Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases.     

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.     

Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.     

Accessing the inaccessible: molecular tools for bifidobacteria

Bifidobacteria are an important group of the human intestinal microbiota that have been shown to exert a number of beneficial probiotic effects on the health status of their host. Due to these effects, bifidobacteria have attracted strong interest in health care and food industries for probiotic applications and several species are listed as so-called "generally recognized as safe" (GRAS) microorganisms. Moreover, recent studies have pointed out their potential as an alternative or supplementary strategy in tumor therapy or as live vaccines. In order to study the mechanisms by which these organisms exert their beneficial effects and to generate recombinant strains that can be used as drug delivery vectors or live vaccines, appropriate molecular tools are indispensable. This review provides an overview of the currently available methods and tools to generate recombinant strains of bifidobacteria. The currently used protocols for transformation of bifidobacteria, as well as replicons, selection markers, and determinants of expression, will be summarized. We will further discuss promoters, terminators, and localization signals that have been used for successful generation of expression vectors.     

Recombinant acellular pertussis vaccine—from the laboratory to the clinic: improving the quality of the immune response

Abstract Vaccination is the most effective way to prevent infectious diseases. Recombinant DNA technologies have provided powerful new tools to develop vaccines that were previously impossible or difficult to make, and to improve the vaccines that were already available but had been developed using old technology. In the case of whooping cough, an effective vaccine (composed of killed bacterial cells) is available, but its use is controversial because of the many side effects that have been associated with it. An improved vaccine against this disease should contain pertussis toxin, a molecule that needs to be detoxified in order to be included in the vaccine. Classical methods of detoxification, such as formaldehyde treatment have been used to inactivate this toxin. We have used recombinant DNA technologies to clone the pertussis toxin gene, express it in bacteria, map the B and T cell epitopes of the molecule, and to identify the amino acids that are important for enzymatic activity and toxicity. Finally, we have used this information to mutate the gene in the chromosome of Bordetella pertussis in order to obtain a strain that produces a molecule that is already non-toxic. This genetically inactivated pertussis toxin was tested extensively in animal models and clinical trials and was found to induce an immune response that is superior in quality and quantity to that induced by the vaccines produced by conventional technologies.     

Overview of vaccines

This article lists the vaccines current available for the control of both viral and bacterial infections. They may be attenuated live or inactivated whole microorganisms, or subunit preparations. Many more are in the pipeline and increasing attention is being given to establishing their safety before registration. Following the earlier eradication of smallpox, good progress is now being made toward the global eradication of poliomyelitis and a new program to eliminate measles from the Americas has begun. A variety of new approaches to vaccine development is now available. The hepatitis B virus surface antigen, made by DNA-transfected yeast or mammalian cells, is the basis of the first genetically engineered vaccine. Early in the 21st century, new vaccines based on oligopeptides, recombinant live viral or bacterial vectors (often existing live vaccines), or recombinant DNA plasmids are likely to be registered for human use. The efficacy of vaccines depends on the immune responses generated, and the recent substantial increase in our understanding of the mammalian immune system now offers great opportunities for manipulation to best obtain desired responses. These include mixing vaccine formulations to maximize immune responses, and combining vaccines to simplify their administration. Despite these advances, some persisting infections, such as those caused by HIV, plasmodia, and mycobacteria, still pose a great challenge to vaccine developers.     

Pre-erythrocytic malaria vaccine: mechanisms of protective immunity and human vaccine trials

In order to provide a rational basis for the development of a pre-erythrocytic malaria vaccine we have aimed at: (a) elucidating the mechanisms of protection, and (b) identifying vaccine formulations that best elicit protection in experimental animals and humans. Based on earlier successful immunization of experimental animals with irradiated sporozoites, human volunteers were exposed to the bites of large numbers of Plasmodium falciparum or P. vivax infected irradiated mosquitoes. The result of this vaccine trial demonstrated for the first time that a pre-erythrocytic vaccine, administered to humans, can result in their complete resistance to malaria infection. However, since infected irradiated mosquitoes are unavailable for large scale vaccination, the alternative is to develop subunit vaccines. The human trials using irradiated sporozoites provided valuable information on the human immune responses to pre-erythrocytic stages and studies on mice an excellent experimental model to characterize protective immune mechanisms. The circumsporozoite protein, the first pre-erythrocytic antigen identified, is present in all malaria species, displaying a similar structure, with a central region of repeats, and two conserved regions, essential for parasite development. Most pre-erythrocytic vaccine candidates are based on the CS protein, expressed in various cell lines, microorganisms, and recently the corresponding DNA. We and others have identified CS-specific B and T cell epitopes, recognized by the rodent and human immune systems, and used them for the development of synthetic vaccines. We used synthetic peptide vaccines, multiple antigen peptides and polyoximes, for immunization, first in experimental animals, and recently in two human safety and immunogenicity trials. We also report here on our work on T cell mediated immunity, particularly the protection of mice immunized with viral vectors expressing CS-specific cytotoxic CD8+ T cell epitopes, and the striking booster effect of recombinant vaccinia virus. To what degree CD8+ T cells, and/or other T cells specific for sporozoites and/or liver stage epitopes, contribute to pre-erythrocytic protective immunity in humans, remains to be determined.     

