Vaccine developers pursuing purer subunit vaccines to reduce reactogenicity, often run into problems of poor vaccine immunogenicity. This has driven a resurgence of interest in newer adjuvants. Aluminium salts (alum) remain the only adjuvants approved by the US FDA for human use, having been first introduced in the 1930s in a far less stringent regulatory environment than the present. The new adjuvant ‘kids on the block’, the Toll-like receptor (TLR) agonists, derive from the highly reactogenic contaminants that were removed to reduce toxicity of old-style vaccines and, not surprisingly, suffer from similar problems. Clinical development of new adjuvants faces a number of roadblocks and difficult questions to be answered. In particular, answers are needed over potential formulation and stability issues arising from substitution of traditional aluminium salts for newer adjuvants; what is likely to be the response of the FDA when vaccines containing these new adjuvants first present themselves for registration? How much human data are required to prove a new adjuvant is safe? Lastly, what do we know about the mechanisms of action of different adjuvants and their potential for human toxicity, and how should this knowledge guide our choice for the best adjuvant for a particular vaccine indication? Despite the recent renaissance in adjuvant development, these and many other important questions need to be answered before new adjuvants become clinically available.
Old-style vaccines made from live or killed whole organisms are highly effective at inducing protective immunity. On the downside, they suffer from a high level of local reactogenicity and systemic toxicity. In light of these problems, there has been pressure on vaccine manufacturers to develop safer and less reactogenic subunit vaccines free of reactogenic contaminants, such as lipopolysaccharide (LPS), DNA or RNA. Examples of reduced reactogenicity vaccines include acellular pertussis, recombinant hepatitis B surface antigen or subunit-inactivated influenza vaccines. Unfortunately, lower reactogenicity has generally come at the expense of reduced vaccine immunogenicity and effectiveness. The need to enhance the immunogenicity of highly pure subunit vaccines has led to recent resurgence in interest in new adjuvants. A particular driver for this adjuvant renaissance has been the concern of an imminent influenza pandemic and recognition of the urgent need for better adjuvants to improve the immunogenicity of H5N1 vaccines. Given inadequate global influenza vaccine-manufacturing capacity, adjuvants could play an important role in antigen dose-sparing, thereby stretching limited H5N1 antigen supplies. The US government, through the NIH, has led the charge to develop new and improved vaccine adjuvants to help counter these threats. Major injections of US government funds have enabled many stalled adjuvant development projects to be dusted off and reinvigorated. Examples of born-again adjuvants include bacterially derived compounds, such as monophosphoryl lipid A (MPL), oil-in-water emulsions, such as MF59, saponin-based adjuvants, such as QS21 and immunostimulating complexes, plus our own Advax inulin-based adjuvants, all products that had their genesis in the 1970s and 1980s but subsequently languished. In the interim, we have seen the discovery of the TLR pathways, providing an explanation for the immune-enhancing effects and reactogenicity of vaccine contaminants. Where is all this adjuvant research leading us?
The desire for new and improved adjuvants stems not only from the need to make existing inactivated vaccines more potent but also to meet specific requirements, such as antigen sparing, more rapid seroprotection, stimulation of T-cell immunity, enhancement of protection in neonates and the elderly and stimulation of longer-lasting protective immunity. Since the empiric search has identified largely highly reactogenic adjuvants, we need to put aside old assumptions and dogmas and, instead, develop a search algorithm to identify safe and effective adjuvants based on actual mechanisms of action.
Ideally, adjuvants should strongly stimulate B- and T-cell immunity while avoiding the excess innate immune system activation and inflammatory cytokine production that mediates adjuvant reactogenicity. This flies in the face of immunological dogma that high levels of inflammatory cytokine production are critical to adjuvant success, as exemplified by the Matzinger ‘danger hypothesis’, which proposes that tissue damage or toxicity as induced by inflammatory cytokines is a necessary prerequisite for an effective immune response Citation[1]. While the ‘danger hypothesis’ might suggest that vaccine immunogenicity and reactogenicity are inseparable, there are exceptions – principally Advax, a polysaccharide-based adjuvant that potently enhances vaccine immunogenicity without increasing reactogenicity Citation[2–4].
