Section I. Innovative Technologies and Approaches in Vaccine Research

1. Deep Sequencing in Vaccine Research, Development and Post-marketing Surveillance

Stefano Censini, Silvia Guidotti, Giulia Torricelli, Rino Rappuoli and Fabio Bagnoli

Since it was established in the ‘70s by Frederick Sanger DNA sequencing has brought enormous contribution to virtually all areas of life and medical sciences. More recently, Next Generation Sequencing technologies (NGS) have gradually flanked and then replaced the Sanger method in most sequencing projects. The major aim of this chapter is to describe the extraordinary technological advances from the first to the third generation sequencing as well as the consequent expanding range of their applications. NGS is enabling affordable large-scale genomics, metagenomics, transcriptomics and epigenomics studies at extremely deep resolution. Another key area of NGS is the analysis of human B- and T-lymphocyte repertoires. The explosion of new sequencing technologies was paralleled by a similar advance in computational science and synthetic biology leading to the era of "teleporting life". Altogether these new technologies and their applications are significantly increasing chances to discover and develop efficacious vaccines.

2. New Bioinformatics Algorithms Applied to Deep Sequencing Projects

Mina Rho

Advances in high throughput sequencing technology have made significant contributions to research in genomics, epigenomics, and metagenomics. With abundant sequencing data currently available, we can now better investigate the dynamics of pathogen and host genomes, as well as their interactions within populations, to provide solid foundations for vaccine development and therapeutic research. In this chapter is provided an overview of bioinformatics algorithms developed for deep sequencing projects, in particular those related to vaccine development. These essential bioinformatics algorithms provide unprecedented opportunities to better understand the pathogens and human genomes, and their variants at single-nucleotide resolution. Many bioinformatics algorithms are also available for de novo identification and assembly of novel pathogen genomes and protein-coding genes. In order to catalogue the diverse immune repertoire, efficient bioinformatics algorithms have also been developed to detect recombination and hypermutations in T-cell receptors and antibodies. Current challenges in combining computational approaches to vaccine development and therapeutic research are also discussed.

3. Comparative Genomics Approaches for Tracking the Emergence and Spread of Disease-associated Bacteria

Tracy H. Hazen and David A. Rasko

The development of effective vaccines hinges on an understanding of the underlying genetic diversity of the disease-causing bacteria targeted by the vaccine. The decreasing cost of genome sequencing has lead to the use of comparative genomics of collections of bacteria associated with recent outbreaks. Large-scale comparative genomics studies have also been used to track the emergence and spread of antibiotic resistant Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), and Clostridium difficile. Also, comparative genomics has been used to understand the occurrence of the Haiti cholera outbreak and identify factors that may contribute to increase virulence in O157:H7 E. coli associated with outbreaks, and the O104:H4 E. coli responsible for the deadly German outbreak in 2011. Approaches used in comparative genomics studies range from gene-based comparisons of diverse groups of isolates, to SNP-based analyses of often clonally-related isolates associated with outbreaks. Both the gene-based and SNP-based analyses have lead to the development of assays for detection and classification of disease-associated bacteria and their virulence-associated genes as indicators of their pathogenic potential. In the following, we provide an overview of the utility of different comparative genomics approaches for investigating the emergence and dissemination of disease-associated bacteria and discuss specific studies that have resulted in the development of assays for rapid identification of clinical bacteria based on insights from comparative genomics studies. The use of whole-genome sequencing and comparative genomics for identifying outbreak-associated bacteria can aid public health decisions regarding the treatment of patients and the appropriate vaccines to distribute in order to prevent further spread of the outbreak.

