Attenuated salmonella and Shigella as carriers for DNA vaccines

The discovery that genes can be functionally transferred from bacteria to mammalian cells has suggested the possible use of bacterial vectors as gene delivery vehicles for vaccines. Attenuated invasive human intestinal bacteria, such as Salmonella and Shigella, have been used as plasmid DNA vaccine carriers and their potency has been evaluated in several animal models. This delivery system allows the administration of DNA vaccines together with associated bacterial immunostimulators directly to professional antigen presenting cells via human mucosal surfaces. Various strategies have been taken to improve the use of this delivery system to achieve robust immune responses at both mucosal and systemic sites of the immunized animals.     

The Use of Live Attenuated Bacteria as a Delivery System for Heterologous Antigens

Live attenuated mutants of several pathogenic bacteria have been exploited as potential vaccine vectors for heterologous antigen delivery by the mucosal route. Such live vectors offer the advantage of potential delivery in a single oral, intranasal or inhalational dose, stimulating both systemic and mucosal immune responses. Over the years, a range of strategies have been developed to allow controlled and stable delivery of antigens and improved immunogenicity where required. Most of these approaches have been evaluated in Salmonella vaccine vectors and, as a result, several live attenuated recombinant Salmonella vaccines are now in human clinical trials. In this review, these strategies and their use in the development of a delivery system for the Yersinia pestis V antigen are described.     

New patents on mucosal delivery of vaccines

Background: Noninvasive mucosal immune responses have been shown to be important in controlling various infections through the mucosal route. Therefore, the appropriate induction of humoral, mucosal and cellular immune response should be elicited after immunization. Objective: The objective of this review is to give an overview of novel strategies and patents for the delivery of vaccines through the mucosal route. Method: Different strategies have been developed and patented to facilitate and enhance the mucosal immunity, including the use of lipid-based delivery systems (i.e., liposomes, virosomes, archaeosomes, chochleated, immune stimulating complexes), entrapment/encapsulation of immunogens into polymeric matrix (poly(lactide-co-glycolide), chitosan, alginates, carbopol, gelatin etc.), admixing of immunogens with mucosal adjuvants (cholera toxin or CT, enterotoxin, lipid A, tetanus toxin or lymphotactin), use of live attenuated bacterial and viral vector encoding antigen of interest and ingestible plant-based mucosal vaccines. Conclusion: Lipid- and polymer-based novel delivery systems have been widely investigated in mucosal vaccine delivery systems. Recent advancement in the molecular technology has also shown great potential of genetic immunization for the delivery of wide range of infectious molecular targets. Effective and selective delivery of vaccines through the mucosal route could provide new therapeutic conduit in the treatment of mucous-associated disease.     

M cell-targeted delivery of vaccines and therapeutics

Background: M (microfold or membranous) cells are specialised epithelial cells responsible for antigen sampling at the interface of mucosal surfaces and the environment. Their high transcytotic ability make M cells an attractive target for mucosally delivered vaccines and therapeutics. Objective: This brief review discusses the current state of M cell-targeted mucosal delivery systems and the potential of such delivery systems for the development of new vaccines and therapeutics against mucosal infectious and inflammatory diseases. Scope: A variety of synthetic microparticles/nanoparticles have been developed and tested as vehicles for M cell-targeted mucosal drug and vaccine delivery. β1 integrins, pathogen recognition receptors, specific carbohydrate residues and other M cell surface antigens have been exploited as potential targets for the delivery of mucosal vaccines and therapeutics. Conclusion: Despite a considerable body of literature, much work still needs to be done before an effective M cell-targeted vaccine or therapeutic is developed.     

