1 . Overview of Vaccine Strategies

Ruth Arnon

This chapter describes the different strategies applied for vaccination against microbial diseases. These include vaccines against bacterial, viral and parasitic infections which led to tremendous improvement in public health. The use of both live attenuated or killed whole organisms and their sub-units is discussed, as well as more novel approaches, such as DNA vaccines, recombinant vaccines and epitope-based, or peptide vaccines. The advantages and disadvantages of each approach are presented, eluding to various considerations, such as efficacy, safety and cost of production. The application of passive vaccination, including the use of pooled IgG (IVIG) is also described in brief. As indicated, these combined strategies led to a long list of vaccines that are presently approved and licensed in the USA, Europe and many other countries, as summarized in a detailed table, which refers also to the pediatric combination vaccines DPT and MMR that are used worldwide, and led to drastic reduction of the incidence of infectious diseases.

2 . Designing Vaccines in the Era of Genomics

Fabio Bagnoli, Nathalie Norais, Ilaria Ferlenghi, Maria Scarselli, Claudio Donati, Silvana Savino, Michèle A. Barocchi and Rino Rappuoli

Genome sequencing has become routine, and modern vaccine design is taking advantage of the accumulating genomic information. Reverse vaccinology, an approach in our institute, is built on genome-based antigen discovery and has largely replaced classical vaccinology methods based on growing and dissecting the microorganism. The main advantage of the approach is the fast prediction of vaccine candidates. Most of the antigens will be surface exposed proteins, since these antigens are most likely accessible to antibodies. This approach can be applied to non-cultivable microorganisms, something difficult or impossible to do with conventional approaches. When the first reverse vaccinology project was started, in the year 2000, antigen identification was mainly based on bioinformatic analysis of one genome. Since then, the technique has shown its full potential, with the first genome-derived vaccine now in clinical trials and several vaccines in preclinical studies. In the meantime the approach has been improved with the support of proteomics, functional genomics and comparative genomics. Herein, we provide a description of the complete process: from antigen prediction to high-throughput purification, screening and selection of the vaccine composition. Furthermore, future applications of structural biology to vaccinology are discussed.

3 . New Analytical Approaches for Measuring Protective Capacity of Antibodies

Moon H. Nahm and Carl E. Frasch

Antibodies to the pneumococcal polysaccharide capsule protect the host by opsonizing pneumococci for host phagocytes, while antibodies to the meningococcal polysaccharide capsule protect by directly killing meningococci in the presence of complement. In vitro measurement of serum bactericidal antibody (SBA) against the meningococcus has been used for a long time as a measure of protective immunity. Technical developments of pneumococcal opsonophagocytosis assays (OPA) in the past decade permit measurements of opsonic capacity of sera from persons immunized with pneumococcal vaccines. Experience with OPAs shows that opsonic capacities of antisera are better than their antibody levels in predicting vaccine efficacy. Thus, measurements of opsonic capacity could be a surrogate of clinical studies of pneumococcal vaccines. By being the surrogate for clinical studies, the assays for protective function of antibodies would reduce the need for large clinical trials and facilitate vaccine developments and improvements.

4 . New Frontiers in the Chemistry of Glycoconjugate Vaccines

David R. Bundle

Methods for single point attachment of polysaccharides and oligosaccharides to protein carriers and T-cell peptides are briefly reviewed with emphasis on contemporary approaches that involve synthetic oligosaccharides with linker or tether chemistry designed for compatibility with synthetic strategies. The synthesis and evaluation of conjugate vaccines designed to combat infectious bacterial and fungal diseases, as well as promising attempts to design and test therapeutic cancer vaccine are summarized. The prevailing dogma that protective B-cell epitopes should be comprised of 10-20 monosaccharides is confirmed for several experimental vaccines including those directed toward Shigell flexneri and Shigella dysenteriae. However, several small epitopes composed of 3-5 monosaccharide residues are sufficient to induce antibody against the whole organism and to confer protection.

