New strategies to overcome the drawbacks of currently available flu vaccines.

Vaccination represents the most efficient tool to control morbidity and mortality resulting from influenza infections in humans. The currently licensed influenza vaccines provide good protection levels in healthy adults, whereas lower protection is generally achieved in ageing individuals who are at a higher risk of developing severe clinical manifestations. Future improvements in influenza vaccines should address the needs of high risk groups including the elderly, small children and chronic patients. Recently, due to the increased incidence of avian influenza pandemic outbreaks, the prevention of a potential human influenza pandemic turned into another crucial issue in the influenza vaccination field. The development and validation of manufacturing processes for efficient and safe pandemic vaccines became one of the top priorities of health, regulatory and funding agencies all over the world. In the pandemic context, the development of novel vaccines administered via the mucosal route may play a significant role by reducing virus shedding from infected individuals. This chapter provides insights in the limitations of existing manufacturing processes, new approaches to overcome limitation in vaccine production, mechanisms of action of current vaccines and discuss potential strategies to improve the immunogenicity and efficacy of influenza vaccines.

Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs.

Lipid-based vesicles are a very promising approach to treat diseases such as cancer, chronic infections and auto-immunity. Modern drug encapsulation methods allow efficient packing of therapeutic substances inside liposomes, thereby reducing the systemic toxicity of the drugs. Specific targeting can enhance the therapeutic effect of the drugs through their accumulation at the diseased site. In the vaccine field, the integration of functional viral envelope proteins into liposomes has led to an antigen carrier and delivery system termed a virosome, a clinically proven vaccine platform for subunit vaccines with an excellent immunogenicity and tolerability profile.

Haemolysin A and listeriolysin--two vaccine delivery tools for the induction of cell-mediated immunity.

Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors. While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery. Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity. In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites.

Live attenuated bacteria as vectors to deliver plasmid DNA vaccines.

Live attenuated bacterial vaccines allow vaccination via the mucosal surfaces and specific targeting to professional antigen presenting cells located at the inductive sites of the immune system. A novel approach exploits attenuated intracellular bacteria as a delivery system for eukaryotic antigen expression vectors (so-called DNA vaccines). Candidate carrier bacteria include attenuated strains of Salmonella, Shigella and Listeria spp, which have been shown, in vitro, to deliver DNA vaccines to human cells. Bacterial DNA vaccine delivery has also demonstrated in vivo efficacy in several experimental animal models of infectious diseases and tumors. The next step should be the clinical assessment of the safety, immunogenicity and efficacy of DNA vaccine delivery by live bacterial vaccines.

Virosomal adjuvanted antigen delivery systems.

The development of novel and increasingly safer vaccines frequently utilizes well-characterized antigens, in particular highly purified proteins or synthetic peptides. In spite of some achievements, this approach is frequently impeded by the fact that such antigens are often poor immunogens when administered alone. This fact has necessitated the development of suitable adjuvants that possess the ability to enhance the immunogenicity of a given antigen, preferably with little or no side effects. This paper discusses one of the successes of vaccinology of the past decade: virosomal vaccines. The principles of the concept, immunoadjuvant action and application of virosomes in two currently licensed vaccines are detailed, with specific reference to the induction of both humoral and cellular immunity.

Nonclinical safety evaluation of Escherichia coli heat-labile toxin mucosal adjuvant as a component of a nasal influenza vaccine.

Conventional influenza vaccines currently in use are administered parenterally and generally confer good protection against systemic disease through the induction of high titers of serum virus-neutralizing antibodies. Parenteral vaccines are suboptimal in that they fail to induce a local mucosal response that may prevent the early stages of virus infection. Thus, the intranasal administration of a vaccine may provide a viable alternative to the parenteral route. Indeed, intranasal administration of vaccine antigens when formulated with an appropriate mucosal adjuvant (e.g., bacterial toxins), results in a vigorous local and systemic immune response. This review discusses the nonclinical safety evaluation of Escherichia coli heat-labile toxin as a mucosal adjuvant for an intranasally administered influenza vaccine.

Morphological and immunocytochemical analysis of Escherichia coli-specific surface antigens in wildtype strains and in recombinant Vibrio cholerae.