4. Quantitative Proteomics in Vaccine Research

Massimiliano Biagini and Nathalie Norais

Functional genomics and proteomics have become power tools for all life science researchers and the application in the vaccinology field is particular intriguing. The urgent need for fast, effective and safer vaccines let a substantial shift towards quantitative approaches that will have a huge impact in all vaccine development stages. Proteomics is deeply contributing in the early phase of candidate discovery and it appears more and more implemented in the following phases of antigen characterization, formulation, safety and conservation in strains circulating in epidemic areas. In this chapter we will virtually retrace the brief history of proteomics, following the technical steps that have characterized the evolution of this "daughter" of functional genomics, deepening the studies that have contributed to the knowledge of pathogen biology and the microbiological and immunological mechanisms, fundamental for the development of new vaccines. And more importantly you will appreciate as precise quantitative analysis can now be carried out thanks to the latest advances in mass spectrometry. We will see how proteomic technologies have become an essential tool for the analysis of vaccines in the manufacturing phase. Finally, we will evocate the last great challenge in the field of vaccinology, the unification of diverse scientific knowledge to have a unitary vision of the relationship between human being immune system and the pathogen by System Biology.

5. Structural Biology in Vaccine Research

Danilo Donnarumma, Matthew J. Bottomley, Enrico Malito, Ethan Settembre, Ilaria Ferlenghi and Roberta Cozzi

The emerging strategy for next-generation vaccine development is the structure-based antigen design approach. To enhance vaccine efficacy the atomic-resolution structural approach, able to generate information on the overall antigen structure, becomes the driving force in the production of engineered antigens with improved immunological properties and biophysical attributes that facilitate manufacturing. The first and most important step of the structure-based approach is the definition of the regions of an antigen that are bound by protective antibodies: the epitopes, core elements in the molecular nature of the host-pathogen interaction. The definition of naturally-occurring epitopes (also called ‘epitope mapping') provides insight into the molecular features recognized by the host immune response upon infection by the pathogen. Information available about such interactions can help in the selection and in the rational-design of an antigen that elicits the desired neutralizing or bactericidal response upon immunization. Moreover, epitope-mapping data defines which parts of an antigen are exposed and accessible on the surface of the pathogen, and thus provides insights into the functional regions of the protein. Going beyond vaccine research, epitope mapping has several applications in development of disease diagnostics and immune-therapeutics. Many different epitope mapping studies have been performed in the last 3 decades, generating a wealth of information that has been analyzed and interpreted in many ways. Although these data are beginning to provide clues about the common themes of antigen-antibody interactions, most recently analyzed via computational studies, an empirical approach is still essential in order to generate reliable information about any specific antibody-antigen interaction. Herein we review different strategies to identify and characterize protective epitopes, from the simplest epitope mapping approach, such as the synthetic library approach, through to well-established approaches like X-ray crystallography, NMR and negative stain single-particle electron microscopy. We also discuss the emerging applications of mass spectrometry and finally the computational prediction of epitopes. We highlight several examples revealing how the combination of epitope mapping and structural biology has already enabled improved antigen design and discuss how such structure-guided antigen design approaches may evolve further in the future.

6. Cellular Screens to Interrogate the Human T and B Cell Repertoires and Design Better Vaccines

Jens Wrammert and Kaja Murali-Krishna

The success of vaccines depends upon their ability to induce memory T cells, B cells and long lasting antibody secreting plasma cells. These memory cells differ in quantity, quality, homing characteristics and persistence depending upon vaccine antigen, adjuvants and delivery systems. A thorough understanding of the receptor repertoire associated with protective immunity, and in some cases infection-driven pathology, or in situations such as autoimmunity, cancer, allergy or transplant rejection, is important for rational design of vaccines and therapeutics. In this chapter we will first briefly introduce general processes involved in the generation of naïve T and B cell receptor repertoire and the complex functional changes that can occur in these cells when they encounter antigen. Following this, we will discuss recent progress in the development of novel tools for B and T-cell receptor repertoire analysis combined with cellular states and functions. The concurrent development of genomics and detailed functional analyses, both on a global as well as at a single cell level, also allows us to gain additional insight into the mechanisms of immunological memory. Together, these cellular screens allow defining optimal antigens and adjuvants and ultimately enable the development of more efficacious vaccines.

