Abstract

1. Introduction

Traditional vaccines such as whole cell pertussis [1] or whole virus influenza vaccines [2] are highly immunogenic albeit at the price of significant local and systemic reactogenicity. To reduce reactogenicity, modern approaches incorporate split, subunit or recombinant antigens from which reactogenic contaminants such as lipopolysaccharide, DNA and RNA are removed. As highlighted by acellular pertussis vaccines, the improved safety of subunit vaccines comes at the price of reduced immunogenicity[1]. The move to subunit vaccines has also resulted in some cases in a shift from a balanced Th1 and Th2 vaccine response to a more Th2-biased response[1]. While reversion to whole cell vaccine approaches could improve immunogenicity[3], it would also recreate excess reactogenicity. Incomplete virus splitting during manufacture was found to be responsible for excess hospitalizations for febrile convulsions caused by a recently withdrawn pediatric inactivated influenza vaccine, thereby highlighting this tradeoff [4]. Thus, there is a close relationship between vaccine immunogenicity and reactogenicity, arising from contaminants such as lipopolysaccharide, DNA and RNA contained in whole cell vaccines that act as both inbuilt adjuvants and reactogens [5]. Both properties reflect the ability of these contaminants to induce inflammation via activation of innate immune receptors, with the consequence that their adjuvant action and reactogenicity are inseparable, with dose-limiting reactions including local swelling and pain plus systemic fever and malaise [6]. Adjuvant reactogenicity can thereby be regarded as a dose-dependent phenomena reflecting local tissue damage and systemic inflammation induced by activation of innate immune receptors [7]. Should an adjuvant induce excess reactogenicity in a vaccine and the problem cannot be solved by lowering the dose of the reactogenic component then the combined adjuvanted vaccine formulation could be regarded as potentially unsafe, although even this is context dependent as, for example, the withdrawn pediatric influenza vaccine mentioned above was still regarded as safe and remained approved for use in older individuals not at risk of febrile convulsions [4], thereby highlighting the extreme complexity of vaccine safety assessment.

An even greater challenge than adjuvant safety assessment which focuses on the chance of immediate adverse effects (pain, swelling, fever), is the assessment of adjuvant risk which refers to the relative possibility of development of any adjuvant-associated problem over the life of the individual being immunized. Hence, the most challenging aspect of assessment of adjuvant risk is determination of the basis, or lack thereof, of various reported associations between use of vaccines containing specific adjuvants and development of rare autoimmune or chronic degenerative disorders, for example, associations between use of squalene emulsion adjuvanted vaccines and narcolepsy [8] or Gulf War Syndrome [9] or between use of aluminum adjuvants and macrophagic myofasciitis [10] or Alzheimer’s disease [11]. Such assessments are made exceedingly difficult by the paucity of data, the inability to do controlled studies in humans to prove causation, and the potentially extremely long time period between immunization and onset of symptoms. Hence, causation in the vast majority of such cases has never been established, leaving uncertainty as to whether any of these associations might be real or are just linked by chance. There is thus a great need for better research tools with which to probe the nature of such associations. This review will focus on current adjuvants that have at least reached the stage of human clinical trial testing to identify what is known and what is still to be learned about all aspects of adjuvant safety.

2. Literature search methods

Articles were identified in PubMed using the key word terms ‘vaccine adjuvant safety’ and ‘vaccine adjuvant toxicity’ with a focus on articles published in the last 10 years. Only human adjuvants for which there was published clinical trial data were included.

3. Adjuvant-associated local toxicity

Local adjuvant-associated side effects range from mild injection site pain, tenderness, redness, inflammation and swelling at one end of the spectrum, to formation of granulomas, sterile abscesses, lymphadenopathy and chronic skin ulceration, at the other (reviewed in [6]). Local vaccine side effects may reflect direct chemical irritation due to non-physiological pH, osmolarity, salt concentrations or direct cell toxicity. Such local irritant effects are typically associated with immediate severe injection site pain followed by an inflammatory response triggered by the tissue damage. Examples of adjuvants that induce local reactogenicity include saponins (e.g. Quil A, QS21, ISCOMS, ISCOMATRIX) and oil emulsions (e.g. complete Freund’s adjuvant, incomplete Freund’s adjuvant, Montanide, MF59, AS03) [7]. Immediate reactions are likely to reflect irritation and inflammation induced by the adjuvant component itself but if delayed by 24–48 hours may reflect an excessive delayed type hypersensitivity (DTH) response against a vaccine component in an already primed individual [12]. Local reactogenicity is not life threatening but could still lead to significant morbidity, e.g. at worst, a sterile abscess needing surgical drainage or skin ulceration requiring skin grafting. Some local reactions such as severe pain, while not directly damaging to physical health, may still strongly impact negatively on the public’s perception of risk-benefits of immunization and hence should be avoided on these grounds.

