Abstract: Since the first mass vaccination against smallpox and its eventual eradication, many more vaccines have been developed based on advances in bacteriology and virology and the use of attenuated live or killed whole pathogens. Immunological discoveries have allowed the development of more refined anti-toxin and conjugate vaccines, while biotechnology provided the tools for rationally designed, genetically engineered vaccines. Many challenges remain in developing safer and more effective vaccines against the more complex diseases such as tuberculosis and HIV-AIDS, and for the rapid protection against newly emerging pathogens or pathogen strains. These vaccines are likely to require the isolation of the "protective" antigenic molecules from the whole pathogen, as well as ways to deliver these to induce effective immune responses with minimal side effects. It has long been recognized that most antigens require the addition of an "adjuvant," an ill-defined substance that non-specifically triggers the innate immune system and boosts an immune response, with aluminum-based adjuvants the most commonly used in most present vaccines. Recent immunological breakthroughs have uncovered that the innate immune system has a much higher degree of complexity than previously thought and can be activated along a wide range of different pathways, depending on the engagement of different innate immune receptors. This in turn determines the type of immune response that will be generated against the vaccine antigens or pathogens. Harvesting the complexity and exquisite specificity of this innate immune system has inspired a new direction in vaccine research, towards the generation of novel adjuvant formulations, tailored to induce defined immune responses effective against specific pathogens. This review gives a brief overview of vaccine development and summarizes different aspects of adjuvant formulation that may influence their activity and specificity.

A Brief History of Vaccination

Edward Jenner is generally credited as the founder of the science of vaccines or “vaccinology,” following his scientific experimentation and worldwide dissemination of the little known practice of inoculating people with cowpox lesions to protect against the devastating human smallpox disease (Allen, 2007; Riedel, 2005). This replaced the common but much more dangerous practice of smallpox inoculation or “variolation.” While Jenner’s first experiment took place in 1796, it was not until the 1880s that an understanding of infectious diseases and the action of vaccines became better known, with the establishment of the “germ theory of disease” by Louis Pasteur and Robert Koch, and the observation that weakened or attenuated germs could protect against the virulent organisms. The progress made in culturing some of these germs or bacteria in relatively simple broths established the science of medical bacteriology and drove the development of a number of bacterial vaccines (Table 1), based on Pasteur’s simple formulation “Isolate, attenuate, vaccinate.” The clinical development of penicillin by Howard Florey in 1940 heralded the age of antibiotic therapies and a decline in bacterial vaccine research. However, by this time viruses had been discovered as separate disease agents and continuous progress made in culturing infectious viruses in animals, cell lines, and embryonated eggs established medical virology and drove the rapid development of viral vaccines along similar principles as the bacterial vaccines (Table 1). Although the danger of live, attenuated bacterial and viral vaccines was in some cases ameliorated with the use of killed, whole organisms, these killed vaccines were generally less effective and posed problems of side reactions and a continuous danger of contamination with live disease agents. The era of biotechnology, and in particular genetic engineering, allowed the production and use of more defined and safer pathogen structures as vaccines, including the recombinant rabies and hepatitis B vaccines, and the rotavirus vaccines produced by artificial reassortment or mixing of the genetic material of different viruses (Table 1). The recent development of the human papilloma virus (HPV) vaccine combined genetic technology to produce recombinant HPV coat proteins with nanotechnology as they spontaneously assemble into submicroscopic virus-like particles (VLP) thereby increasing their antigenicity.

Contributions of Immunology to Vaccine Development

Although the discoveries of Jenner and Pasteur are often credited with the establishment of the field of Immunology (Silverstein, 2009), until recently the vast advances in our understanding of the immune system in health and disease have not led to many practical applications in vaccine development (Table 1). A major exception is the discovery of humoral immunity and the isolation and characterization of antibodies, championed by Paul Ehrlich in the 1890s. The realization that antibodies could neutralize bacterial toxins, the major disease agents in diphtheria, tetanus, and botulism, led to the development of toxin-antitoxin and later toxoid (deactivated toxin) vaccines (Allen, 2007; Marrack et al., 2009). As protection in most vaccines depends on the presence of appropriate antibodies (Zinkernagel and Hengartner, 2006), the development of antibody-based assays did and continues to play a crucial role in the evaluation and testing of new and existing vaccines (Silverstein, 2009). The discovery of the role of maternal antibodies in protection of the young from infectious diseases also had significant implications for the reduced effectiveness of vaccines and the timing of vaccine delivery in this population.

