Vaccinology: Principles and Practice

Covering all aspects of vaccine research and development in one volume, this authoritative resource takes a comprehensive and systematic approach to the science of vaccinology focusing not only on basic science, but also on the many stages required to commercialize and navigate the regulatory requirements for human application, both in the United States and Europe.

• Reviews in detail the process of designing a vaccine, from the initial stages of antigen discovery to human application
• Includes evaluation of vaccine efficacy and safety
• Details clinical trial design, including regulatory requirements
• Discusses the emerging field of active cellular immunotherapy

Vaccinology: Principles and Practice provides an invaluable resource for clinicians, scientific and medical researchers, lecturers and postdoctoral fellows working in the field of vaccines.

• Language: English
• Publisher: Wiley
• Release date: Jun 12, 2012
• ISBN: 9781118345344

Book preview

PART 1

Introduction

CHAPTER 1

Concept and Scope of Modern Vaccines

D. Huw Davies¹, Clint S. Schmidt², & Nadeem A. Sheikh³

¹School of Medicine, University of California at Irvine, Irvine CA, USA

²NovaDigm Therapeutics, Inc., Grand Forks, ND, USA

³Clinical Immunology, Research, Dendreon Corporation, Seattle, WA, USA

Introduction

Historically, vaccination has probably had the greatest impact on human health of any medical intervention technique. Immunization is the only cost effective solution that can arrest and even eradicate infectious diseases. The science of vaccinology can be traced to the ancient Chinese, who protected against smallpox by the process of variolation, in which small quantities of scabs from a lesion of an infected person were intranasally inoculated [1]. This process was revived in the early 18th century when Lady Mary Montagu, who had observed variolation being practiced in Turkey, advocated its use to prevent smallpox. Modern vaccinology started as a proper scientific endeavor by Edward Jenner's findings that cowpox pustules would prevent smallpox infection [2]. His work was the first to be evaluated scientifically and established the scientific basis for using a related but less dangerous pathogen to engender immune responses that are cross-protective against the more virulent pathogen [3]. The seminal work and findings of Jenner lay unexploited for nearly a century until Louis Pasteur demonstrated that chickens could be protected from cholera by inoculation with attenuated bacteria [4]. Similar experiments also showed that sheep could be protected from anthrax [5]. This concept of weakening a pathogen to invoke the immune system to produce a response forms the basis of immunity elicited by the Bacille Calmette-Guérin (BCG) tuberculosis vaccine, first administered in 1921 [6] and still in wide use today.

Vaccines are defined as immunogenic preparations of a pathogen that evoke an immune response without causing disease. While attenuation and inactivation of pathogens are conventional approaches, and are still used, modern vaccines also exploit recent developments in immunology, genomics, bioinformatics, and structural and protein chemistry. At the heart of all vaccines is antigen – the ligand of the receptors of T and B lymphocytes. Lymphocytes are the effector cells of the adaptive immune system that mediate immunologic memory responses – the very hallmark of vaccination – which set vaccination apart from other forms of modern immune system manipulation, such as broad-spectrum immunopotentiators, cytokine therapy, or passive transfer of specific hyperimmune globulins derived from human plasma.

Table 1.1 Incidence of disease and the year of peak rate in the USA prior to and after mass immunization programs were initiated.

Table 1-1

The scope of modern and future vaccines has widened considerably since the empirical approaches of the pre-genomic era. Vaccines can now be designed rationally, even customized to individual needs. Developments in many areas of vaccinology, from adjuvants, proteomics, expression library immunizations (ELI), and sub-unit vaccines, to innovative funding and philanthropy, continue to reach new milestones. However, there are challenges in the road ahead. The vaccines that have not yet been made either exceed the limits of current technology or there is a lack of incentive. Here we outline the limitations of current vaccine technology and, through the following chapters, identify technologies that may help the field of vaccinology to advance.

Triumphs and limitations of current vaccination

After access to affordable nutrition, clean drinking water, and sanitation, low cost vaccines are the single most cost effective healthcare measure that can be taken to protect human health. This is highlighted by the fact that mass immunization programs have directly resulted in the control of several infectious diseases. For example, rates of incidence of diphtheria, measles, mumps, pertussis, and a number of other common diseases have been reduced by over 99% in the United States (Table 1.1). In the case of smallpox, global eradication was achieved through a concerted effort led by the World Health Organization (WHO). For polio, a concerted eradication program has reduced the incidence year after year from approximately 35 000 cases annually to fewer than 4000 in 1996 (Figure 1.1). Similarly, as the number of immunizations against measles has risen over the past two decades, the number of reported measles infections has fallen (Figure 1.2).

Figure 1.1 Impact of polio eradication program upon cases of polio infection worldwide. Each year there has been a gradual decrease in the numbers of polio cases reported. Adapted from Vaccine & Immunization News No. 5, 1997 (WHO Publications).

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Figure 1.2 Impact of measles eradication program upon cases of measles infection worldwide. Shaded bars, coverage; line, number of deaths. Each year there has been a gradual decrease in the numbers of deaths due to measles. Adapted from Vaccine & Immunization News No. 4, 1997 (WHO Publications).

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In addition, by controlling infections, vaccines reduce expenditure on future treatment (Table 1.2). Such costs are highlighted by the Centers for Disease Control (CDC) [8], which estimates that for every dollar invested in immunization, between $2 and $29 are saved. In addition the entire cost of the global smallpox eradication program, approximately $32 million, is returned every 20 days in not having to vaccinate travelers. A specific case in point is made by the combined measles, mumps, and rubella (MMR) vaccine. Immunization with this combined MMR vaccine was estimated to provide $5.1 billion direct and indirect cost savings in the USA for 1992 alone [9].

Table 1.2 Cost effectiveness of childhood vaccines in the United States and the estimated returned savings, both direct and indirect, from vaccination.