聚氨酯塑料的微生物降解

未被合理处置的废塑料污染已成为全球性的环境问题,探索塑料废弃物的无害化处理技术势在必行。近来,研究证实了自然界中存在可以降解塑料的微生物及酶。利用微生物或酶对废塑料进行生物处理成为可能。聚氨酯塑料(Polyurethane,PUR)是广泛应用的通用塑料之一,其废弃物量已占到所有废塑料总体积的30%。文中将PUR塑料发明应用70年来有关微生物降解的研究进行了全面综述,对PUR塑料降解真菌、细菌、降解基因与酶、降解产物及相关的生物处理技术系统等进行了总结与分析,并对实现PUR废塑料高效生物处理需解决的关键科学问题进行了展望。     

Recombinant peptide replicates immunogenicity of synthetic linear peptide chimera for use as pre-erythrocytic stage malaria vaccine

Synthetic linear peptide chimeras (LPCs_cys+) show promise as delivery platforms for malaria subunit vaccines. Maximal immune response to LPCs_cys+ in rodent malaria models depends upon formation of cross-linkages to generate homopolymers, presenting challenges for vaccine production. To replicate the immunogenicity of LPCs_cys+ using a recombinant approach, we designed a recombinant LPC (rLPC) based on Plasmodium yoelii circumsporozoite protein-specific sequences of 208 amino acids consisting of four LPC subunits in series. BALB/c or CAF1/J mice were immunized with synthetic or recombinant LPCs. Antibody concentrations, cytokine production and protection against challenge were compared. Recombinant peptide replicated the robust, high avidity antibody responses obtained with the synthetic linear peptide chimera. After in vitro stimulation spleen cells from mice immunized with rLPC or synthetic LPC_cys+ produced gamma interferon and IL-4 suggesting the efficient priming of T cells. Immunization of mice with either recombinant or synthetic LPC_cys+ provided comparable protection against experimental challenge with P. yoelii sporozoites. Recombinant LPCs reproduced the immunogenicity of synthetic LPC_cys+ without requiring polymerization, improving prospects for use as malaria vaccines.     

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。     

基因组装技术在合成生物学中的应用

基因组装技术是合成生物学领域近年来发展起来的新型技术。它基于大规模基因组数据分析,发现新型的或隐藏的生物活性物质合成基因簇。利用基因组装技术,可提高或激活沉默的生物合成基因簇在微生物中的表达,从而合成潜在的、有价值的生物活性物质。本文旨在阐明最新的体内和体外基因组装技术的设计原理、关键策略及其应用。基因组装技术是合成生物学、代谢工程和功能基因组学研究的重要工具,对生物活性物质的高效生产及合成具有重要意义。     

基因疫苗研究进展

随着免疫学,分子生物学的进展以及对发病机理的深入理解,大量疾病可能被接种疫苗所控制,大量新的方法被用于开发更为安全有效的疫苗,这些方法包括亚单位疫苗,基因修饰活疫苗以及最近的基因疫苗,本文重点介绍使用基因疫苗的新技术以及一些相关的例子。     

Protein microarrays: from serodiagnosis to whole proteome scale analysis of the immune response against pathogenic microorganisms

Microbial diseases remain the most common cause of global mortality and morbidity. Scientific and technical achievements have dramatically improved the possibilities of investigating the humoral immune response against the whole proteome of microbial organisms. A number of genomes of microbial organisms responsible for diseases of worldwide medical importance such as Plasmodium, Toxoplasma, Mycobacterium, Streptococcus, Neisseria, Salmonella, Borrelia, and Rickettsia species have already been sequenced or will be available in the very near future. High-throughput assays such as protein microarrays have been clinically validated in serum for detecting the presence of antibodies directed against microbial antigens. Computational technologies for processing large sets of data are rapidly being developed. Such a powerful combination of genomic information and assays now offers the opportunity to identify the microbial antigens that, either alone or in combination, function as targets of natural acquired immunity against infectious diseases. This information will prove invaluable for developing vaccines against a series of microorganisms of medical relevance that are urgently needed, e.g., malaria. Additional applications of these technologies include the development of a microbial antigen array for the early serodiagnosis of both common and rare infectious diseases. This review will focus on technical and scientific issues concerning the use of antigen microarrays for vaccine development and the serodiagnosis of infectious diseases.     