As early as 1918, the clinical immunochemist Karl Lansteiner discovered that small, non-immunogenic molecules (haptens) could be made to elicit specific antibodies if they were artificially linked to larger, immunogenic “carrier” molecules before injection. This “hapten-carrier” concept was not taken up by the vaccine world until the 1980s when the polysaccharides of Haemophilus influenzae type B (Hib), which are poorly immunogenic in young children, were linked to tetanus toxoid, thereby producing the first of a series of “conjugate-vaccines” that were effective in stimulating the immature immune system of the very young to produce antibodies that could protect this most susceptible population (Avery and Goebel, 1929; Pulendran and Ahmed, 2011).

Another immunological breakthrough in vaccine development followed the observation by Glenny and colleagues, that when diphtheria toxoid was precipitated with aluminum sulphate or potash alum (now generally referred to as alum) and injected into guinea pigs, a vastly greater anti-toxin (antibody) response was produced compared to untreated toxoids (Glenny, 1930; Marrack et al., 2009). Glenny postulated that this was due to the slow release of the antigen into the tissue thereby providing prolonged stimulation of the immune system (Glenny, 1930). This property of alum in “helping” (Latin: adjuvare) the immune response to an adsorbed antigen inspired the term “adjuvant.” While other adjuvants have been a routine and essential component in experimental immunology research, most notably Freund’s complete and incomplete adjuvant, their mode of action is still incompletely understood and, until recently, alum has been the only approved adjuvant used in human vaccines. This has led immunologist Charles Janeway, Jr., to call adjuvants the “immunologist’s dirty little secret” (Gayed, 2011). Ironically, it is this long-neglected component of vaccines that is now reviving an old branch of Immunology, innate immunity, and shows great promise as the next breakthrough in vaccine development.

Why Do We Need Adjuvants?

Attenuated or killed whole organism vaccines can induce long lasting immunity, often life-long, with few or no booster vaccination required to maintain protective immunity. A complication with some of these vaccines is that they can be quite reactogenic, induce mild disease, and in severe cases revert to virulence. The realization that immune cells recognize specific molecules (antigens) on the surface of, or secreted by, pathogens led to a focus on antigen identification, purification, and formulation. The protective antigens of many diseases were identified and incorporated into vaccines, leading to the anti-toxin vaccines, polysaccharide vaccines, and, more recently, the recombinant antigen vaccines. These “subunit” vaccines are much safer and generally have fewer side effects; however, they also tend to be less immunogenic, requiring multiple doses and periodic booster shots. The critical role adjuvants played in the effectiveness of these vaccines was already proven by the original Glenny experiments, demonstrating up to 1,000 fold increase in antibody production when alum was added to the purified toxoid vaccine (Glenny, 1930). More recently, the addition of more potent adjuvant systems has been promoted to stimulate the immune response in individuals with weakened immune systems (e.g., the elderly), and to reduce the amount of antigen needed to vaccinate the general population in a pandemic. To this effect, a new oil and water emulsion adjuvant (MF59, Novartis) has been approved for seasonal flu following the avian and swine influenza pandemic threats.

The protective immune response induced by all current killed and subunit vaccines relies solely on the production and maintenance of specific antibodies (Guy, 2007; Zinkernagel and Hengartner, 2006). Antibodies can neutralize toxins, inhibit cell invasion, and contribute to antibody-mediated cellular cytotoxicity. However, some pathogens live or hide within our cells and are not susceptible to antibodies; for these infections it may be necessary to induce cell-mediated immunity and/or cytotoxic T cells to kill the pathogen-infected cell or its resident microbes. This cell mediated immunity cannot be achieved by adjuvants currently used for killed or subunit vaccines. New adjuvant systems that can modulate or shape the type of immune response induced against these pathogens may generate vaccines with different modes of action (Germain, 2010). These may be crucial to combat the difficult diseases such as HIV-AIDS, malaria, tuberculosis, and cancers. In addition, not all antibodies are protective and the induction of different antibody classes may determine the outcome of disease or cure, as for example the anaphylactic antibodies produced by allergens versus the blocking antibodies produced after allergen immunotherapy (Akdis, 2006).

How Do Adjuvants Work?

Immunological adjuvant is generally defined as substances that act to accelerate, prolong, and/or enhance the antigen-specific immune responses when added to purified or complex antigens (Coffman et al., 2010; Guy, 2007; Pulendran and Ahmed, 2011). This simple definition hides a lot of ambiguity as widely divergent and unrelated mechanisms may be involved in strengthening and directing an immune response to vaccination. The most recognized of these are summarized in the following sections.