Despite the impact of vaccines on childhood infectious diseases such as measles, diphtheria, polio, and meningitis, there are many infectious diseases that continue to thwart vaccination programs, particularly in resource-poor countries, such as malaria, salmonellosis, and tuberculosis. There are several reasons why we lack vaccines to these diseases:

Genetic instability. A major roadblock to vaccination against many pathogens is unpredictable antigenic variation. Antigenic variation per se is not an insurmountable challenge: current vaccine technology already protects us from pathogens with relatively small numbers of serotypes, for example polio (three serotypes) and rotavirus (four serotypes), and in principle scaling to dozens or even hundreds of strains of a pathogen, such as Streptococcus pneumoniae (ninety known serotypes), is possible. It is well known that an individual can become immune to a formidable number of strains of a particular pathogen, as evidenced by the acquisition of immunity to malaria or the common cold over the course of two or three decades of natural exposure. This could be replicated in a compressed time frame with the appropriate vaccine. The real challenge is to provide pre-existing immunity to pathogens or strains of pathogens that do not yet exist. While the pre-existing repertoire of the adaptive immune system clearly has the capacity to respond to and retain immunologic memory of any antigen, current vaccine production methods require these antigens to be known beforehand. HIV is the worst-case scenario of such a pathogen. Although we know antibodies to gp120 can confer protection, the ability of the virus to generate a seemingly infinite number of antigenically distinct molecules as it replicates has thwarted most attempts to develop a vaccine. This intractable problem remains a major hurdle to development of vaccines against organisms with unpredictable antigenic variation.

Complexity. This generally goes hand-in-hand with the size of the genome of a pathogen and the number of distinct stages in its life cycle. Most successful vaccines are against viruses, much fewer are against bacteria, and few are in development against parasites or fungi. In order to achieve immunity by vaccination, a vaccine has to be able to emulate the immunogenic components of natural infection without causing the disease. Attenuated organisms lack the pathogenicity of the parent pathogen and retain the ability to engender protective immunity. Often only a single immunization is required, and immunity is frequently life long. Using these criteria, the best vaccines are arguably therefore live, attenuated organisms. However, the trend today is toward the development of killed or subunit vaccines because they pose no risk of reversion. Killed organisms may retain some of the inherent immunogenic properties of the live organism (e.g., components in the cell wall) although such vaccines may contain live organisms if not prepared correctly. These risks mean killed organisms are also gradually being replaced in favor of safer subunit or recombinant protein-based vaccines or nucleic acid vaccination. Unfortunately, recombinant protein vaccines face the greatest challenges, particularly those aimed against bacteria or parasites. Unless pathogenicity is mediated by a single component (such as an exotoxin, which can be protected against using a simple toxoid vaccine) protective immunity appears to be mediated by responses to multiple antigens. To date, single recombinant protein vaccines have performed poorly in providing protection against bacteria.

Correlates of protection. A related roadblock is a lack of knowledge of the antigen(s) required to engender protection. We measure immune responses to a pathogen using in vitro tools, such as ELISAs, neutralization assays, and γ-interferon release assays. These can be very sophisticated and map the antigens recognized, and even the epitopes within, in fine detail. The assumption is made that these detectable responses overlap, at least in part, with the antibodies and T cells that mediate protection. However, many may be immunologically irrelevant. Simply because we can detect a response to a particular antigen does not always connote a primary role in protection. Conversely, an antigen that is critical for engendering a protective response may not be detected by our in vitro assays. Thus the antigen(s) used for a vaccine do not necessarily have to be particularly immunogenic in natural infection. The overlap between the measurable reactome and the protectome is a largely unexplored area.

Adjuvants. Expectations of modern vaccines can sometimes be unrealistic. Current vaccines are prophylactic and are administered to healthy individuals. Therefore any safety issues have to be weighed carefully against the benefit. While subunit vaccines are the safest of all our options, the gain in safety is a tradeoff in efficacy. Most subunit vaccines and recombinant protein vaccines lack the inherent proinflammatory properties of attenuated organisms, which have to be replaced by the inclusion of an adjuvant. For reasons that are still not fully understood, immunity generated by proteins formulated in adjuvants decays more rapidly than that generated from live organisms, thereby requiring booster immunizations. Swelling, aching, and fever – the very proinflammatory properties required of a good adjuvant – are considered unacceptable side effects. (The stress suffered by a parent when their child has a fever will serve as a reminder of this.) These are trivial compared to the disease itself, yet relatively few adjuvants are approved for human use, and those that have been approved are all mild. Ultimately it may be impossible to engender complete immunity by vaccination without causing disease of some sort, and the best vaccines are likely to be a compromise.

Modern approaches that impact vaccine design

Genomics

In the pre-genomic era, vaccines were made from animal pathogens, or human pathogens either attenuated by abnormal growth conditions or killed by chemical inactivation. Many successful vaccines were developed using this empirical approach. This gave way to extracts of pathogens – or subunit vaccines – where components of the pathogen were used in place of the whole organism. In the post-genomic era, the production of subunit vaccines has become more rational and the preparations of antigens more precisely controlled. Despite their drawbacks, recombinant protein vaccines have had, and will continue to have, a major impact on diseases caused by simple pathogens, especially viruses, where a single antigen is often enough to provide immunity (e.g., human papilloma virus, HPV). Even for more complex pathogens such as bacteria and parasites, there is still the expectation that recombinant protein vaccines can provide protection, particularly if adjuvanted cocktails of protective antigens are used. Continued progress in this area has been hampered by the identification of candidate antigens. The problem has been the sheer size of the genome and the number of potential antigens available, and until recently the discovery of potential subunit vaccine antigens have been piecemeal and non-systematic.

Modern high throughput approaches to proteome-wide expression and screening technologies promise to revolutionize the discovery of new vaccine antigens for old diseases. A recent antibody profiling study of acquired immunity to malaria in the Gambia, for example, identified antibodies to several antigens present in children with acquired immunity that are absent from children who were still undergoing seasonal bouts of malaria [10]. These antigens would be considered prime targets for vaccine development. Importantly, the same study revealed the antigens currently being evaluated in clinical trials were not among these discriminatory antigens. The conclusion from studies like this is that non-biased screening approaches may lead to the discovery of different antigen sets than conventional intuitive approaches. It remains to be determined whether these new antigens lead to better vaccines, and Part 3 of this book focuses on these new tech-nologies.