Adhesion is the first step in the pathogenesis of enterotoxigenic Escherichia coli infections. The genes encoding the most prevalent adhesion factors CFA/I, CS3 and CS6 were cloned into Vibrio cholerae strain CVD 103-HgR and expression of fimbriae was investigated in wildtype and recombinant strains by transmission electron microscopy in conjunction with immunolabelling and negative staining. Negative staining was effective in revealing CFA/I and CS3, but not CS6. Although morphology of fimbriae differed between wildtype and recombinant strains, corresponding surface antigens were recognized by specific antibodies. The present study provides evidence that ETEC-specific fimbriae can adequately be expressed in an attenuated V. cholerae vaccine strain and that immunoelectron microscopy is a critical tool to validate the surface expression of antigens in view of their possible suitability for recombinant vaccines.

A clinical study to assess the safety and immunogenicity of attenuated measles vaccine administered intranasally to healthy adults.

BACKGROUND: Despite the availability of a safe and effective vaccine for over four decades, measles remains one of the most common infectious disease killers of children in the world. Mucosal administration of currently licensed measles vaccine has been proposed to address issues of needle safety and improve vaccine uptake. METHODS: Healthy adult volunteers were randomized to receive live-attenuated monovalent measles virus vaccine (Moraten Berna) via the standard subcutaneous (SQ) or the experimental intranasal (IN) route in a randomized, double-masked fashion. Safety, reactogenicity, immunogenicity, and shedding were assessed. RESULTS: Safety, reactogenicity, and viral shedding were not significantly different in the two study groups. Immunogenicity was markedly lower in the group of volunteers that received vaccine via the IN route. Plaque reduction neutralization (PRN) geometric mean titers (GMT) were 125 (95% confidence interval [CI] 68-228) milli International Units per milliliter (mIU/mL) on day 28 in recipients of IN vaccine versus 645 (95% CI 468-889) mIU/mL in recipients of vaccine SQ; p< 0.001 by Mann-Whitney Rank Sum. 50% of measles non-immune individuals mounted titers above the protective threshold of PRN 200 mIU/mL after IN administration versus 100% of volunteers who received the vaccine SQ. CONCLUSION: Intranasal administration of live-attenuated measles vaccine was safe and well tolerated, but failed to mount significant immune responses when compared to subcutaneous administration. It is possible that higher doses or smaller particle size are necessary for successful intranasal measles vaccination and boosting.

Attenuated measles virus as a vaccine vector.

Live attenuated measles virus (MV) vaccines have an impressive record of safety, efficacy and ability to induce life-long immunity against measles infection. Using reverse genetics technology, such negative-strand RNA viruses can now be rescued from cloned DNA. This technology allows the insertion of exogenous genes encoding foreign antigens into the MV genome in such a way that they can be expressed by the MV vaccine strain, without affecting virus structure, propagation and cell targeting. Recombinant viruses rescued from cloned cDNA induce immune responses against both measles virus and the cloned antigens. The tolerability of MV to gene(s) insertion makes it an attractive flexible vector system, especially if broad immune responses are required. The fact that measles replication strictly occurs in the cytoplasm of infected cells without DNA intermediate has important biosafety implications and adds to the attractiveness of MV as a vector. In this article we report the characteristics of reporter gene expression (GFP, LacZ and CAT) and the biochemical, biophysical and immunological properties of recombinant MV expressing heterologous antigens of simian immunogeficiency virus (SIV).

Viral vectors for malaria vaccine development.

A workshop on viral vectors for malaria vaccine development, organized by the PATH Malaria Vaccine Initiative, was held in Bethesda, MD on October 20, 2005. Recent advancements in viral-vectored malaria vaccine development and emerging vector technologies were presented and discussed. Classic viral vectors such as poxvirus, adenovirus and alphavirus vectors have been successfully used to deliver malaria antigens. Some of the vaccine candidates have demonstrated their potential in inducing malaria-specific immunity in animal models and human trials. In addition, emerging viral-vector technologies, such as measles virus (MV), vesicular stomatitis virus (VSV) and yellow fever (YF) virus, may also be useful for malaria vaccine development. Studies in animal models suggest that each viral vector is unique in its ability to induce humoral and/or cellular immune responses. Those studies have also revealed that optimization of Plasmodium genes for mammalian expression is an important aspect of vaccine design. Codon-optimization, surface-trafficking, de-glycosylation and removal of toxic domains can lead to improved immunogenicity. Understanding the vector's ability to induce an immune response and the expression of malaria antigens in mammalian cells will be critical in designing the next generation of viral-vectored malaria vaccines.