The mechanism by which aluminium salts act as adjuvants remains uncertain, and does not correlate with alum’s ability to absorb proteins Citation[5]. Most probably, alum works by stimulating its own uptake by antigen-presenting cells (APCs), which it then poisons Citation[6,7]. Therefore, the toxicity of aluminium satisfies Matzinger’s ‘danger signal’ model and helps explain the reactogenicity of alum-based vaccines. Interestingly, a similar mechanism was proposed many years ago to explain the action of beryllium salt, a related metal salt, which is even more toxic to macrophages than aluminium and, similarly, has potent adjuvant activity Citation[8]. If live APCs are required to activate T cells, then this might explain why alum fails to induce cytotoxic T cells. If alum largely works via cellular toxicity, then how do oil and water emulsions, such as Montanide or MF59, work Citation[9]? Emulsions are highly irritant and cause local inflammation, attracting a monocytic and neutrophil infiltrate and, thereby, satisfing the ‘danger hypothesis’. Another effective, albeit toxic, adjuvant is QS21, a collection of triterpenoid glycosides (saponins), derived from the bark of Quillaja saponaria. Saponins dissolve cell membranes and induce major local tissue damage Citation[10], providing a strong danger signal and major injection site pain Citation[11–13]. A major recent breakthough in immunology has been the identification of TLRs. TLRs are highly conserved throughout nature and are part of the early warning system of the innate immune system. While a multitude of agonists of various TLRs are in preclinical testing, the most advanced human candidates are compounds activating TLR4 or TLR9. LPS is highly potent activator of innate immunity Citation[14], binding and activating TLR4. MPL is a chemically modified derivative of LPS from Salmonella minnesota R595, which is used in complex adjuvant formulations with alum, QS21, liposomes and emulsions, and is a component of GlaxoSmithKline’s proprietary AS02 and AS04 adjuvants Citation[15–17]. Similar to LPS, MPL interacts with TLR4, resulting in the release of proinflammatory cytokines, including TNF, IL-2 and IFN-γ, thereby, providing a danger signal and enhancing antibody and T-cell responses Citation[18,19] with associated increases in reactogenicity. TLR9 agonists, based on unmethylated CpG dinucleotides Citation[20–22] are also under study in human vaccines. Binding of CpG to TLR9 leads to activation of NFκB triggering the release of inflammatory cytokines Citation[23], which provide a danger signal stimulating Th1 immunity but which also mediate adjuvant toxicity Citation[24].
Adjuvant side effects can be separated into local and systemic. Local side effects range from increased injection site pain, inflammation and swelling, to granulomas, sterile abscess formation, lymphadenopathy and ulceration. Systemic vaccine reactions may include nausea, fever, adjuvant arthritis, uveitis, eosinophilia, allergic reactions, organ-specific toxicity, anaphylaxis or immunotoxicity mediated by liberation of cytokines, immunosuppression or induction of autoimmune diseases Citation[25,26]. Given that most candidate adjuvants, including TLR agonists, work via generation of tissue damage and, thereby, immune ‘danger signals’, not surprisingly, their inclusion in vaccines results in major increases in local reactogenicity and systemic toxicity. Significant regulatory and other hurdles exist for development of new adjuvants. For FDA approval, new adjuvants must convincingly demonstrate safety, tolerability and a clear benefit that outweights any increased reactogenicity. Few adjuvants have been able to satisfactorily pass this hurdle, particularly for prophylactic vaccines where the regulatory bar is at its highest. Hence, a major challenge in adjuvant development is how to achieve a potent adjuvant effect while avoiding reactogenicity or toxicity Citation[27]. All the previously described candidate human adjuvants, including alum Citation[28], MF59 Citation[29], ISCOMS Citation[30], QS21 Citation[31], AS02 Citation[16] and AS04 Citation[17] suffer from substantial local reactogenicity and systemic toxicity. Although most vaccine reactions are not life threatening and resolve over time, even rare serious adverse events discourage better community acceptance of routine vaccination.
Given the strong evidence supporting the danger or tissue damage model of adjuvant action, it is surprising to find one major exception to the rule; Advax, which is an adjuvant based on nanoparticles of inulin. Inulin is a natural, plant-derived polysaccharide derived from fructose and glucose. Specific isoforms of inulin have the unique ability to enhance antigen-specific humoral and cellular immune responses without inducing reactogenicity or other evidence of a danger signal Citation[2–4,32–36]. Advax does not induce inflammatory cytokine production, thereby, apparently refuting the idea that danger signals are essential for potent adaptive immune responses. These favorable results have been confirmed in multiple animal models, as well as in recent human clinical trials. The conclusion is that there is still much we do not understand about adjuvants and how they work. Vaccine-funding agencies should consider initiating specific funding programs for adjuvant science and clinical development as this remains a major roadblock in new vaccine development.
Financial & competing interests disclosure
This publication was supported by funds provided by Grant No. 5U01AI061142-02 from the National Institutes of Health, National Institute of Allergy and Infectious Diseases. Its contents are solely the responsibility of the author and do not necessarily represent the official views of the National Institutes of Health, National Institute of Allergy and Infectious Diseases. The author performs consultancy work for Vaxine Pty Ltd, the company developing Advax and other adjuvants. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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