Mucosal delivery of nanovaccine strategy against COVID-19 and its variants

Despite the global administration of approved COVID-19 vaccines (e.g., ChAdOx1 nCoV-19®, mRNA-1273®, BNT162b2®), the number of infections and fatalities continue to rise at an alarming rate because of the new variants such as Omicron and its subvariants. Including COVID-19 vaccines that are licensed for human use, most of the vaccines that are currently in clinical trials are administered via parenteral route. However, it has been proven that the parenteral vaccines do not induce localized immunity in the upper respiratory mucosal surface, and administration of the currently approved vaccines does not necessarily lead to sterilizing immunity. This further supports the necessity of a mucosal vaccine that blocks the main entrance route of COVID-19: nasal and oral mucosal surfaces. Understanding the mechanism of immune regulation of M cells and dendritic cells and targeting them can be another promising approach for the successful stimulation of the mucosal immune system. This paper reviews the basic mechanisms of the mucosal immunity elicited by mucosal vaccines and summarizes the practical aspects and challenges of nanotechnology-based vaccine platform development, as well as ligand hybrid nanoparticles as potentially effective target delivery agents for mucosal vaccines.     

Recent advances in immunological adjuvants: the development of particulate antigen delivery systems

New generation vaccines, including those based on recombinant proteins, are safer than traditional vaccines, but are less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. A number of potent immunostimulatory molecules obtained from bacterial cells or plants have been extensively evaluated as adjuvants. However, a number of these molecules have displayed significant toxicity, both in preclinical animal models and in human clinical trials. An alternative approach to the development of novel adjuvants involves the preparation of particulate antigen delivery systems of similar dimensions to natural pathogens. In the absence of additional immunostimulatory molecules, emulsion droplets and microparticles have been shown to be potent adjuvants for the induction of both humoral and cell-mediated immune responses following systemic administration. Moreover, particulate delivery systems have been shown to display an acceptable toxicity profile in a number of clinical trials. Particulate antigen delivery systems also have the potential to function as potent adjuvants following administration by mucosal routes, including oral and intranasal. An alternative approach to the mucosal delivery of vaccines involves the use of genetically detoxified mutant toxins, e.g., LT-K63, as mucosal adjuvants. The use of novel adjuvants and antigen delivery systems is likely to extend the use of vaccines into the area of therapeutics, involving the eradication of infectious diseases and cancers, or the amelioration of autoimmune disorders.     

Intranasal formulations: promising strategy to deliver vaccines

Introduction: The emergence of new diseases and the lack of efficient vaccines against numerous nontreatable pathogens require the development of novel vaccination strategies. To date, only a few mucosal vaccines have been approved for humans. This was in part due to i) the use of live attenuated vaccines, which are not suitable for certain groups of individuals, ii) safety concerns derived from implementation in humans of some mucosal vaccines, iii) the poor stability, absorption and immunogenicity of antigens delivered by the mucosal route and iv) the limited number of available technologies to overcome the bottlenecks associated with mucosal antigen delivery. Recent advances make feasible the development of efficacious mucosal vaccines with adequate safety profile. Thus, currently intranasal vaccines represent an attractive and valid alternative to conventional vaccines.

Areas covered: The present review is focused on the potentials and limitations of market-approved intranasal vaccines and promising candidates undergoing clinical investigations. Furthermore, emerging strategies to overcome main bottlenecks including efficient breaching of the mucosal barrier and safety concerns by implementation of new adjuvants and delivery systems are discussed.

Expert opinion: The rational design of intranasal vaccines requires an in-depth understanding of the anatomic, physicochemical and barrier properties of the nasal mucosa, as well as the molecular mechanisms governing the activation of the local innate and adaptive immune system. This would provide the critical knowledge to establish effective approaches to deliver vaccine antigens across the mucosal barrier, supporting the stimulation of a long-lasting protective response at both mucosal and systemic levels. Current developments in the area of adjuvants, nanotechnologies and mucosal immunology, together with the identification of surface receptors that can be exploited for cell targeting and manipulating their physiological properties, will become instrumental for developing a new generation of more effective intranasal vaccines.     