7. Novel Strategies of Vaccine Administration: The Science Behind Epidermal and Dermal Immunization

Béhazine Combadière and Hélène Perrin

Following the era of vaccinia virus intradermal vaccination, the past two decades have seen interest focusing once again on the cutaneous routes of vaccination. The definition of the skin antigen-presenting cell (APC) phenotype and its function, gene regulation and interaction with the skin microenvironment and inflammatory reactions has added further complexity to the skin immune system. Indeed, activating the appropriate arm of the immune system, that which induces protection, is now one of the major challenges to be faced in the development of vaccines against infectious diseases. Numerous concepts for vaccine delivery to the skin have been developed. However, they have not yet met expectations: this challenge remains to be met by using innovative approaches in immunology and vaccine design. Therefore, understanding the tight skin molecular and cellular network might refresh our knowledge on skin immunization procedures and vaccine development and have important impact on future vaccination strategies.

8. TLRs as Targets to Develop Novel Adjuvants

Şefik Ş. Alkan

This chapter starts with a summary of our current understanding of innate immunity, which is the rapid responding arm of the immune system. Innate cells recognize microorganisms via molecular sensors collectively called pattern-recognition receptors (PRR). These evolutionarily conserved sensors consist of four families based on their molecular structures: C-type lectin receptors, NOD-like receptor, and RIG-I-like receptors and Toll-like receptors (TLRs). We will focus on the latter, as TLR agonists are molecules most advanced as vaccine adjuvants. Various degrees of information are available about TLR (TLR2-TLR9) agonists that are under development for the treatment of infectious diseases and cancer: while some are at very early stage, many are in clinical trials and a few are approved as vaccine adjuvants. Decades of TLR research have taught us several lessons. Just to name a few: There is variation in the cellular distribution of TLRs in different species, which makes the translation of animal data into the clinic difficult; simultaneous delivery of TLR agonist and the antigen is more efficacious; multiple TLR activation produces much better adjuvant effects. Equipped with these lessons, we are not far from utilizing multiple TLR agonists as adjuvants in 21st century vaccines against infectious diseases and cancer.

9. The Importance of Cell Mediated Immunity for Bacterial Vaccines

Alison G. Murphy and Rachel M. McLoughlin

Despite the fact that vaccines have been pivotal in controlling many serious diseases, traditional vaccine strategies are not without limitations. Current challenges include the development of novel strategies to confer protection against antigenically hypervariable pathogens, opportunistic pathogens, rapidly evolving anti-microbial resistant pathogens and non-cultivatable pathogens. In addition, some recently licensed vaccines offer only limited coverage (e.g. pneumococcal vaccines) and well-established vaccines have displayed decreasing immunogenicity (e.g. pertussis vaccine). Continued development of effective anti-bacterial vaccines will rely upon a clear understanding of the correlates of immunity against distinct pathogens. During the course of natural infection both cellular and humoral immune responses are utilised, therefore vaccines should induce antigen specific responses by both arms of immunity. Novel anti-bacterial vaccines should strive to activate adaptive cellular immunity, i.e. antigen-specific T cells, due to their well documented roles in induction of antibody isotype switching, cytolytic functions and regulation of phagocyte responses. The development of such vaccines will require a more lucid understanding of the contribution played by specific T-cell subsets in mediating immunity to natural infection. Additionally, inducing T cell responses via immunisation will require further investigation into the use of appropriate adjuvants, which have the capacity to direct specific T cell responses. New vaccines, which specifically target cellular immunity in addition to humoral immunity, maybe key to providing protection against bacterial infections for which antibiotics are currently the only available treatment. Furthermore, the incorporation of elements targeting cellular immunity in currently licensed vaccines could potentially improve their efficacy.

10. T Cell Inducing Vaccines

Sarah Gilbert

For some infectious pathogens a protective T cell response as well as or instead of a functional antibody response is necessary to protect against disease. The development of vaccines designed to induce T cell responses has been hampered by difficulties in finding appropriate technologies for measuring and characterizing the immune response as well as defining safe and highly immunogenic methods of vaccination. This chapter will review progress in both of these areas, and report on advances in development of vaccines for malaria, HIV, influenza, tuberculosis and cancer that rely on inducing T cell responses. Key publications are listed in Table 1.