Role of transgenic plants in agriculture and biopharming

At present, environmental degradation and the consistently growing population are two main problems on the planet earth. Fulfilling the needs of this growing population is quite difficult from the limited arable land available on the globe. Although there are legal, social and political barriers to the utilization of biotechnology, advances in this field have substantially improved agriculture and human life to a great extent. One of the vital tools of biotechnology is genetic engineering (GE) which is used to modify plants, animals and microorganisms according to desired needs. In fact, genetic engineering facilitates the transfer of desired characteristics into other plants which is not possible through conventional plant breeding. A variety of crops have been engineered for enhanced resistance to a multitude of stresses such as herbicides, insecticides, viruses and a combination of biotic and abiotic stresses in different crops including rice, mustard, maize, potato, tomato, etc. Apart from the use of GE in agriculture, it is being extensively employed to modify the plants for enhanced production of vaccines, hormones, etc. Vaccines against certain diseases are certainly available in the market, but most of them are very costly. Developing countries cannot afford the disease control through such cost-intensive vaccines. Alternatively, efforts are being made to produce edible vaccines which are cheap and have many advantages over the commercialized vaccines. Transgenic plants generated for this purpose are capable of expressing recombinant proteins including viral and bacterial antigens and antibodies. Common food plants like banana, tomato, rice, carrot, etc. have been used to produce vaccines against certain diseases like hepatitis B, cholera, HIV, etc. Thus, the up- and down-regulation of desired genes which are used for the modification of plants have a marked role in the improvement of genetic crops. In this review, we have comprehensively discussed the role of genetic engineering in generating transgenic lines/cultivars of different crops with improved nutrient quality, biofuel production, enhanced production of vaccines and antibodies, increased resistance against insects, herbicides, diseases and abiotic stresses as well as the safety measures for their commercialization.     

Genetic Engineering and Nitrogen Fixation

Molecular cloning is based on isolation of a DNA sequence of interest to obtain multiple copies of it in vitro. Application of this technique has become an increasingly important tool in clinical microbiology due to its simplicity, cost effectiveness, rapidity, and reliability. This review entails the recent advances in molecular cloning and its application in the clinical microbiology in the context of polymicrobial infections, recombinant antigens, recombinant vaccines, diagnostic probes, antimicrobial peptides, and recombinant cytokines. Culture-based methods in polymicrobial infection have many limitation, which has been overcome by cloning techniques and provide gold standard technique. Recombinant antigens produced by cloning technique are now being used for screening of HIV, HCV, HBV, CMV, Treponema pallidum, and other clinical infectious agents. Recombinant vaccines for hepatitis B, cholera, influenza A, and other diseases also use recombinant antigens which have replaced the use of live vaccines and thus reduce the risk for adverse effects. Gene probes developed by gene cloning have many applications including in early diagnosis of hereditary diseases, forensic investigations, and routine diagnosis. Industrial application of this technology produces new antibiotics in the form of antimicrobial peptides and recombinant cytokines that can be used as therapeutic agents.     

Plant‐based oral vaccines against zoonotic and non‐zoonotic diseases

The shared diseases between animals and humans are known as zoonotic diseases and spread infectious diseases among humans. Zoonotic diseases are not only a major burden to livestock industry but also threaten humans accounting for >60% cases of human illness. About 75% of emerging infectious diseases in humans have been reported to originate from zoonotic pathogens. Because antibiotics are frequently used to protect livestock from bacterial diseases, the development of antibiotic‐resistant strains of epidemic and zoonotic pathogens is now a major concern. Live attenuated and killed vaccines are the only option to control these infectious diseases and this approach has been used since 1890. However, major problems with this approach include high cost and injectable vaccines is impractical for >20 billion poultry animals or fish in aquaculture. Plants offer an attractive and affordable platform for vaccines against animal diseases because of their low cost, and they are free of attenuated pathogens and cold chain requirement. Therefore, several plant‐based vaccines against human and animals diseases have been developed recently that undergo clinical and regulatory approval. Plant‐based vaccines serve as ideal booster vaccines that could eliminate multiple boosters of attenuated bacteria or viruses, but requirement of injectable priming with adjuvant is a current limitation. So, new approaches like oral vaccines are needed to overcome this challenge. In this review, we discuss the progress made in plant‐based vaccines against zoonotic or other animal diseases and future challenges in advancing this field.     