Manipulating the immune system: humoral versus cell-mediated immunity

Many of the vaccines in use today were designed on an empirical basis with little understanding of the mechanism of protective immunity or knowledge of the protective antigens. Certain of these vaccines, based on killed or attenuated bacteria or viruses, are associated with unacceptable side-effects. New generation vaccines based on recombinant proteins or naked DNA have considerably improved safety profiles, but are often poorly immunogenic, especially when administered by mucosal routes. This is a particular problem with oral delivery; where high doses of antigen are required to generate even modest immune responses. In contrast, nasal delivery of antigens with a range of adjuvants or delivery systems has been shown to generate relatively potent immune responses and to protect against infection in animal models. Advances in immunology have demonstrated that a variety of cellular and humoral immune effector mechanisms, that are regulated by distinct Th1 and Th2 subtypes of T cells, mediate protection against different infectious diseases. The identification of adjuvants and immunomodulators, that can promote the selective induction of these distinct populations of T cells, has now made it possible to rationally design safe and effective mucosal vaccines against a range of infectious diseases of man.     

Mucosal immunisation: adjuvants and delivery systems

The mucosal administration of vaccines is an area currently receiving a high level of interest due to potential advantages offered by this technique. These advantages include the ability to administer vaccines without need for needles, thus improving patient compliance with vaccination schedules, and the capacity to induce immune responses capable of preventing infections at the site of acquisition. Despite these advantages a number of limitations exist which currently inhibit our ability to successfully develop new mucosal vaccines. As such, much research is currently focused on developing new adjuvants and delivery systems to overcome these difficulties. However, despite high levels of interest in this area, relatively few mucosal vaccine candidates have successfully progressed to human clinical trials. In the review that follows, we aim to provide the reader with an overview of the immune system with respect to induction of mucosal immune responses. Furthermore, the review provides an overview of a number of microbial (bacterial toxins, CpG DNA, cytokines/chemokines, live vectors, and virus like particles) and synthetic (microspheres, liposomes, and lipopeptides) strategies that have been investigated as adjuvants or delivery systems for mucosal vaccine development, with a focus on the delivery of vaccines via the oral route.     

The use of live attenuated bacteria as a delivery system for heterologous antigens

Live attenuated mutants of several pathogenic bacteria have been exploited as potential vaccine vectors for heterologous antigen delivery by the mucosal route. Such live vectors offer the advantage of potential delivery in a single oral, intranasal or inhalational dose, stimulating both systemic and mucosal immune responses. Over the years, a range of strategies have been developed to allow controlled and stable delivery of antigens and improved immunogenicity where required. Most of these approaches have been evaluated in Salmonella vaccine vectors and, as a result, several live attenuated recombinant Salmonella vaccines are now in human clinical trials. In this review, these strategies and their use in the development of a delivery system for the Yersinia pestis V antigen are described.     

Conference Science Medal Lecture: Recent Advances in Vaccine Adjuvants for Systemic and Mucosal Administration*

Although vaccines produced by recombinant DNA technology are safer than traditional vaccines, which are based on attenuated or inactivated bacteria or viruses, they are often poorly immunogenic. Therefore, adjuvants are often required to enhance the immunogenicity of these vaccines. A number of adjuvants which are particulates of defined dimensions (< 5 μm) have been shown to be effective in enhancing the immunogenicity of weak antigens in animal models. Two novel adjuvants which possess significant potential for the development of new vaccines include an oil-in-water microemulsion (MF59) and polymeric microparticles. MF59 has been shown to be a potent and safe adjuvant in human subjects with several vaccines (for example HSV-2, HIV-1 and influenza virus). An MF59 adjuvanted influenza has been recommended for approval in Italy. Microparticles prepared from the biodegradable polymers the poly(lactide-co-glycolides) (PLG) are currently undergoing extensive pre-clinical evaluation as vaccine adjuvants. Because of their controlled release characteristics, microparticles also possess considerable potential for the development of single dose vaccines. The development of single dose vaccines would offer significant advantages and would improve vaccination uptake rates in at risk populations, particularly in the developing world. In addition to systemic administration, microparticles have also also been shown to enhance the immunogenicity of vaccines when administered by mucosal routes. Therefore microparticles may allow the development of novel vaccines which can be administered by non-parenteral routes. Mucosal administration of vaccines would significantly improve patient compliance by allowing immunization to be achieved without the use of needles. An alternative approach to the development of mucosally administered vaccines involves the production of genetically detoxified toxins. Heat labile enterotoxin (LT) from Escherichia coli and cholera toxin from Vibrio cholerae are two closely related bacterially produced toxins, which are the most potent adjuvants available. However, these molecules are too toxic to be used in the development of human vaccines. Nevertheless, these toxins have been modified by site-directed mutagenesis to produce molecules which are adjuvant active, but non-toxic. The most advanced of these molecules (LTK63), which has a single amino acid substitution in the enzymatically active subunit of LT, is active as an adjuvant, but non-toxic in pre-clinical models. The approach of genetically detoxifying bacterial toxins to produce novel adjuvants offers significant potential for the future development of mucosally administered vaccines.     