Improved delivery systems and adjuvants

Recombinant proteins are, for the most part, poorly immunogenic and require delivery in an immunogenic package. The most successful delivery systems for recombinant proteins are often based on macromolecular assembles of one sort or another, and can take the form of immune stimulating complexes (ISCOMs), liposomes, or virus-like particles. Suspensions of antigen bound to inorganic particles such as alum are also immunogenic. It seems dendritic cells are particularly efficient at ingesting and responding to insoluble, particulate antigens, but less so to soluble proteins.

Other steps can be taken to improve the immunogenicity of existing vaccines. For example, peptide vaccines suffer from short half life in vivo, which can be improved by chemical modification to improve stability. Nucleic acid vaccines, although showing great promise in animal models, currently have had less developmental success in humans. The reasons are still unclear but their efficacy can be improved by using live vectors to boost them. The immunogenicity of recombinant vectors such as vaccinia or adenovirus is blunted by pre-existing immunity. This can be overcome by using animal viruses as vectors, such as fowlpox, where pre-existing immunity does not exist. These examples and other antigen engineering technologies are examined in Part 4.

Therapeutic vaccination

Currently none of the licensed traditional vaccines for use in humans are therapeutic, but instead are prophylactic and depend on antibodies to block initial infection. A vaccine administered after infection in order to treat (not prevent) disease is a realistic goal of modern vaccination. Once a pathogen has established an infection, the type of immune response required to eliminate the infection depends largely on whether the pathogen remains extracellular or gains entry into cells, where it becomes inaccessible to antibody. The optimism for therapeutic vaccines comes from great strides in the 1980s and 1990s in understanding T-cell recognition and antigen processing/presentation, and the realization that vaccines specifically targeting cell-mediated immunity could engender protection against pathogens that reside within cells. Both CD8 and CD4 T cells can mediate killing of cells harboring intracellular pathogens, particularly viruses (CD8) and bacteria that reside in endosomal compartments (CD4). Many model systems in animals have shown proof-of-principle of therapeutic vaccination. The bottleneck to translating this to vaccine development is, as with antibody vaccines, the size of the pathogen genome. Uniquely with T cells, the problem is amplified if synthetic peptides are desired for vaccination. Again, high throughput proteomic screening platforms and ever improving predictive algorithms promise to define the antigens needed, while carefully selected delivery vehicles or adjuvants will ensure the correct T cell subset(s) is stimulated.

Although the field of therapeutic vaccination is still developing for infectious disease, some promising inroads have been made in the cancer immunotherapy field. These technologies are based on the ex vivo activation [11] and amplification of the specific cellular immune response, followed by re-infusion of immune cells to the patient, as opposed to the in vivo activation hopefully achieved by traditional vaccines. The oncology targets for this approach are many. However, the field is gaining momentum with the FDA approval of Dendreon Corporation's Provenge™ (sipuleucel-T) for asymptomatic, or minimally symptomatic metastatic, androgen-independent prostate adenocarcinoma [12].

A return to attenuated organisms?

With the notable exception of toxoids, the disappointing previous performances of single recombinant protein subunit vaccines against complex pathogens (bacteria, fungi, and parasites) compel us to continue the development of live attenuated vaccines alongside subunit vaccine development, to ensure the highest probability of discovering a successful vaccine against any particular pathogen. Live attenuated vaccines have many advantages over killed or subunit vaccines, although the safety requirements are more stringent owing to the risk of reversion to a pathogenic phenotype. Attenuated live bacterial vaccines currently licensed for human use include Mycobacterium bovis strain Bacille Calmette-Guérin (developed in the 1920s), Salmonella typhi Ty21a (1970s), and Vibrio cholerae CVD 103-HgR (1980s) [13]. The latter was derived by site-directed mutagenesis of the cholera toxin A gene (ctxA), and in some respects it represents the flipside of the traditional cholera toxoid vaccine. Although low hanging fruit for an attenuated vaccine, it points toward the rational way in which such vaccines may be made in the future. For most bacteria, multiple virulence factors are linked to pathogenesis. Traditional approaches to attenuation, such as forced adaptation to unusual culture conditions or radiation/chemical mutagenesis, are too hit and miss for modern rational approaches. With increasingly rapid annotation of sequenced pathogens comes the potential for systematic identification of virulence factors and their targeting for mutagenesis or deletion. Technologies for screening large numbers of mutants for attenuation and immunogenicity need to be developed, and will likely involve in vitro models.

Allied to this are vaccines based on animal pathogens – the Jennerian approach. The smallpox vaccine is often described as the prototype of all vaccines, and the only vaccine to have approached the eradication of a human disease. The principle of the original smallpox vaccine (which was cowpox) is somewhat different to the attenuated and killed vaccines that have followed. Cowpox is not an attenuated version of the human pathogen, but a closely related, less pathogenic, species of orthopoxvirus. The origins of vaccinia are not clear but modern phylogenetic analyses indicate it is a domesticated version of cowpox. Although vaccines based on animal pathogens are less pathogenic, attenuated strains are preferable. Replication-competent smallpox vaccines are being replaced by attenuated vaccinia strains such as MVA. The attenuated Mycobacterium bovis strain BCG, first produced as a vaccine against M. tuberculosis in the 1920s, also works on this Jennerian principle. More recent examples include the human-animal reassortant rotavirus vaccines that have been developed using animal rotaviruses engineered to contain antigens from the human rotavirus [14].