5 . Bacterial Protein Toxin Used in Vaccines

Jerry M. Keith

At first glance, the idea of using protein toxins as vaccines against bacterial human diseases seems somewhat of a paradox. However, in some diseases, the severe pathological effects manifested by the causative agents are mediated entirely by protein toxins. Thus, it seems reasonable to expect that if antibodies could be induced against the protein toxin, they should be effective at preventing severe disease. Of course, the obvious challenge is to detoxify the protein toxin activity without destroying its ability to induce neutralizing antibodies. From an academic point of view, it is ironic that early vaccines against diphtheria, tetanus, and whooping cough were successful without understanding what made them work. One of the keys to this puzzle was uncovered quite by accident when it was discovered that diphtheria toxin stock preparations stored in large earthenware jars too large to be autoclaved were being detoxified by the residual formalin that leached into the preparations from the formalin-sterilized jars. It took two decades for this discovery to be understood and appreciated to a point where formalin-treatment could be applied to produce toxoid preparations for vaccination. It then took another half a century to develop the scientific tools and knowledge needed to bring forth the new generation of vaccines, which are highly effective and less reactogenic. This chapter traces the scientific history, controversies, and development of diphtheria, tetanus, and pertussis vaccines.

6 . Adjuvants

David A. G. Skibinski and Derek T. O'Hagan

The development of new effective vaccines, especially those consisting of highly purified antigens, will increasingly require the inclusion of an adjuvant. With over half a century of experience, aluminium containing adjuvants (alum) will continue to be widely used and until very recently remained the only vaccine adjuvant approved for human use in the US. In recent years a number of studies have started to reveal a more detailed understanding of alum's mechanism of action. Here we review these recent advances as well as discussing considerations for optimal formulation of the adjuvant. We will also address the need for more potent adjuvants than alum, with particular emphasis on the discovery and development of MF59, an emulsion based vaccine adjuvant which as been licensed for more than ten years in more than 20 countries, for use in an influenza vaccine focused on elderly subjects (Fluad®).

7 . Mucosal Vaccines

Rajesh Ravindran and Bali Pulendran

The term "mucosal vaccination" has traditionally been used to describe strategies in which a vaccine is administered via the mucosal route. Unlike parenteral vaccination, mucosal vaccines do not require the use of needles, thus enabling vaccine compliance and reducing logistical challenges and the risks of acquiring blood borne infections. However, despite the great success of mucosal vaccines such as the polio vaccine, several formidable challenges hinder the effective elicitation of immunity against pathogens that invade mucosal sites. First, in humans the mucosal surfaces of the gut, lung, oral cavity and reproductive tracts are estimated to cover an area of 400 square meters, and thus represent the largest portal of entry for pathogens. Second, the acidic environments of many mucosal sites, and the delineation of mucosal sites by the epithelial barrier, pose challenges to the effective delivery of vaccines. Third, the mucosal immune system is faced with a somewhat schizophrenic challenge of having to launch robust immunity against mucosal pathogens, whilst restraining immune reactivity to commensals and food antigens. Fourth, the induction of the appropriate type of immune response is critical for effective protection against different pathogens. Fifth, the accurate quantitation of mucosal T and B cell responses pose unique challenges. Despite these challenges, recent advances in our understanding of the innate immunity and its regulation of adaptive immunity at mucosal sites, are beginning to offer new insights into strategies that result in immune protection at mucosal surfaces. In particular, several recent studies demonstrate that parenteral vaccination with the appropriate adjuvants can induce migration of antigen-specific T and B lymphocytes to mucosal sites. These advances promise to accelerate the development and testing of new mucosal vaccines against many diseases including HIV/AIDS. In this chapter, we will review the present mucosal immunization strategies and look at opportunities for exploiting newer developments for devising effective oral vaccines. Most infectious agents that infect humans do so via mucosal sites, principally the digestive, respiratory and genitourinary tracts. Immune defenses at mucosal surfaces therefore constitute a very vital part of the overall protective responses against these invading pathogens. Vaccines that are administered via the oral routes most proficiently induce the mucosal immune responses. In contrast, parenterally administered vaccines are generally poor inducers of mucosal immunity and are therefore less efficient against infections originating at mucosal surfaces (Lamm, 1997; Levine, 2000). However, only a few mucosal vaccines have been approved for human use (Table 1) (Levine, 2000). However, progress in research aimed at understanding the molecular and cellular mechanisms of the mucosal system is presently accelerating, allowing us to design innovative strategies for the development of mucosal vaccines.