11. Exploiting the Mutanome for Personalized Cancer Immunotherapy

Ugur Sahin, Sebastian Kreiter, John Castle, Martin Löwer, Cedrik Britten and Özlem Türeci

Cancer mutations conceptually represent ideal targets for cancer immunotherapy as they combine a favorable safety due to the lack of their expression in healthy tissues and capability of supreme immunogenicity as they are not affected by central tolerance mechanisms. However, the systematic immunotherapeutic targeting of cancer mutations is hampered so far as 95% of the mutations in a tumor are unique to that single patient and only a small number of mutations are shared between patients. We have recently introduced a personalized immunotherapy approach targeting the spectrum of individual mutations by joining innovations from different technology fields. Next generation sequencing (NGS) is applied to enable the rapid identification of somatic mutations in individual tumors (the mutanome). Immunoinformatic tools are applied for prioritization of mutated epitopes that are predicted to be highly immunogenic and presented on MHC molecules. RNA-based vaccines encoding multiple mutations can be rapidly and affordably synthesized as custom GMP drug products. Integration of these cutting edge technologies into a clinically applicable process holds the promise of successful targeting of cancer heterogeneity by the multi-epitope approach.

Section II. Challenges for the Decade of Vaccines

12. Malaria Vaccine Development: Progress to Date

Kevin Marsh

Malaria is a major global health problem with substantial morbidity and mortality. Some progress in reducing this burden has been made with existing malaria control measures and a vaccine would be an important addition. The malaria parasite is complex, presenting several thousand possible antigens expressed in various lifecycle stages. We examine the difficulties in translating the findings from immuno-epidemiology to vaccination strategies, and the difficulties in selecting an appropriate animal model. Despite the apparent complexity of the problem a partially successful vaccination approach has been progressed through to Phase III trials and a license application is expected. Furthermore whole parasite vaccination approaches appear highly effective in experimental challenge studies. Further improvements are expected as antigen selection and antigen delivery are optimized with rapid down-selection of approaches using in-vitro functional studies, global genetic parasite diversity, appropriately designed and interpreted animal models before proceeding to Controlled Human Malaria Infection studies and field trials. A highly effective malaria vaccine based on a multi-stage, multi component approach is technically feasible.

13. Tuberculosis

Else Marie Agger and Peter Andersen

Every four seconds in the world one person develops tuberculosis and someone dies from the disease every 20-30 seconds. Once considered a conquered plague, tuberculosis remains a major cause of human mortality and morbidity. The protective efficacy of the currently licensed vaccine, BCG, is highly variable and a new effective vaccine is considered the most effective way of controlling the disease. In 1997, NIAID hosted a workshop entitled "Tuberculosis vaccines: how close to human testing". In only fifteen years we have witnessed remarkable progress with thirteen new tuberculosis vaccines currently in various stages of clinical development and a plethora of other candidates in preclinical development. The vaccine candidates are designed either as whole-organism live mycobacterial vaccines to replace BCG or as subunit vaccines to boost BCG-induced immunity. With some of these second-generation tuberculosis vaccines approaching phase III efficacy testing in humans, the coming years will be critical for the prospects of a new tuberculosis vaccine.

14. HIV-1 Vaccine Development

Barton F. Haynes, Georgia D. Tomaras, Hua-Xin Liao and Andrew J. McMichael

Development of an AIDS vaccine is a global priority. In spite of one vaccine efficacy trial that showed an estimated 31% efficacy, other efficacy trials have failed. Some chronically HIV-infected individuals are able to make antibodies that broadly neutralize many HIV strains, but these antibodies are not readily made in response to vaccination. Broadly neutralizing antibodies have unusual traits such as autoreactivity and long heavy chain third complementarity determining regions, traits in B cell receptors that lead to tolerance control of B cell expansion. Another roadblock for HIV vaccine development comes from HIV integration into the host genome soon after transmission, and establishment of a latent pool of infected CD4 T cells that is resistant to both immune responses and anti-retroviral drugs. Finally, the high mutation rate of HIV leads to escape from both T and B cell responses, and necessitates vaccine induction of a polyclonal protective response. Thus, development of an HIV vaccine faces unprecedented problems, and is requiring the implementation of novel strategies to attempt to circumvent the extraordinary challenges being faced in HIV vaccine development.