Improve existing vaccines and vaccine uptake

Most attenuated live organisms have limited efficacy, in part because the attenuation is so severe. Attempts to improve existing vaccines, such as with more potent adjuvants or adjuvant combinations, or improved manufacturing methods, is therefore another approach upon which modern technologies can be brought to bear. Basic research in immunologic processes will undoubtedly continue to reveal novel approaches to improving the immunogenicity of existing vaccines. The discovery of the role of Toll-like receptors [15,16] and the application of contemporary immunologic techniques [17] have helped our understanding of the basis of adjuvanticity. Likewise, our understanding of antigen processing pathways and different regulatory and effector T cell subsets has revealed the importance of antigen delivery in the type of immune response elicited. In the future, immunomodulators that switch off suppressive pathways and promote proinflammatory pathways, or ligands that target antigens to specific cells and tissues of the immune system, may be routinely engineered into vaccines. It is likely that our understanding of other critical processes, such as immunologic memory and immunodominance, will also become clearer in the near future and influence our design of vaccines and the adjuvants used.

It is worth remembering we do not need to discover new vaccines to make an impact on global health. The WHO estimates that 2.7 million children die annually from diseases that could be prevented with existing vaccines, almost half of which are caused by rotavirus and Streptococcus pneumoniae [18]. The majority of these are in resource-poor countries. The WHO's Expanded Programme on Immunization (EPI), first introduced in 1974, aims to bring vaccination to children throughout the world. The scheme was recently expanded to cover the world's poorest nations through the Global Alliance for Vaccines and Immunisation (GAVI) (www.gavialliance.org). Complacency and misinformation are problems in developing countries, and threaten to undermine vaccine-induced protection. Simply because a disease is no longer as common as it once was creates the illusion it is eradicated, allowing re-emergence if vaccination is not maintained. Clearly, mandatory childhood vaccination is important but remains essentially optional in most countries. Regardless of whether or not one believes there is a role for the MMR vaccine in the development of autism, the reduction in uptake of the MMR vaccine in response to the recent hysteria had a direct effect on the rise in cases of childhood measles [19].

Hurdles and challenges for the future

Non-infectious diseases as targets for modern vaccines

The identification of autoantigens associated specifically with cancer and autoimmune disease has opened up new opportunities for vaccination. These are predominantly therapeutic T cell-based vaccines administered to individuals who already have disease. This considerably extends the concept of a vaccine beyond the traditional immunogenic preparation of a pathogenic microorganism, and indeed the recently approved HPV vaccines are a significant advance in the prophylactic vaccination against a virus-associated cancer [20].

Transition from research to trial

The pages of vaccine journals (and indeed this very book) are full of novel and ingenious vaccines, delivery systems, adjuvants, vectors, and scientific methods. Yet only the simplest and safest vaccines are ever considered for clinical trial. The realities of obtaining necessary approvals, producing a vaccine to current good manufacturing practice (cGMP) standards, and finding funding are far removed from most academic laboratories where basic vaccine research is conducted. Even if a candidate is evaluated in Phase I or II clinical studies, the investment required to enter Phase III trial is beyond the scope of most government funding agencies and requires the involvement of industry. For example, it is estimated that the research and development costs of bringing Gardisil™, an HPV vaccine comprising four recombinant proteins, to market was in excess of $1 billion. There have been several attempts to overcome the economic barrier against the development of less lucrative vaccines and diagnostics, such as with tax incentives and guaranteed government purchases. Additionally, non-profit organizations, such as the Wellcome Trust, and more recently the Bill & Melinda Gates Foundation, have become pivotal drivers for vaccine development. Thus, with the cooperation between scientific, industrial, non-profit, and political entities, the field of vaccinology will continue to advance, meeting the world's unmet medical needs.

References

1 Fenner F, Henderson DA, Arrita L, Jezek Z, Ladnyi ID. Smallpox and its Eradication. World Health Organization, Geneva, 1988, p. 4.

2 Jenner E. On the Origin of the Vaccine Inoculation. Printed by D. N. Shury, London, 1801.

3 Jenner E. An inquiry into the causes and effects of the variolae vaccinae, a disease discovered in some of the western counties of England, particularly Gloucestershire, and known by the name of the cow pox, London 1798. In CNB Camac (Ed.) Classics of Medicine and Surgery. Dover Press, London, 1959, pp. 213–40.

4 Pasteur L, Chamberland CE, Roux E. Sur la vaccination charbonneuse. CR Acad Sci Paris 1881;92:1378–83.

5 Pasteur L. Une statistique au sujet de la vaccination préventive contre le charbon portant sur quatre-vingt-cinq-mille animaux. CR Acad Sci Paris 1882;95:1250–52.

6 Calmette A. La Vaccination préventive contre la tuberculose par le BCG. Masson, Paris, 1927.

7 Saldarini RJ. For vaccines, the future is now. Nat Med 1998;4:485–91.

8 Bloom BR, Widdus R. Vaccine visions and their global impact. Nat Med 1998;4:480–84.

9 Hatziendru EJ, Brown RF, Halpern MT. Report to the Centers for Disease Control and Prevention. Cost benefit analysis of the measles-mumps-rubella (MMR) vaccine. Batelle, Arlington, VA, 1994.

10 Crompton PD, Kayala MA, Traore B, et al. A prospective analysis of the Ab response to Plasmodium falciparum before and after a malaria season by protein microarray. Proc Natl Acad Sci U S A 2010;107:6958–63.

11 Schmidt CS, Morrow WJW, Sheikh NA. Smart adjuvants. Expert Rev Vaccines 2007;6:391–400.

12 Wesley JD, Whitmore JB, Trager JB, Sheikh NA. An overview of sipuleucel-T: autologous cellular immunotherapy for prostate cancer. Hum Vaccin Immunother 2012;8(4).

13 Levine MM, Kaper JB, Herrington D, et al. Safety, immunogenicity, and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103-HgR. Lancet 1988;2:467–70.

14 Dennehy PH. Rotavirus vaccines: an overview. Clin Microbiol Rev 2008;21:198–208.

15 Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996;86(6):973–83.

16 Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388:394–7.

17 Marrack P, McKee AS, Munks MW. Towards an understanding of the adjuvant action of aluminium. Nat Rev Immunol 2009;9:339–48.