8 . Intralymphatic Vaccination

Thomas M. Kündig, Pål Johansen, and Gabriela Senti

The immune response is initiated by dendritic cells (DCs) and other antigen-presenting cells. These cells are present in nearly all organs and tissues of the body, so that theoretically any organ or tissue could serve as a route for vaccine administration. The choice of route is therefore mainly based on practical aspects. Using conventional needle and syringe the subcutaneous or intramuscular route are standard. The dermis and especially the epidermis are technically more difficult to target, but are likely to gain more interest due to the recent development of micro-needle patches and needle free injection devices. Vaccine administration via mucosal surfaces such as nasal or oral vaccination represents another option for needle free vaccine administration.While all the above mentioned routes of administration have been proven to work and protect against childhood diseases, influenza and many other infectious agents, the discussion and comparison of these different routes usually focuses on patient convenience, reduction of pain and distress for children, cost and on the possibility for mass vaccination. In this review, however, we would like to focus on how the route of administration can enhance the efficacy of vaccination. Especially in therapeutic vaccination, i.e., in a smaller patient number that already suffers from a disease, vaccination efficiency rather than convenience is the main issue. This is particularly the case in therapeutic cancer vaccines and in allergen specific immunotherapy. Intralymphatic vaccination is a strategy to maximize immunogenicity and therefore vaccine efficacy. The main part of this review will discuss this long known vaccination route and its clinical applicability in therapeutic vaccination.

9 . The First Vaccine Obtained Through Reverse Vaccinology: The Serogroup B Meningococcus Vaccine

Jeannette Adu-Bobie, Beatrice Aricò, Marzia M. Giuliani and Davide Serruto

Neisseria meningitidis was isolated over one hundred years when Anton Weicshelbaum identified the causative agent of cerebrospinal meningitis. Since its isolation in 1887, N. meningitidis has been recognized to cause endemic cases, case clusters, epidemics and pandemics of meningitis and devastating septicaemia. Despite over one century since its discovery, scientists have yet to identify a universal vaccine for this deadly bacterium. Although vaccines exist for several serogroups of pathogenic N. meningitidis, serotype B (MenB) has eluded scientists for decades, until the advent of genomics. The genome era has completely changed the way to design vaccines. The availability of the complete genome of microorganisms combined with a novel advanced technology has introduced a new prospective in vaccine research. This novel approach is now known as "Reverse Vaccinology" and N. meningitidis can be considered the first successful example of its application. This chapter will describe the successful story of the development of the serogroup B vaccine, starting from the analysis of genome and finishing with the results obtained in clinical trials.

10 . Vaccines for Neglected Diseases

Allan Saul

Infectious diseases exert a major burden of disease in developing countries. While better use of existing vaccines would make an appreciable difference, the greatest burden is caused by diseases for which we currently have no vaccines. The picture, especially in children, is dominated by diarrheal and respiratory diseases. Paradoxically diseases have relatively low priority for funding in absolute terms, and especially in relationship to the burden of disease. Thus, new vaccines for these neglected diseases need both innovative scientific solutions and innovative development schemes involving scientific institutes, public financing and industrial input. The industrial input is critical: not only will vaccine manufacture require an industrial partner, but the knowledge to efficiently undertake the technical and clinical development leading to vaccine production largely resides in industry. A potentially important development in this area has been the recent formation of Industry Linked Vaccine Institutes: For example, The Novartis Vaccines Institute for Global Health and the Hilleman Laboratories. These are an important conduit for applying industrial know how for developing commercial vaccines to the pressing need for vaccines for neglected diseases of developing countries.

11 . Vaccines to Combat Pseudomonas aeruginosa Infections in Immunocompromised Patients

Jennifer M. Scarff and Joanna B. Goldberg

Pseudomonas aeruginosa is an important opportunistic pathogen that causes an array of nosocomial infections, such as ventilator-associated pneumonia and infections in cancer patients. P. aeruginosa infections are difficult to treat with antibiotics, making the need for other therapeutic options, such as vaccination, critical. Animal models, such as immunocompromised mice and dogs, have been used to investigate the efficacy of P. aeruginosa-specific vaccines. The main target antigen for these vaccines has been the lipopolysaccharide (LPS) of P. aeruginosa. These animal models have demonstrated that vaccination may be partially protective, but that a combination of vaccination with either antibiotic treatment or cell transfusion protocols typically works best. The efficacy of vaccination, particularly against LPS, has been investigated in human cancer patients. These patients were capable of mounting an immune response, but it was often short-lived or accompanied by severe side effects. An anti-Pseudomonas vaccine could be beneficial to aid in treatment of nosocomial infections caused by this bacterium, but would need optimization for better efficacy.