合成生物学技术在环境保护中的应用

合成生物学是一个基于生物学和工程学原理的科学领域,其目的是重新设计和重组微生物,以优化或创建具有增强功能的新生物系统。该领域利用分子工具、系统生物学和遗传框架的重编程,从而构建合成途径以获得具有替代功能的微生物。传统上,合成生物学方法通常旨在开发具有成本效益的微生物细胞工厂进而从可再生资源中生产化学物质。然而,近年来合成生物学技术开始在环境保护中发挥着更直接的作用。本综述介绍了基因工程中的合成生物学工具,讨论了基于基因工程的微生物修复策略,强调了合成生物学技术可以通过响应特定污染物进行生物修复来保护环境。其中,规律间隔成簇短回文重复序列(Clustered Regularly Interspersed Short Palindromic Repeats, CRISPR)技术在基因工程细菌和古细菌的生物修复中得到了广泛应用,生物修复领域也出现了很多新的先进技术,包括生物膜工程、人工微生物群落的构建、基因驱动、酶和蛋白质工程等。有了这些新的技术和工具,生物修复将成为当今最好和最有效的污染物去除方式之一。     

Pharmaceutical discoveries based on Ethnomedicinal plants: 1985 to 2000 and beyond

Fifteen years after a symposium on economic plants projecting to the year 2000, what predictions and remarks of the speakers with reference to ethnobotany and medicinal plants proved prophetic and what others fell short? Certainly they predicted a continuation of acculturation, biodiversity loss, and technology advances. They also foresaw the need for collaborative, multidisciplinary research and major government funding, and changes in regulations governing an expanded industry in botanicals, nutriceuticals, and phytopharmaceuticals, all of which occurred. However, many technologies were in their infancy or unknown, such as gene amplification and recombinant procedures, high-throughput screening, gene chip technology, and combinatorial chemistry, and are only now being used in research and development in the discovery of new therapeutics. Likewise, who would have foreseen worldwide devastating effects of mutant microorganisms resistant to drugs of choice for treating important diseases, such as malaria and tuberculosis? Research data involving these two infections illustrate the value of finding activity based on collections specifically targeted by indigenous users (antimalaria) and those generally used medicinally together with chemotaxonomic selections (antituberculosis). A discussion of research beyond 2000 addresses the future of ethnobotanical research, acculturation and biodiversity losses, collaborative research, regulation and/or standardization of Pharmaceuticals, nutriceuticals, and phytopharmaceuticals, and new technologies.     

Extracellular-matrix-binding proteins as targets for the prevention of Staphylococcus aureus infections

Staphylococcal infections cause a number of serious diseases, ranging from acute septicaemia to chronic problems such as osteomyelitis and septic arthritis. Resistance to antibiotics is a growing problem and has re-ignited interest in vaccines and in passive immunization with antibodies. Natural infections and vaccines based on whole bacteria lead to poor antibody responses, but recent research using animal models of several staphylococcal diseases reveals that vaccines based on recombinant staphylococcal extracellular-matrix-binding proteins are much more protective. Passive immunization with antibodies against one of these proteins (collagen-binding protein) also shows promise in a mouse model of sepsis.     

Site-specific immobilization of recombinant antibody fragments through material-binding peptides for the sensitive detection of antigens in enzyme immunoassays

The immobilization of an antibody is one of the key technologies that are used to enhance the sensitivity and efficiency of the detection of target molecules in immunodiagnosis and immunoseparation. Recombinant antibody fragments such as VHH, scFv and Fabs produced by microorganisms are the next generation of ligand antibodies as an alternative to conventional whole Abs due to a smaller size and the possibility of site-directed immobilization with uniform orientation and higher antigen-binding activity in the adsorptive state. For the achievement of site-directed immobilization, affinity peptides for a certain ligand molecule or solid support must be introduced to the recombinant antibody fragments. In this mini-review, immobilization technologies for the whole antibodies (whole Abs) and recombinant antibody fragments onto the surfaces of plastics are introduced. In particular, the focus here is on immobilization technologies of recombinant antibody fragments utilizing affinity peptide tags, which possesses strong binding affinity towards the ligand molecules. Furthermore, I introduced the material-binding peptides that are capable of direct recognition of the target materials. Preparation and immobilization strategies for recombinant antibody fragments linked to material-binding peptides (polystyrene-binding peptides (PS-tags) and poly (methyl methacrylate)-binding peptide (PMMA-tag)) are the focus here, and are based on the enhancement of sensitivity and a reduction in the production costs of ligand antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.