18 Immunization Summary: The 2007 Edition. Geneva: United Nations Children's Fund & World Health Organization, Geneva, 2007.

19 Eaton L. Measles cases in England and Wales rise sharply in 2008. BMJ 2009;338:b533.

20 Hariri S, Unger ER, Powell SE, et al. The HPV vaccine impact monitoring project (HPV-IMPACT): assessing early evidence of vaccination impact on HPV-associated cervical cancer precursor lesions. Cancer Causes Control. 2011, Nov 23 [Epub ahead of print].

PART 2

Principles of Vaccine Design

CHAPTER 2

Strategies to Stimulate Innate Immunity for Designing Effective Vaccine Adjuvants

Heather L. Wilson¹,², Scott Napper¹,², George K. Mutwiri¹,³, Sylvia van Drunen Littel-van den Hurk¹,⁴, Hugh Townsend¹,⁷, Lorne A. Babiuk⁵, Andrew A. Potter¹,⁶ & Volker Gerdts¹,⁶

¹Vaccine & Infectious Disease Organization, Saskatoon, Canada

²Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada

³School of Public Health, University of Saskatchewan, Saskatoon, Canada

⁴Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada

⁵Office of Vice President Research, University of Alberta, Edmonton, Canada

⁶Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada

⁷Department of Large Animal Clinical Sciences, University of Saskatchewan, Canada

Principles of vaccine design: stimulation of innate immunity

Stimulation of innate immunity is an important requirement for the induction of effective immune responses following vaccination. The majority of today's vaccines contain adjuvants that were added for the purpose of enhancing the magnitude, type, onset, and duration of the acquired immune response. The recognition of the role and importance of adjuvants in the stimulation of innate immunity and the relationship between innate and acquired immunity are more recent.

Innate and acquired immunity are intimately linked through antigen-presenting cells (APCs), in particular macrophages and dendritic cells (DCs). In an immature stage, these cells specialize in uptake of antigens and are equipped with a variety of pattern recognition receptors (PRRs), which facilitate the recognition of highly conserved pathogen-associated molecular patterns (PAMPs) such as bacterial and viral DNA, lipopolysaccharide (LPS), and flagellin (Table 2.1) [1]. Signaling through PRRs results in activation of multiple signaling pathways and the subsequent increase in expression of a plethora of effector molecules, including major histocompatibility complex (MHC), co-stimulatory molecules, and proinflammatory chemokines and cytokines (see Chapter 4 for more detail). Once activated, DCs begin to mature and home to the draining lymph node, where they present the antigen to naïve lymphocytes as part of the specific or acquired immune response (Figure 2.1). This maturation process is characterized by a loss of endocytic and phagocytic capacities and an increase in the surface expression of co-stimulatory molecules such as CD80, CD86, and CD40 [2,3]. With maturation, DCs also change expression of chemokine receptors (CCR) from those that are expressed in the peripheral tissues (CCR1, CCR2, CCR5, and CCR6) toward expression of CCR7, which recognizes CCL19 and CCL21. These two chemokines are constitutively expressed in T-cell zones of secondary lymphoid organs and facilitate the migration of mature DCs into the lymph nodes for antigen presentation to T cells [4-6].

Figure 2.1 Activation and maturation of dendritic cells. CCR, C-chemokine receptor; TLR, Toll-like receptor; CLR, C-type lectin receptor; NLR, NOD-like receptor; MHC II, type II major histocompatibility complex; TCR, T cell receptor.

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Table 2.1 PRRs and their activating PAMPs.

Table 2-1Table 2-2Table 2-3

Effective antigen presentation is mediated by a sequence of signals, the first being the antigen itself. The second and third signals are provided by co-stimulatory molecules and secreted cytokines, which ultimately determine the nature of the acquired immune response. Co-stimulatory molecules have both positive and negative modulatory effects on T- and B-cell activation and include members of the B7/CD28 family, tumor necrosis factor (TNF) superfamily, and the signaling lymphocyte activation molecule family (see Chapter 27). Depending on the nature of the maturation stimulus, the subset of dendritic cells, and the local environment in which antigen is recognized, dendritic cells are able to prime naïve T cells and then induce clonal expansion and differentiation into T helper 1 (Th1), Th2, or Th17 cells, all of which are distinguishable on the basis of their receptors and subsequent cytokine production profile [7,8]. Dendritic cells that induce a proinflammatory immune response are referred to as DC1 or DC2. These are the cells that ultimately induce Th1-type or Th2-type immune responses, respectively. Conversely, tolerogenic dendritic cells, referred to as DC0, are required for induction of tolerance and immune suppression and are characterized by expression of CD154. Thus, it is the nature of the danger signal at the initial site of infection that provides the immune system with the necessary information regarding the nature of the antigen and instructs the type of immune response needed to control the infection.

In the absence of inflammation or infection, dendritic cells are considered quiescent, not fully mature, and present self-antigens or non-immunogenic proteins leading to T cell deletion, anergy, or differentiation into regulatory cells [9]. This important immunologic process is designed to purge the peripheral T-cell repertoire of autoreactive T cells that have escaped thymic depletion and could potentially give rise to autoimmunity.

Innate immune stimulators

The stimuli involved in the activation and maturation of dendritic cells can act either independently or synergistically to promote cytokine secretion as well as upregulation of PRR expression. Integration of the host response to several PAMPs or damage-associated molecular patterns (DAMPs) allows for a highly tailored immune response [10,11]. Most pathogens contain several PAMPs that are recognized by the host cell PRRs, suggesting that the immune responses do not act in isolation but instead act in concert to elicit protective immune responses. Therefore, it is logical to design vaccines with multiple PAMPs/DAMPs to stimulate complementary and/or redundant PRR signaling pathways to mimic what occurs in nature. Vaccine components should instruct dendritic cells at the site of vaccination as to the type of immune response required to establish effective immunity and immunologic memory to combat subsequent, natural infection. Here we will review Toll-like receptors (TLRs) and non-TLRs for their potential use as vaccine adjuvants, alone and in combination, and describe how activation of their corresponding innate immune receptors may contribute to instruction of adaptive immunity and/or vaccine efficacy.