Biosafety evaluation of recombinant live oral bacterial vaccines in the context of European regulation.

Live bacterial vaccines represent a highly valid preventive strategy in the fight against infectious disease. However, the road from research to market is peppered with hurdles, one of which is the requirement for high biosafety characteristics, which the candidate vaccine has to display. In Europe, the European Agency for the evaluation of medicinal products (EMEA) is the relevant authority regulating the licensure of genetically engineered vaccines. For this purpose, the agency may rely on several directives and guidelines defined in the past 15 years. As for live vaccines containing genetically modified organisms (GMOs) susceptible to be released into the environment, Directive 2001/18/EC determines the framework and principles of an environmental risk assessment (ERA) process, the results of which constitute an important section of the vaccine registration package submitted to registration authorities. In this article, we address the implications of current European regulations for the approval of live oral bacterial vaccines with emphasis on the assessment of potential risks associated with environmental release. Biosafety aspects of already registered and some promising live bacterial vaccine strains will be briefly discussed.

Expression of enterotoxigenic Escherichia coli colonization factors in Vibrio cholerae.

As a first step towards a vaccine against diarrhoeal disease caused by enterotoxigenic Escherichia coli (ETEC), we have studied the expression of several ETEC antigens in the live attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Colonization factors (CF) CFA/I, CS3, and CS6 were expressed at the surface of V. cholerae CVD 103-HgR. Both CFA/I and CS3 required the co-expression of a positive regulator for expression, while CS6 was expressed without regulation. Up-regulation of CF expression in V. cholerae was very efficient, so that high amounts of CFA/I and CS3 similar to those in wild-type ETEC were synthesized from chromosomally integrated CF and positive regulator loci. Increasing either the operon and/or the positive regulator gene dosage resulted in only a small increase in CFA/I and CS3 expression. In contrast, the level of expression of the non-regulated CS6 fimbriae appeared to be more dependent on gene dosage. While CF expression in wild-type ETEC is known to be tightly thermoregulated and medium dependent, it seems to be less stringent in V. cholerae. Finally, co-expression of two or three CFs in the same strain was efficient even under the control of one single regulator gene.

Plasmid maintenance systems suitable for GMO-based bacterial vaccines.

Live carrier-based bacterial vaccines represent a vaccine strategy that offers exceptional flexibility. Commensal or attenuated strains of pathogenic bacteria can be used as live carriers to present foreign antigens from unrelated pathogens to the immune system, with the aim of eliciting protective immune responses. As for oral immunisation, such an approach obviates the usual loss of antigen integrity observed during gastrointestinal passage and allows the delivery of a sufficient antigen dose to the mucosal immune system. Antibiotic and antibiotic-resistance genes have traditionally been used for the maintenance of recombinant plasmid vectors in bacteria used for biotechnological purposes. However, their continued use may appear undesirable in the field of live carrier-based vaccine development. This review focuses on strategies to omit antibiotic resistance determinants in live bacterial vaccines and discusses several balanced lethal-plasmid stabilisation systems with respect to maintenance of plasmid inheritance and antigenicity of plasmid-encoded antigen in vivo.

Improved immune responses to influenza vaccination in the elderly using an immunostimulant patch.

The elderly have greater morbidity and mortality due to influenza, and respond poorly to influenza vaccination compared to younger adults. This study was designed to determine if the adjuvant heat-labile enterotoxin from Escherichia coli (LT), administered as an immunostimulant (IS) patch on the skin with influenza vaccination, improves influenza immune responses in the elderly. Three weeks following vaccination, hemagglutination inhibition (HAI) responses in LT IS patch recipients showed improvement over those of elderly receiving vaccine alone, as demonstrated by significance or trends in fold rise [A/Panama (P = 0.004), A/New Caledonia (P = 0.09)], seroconversion [A/New Caledonia (63% versus 40%, P = 0.01), A/Panama (54% versus 36%, P = 0.08)] and seroprotection [26%, 20% and 16% greater for the patch group for A/New Caledonia, A/Panama and B/Shandong strains, respectively]. The data suggest that an LT IS patch may further enhance influenza vaccine immune responses in the elderly.