Immunité et vaccinations antivirales : exemple de la muqueuse respiratoire

ObjectiveAs the mucosal surfaces of the respiratory tract represent a major portal of entry for most human viruses and many bacteria, they seem to be a critical component of the mammalian immunologic repertoire. Thus, vaccines stimulating this local immunity could represent an interesting approach to prevent these infections. After detailing the different mechanisms implied in this mucosal immunity, the aim of this study is to analyze the basis of such a vaccination and the different vaccines available to mucosal respiratory tract use.Mucosal immunityThe major antibody isotype in external secretions is secretory immunoglobin A (S-IgA); the role of IgM (S-IgM) and IgG (S-IgG) are actually questionned. It is, however, interesting that the major effector cells in the mucosal surfaces are not IgA B cells, but T lymphocytes that may represent up to 80% of the entire mucosal lymphoid cell population.Immunoprophylaxis by the mucosal routePassive antibodies were shown to protect against mucosal viral infections, such as those caused by RSV, but very high quantities of passive antibodies are needed to restrict virus replication on mucosal surface.In general, factors which favor development of mucosal antibody and cell mediated immune responses include the oral or respiratory immunization and the replicating nature of the vaccine agents. However, to date only a few vaccines have become available to mucosal respiratory tract use, and cold-adapted influenza virus vaccines is the only one available using nasal route. Other parenteral licensed vaccines have not been recommended for mucosal administration. Some of them have been experimentally used with nasal administration of replicating agents (varicella and measles vaccines) or non replicating agents (influenza inactivated vaccine), but have been found to induce a very low mucosal response.ConclusionBased on the experience with existing vaccines, the development of mucosal immunity or administration of vaccines via the mucosal route is clearly not a prerequisite today for control or prevention of most viral infectious respiratory diseases or diseases with respiratory tract as a route of contamination. But the example of live attenuated intranasal influenza vaccine inducing both systemic and local immune response without immunopathology, is promising for the future of the mucosal immunization against respiratory viral infections.     

DNA vaccines

DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention and therapy of disease. Regarding the inoculation method of DNA vaccine, we recommend the gene gun delivery system, which is a highly reliable method compared to intramuscular inoculation. DNA vaccines could have potential advantages over other types of vaccines in that these vaccines can induce strong cellular immune responses, cytotoxic T lymphocytes and type 1 helper T cells, without resorting to live organisms or complicated protein formulation. The cellular immune responses are especially required for the protection against infections with intracellular pathogens such as viruses and Mycobacterium tuberculosis and protection against cancers, suggesting that they seem to be suitable targets of DNA vaccines. We describe here that their application to bacterial infections requires optimization of codon usage in the DNA vaccines to the host animal to improve translational efficiencies of the bacteria genes. DNA vaccines for a variety of pathogens and cancers have now entered phase I/II human clinical trials.     

Nucleic acid vaccines: prospects for non-viral delivery of mRNA vaccines

Introduction: Nucleic acid-based vaccines are being developed as a means to combine the positive attributes of both live-attenuated and subunit vaccines. Viral vectors and plasmid DNA vaccines have been extensively evaluated in human clinical trials and have been shown to be safe and immunogenic, although none have been licensed for human use. More recently, mRNA-based vaccine alternatives have emerged and might offer certain advantages over their DNA-based counterparts.

Areas covered: This review describes the two main categories of mRNA vaccines: conventional non-amplifying and self-amplifying mRNA. It summarizes the initial clinical proof-of-concept studies and outlines the preclinical testing of the next wave of innovations for the technology. Finally, this review highlights the versatile functionality of the mRNA molecule and introduces opportunities for future improvements in vaccine design.