Toll-like receptors

TLRs are germline encoded PRRs that recognize a variety of PAMPs associated with bacteria, viruses, parasites, and fungi to initiate innate immune responses and to instruct adaptive immunity [12,13] (see Chapter 28). TLRs are highly conserved type I integral membrane proteins that share a conserved Toll/interleukin 1 (IL1) receptor (TIR) domain with IL1 receptors [14]. The ligand-binding domain consists primarily of a repeating pattern of a leucine-rich repeat (LRR) motif, which provides an adaptable structural matrix for biomolecular interactions with a variety of distinct ligands [15,16].

Signaling through TLRs involves an intracellular cascade that includes the myeloid differentiation primary response gene 88 (MyD88), IL1 receptor activated kinase (IRAK), TIR-associated-protein (TIRAP), Toll receptor-associated activator of interferon (TRIF), Toll receptor-associated molecule (TRAM), and TNF receptor-associated factor 6 (TRAF6), leading to activation of NF-κB [12] (Figure 2.2). NF-κB activation results in a proinflammatory response through the induction of proinflammatory cytokines, and induction of MHC molecules and co-stimulatory signals that provide a link from pathogen recognition by the innate immune system to activation of the adaptive immune system (see Chapters 3 and 4) [17]. Some of the specific mechanisms by which activation of the TLR system promotes adaptive immune responses include: (i) antigen internalization and maturation of DCs [18]; (ii) influencing migration of DCs [19]; (iii) promoting Th1 responses [20]; (iv) cross-priming and -presentation [21,22]; (v) reversal of tolerance [23-26]; and (vi) upregulation of MHC and co-stimulatory molecules [27,28] (see Chapter 27). Comparative evaluation of various TLR ligands as adjuvants must consider the appropriateness of the resulting responses as well as properties of the ligand, such as stability and ease of production.

Figure 2.2 PRR signaling events lead to production and maturation of proinflammatory cytokines. The innate immune response responds in a general manner to factors present in invading pathogens. Pathogen-associate patterns activate the innate immune response, trigger an inflammatory response, and ultimately stimulate antigen-specific immunity. Both ligands and simplified pathways are shown. Adapted from Biocarta (www.biocarta.com/pathfiles/h_tollPathway.asp) with permission.

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TLR1/2/4/6 agonists

The bacterial outer-surface lipoprotein (OspA) has been shown to possess adjuvant activity in a manner that is dependent upon the presence of TLR1 in humans, and both TLR1 and TLR2 in mice [29]. In humans, the efficacy of an induced response correlates with levels of TLR1 expression [29]. In mice, a tri-palmitoyl-S-glyceryl-cysteine (Pam3Cys)-modified OspA vaccine offered protection even in TLR2−/− mice, indicating the potential presence of additional adjuvants [30]. Similarly, Haemophilus influenzae type b (Hib) outer membrane protein complex (OMPC) vaccine-induced proinflammatory cytokines, but not antigen-specific IgG titers, were also TLR2-dependent, indicating that other adjuvant factors may be present in this vaccine formulation [31]. Two bacterial membrane components, macrophage-activating lipopeptide 2 (MALP-2) of Mycoplasma spp. and a synthetic bacterial lipopeptide PAM3CSK4, are potent adjuvants [32,33] recognized by TLR2/6 and TLR2/1 heterodimers, respectively [34,35].

TLR3 agonists

TLR3 plays a critical antiviral role through recognition of viral double-stranded RNA (dsRNA) as well as nucleic acid intermediates of viral replication. A synthetic analog of dsRNA, polyinosinic:polycytidylic acid (poly I:C), has been investigated as a therapeutic agent in patients for treatment of leukemia [36]. Poly I:C, in combination with chitin microparticles, offers protective immunity against pathogenic strains of influenza virus [37]. Mechanistically, the ability of dsRNA to promote maturation of CD8α+ DCs and induction of CD4+ and CD8+ T cell responses through interferon-mediated cross-priming is thought to be critical to the ability of TLR3 to function as an effective target for adjuvants [21].

TLR4 agonists

LPS, the ligand of TLR4, has been shown to be a potent vaccine adjuvant (see Chapter 28) [38,39]. LPS has been modified to monophosphoryl lipid A (MPL), which is much less toxic and has been used as a vaccine adjuvant in human clinical studies [40,41]. An aqueous formulation of MPL1 and alum, AS04™ (GlaxoSmithKline), achieves higher antibody titers with fewer injections and was employed as an adjuvant for a licensed hepatitis B (HBV) vaccine (Fendrix1). Other TLR4 agonists, such as lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [39], glucopyranosyl lipid A [GLA] and E6020), induced immune responses compatible with their application as adjuvants [42].

TLR5 agonists

Recombinants of the TLR5 agonist flagellin exert potent adjuvant activity that enhances protection from Listeria monocytogenes (p60 and listeriolysin O) and influenza (matrix 2 proteins, M2e [43]).

TLR7 and 8 agonists

Viral single-stranded RNA (ssRNA) and synthetic imidazoquinolins are potent ligands for TLR7 and TLR7/8 in mice and humans, respectively, for induction of type I interferons (IFNs) and for the promotion of cellular immune responses [44,45]. The distinct pattern of expression of TLR7 and TLR8 in humans (where TLR7, but not TLR8, is highly expressed in plasmacytoid DCs, and TLR8, but not TLR7, is highly expressed in monocytes) explains the differential responses of particular cells to these ligands. Activation of plasmacytoid DCs through TLR7 results in production of type I IFNs, while activation of monocytes through TLR8 induces synthesis of proinflammatory cytokines [46]. TLR7 agonists, such as imiquimod, have been used as immunotherapeutics for treatment of a variety of disorders such as genital warts, actinic keratosis, hepatitis B/C, and cancer [41,47,48,49]. TLR7/8 agonists enhance the generation of Th1 responses and CD8 T cell proliferation when used as adjuvants with HIV Gag protein vaccine [50].

