has occurred in parallel to the remarkable growth in the development of therapeutic proteins as products; therefore, recent vaccine innovations can leverage the progress made in the broader biotechnology industry. Numerous important legacy vaccines are still in use today despite their traditional manufacturing processes, with further development focusing on improving stability (e.g., novel excipients) and updating formulation (e.g., combination vaccines) and delivery methods (e.g., skin patches). Modern vaccine development is currently exploiting a wide array of novel technologies to create safer and more efficacious vaccines including: viral vectors produced in animal cells, virus-like particles produced in yeast or insect cells, polysaccharide conjugation to carrier proteins, DNA plasmids produced in E. coli, and therapeutic cancer vaccines created by in vitro activation of patient leukocytes. Purification advances (e.g., membrane adsorption, precipitation) are increasing efficiency, while innovative analytical methods (e.g., microsphere-based multiplex assays, RNA microarrays) are improving process understanding. Novel adjuvants such as monophosphoryl lipid A, which acts on antigen presenting cell toll-like receptors, are expanding the previously conservative list of widely accepted vaccine adjuvants. As in other areas of biotechnology, process characterization by sophisticated analysis is critical not only to improve yields, but also to determine the final product quality. From a regulatory perspective, Quality by Design (QbD) and Process Analytical Technology (PAT) are important initiatives that can be applied effectively to many types of vaccine processes. Universal demand for vaccines requires that a manufacturer plan to supply tens and sometimes hundreds of millions of doses per year at low cost. To enable broader use, there is intense interest in improving temperature stability to allow for excursions from a rigid cold chain supply, especially at the point of vaccination. Finally, there is progress in novel routes of delivery to move away from the traditional intramuscular injection by syringe approach.     

Bacterial ghosts--biological particles as delivery systems for antigens, nucleic acids and drugs

Despite the exponential rate of discovery of new antigens and DNA vaccines resulting from modern molecular biology and proteomics, the lack of effective delivery technology is a major limiting factor in their application. The bacterial ghost system represents a platform technology for antigen, nucleic acid and drug delivery. Bacterial ghosts have significant advantages over other engineered biological delivery particles, owing to their intrinsic cellular and tissue tropic abilities, ease of production and the fact that they can be stored and processed without the need for refrigeration. These particles have found both veterinary and medical applications for the vaccination and treatment of tumors and various infectious diseases.     

mRNA疫苗在疾病预防与治疗中的研究进展与展望

基于信使RNA(messenger RNA, mRNA)的核酸疫苗是近年来兴起的一种mRNA技术。mRNA疫苗比传统疫苗有许多优点,能够实现快速、经济、高效的生产。单个mRNA疫苗可以编码多种抗原,增强对特定病原体的免疫反应,提高疾病的治疗效率,以单一配方针对多种病原微生物或疾病。mRNA疫苗相关技术在新型冠状病毒肺炎疫情防控中被视作一种革命性的疫苗技术,以创纪录的速度完成研发并成功应用。由于mRNA自身稳定性差,新型递送系统的开发与应用至关重要。随着mRNA相关药理学的深入研究,mRNA疫苗的临床应用进入了一个崭新的阶段。近年来。mRNA疫苗在传染性疾病预防、肿瘤治疗等方面获得充分发展并取得了一定的研究成果,对其进行概述并进行一定程度的展望。     

Bioengineering bacterial outer membrane vesicles as vaccine platform

Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.     

Research Advances on Transgenic Plant Vaccines

In recent years, with the development of genetics molecular biology and plant biotechnology, the vaccination (e.g. genetic engineering subunit vaccine, living vector vaccine, nucleic acid vaccine) programs are taking on a prosperous evolvement. In particular, the technology of the use of transgenic plants to produce human or animal therapeutic vaccines receives increasing attention. Expressing vaccine candidates in vegetables and fruits open up a new avenue for producing oral/edible vaccines. Transgenic plant vaccine disquisitions exhibit a tempting latent exploiting foreground. There are a lot of advantages for transgenic plant vaccines, such as low cost, easiness of storage, and convenient immune-inoculation. Some productions converged in edible tissues, so they can be consumed directly without isolation and purification. Up to now, many transgenic plant vaccine productions have been investigated and developed. In this review, recent advances on plant-derived recombinant protein expression systems, infectious targets, and delivery systems are presented. Some issues of high concern such as biosafety and public health are also discussed. Special attention is given to the prospects and limitations on transgenic plant vaccines.     

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.     

A Vibrio cholerae ghost-based subunit vaccine induces cross-protective chlamydial immunity that is enhanced by CTA2B, the nontoxic derivative of cholera toxin

The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E -mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro . Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-γ, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity.     

转基因植物疫苗的研究进展

近些年,随着遗传技术和植物基因工程的发展进步,疫苗（亚单位疫苗、活载体疫苗和核酸疫苗等）的研究迅速发展起来。尤其是利用转基因植物技术生产植物疫苗的研究受到了广泛的关注,在转基因植物（蔬菜、水果、农作物）的可食用部位表达抗原生产人或动物治疗用重组蛋白和疫苗的技术为可食性疫苗的研制开辟了新途径,展现了诱人的开发前景。植物来源的疫苗具有很多优势,如生产成本低、易于保存、免疫接种方便、甚至不需提取纯化等处理而直接食用。目前已有很多转基因植物疫苗产品投入开发和生产。文章综述了近几年转基因植物疫苗在表达系统、生产、生物安全／管理、公众健康等方面的研究进展,对转基因植物疫苗存在的问题进行了分析,并对其研究前景提出了展望。     

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.     

Risk analysis for plant-made vaccines

The production of vaccines in transgenic plants was first proposed in 1990 however no product has yet reached commercialization. There are several risks during the production and delivery stages of this technology, with potential impact on the environment and on human health. Risks to the environment include gene transfer and exposure to antigens or selectable marker proteins. Risks to human health include oral tolerance, allergenicity, inconsistent dosage, worker exposure and unintended exposure to antigens or selectable marker proteins in the food chain. These risks are controllable through appropriate regulatory measures at all stages of production and distribution of a potential plant-made vaccine. Successful use of this technology is highly dependant on stewardship and active risk management by the developers of this technology, and through quality standards for production, which will be set by regulatory agencies. Regulatory agencies can also negatively affect the future viability of this technology by requiring that all risks must be controlled, or by applying conventional regulations which are overly cumbersome for a plant production and oral delivery system. The value of new or replacement vaccines produced in plant cells and delivered orally must be considered alongside the probability and severity of potential risks in their production and use, and the cost of not deploying this technology – the risk of continuing with the status quo alternative.