Antigen clustering

The surface of most pathogens consist of highly ordered arrays of repeat structures: nematodes and parasites often have a glycocalyx composed of mucins and large repetitive glycans, gram positive bacteria have a glycolipid cell wall while gram negative bacteria have lipopolysaccharide (LPS) on the surface, and the outer proteins of viruses form a regular matrix. The immune system has evolved to recognize these ordered structures through multiple mechanisms all of which enhance antigen uptake and recognition and activation of B-cells. Pentraxins, natural IgM, complement, mannose binding lectin, and C-type lectins all recognize multimeric highly repetitive antigens and enhance uptake by, or activation of, B-cells. The human papilloma virus (HPV) vaccines (Cervarix^TM and Gardasil^TM) are examples of vaccines where the viral antigen proteins self-assemble into an orderly array to form virus-like particles (VLP). Clustered antigens are very efficient at activating B-cells through their surface immunoglobulin receptors, leading to their increased proliferation and differentiation and subsequently higher antibody titers (Bachmann and Zinkernagel, 1997). It has recently been shown that ordered clustered arrays of beta-glucan, the cell wall component of yeast and fungi, activate dectin-2 signalling when present as a particulate coat, which triggers phagocytosis and immune activation (Goodridge et al., 2011; Kerrigan and Brown, 2011). Ordered, repetitive, particulate surfaces also bind natural IgM and complement components and are more efficiently taken up by lymph node macrophages. These can then transfer the antigens to follicular dendritic cells that maintain the antigen for long-term presentation to B-cells within the lymph node, thereby increasing immunity (Gonzalez et al., 2010).

Size of vaccine particles

The particle size of a vaccine affects its migration patterns and cellular uptake. Small 40-80 nm structures are more efficiently taken up by dendritic cells, the most potent antigen presenting cells (APCs), while smaller sub 25 nm particles drain directly into the lymphatic vessels and access APCs in the lymph nodes (Bachmann and Jennings, 2010; Fifis et al., 2004; Gamvrellis et al., 2004). Larger particles (>200 mm) remain trapped within the tissue injection site and need to be carried to the lymph node by monocytes, neutrophils, and dendritic cells (Bachmann and Jennings, 2010; de Veer et al., 2010; 2007). The size of the particles also influences the immune response, with smaller (~40 nm) nanoparticles more likely to induce IFN-gamma and larger sized particles (>200 nm) inducing more interleukin-4 expression from helper T-cells (Fifis et al., 2004). Modern real time in vivo imaging techniques have revealed a detailed picture of exactly where different sized antigens are transported to once they reach the lymph node (Gonzalez et al., 2010). Small antigens (<70kD) directly enter the lymphatic conduits while larger antigens remain in the subcapsular sinus and are taken up by sinus lining macrophages. Antigens are transferred to follicular dendritic cells and subsequently to B-cells present within the B-cell zone of the lymph node. The lymph node also contains dendritic cells which can capture viral-sized antigen directly, as was recently shown for influenza virus (Gonzalez et al., 2010). Modern manufacturing techniques mean that vaccine developers now have a large assortment of particles, chemical structures, polymers, and liposomes to choose from and test as particulate vaccine delivery vehicles (Guy, 2007; Heegaard et al., 2011).

Maintaining an antigen depot

Nearly all non-replicating vaccines require a prime-boost strategy where repeat doses of antigen and adjuvant are administered months following the initial vaccination. It is not entirely clear why some vaccines require multiple boosts; it may be to strengthen immune responses by introducing more antigen as the initial dose is quickly depleted, or because of insufficient immune reactogenicity of the vaccine. While follicular dendritic cells can maintain antigen within the lymph node, the signals that cause this to persist are unknown. With this in mind, the importance of antigen persistence or “depot” is seen as a desirable trait in an adjuvant system and more likely to reduce the number of booster shots required. Many methodologies have been developed to contain antigen, from degradable micro-particles to slow release implants, and these depot forming vaccines tend to perform better than non-depot forming formulations (Kemp et al., 2002). Alum adjuvant was initially thought to be a depository or depot adjuvant, acting through slow release or “leaking” of absorbed antigen from the injection bolus. However, recent studies using a lymphatic cannulation model that allows real-time collection of fluid and cells from a vaccine injected tissue site, showed for the first time that approximately 50% of the antigen absorbed to alum is immediately released as soluble antigen after injection and freely flows into the lymph node over a 24-hour period, with similar kinetics as non-adjuvanted antigen injections (de Veer et al., 2010). After this, antigen is taken up and transported by cells as particulate antigen conjugated to alum particles. It is likely that this cellular transport, together with the unique interaction of alum with dendritic cells and its retention on alum particles at the site of injection, is responsible for alum’s adjuvant properties. A minimum duration of antigen supply to the immune system is, however, likely to be required for an effective immune response (Zinkernagel, 2000).