TLR9 agonists

Toll-like receptor 9 has been the focus of considerable research for the ability to modulate its activity, and subsequent innate immune responses, through DNA-based immunotherapeutics. The immunostimulatory action of bacterial DNA can be effectively mimicked with synthetic, single-stranded oligodeoxynucleotides (ODNs) that are typically 24–30 nucleotides in length. These ODNs are attractive as therapeutics because of their low cost, chemical stability, and ease of production. In macrophages, dendritic cells, and B cells, CpG ODNs induce production of proinflammatory cytokines and chemokines to shift the host's immune response to favor a Th1 response, accounting for their action as vaccine adjuvants for a variety of bacterial and viral antigens in a large number of species [51].

Non-Toll-like receptors

Neutrophil serine proteases

Neutrophils are the first cells recruited to the site of infection whereupon they can directly initiate an attack against the invading pathogens or modify the local environment to promote increased immune cell recruitment. To kill pathogens, neutrophils phagocytose and sequester pathogens into the phagolysosome, where they release large quantities of reactive oxygen species (ROS), antimicrobial peptides, and serine proteases such as cathepsin G, neutrophil elastase, and proteinase 3 [52]. Upon activation, these zymogens are released through exocytosis to assist in extracellular killing. Upon cleavage by serine proteases, CCR1 ligands show up to 1000-fold increase in monocyte recruitment [53]. Similarly, cleavage of chemerin by cathepsin G and elastase triggers increased dendritic cell recruitment [54]. Neutrophils release granule proteins and chromatin, which together form extracellular traps (NETS) that bind bacteria within minutes of activation and release [55]. These localized areas are exposed to a high concentration of serine protease, which degrades virulence factors and kills Gram-positive and Gram-negative bacteria [55]. The importance of these proteases is evident in experiments in which mice with normal superoxide production but deficient in neutrophil-granule proteases such as cathepsin G and elastase cannot effectively combat and clear staphylococcal and candida infections [56,57]. Serine proteases provide the host with a mechanism to control and/or fine tune the immune response, making these proteins very attractive as vaccine adjuvants [54].

NOD-like receptors

NOD-like receptors (NLRs) are PRRs comprised of three domains: an N-terminal effector binding region (such as caspase recruitment domain [CARD], pyrin domain [PYD], and baculovirus IAP [inhibitor of apoptosis protein] repeat [BIR]), which mediate protein-protein interactions; a NOD domain, which is responsible for nucleotide binding and self-oligomerization; and a C-terminal leucine rich repeat (LRR), which detects conserved microbial patterns and modulates NLR activity (reviewed in [58]). Upon ligand binding, these receptors undergo oligomerization, associate with accessory proteins, and ultimately induce NF-κB and MAPK signaling. NOD1 is an NLR that is present in a variety of cell types, but NOD2 appears to be restricted to macrophages, DCs, Paneth cells, keratinocytes, and epithelial cells [58]. NOD1 recognizes the peptidoglycan fragments FK156 and meso-DAP (iE-DAP) [59] whereas NOD2 recognizes muramyl dipeptide (MDP) [60], a component of Freund's Complete Adjuvant (FCA) [61]. When used in conjunction with lipophilic carrier systems such as liposomes, oil-in-water emulsions, or some lipophilic derivatives, MDP induces strong cellular immunity (reviewed in [62]). MDP is too pyrogenic and arthritogenic to be used as an adjuvant in humans.

The inflammasome

Members of the NLR family such as Nalp1, Ipaf, and cryopyrin respond to damage and inflammatory signals by complexing with caspase 1 and an adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD domain) to form the inflammasome, which mediates activation of caspase 1 [63,64,65]. Activation of the inflammasome requires DAMPs such as ATP, toxins, HMGB1, crystals, or membrane-damaging molecules. Ligands for Ipaf include flagellin from Salmonella typhimurium, Legionella pneumonia, and Pseudomonas aeruginosa, which are delivered by the type III or type IV secretion system into the cytosol [66,67,68]. Cryopyrin inflammasomes may respond to changes in cellular potassium, MDP [69], LPS [70], and uric acid [71]. Activated caspase 1 processes immature proinflammatory cytokines pro-IL1β, pro-IL18, and pro-IL33 into active proteins. IL1β and IL18 in turn recruit inflammatory cells to sites of infection, and IL33 promotes Th2-biased immunity [72,73]. Thus, IL1β, one of the key players in the innate immune response, requires TLR signaling through various PAMPs for production and secretion, but it requires DAMP-dependent activation of the inflammasome to become activated. Inflammasome agonists should be regarded as useful components of vaccines and immunostimulators.

One of the most recognized inflammasome agonists is alum, an adjuvant that has been used in humans for over 50 years. However, its mechanism of action has only recently been discovered. Alum was originally thought to assist in the depot effect of the vaccine; that is, to cause the antigen and other vaccine components to reside for an extended period of time at the site of injection. It has since been shown to increase antigen uptake by dendritic cells in vitro [74], and to induce myeloid cell migration to mouse spleen, where the myeloid cells may play a role in priming and expansion of antigen-specific B cells [75]. Intraperitoneal injection of alum induces monocyte recruitment and migration to local lymph nodes, where they differentiate into inflammatory dendritic cells capable of priming T cells [76]. Alum has been shown to synergize with TLR agonists to enhance both cellular and humoral immune responses compared to each adjuvant alone [77]. Recent studies have shown that alum is phagocytosed by macrophages and is responsible for activation of the capase 1 inflammasome [78,79].