Expert opinion: The prospects for mRNA vaccines are very promising. Like other types of nucleic acid vaccines, mRNA vaccines have the potential to combine the positive attributes of live attenuated vaccines while obviating many potential safety limitations. Although data from initial clinical trials appear encouraging, mRNA vaccines are far from a commercial product. These initial approaches have spurred innovations in vector design, non-viral delivery, large-scale production and purification of mRNA to quickly move the technology forward. Some improvements have already been tested in preclinical models for both prophylactic and therapeutic vaccine targets and have demonstrated their ability to elicit potent and broad immune responses, including functional antibodies, type 1 T helper cells-type T cell responses and cytotoxic T cells. Though the initial barriers for this nucleic acid vaccine approach seem to be overcome, in our opinion, the future and continued success of this approach lies in a more extensive evaluation of the many non-viral delivery systems described in the literature and gaining a better understanding of the mechanism of action to allow rational design of next generation technologies.     

Chitosan-based delivery systems for mucosal vaccines

Introduction: Mucosal vaccine development faces several challenges and opportunities. Critical issues for effective mucosal vaccination include the antigen-retention period that enables interaction with the lymphatic system, choice of adjuvant that is nontoxic and induces the required immune response and possibly an ability to mimic mucosal pathogens. Chitosan-based delivery systems are reviewed here as they address these issues and hence represent the most promising candidates for the delivery of mucosal vaccines.

Areas covered: A comprehensive literature search was conducted, to locate relevant studies published within the last 5 years. Mucosal delivery via nasal and oral routes is evaluated with respect to chitosan type, dosage forms, co-adjuvanting with novel adjuvants and modulation of the immune system.

Expert opinion: It is concluded that chitosan derivatives offer advantageous opportunities such as nanoparticle and surface charge manipulation that facilitate vaccine targeting. Nevertheless, these technologies represent a longer-term goal. By contrast, chitosan (unmodified form) with or without a co-adjuvant has significant toxicology and human data to support safe mucosal administration, and thus has the potential for earlier product introduction into the market.     

Proteomics of bacterial pathogens

Background: Significant progress has been made in the characterisation of bacterial pathogens using a combination of genomic and proteomic technologies. The data generated have improved our understanding of how these microbes interact with the human host to cause disease. Objective: Practical outcomes include the identification of putative vaccines and new drug targets. Method: This review highlights those developments achieved through the use of proteomic technologies including established electrophoretic methods as well as state-of-the-art mass spectrometry based techniques. Conclusion: Proteomics has been used at diverse levels to investigate microbial physiology, gene expression and the complex interactions between bacteria and their hosts. Pathogenic determinants are identified through comparative proteomics between virulent and avirulent isolates whereas complex disease phenotypes can be correlated with specific proteomic signatures identified through the analysis of large collections of natural isolates. Initial progress has been achieved on defining the bacterial proteome during in vivo infection, which will probably be a key research area over the coming years.     

Section Review: Biologicals & Immunologicals: Human papillomavirus infection,genital warts and cervical cancer: prospects for prophylactic and therapeutic vaccines

Papillomaviruses cause warts of the skin, anogenital mucosa, and bronchial mucosa, and also pre-neoplastic lesions of the cervical and vulval mucosae, which in a proportion of women progress to invasive carcinomata. Papillomaviruses cannot be propagated in vitro, which has hindered the development of prophylactic vaccines, but recent production of synthetic virus like particles (VLPs) in vitro using recombinant DNA technology has resulted in vaccines which prevent papillomavirus infection in animal models, and has given rise to commercial interest in human prophylactic vaccines. Papillomavirus proteins are generally poorly presented to the immune system in the course of natural infection and, therefore, therapeutic immunity may be produced by appropriate choice of viral protein and delivery system. Immunotherapy for PV associated cancer is targeted at two non-structural PV proteins expressed in cancer cells (E6 and E7), which have been shown to be effective targets for immunotherapy in experimental tumour models: Phase I/II clinical trials are now underway. Immunotherapy for pre-neoplastic lesions presents a greater choice of potential viral antigenic targets: as animal models give conflicting data, clinical trials in man may be necessary to choose the correct antigens.     