Novel vaccination strategies based on recombinant Mycobacterium bovis BCG.

In this manuscript, we will review the utilization of Mycobacterium bovis Bacille Calmette-Guerin (BCG) as a vaccine against tuberculosis (TB) and as a carrier system for heterologous antigens. BCG is one of the most widely used vaccines. Novel techniques in genome manipulation allow the construction of virulence-attenuated recombinant (r)-BCG strains that can be employed as homologous vaccines, or as heterologous antigen delivery systems, for priming pathogen-specific immunity against infectious diseases, including TB. Several approaches are available for heterologous antigen expression and compartmentalization in BCG and recent findings show the potential to modulate and direct the immune responses induced by r-BCG strains as desired. Recent achievements in complete genome analysis of various target pathogens, combined with a better understanding of protective pathogen-specific immune responses, form the basis for the rational design of a new generation of recombinant mycobacterial vaccines against a multitude of infectious diseases.

Biosafety aspects of the recombinant live oral Vibrio cholerae vaccine strain CVD 103-HgR.

The development of live attenuated vaccines, allowing for the safe and effective immunisation at mucosal surfaces, is a strategy of great interest for vaccinologists. The main advantage of this approach over conventional parenteral vaccines is the induction of strong mucosal immune responses, allowing targeting of the pathogen at the initial point of contact with the host. Further advantages include the ease of administration, high acceptance by vaccines, and relatively low production costs. Finally, well-characterised, safe and immunogenic vaccine strains are well suited as vectors for the mucosal delivery of foreign vaccine antigens and of DNA vaccines. However, such vaccines, when based on or containing genetically modified organisms (GMOs), are facing new and specific regulatory hurdles, particularly regarding the potential risks for humans and the environment. In this contribution we address selected aspects of the risk assessment of live attenuated bacterial vaccines covered in the course of the registration of vaccine strain CVD 103-HgR as a recombinant live oral vaccine against cholera.

Mycobacterium bovis BCG-based vaccines against tuberculosis: novel developments.

Mycobacterium bovis Bacille Calmette-Guérin (BCG) is one of the most widely used vaccines. Modern techniques in genome manipulation allow the construction of recombinant (r)-BCG strains that can be employed as highly immunogenic vaccines against tuberculosis (TB) with an enhanced safety profile. In addition, the development of novel procedures to cultivate BCG will allow the large-scale production of future BCG-based vaccines.

Experience with registered mucosal vaccines.

Most pathogens gain access to their host through mucosal surfaces. It is therefore desirable to develop vaccination strategies that lead to mucosal immune responses. Ideally, a vaccine should be administered mucosally in order to elicit mucosal protection. Several attenuated live viral and bacterial pathogens are registered as oral vaccines for human use, including the oral polio vaccine (Sabin) as well as attenuated strains of Salmonella typhi and Vibrio cholerae. These attenuated bacterial live vaccines-S. typhi Ty21a as well as V. cholerae CVD 103-HgR-are employed as vaccines against typhoid and cholera, respectively. In this manuscript, we review the immune responses that are induced by these vaccines, with a focus on mucosal immunity.

Strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine.

The proliferation of influenza viruses causes costly, recurrent, annual epidemics. Current vaccines, mainly administered parenterally, have been shown to be suboptimal in terms of efficacy, particularly where local IgA responses are concerned. Recent investigations of virosomes as delivery systems for viral HA and NA antigens have demonstrated an improved immune response. This paper investigates the efficacy of a novel virosome-based intranasal influenza vaccine by its ability to reduce disease symptoms and its effect on viral shedding in nasal secretions of immunised ferrets. The use of ferrets in the study of influenza vaccines is based on the good comparability between ferret and human response to the disease. Intranasal, as opposed to parenteral, administration of a trivalent virosome-based subunit vaccine adjuvanted with HLT provides an almost total prevention of virus shedding combined with a high level of immunological protection against homologous virus challenge. The ease of application of an intranasal vaccine may have positive repercussions in the adoption of influenza vaccinations, particularly in 'at-risk' groups.