RIG-1-like receptors

dsRNA generated during viral replication is recognized by the cytoplasmic RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), which associate with interferon-β promoter stimulator 1 (IPS-1) and signal through NF-κB signaling cascade and IFN regulatory factor 3 (IRF3) in a caspase 8 and caspase 10 dependent pathway [80]. Activation of caspase 8 and caspase 10 induces inflammatory cytokine expression [80,81], inhibition of translation and viral replication [82], and direct activation of DCs and natural killer (NK) cells, which subsequently promote the survival and effector functions of T and B cells [83,84]. Understanding how these viral sensors mediate immunity will be vital to the development of designer vaccines.

C-type lectin receptors

C-type lectin-like receptors are cell-surface receptors that bind carbohydrate structures and facilitate uptake of pathogens into dendritic cells. Examples of these lectin-like receptors are dectin-1, the dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN), and mannose receptor (MR). MR plays a role in phagocytosis, cellular migration, intracellular signaling (such as IFN-γ production, NF-κB production, etc.), and MHC class II presentation, and it may play a role in resolution of inflammation [85,86,87]. DC-SIGN can activate NF-κB directly to induce proinflammatory cytokine signaling [88]. Dectin-1 recognizes fungal pathogens and, in conjunction with TLR2, promotes production of TNF-α to control fungal pathogenesis [89]. C-type lectin receptors therefore recognize pathogens via their carbohydrate moieties and promote an inflammatory response.

Practical applications for adjuvants

Action

Adjuvants enhance or modulate the immune response in several ways, including formation of an antigen-adjuvant depot, chemoattraction of appropriate immune cells to the site of antigen administration, targeting or delivery to APCs, and direct or indirect immunomodulation [90] (see Chapter 25). Redundancy of PRRs suggests that the host can sense pathogens in many different tissues through a variety of cells and within various cellular compartments. There is evidence that TLRs and NLRs may regulate and/or compensate for each other to prevent overproduction or underproduction of proinflammatory cytokines, respectively. NOD1 and NOD2 agonists synergize with TLR ligands to promote a strong proinflammatory response (reviewed in [58]; [91]. For example, OVA and FK156 prime antigen-specific T-cell and B-cell immunity with a predominant T helper (Th2) polarization profile; however, in the presence of TLR agonists, FK156 can promote increased Th1, Th2, and Th17 responses [92]. The live-attenuated yellow fever vaccine 17D (YF-17D), one of the most successful vaccines available, activates TLR2, 7, 8, and 9 [93], suggesting that the success of at least some of the live vaccines may be due to their ability to activate a number of TLR combinations. Thus, the requirement for multiple agonists to induce potent responses may be a mechanism by which the immune system exerts a stringent combinatorial security code whereby at least two microbial products are required to stimulate a strong immune response to pathogen invasion [94].

Safety

Regardless of the mechanism of adjuvanticity, a certain level of inflammation at the injection site is required for recruitment of immune cells, in particular APCs, and generally is considered necessary for vaccines to be effective. A relationship exists between the immune response and tissue reactions induced by adjuvants, with stronger adjuvants often generating more tissue damage. However, for new adjuvants to become licensed, a balance between safety and adjuvanticity leading to maximal immunogenicity is essential. Although multiple compounds have adjuvant activity under experimental conditions, many of them cause significant tissue damage or other adverse effects following immunization. FCA, a very effective and commonly used adjuvant in experimental animal models, causes significant side effects, including pyrogenicity, leukocytosis, uveitis, and adjuvant arthritis. EMULSIGEN®, an oil-in-water adjuvant licensed for veterinary use, can cause cellulitis and myositis after intramuscular injection when used at 30% (v/v) [95]. Quil-A and ISCOMs can cause an acute hypersensitivity reaction, hemolytic activity, and minor local reactions due to the detergent activity of Quil-A [90], although QS-21, the less toxic purified fraction of Quil-A, is currently used in the recently licensed adjuvant AS02 [96]. Alum may cause side effects in some instances, including erythema, subcutaneous nodules, contact hypersensitivity, and granulomatous inflammation [97]. It is generally accepted that cats immunized with vaccines formulated with alum show a prevalence for the development of sarcomas although whether alum is the direct cause is still a matter of conjecture [98].

The safety and suitability of an adjuvant is also determined by its ability to direct the immune response to be Th1- or Th2-biased, or balanced immunity (see Chapter 25). The tendency of conventional adjuvants such as alum to induce a Th2-type biased immune response may result in failure of protection or even immunopathologic responses following intracellular infections with organisms such as Leishmania major [99] and Schistosoma mansoni [100], both of which require a Th1-type immune response for control. Formalin-inactivated respiratory syncytial virus (FI-RSV) vaccine given to children in the 1960s [101-103] was not protective and even enhanced clinical disease, sometimes resulting in death, after subsequent exposure to RSV. In this case both the inactivation procedure and the adjuvant likely played a role in the induction of a non-neutralizing Th2-biased response. T-helper type 2-dominant immune responses are also associated with allergy, asthma, and autoimmune disease [104,105]. Thus, safe but effective adjuvants are urgently needed for future vaccines.

Summary

Adjuvants have long been used to improve the immune responses to vaccines. Historically, the choice of substances for use as adjuvants was highly intuitive, a process that resulted in the identification of only a few adjuvants that have proven safe and effective for use in human and animal vaccines. To be efficacious, vaccines must stimulate the development of both innate and adaptive immunity. Therefore, a deeper understanding of the complex and intimate links between innate and adaptive immune responses and the ways in which adjuvants can be used to enhance and refine these responses is required for the efficient development of vaccines. The research summarized in this chapter has done much to help us understand the mechanisms by which adjuvants work. Investigations along these lines will continue to be essential to the process of identifying and developing safer and more effective vaccine adjuvants.

Acknowledgements

The authors’ laboratories are funded through grants from the Bill & Melinda Gates Foundation, the Krembil Foundation, Merial Ltd, Genome Canada, the Canadian Institutes for Health Research, the Natural and Engineering Science Council of Canada, Alberta Funding Consortium, Saskatchewan Agriculture Development Fund, Alberta Beef Producers, Beef Cattle Producers Industry Development Fund of British Columbia, and Agriculture and Food Council of Alberta.

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