12 . Nosocomial infections: Staphylococcus aureus

Alice G. Cheng, Olaf Schneewind and Dominique Missiakas

Staphylococcus aureus is the most frequent cause of human skin and soft tissue, bloodstream and respiratory tract infections. Staphylococcal strains have acquired antibiotic resistance traits against available therapies and drug-resistant strains (MRSA, methicillin-resistant S. aureus) are currently isolated in up to 80% of hospital and 60% of community-acquired infections (CA-MRSA). Unlike pneumococci and group A streptococci; S. aureus infections do not raise immunity against subsequent infections. Consistent with this observation, early efforts to develop vaccines from whole-cell killed preparations of staphylococci have failed. More recent work characterized proteins and carbohydrates in the staphylococcal envelope and examined these molecules as protective antigens in vaccine studies. This article reviews the pathogenesis of S. aureus infections as well as past and current efforts that have been pursued to develop effective vaccines.

13 . Toward the Development of a Universal Vaccine Against Group B Streptococcus

Roberta Cozzi, John L. Telford and Domenico Maione

Group B Streptococcus (GBS) is one of the most common cause of life-threatening bacterial infections in infants and is also an emerging pathogen among adult humans, especially in the elderly, immunocompromised and diabetic adults. Capsular polysaccharide based vaccines of the most common serotypes present in the United States and Europe are in an advanced stage of development but they are not effective against serotypes present in other parts of the world. Many protein antigens have been studied for the discovery of an effective universal vaccine that could overcome serotype specificity. Thanks to reverse vaccinology and new technologies, a vaccine combination based on the pilus proteins has been discovered for the development of a universal GBS vaccine that is potentially capable of preventing all GBS infections.

14 . Vaccines against Streptococcus pneumoniae

James C. Paton

Existing vaccines against Streptococcus pneumoniae are targeted at the capsular polysaccharide (PS) of which there are 91 distinct serotypes. Polyvalent purified PS vaccines are immunogenic in healthy adults, but not in high risk groups such as young children and the elderly. Development of PS-protein conjugate vaccines has overcome the poor immunogenicity of PS in children, but the protection imparted is strictly serotype-specific, and the number of included serotypes is even more restricted than in the PS vaccine formulations. Widespread introduction of conjugate vaccines in developed countries has dramatically reduced the incidence of invasive pneumococcal disease due to serotypes included in the vaccine. However, these benefits are being eroded by increases in the incidence of disease caused by non-vaccine serotypes. Conjugate vaccines are also expensive, limiting their use in developing countries, where the burden of pneumococcal disease is greatest. Clearly, there is an urgent need to develop alternative pneumococcal vaccines that are (i) inexpensive, (ii) immunogenic in young children, and (iii) provide protection against all pneumococci regardless of serotype. Advances towards this goal are discussed herein, with particular emphasis on vaccines comprising pneumococcal proteins that contribute to virulence and are common to all serotypes.

15 . Veterinary Vaccines with a Focus on Bovine Mastitis

John R. Middleton

While novel approaches to vaccination against diseases of veterinary importance are being explored, currently marketed products, in general, employ old technology with the majority of products still being killed, modified live, or toxoid preparations. Due to the breadth of diseases encountered in veterinary medicine and the large number of vaccines marketed and under development, this chapter will focus on vaccines aimed at preventing bovine mastitis with a particular focus on Staphylococcus aureus, a bacterium that not only causes mastitis in cattle, but is a leading cause human infection. Vaccine developments for S. aureus in cattle will be compared with research aimed at preventing staphylococcal infection in humans. The remainder of the chapter will discuss other available vaccines aimed at preventing bovine mastitis and serve to illustrate that the goals of vaccination may differ depending on the type of infection being prevented.

16 . Vaccines Against Newly Emerging Viral Diseases: The Example of SARS

Bart L. Haagmans

Several newly emerging viral diseases in humans have been reported recently. The ability to identify and characterize the relevant pathogen and develop safe and effective vaccines against these newly emerging pathogens in a timely manner is utmost importance. In this respect, the global response to the SARS epidemic provided valuable experience which can be utilized to respond quickly to future emerging viral infections. In only few weeks time the nucleotide sequence of this virus was available and through computational analysis of gene sequences diagnostic tests and vaccine candidates were identified and subsequently developed. Eight years after the first SARS outbreak several candidate SARS-CoV vaccines are at various stages of pre-clinical and clinical development. The "classical" inactivated whole virus vaccine as well as a DNA vaccine expressing the spike gene ultimately reached the phase 1 clinical trial testing. These vaccines induce neutralizing antibodies to SARS-CoV and protect against SARS-CoV challenge in diverse animal models. However, these vaccines still need to be further tested against viruses closely related to SARS-CoV that potentially may emerge and for the absence of significant side effects. The lessons learned from this outbreak combined with more recently developed techniques may aid the development of effective vaccines against future emerging viral diseases.