Targeting of vaccine antigens to specific cell types

The engineering of particles or proteins that specifically target individual cell types is possible by fusion of the antigen to a triggering molecule (ligand) that binds to a specific cell type receptor. The antigen can be fused with an antibody or receptor ligand that specifically binds a surface molecule expressed on the target cells. Most studies using this methodology aim to target dendritic cells as these are the most effective antigen presenting cells (Bonifaz et al., 2004). Other methods being developed utilize purified dendritic cells themselves which are treated with antigens and immunomodulators ex-vivo and then injected back into the host to generate the desired immune response. Due to the high expense of this process, its clinical use is limited and is primarily being investigated in anti-cancer vaccine trials (Le et al., 2010). One such vaccine (Provenge, sipuleucel-T) has recently been licensed for a subset of prostate cancer.

Stimulating innate immune pathways

The innate immune system, sometimes referred to as the primitive immune system, has long been an unpopular branch of immunology, generally associated with non-specific complement fixation and phagocytosis. While it is the dominant immune system found in lower organisms (plants, fungi, insects), it was seen to be superseded in importance by the sophisticated adaptive immune responses, orchestrated by B and T lymphocytes, in higher organisms. The innate immune response could be compared to the resident garrison of foot soldiers fighting off the first signs of invasion until the modern “cavalry” arrives with its sophisticated precision weapons. However, recent immunological breakthroughs have indicated a much wider role for the innate immune response; it not only alerts the adaptive immune system to any foreign invaders, but also dictates what weapons it should use to combat a particular infectious agent. This new knowledge has wide reaching implications for infectious disease and vaccine studies, in particular for providing a rational explanation of how adjuvants may work in promoting vaccine protection or pathology.

The re-evaluation of the importance of innate immunity largely began with the discovery of the family of Toll Like Receptors (TLR) and the demonstration that these innate, germline encoded immune receptors, permanently present on a variety of cells and tissues, were specific for conserved, “pathogen-associated molecular patterns” (PAMPS). The TLRs were first discovered in Drosophila, through a screen designed to identify molecules involved in fungal resistance (Lemaitre et al., 1996). The human homologue was identified (Medzhitov et al., 1997) and then a genetic screen to identify genes involved in the recognition of the gram negative bacterial cell wall component, lipopolysaccharide (LPS), identified the human TLR4 gene, which is essential for LPS signaling (Poltorak et al., 1998). The TLRs are members of an increasing family of “pattern recognition receptors” (PRRs) which detect a vast array of infection or “danger” signals, including pathogen molecules, host stress molecules, endosomal/phagosomal nucleic acids, lipids, and glycolipids (Table 2). Recently discovered PRRs include the NALP family of proteins which form the inflammasome and recognize cytoplasmic peptidoglycan, crystalline substances such as uric acid and alum, and general cellular stress through reactive oxygen species (Bauernfeind et al., 2011; Franchi et al., 2009). Nucleic acid recognition receptors include the RIG-I helicases which recognize cytoplasmic double stranded RNA, the IFIT proteins which recognize tri-phosphorylated RNA species (Pichlmair et al., 2011) within the cytoplasm, and the HIN-200 (Roberts et al., 2009) and AIM2 families of proteins which recognize cytoplasmic DNA (Table 2). The ligands and signalling pathways for the C-type lectin receptors and other scavenger receptors demonstrate how fungi, parasites, and other pathogens are recognized and activate innate pathways (Marakalala et al., 2011).

This newly discovered variety of innate immune receptors can dictate the expression or activation of distinct intracellular transcription factors in immune cells, which ultimately induce the production of immunomodulatory molecules including cytokines, leukotrienes, prostaglandins, and chemokines. These molecules in turn promote activation and recruitment of cells to the site of infection and also dictate subtle or dramatic differences in the activation of specific T-helper subsets and the subsequent adaptive immune response (Table 2). Additionally, activation of innate immune pathways often upregulates molecules involved in antigen presentation thereby accelerating and strengthening the development of an adaptive immune response. It is this ability of the innate immune system to modulate the strength, type, and direction of the adaptive immune response that is of interest to vaccinologists. It is thought that the purification or artificial production procedures used to obtain defined antigenic components of a pathogen for modern vaccines discard most of these innate stimulators inherent in the complete microbe. Manipulation of desirable innate pathways by re-incorporating selected innate stimulators into vaccine adjuvants raises the exciting possibility that we will be able to generate vaccines that induce much safer, more effective, and sustained immune responses tailored to specific infections. Already TLR agonists have been incorporated into successful vaccine formulations such as the new Cervarix^TM vaccine which prevents infection with the human papillomavirus (HPV) and has proven effective in preventing cervical cancer (Harper et al., 2006). The addition of the TLR ligand monophosphoryl lipid A (MPL) complexed with alum increases the immunogenicity of this vaccine and reduces the number of doses required to elicit sufficient antibody production. The specific recognition of defined ligands by innate immune receptors and their ability to modulate both the strength and direction of the subsequent adaptive immune response are delivering many potential new vaccine adjuvant candidates.