15. Cancer Immunotherapy: The Road to Rejection

Peter E. Fecci, Christina Chen, Susanne Baumeister and Glenn Dranoff

Immunotherapy continues to gain both momentum and legitimacy as a rational mode of cancer therapy. The idea that the immune system, if correctly manipulated, might be capable of surveying, identifying, and eradicating, in precise fashion, those cells that have mutant or newly expressed proteins en route to neoplastic growth patterns has long proved intriguing. This idea has been gradually shaped into a variety of immunotherapy approaches ranging from antibodies to cell transfers to vaccines. A large number of tumor types are also under study, including melanoma, glioma, lung, prostate, breast, head/neck, cervical, ovarian, pancreas, renal cell, colorectal cancers, and lymphoma. This chapter will review the current state, applications, adjuncts, and challenges to the successful immune-based treatment of cancer, emphasizing clinical trials.

16. The Challenge of Developing Global Health Vaccines Against the Invasive Salmonelloses: Enteric Fever and Invasive Nontyphoidal Salmonella Disease

Calman A. MacLennan

There is a growing awareness of the significance of Salmonella disease as a major public health concern, particularly in the developing world. This encompasses both enteric fever, caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A), and gastrointestinal and invasive disease caused by Salmonella serovars collectively known as nontyphoidal Salmonellae (NTS). While responsible for gastroenteritis in high-income countries, NTS are a common cause of fatal invasive disease in low-income countries, particularly in Africa. Currently available licensed vaccines for use in man are Vi capsular polysaccharide (Vi CPS) and the Ty21a live attenuated vaccine. Both vaccines are targeted against S. Typhi and offer limited or no cross-protection against other serovars of Salmonella. Even against S. Typhi, these vaccines have some significant draw-backs, the main one being their lack of efficacy in children under two years of age. There is increased recognition that new vaccines are required to deal effectively with the global problem of Salmonella. A proper understanding of the modalities of protective immunity against Salmonella is required for the rational development of such vaccines, along with an appreciation of the targets of protective acquired immunity. At an epidemiological level, it is critical to know which population groups most need vaccination against salmonellosis. This chapter deals with the challenges posed by Salmonella to vaccine development. It considers the optimal requirements for new vaccines, particularly those with broad specificity that can be used across a spectrum of ages. The chapter also reviews vaccines against Salmonella that are currently in development and discusses innovations and prospects for vaccines of the future.

17. The Path to an RSV Vaccine

Christine A. Shaw, Max Ciarlet, Brian W. Cooper, Lamberto Dionigi, Paula Keith, Karen B. O'Brien, Maryam Rafie-Kolpin and Philip R. Dormitzer

Respiratory syncytial virus (RSV) is the greatest remaining unmet infant vaccine need in developed countries and an important unmet infant vaccine need worldwide. More than 40 years of effort have yet to result in a licensed RSV vaccine for humans. Key challenges to RSV vaccine development include a peak of severe disease at 2-3 months of age, problematic biochemical behavior of key vaccine antigens, a history of vaccine-mediated disease enhancement, and reliance on animal models that may not accurately reflect human disease processes. Potential paths to overcome these challenges include maternal immunization, structure-based engineering of vaccine antigens, the design of a novel platform for safe infant immunization, and the development of improved animal models for vaccine-enhanced disease.

18. Staphylococcus aureus

Linhui Wang and Jean C. Lee

Development of an effective vaccine to prevent Staphylococcus aureus disease in humans continues to be a major challenge for the research community. There have been four phase III clinical trials aimed at proving efficacy with a single component vaccine or immunotherapeutic, but each has failed at different stages of development. Vaccines comprising multiple staphylococcal proteins and polysaccharides and vaccines aimed at eliciting a T cell response are currently being explored, but there is little agreement on the ideal formulation. Major limitations include our failure to identify convincing correlates of protective immunity to S. aureus, as well as less than optimal models of staphylococcal infection in rodents. Despite these challenges, there are new approaches and ongoing efforts by both industry and academic labs to design an effective vaccine to contain this formidable pathogen.