Prophylaxis and therapy of allergy by mucosal tolerance induction with recombinant allergens or allergen constructs

The mucosal immune system, present along the respiratory, gastrointestinal and genitourinary tract, has to discriminate between harmful pathogens and innocuous antigens, such as food, airborne antigens or the commensal bacterial flora. Therefore the mucosal immune system has acquired two opposing immunological functions, i.e. the induction of immunity and defence of mucosal pathogens, and the induction and maintenance of tolerance to environmental antigens and bacterial flora. As described for autoimmunity a breakdown or failure of tolerance induction is believed to lead also to allergies and food enteropathies. Based on the physiological role to prevent hypersensitivity reactions, tolerance induction via the mucosa has been proposed as a treatment strategy against inflammatory diseases, such as allergies. The aim of our research is to develop mucosal allergy vaccines based on the induction of mucosal tolerance and/or the induction of counter-regulatory immune responses with or without the use of certain mucosal antigen delivery systems, such as lactic acid bacteria. The use of recombinant allergens instead of allergen extracts with varying allergen content and composition may be essential for improvement of the treatment efficacy. In the present review we give examples of different animal models of type I allergy/asthma. Using these models we demonstrate that recombinant allergens or hypoallergenic variants thereof can be successfully used to induce mucosal tolerance in a prophylactic as well as a therapeutic treatment regime. That the concept of mucosal tolerance induction/mucosal vaccine delivery may in principal also function in humans is supported by recent clinical trials with locally (sublingual) applied immunotherapy.     

Recent advances in mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymeric carriers

Importance of the field: The mucosal delivery of vaccines provides the basis for induction of humoral, cellular and mucosal immune responses against infectious diseases. The delivery of antigens to and through mucosal barriers always remains challenging due to adverse physiological conditions (pH and enzymes) and biological barriers created by tight epithelial junctions restricting transportation of macromolecules. Mucoadhesive and biodegradable polymers offer numerous advantages in therapeutic delivery of proteins/antigens particularly through the mucosal route by protecting antigens from degradation, increasing concentration of antigen in the vicinity of mucosal tissue for better absorption, extending their residence time in the body and/or targeting them to sites of antigen uptake. Furthermore, antigen can be delivered more effectively to the antigen presenting cells by anchoring the ligand having affinity on the surface of carrier for the receptors present on the mucosal epithelial cells.

Areas covered in this review: The present review covers various polymeric carriers, which allow the possibility of modification and manipulation of their properties, thereby, enhancing the effectiveness of mucosal vaccines. This article reviews the recent literature and patents in the field of vaccine delivery using mucoadhesive polymeric carriers.

What the reader will gain: The reader will gain insights into various natural polymers, synthetic polymers and ligand derived polymeric carrier systems studied to enhance mucosal immunization.

Take home message: Biodegradable polymeric carriers represent a promising approach for mucosal delivery of vaccine.     

A targeting approach for delivery of polymer microparticle-antibody conjugate against Vibrio parahaemolyticus-induced cytotoxicity to human intestinal epithelial cells

A major traditional of antibacterial drugs is antibiotic which promotes more rapid release of the toxins from bacteria cells in human body, which causes severe infection. The thermostable direct hemolysin (TDH) has been proposed as a major virulence factor of Vibrio parahaemolyticus (Vp). This study covers the preparation of polymer microparticle-antibody conjugate for the development of a drug targeting approach for antibacterial drug delivery. The chemical binding of antibodies (ab) to latex bead of 0.2 μm diameter was performed by using a water-soluble carbodiimide technique. Confocal microscopy revealed that the bacteria were strongly absorbed by the latex beads with bound anti-Vp polyclonal antibody (pAb). Treatment with a latex bead bound both anti-Vp pAb and anti-TDH monoclonal antibody (mAb) significantly inhibited bacterial adherence to the Caco-2 cells (p < 0.01), and reduced TDH-induced cytotoxicity in histology. These preliminary results suggest that it may be possible to effectively protect against Vp infection by using this microparticle-antibody conjugate delivery system.