Animal Models for Vaccine Research

The use of mouse models has dominated modern infectious disease and vaccine research. However, there is increasing concern that mouse models have shown little translational value and may even hinder the development of vaccines against human pathogens (Germain, 2010; Khanna and Burrows, 2011; Pulendran and Ahmed, 2011). Murine models differ significantly in physiology and immune regulatory pathways (including innate immune pathways) from humans, and are not natural hosts for most human pathogens. While vaccine and infectious disease studies in the final human host would be ideal for achieving practical outcomes, there are considerable ethical limitations in human studies and they would benefit from appropriate animal models that allow more rigorous examination and interventions. In parallel to human vaccine development, veterinary vaccines are vigorously pursued, but their achievements are mostly ignored in the general immunology and vaccine literature. This is surprising considering the direct impact of animal infections on human health. More than half of the human pathogens are zoonotic, i.e., also infect farm and wild animals which can serve as reservoirs for human infections, and almost all have closely related homologues in veterinary species (Woolhouse and Gowtage-Sequeria, 2005). Infectious disease and vaccine studies can therefore be performed in natural animal disease models that are also more similar to humans in physiology and anatomy than mice. In addition, many of the emerging human diseases are derived from farm or wildlife animals (e.g., SARS and Hendra virus infections, avian and swine flu) and a closer examination of animal infections may guard against future outbreaks.

Advances in veterinary vaccines have overtaken the human vaccine field with many more licensed veterinary vaccines, adjuvants, and delivery systems on the market, partly due to the reduced safety concerns and registration costs (Heegaard et al., 2011; Meeusen et al., 2007). Thousands of animals are continuously studied by veterinary scientists and animal health companies in vaccine trials under different conditions and provide valuable information on how outbred populations react to different vaccination procedures. It has been advocated that farm animals also provide appropriate biological models to study infectious-disease population dynamics and the effect of vaccination at a population level (Lanzas et al., 2011). A closer collaboration between human and veterinary vaccinologists and the study of natural host-animal models are therefore likely to lead to more translational findings in vaccine research. In this respect, it should be remembered that the first vaccine breakthroughs were derived from farm animals (cowpox by Jenner, cholera in chickens, and anthrax in sheep by Pasteur), and the only vaccine against tuberculosis is derived from natural infections in cattle, which are likely to be much more suitable models to study improvements of this vaccine than currently used mouse models (Van Rhijn et al., 2008).

Conclusion

The empirical approach in vaccine development has taken us as far as it can and new approaches are required to develop the next generation of vaccines to combat existing diseases such as HIV-AIDS, dengue fever, malaria, and helminth infections, and to speed up vaccine development for new emerging diseases. The exciting discoveries in innate immunity have elucidated how vaccine adjuvants may control immune responses and are supplying a steady stream of new adjuvant candidates. However, the next generation of effective vaccines will require consideration of many more factors than just which innate immune pathways to target. Indeed, most approved adjuvants and the VLP vaccines currently in use induce effective immunity and yet contain no substances known to directly trigger innate immune pathways through specific receptor recognition. In addition, many of the innate receptors that most potently activate innate immune pathways are too reactive to be considered safe additions to vaccine formulations. Some innate immune receptors are restricted to endosomes, phagosomes, or the cytoplasm, so adjuvants targeting these receptors require the ligand to be internalized for activity. It is the combination of effective delivery vehicles with defined immunostimulatory components, and a much greater understanding of how these interact in relevant animal models, that promises to deliver the next generation of vaccines.

While the recent unveiling of the mechanisms underlying the action of adjuvants is transforming the direction of vaccine research, it should be kept in mind that vaccine licensing and marketing has to follow stringent regulatory guidelines and requires cost effective production methods (Goetz et al., 2010; Meeusen et al., 2007). These considerations may limit translation of many experimental vaccines into clinical practice and should therefore form part of the initial evaluation of vaccine research programs.

Disclosure

The authors report no conflicts of interest.

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