4. Adjuvant-associated systemic toxicity

Systemic reactogenicity typically includes symptoms such as fever, headache, malaise, nausea, diarrhea, arthralgia, myalgia, and lethargy, largely reflecting adjuvant-associated innate immune activation and downstream inflammation. Adjuvants that strongly activate innate immune receptors, e.g. adjuvants based on pathogen-associated molecular patterns (PAMPs), may thereby be most prone to systemic reactogenicity. This includes toll-like receptor (TLR) ligands such as monophosphoryl lipid A (MPL), flagellin, lipoarabinomannan, peptidoglycan, or acylated lipoprotein (reviewed in[7]). Systemic reactogenicity is also an issue for adjuvants that induce local tissue damage, e.g. oil emulsions and saponins, as this results in release of endogenous damage-associated molecular patterns (DAMPs) that activate innate immune receptors and induce inflammation [13]. Typically, such inflammation-associated adjuvant reactogenicity would be expected to settle once the innate immune response subsides but could potentially last up to several weeks post-immunization.

At the more serious end of the adjuvant systemic toxicity spectrum is the potential for rare immunological toxicities resulting from aberrant immune activation driven by the adjuvant. This includes problems such as immune bias, e.g. the eosinophilia, allergic reactions and anaphylaxis caused by Th2 bias imparted by aluminum adjuvants [14]. It also includes the potential for adjuvants to induce chronic immune activation and inflammation that does not settle post-immunization. An example would be the syndrome of macrophagic myofasciitis whereby long-lasting tissue depots of aluminum adjuvant have been linked to symptoms of chronic fatigue syndrome [15] although, as discussed later, this association is questioned by bodies such as the WHO Global Advisory Committee on Vaccine Safety (GACVS) [16].

Finally, there is the risk that an adjuvant may either act as the trigger or increase the likelihood of a vaccine causing an autoimmune disease. An example is the ability of inflammatory oil emulsion adjuvants to induce adjuvant arthritis in genetically-susceptible animal models [17]. Adjuvant-associated immune dysregulation and the potential to cause autoimmune disorders represent the most widely debated aspect of adjuvant risk assessment. Spontaneous autoimmune conditions only affect a small number of genetically-susceptible individuals in the general population [18]. Hence, even if a vaccine/adjuvant combination was thought to cause autoimmune disease, this would be very hard to prove, particularly if everyone in the population had received the vaccine. The case of narcolepsy and pandemic influenza vaccine was an exception as this was a single once off vaccine use across a very discrete period of time and not everyone had the vaccine so good control groups were available to show use of the particular vaccine was almost certainly the cause of the increased risk of disease[8].

Also included within the spectrum of potential adjuvant systemic toxicity is the potential for chronic organ toxicity of the compounds themselves. For example, aluminum or oil emulsions can form long-term tissue depots and this has been postulated to cause chronic toxic effects. However, detection of chronic toxicity and determination of any causal relationship can be extremely difficult, if not impossible, due to the long delays between disease onset and environmental exposure, e.g. immunization, which may have occurred decades later.

5. Making sense of adjuvant-associated adverse events

There have been periodic reports highlighting potential temporal associations between immunization with adjuvanted vaccines and the occurrence of adverse events. Needless to say, an association may not represent causation, which needs to be established in each individual case. Examples of such associations include reports of macrophagic myofasciitis in patients previously receiving vaccines containing aluminum adjuvant [15], and narcolepsy in children immunized with pandemic influenza vaccine containing squalene emulsion adjuvant [19, 20]. Notably, the incidence of reported adverse events within the context of the total immunized population is often extremely small. Thus, the vaccine-attributable risk of developing narcolepsy was estimated at 1: 16,000 vaccinated Finnish 4 to 19-year-olds [19], but if expressed as a ration of the total immunized Finnish population irrespective of age would be closer to 1:100,000. Although the prevalence of macrophagic myofasciitis is not known the Henri Mondor hospital which identified and specializes in this syndrome reported that 600 cases were diagnosed over a 10 year period [21], but this needs to be put in the perspective of the total French population numbering over 64 million. Hence, the media and anti-vaccine lobby groups are often biased towards reporting and focusing on rare vaccine adverse effects while generally ignoring the extremely large denominator of the total immunized population from which such cases are drawn.

The problem of rare vaccine adverse events from a regulatory perspective is that it is often extremely difficult, if not impossible, to ever establish proof of causality. Hence, the best that can be done is to assess whether causation is plausible or not using knowledge of a particular vaccine’s or adjuvant’s mechanism of actions. Even in situations where causation is held to be probable, such as in the case of the specific pandemic influenza vaccine and childhood narcolepsy, it is still not possible to identify which vaccine component(s) is responsible, such as the relative contribution of the antigen or adjuvant, if present. Whilst animal models might seem the best solution for testing causation of adverse reactions, direct extrapolation from such models is difficult with no guarantee they accurately reflect the human context. Hence all vaccine adjuvant safety assessments are subjective in nature. This indicates an urgent need for more research into methods to better assess adjuvant safety and to investigate rare adverse events that may possibly be vaccine and/or adjuvant related. To better understand these adjuvant safety issues it is useful to individually examine each of the adjuvants for which human data is available.

6. Aluminum adjuvants

After almost a century, aluminum salts maintain their dominance as adjuvants in human vaccines. This reflects the fact that aluminum adjuvants are extremely effective at enhancing antibody responses, are well tolerated, do not cause pyrexia and have the strongest safety record of any human adjuvants [7]. Hence, aluminum adjuvants remain the gold standard against which all new adjuvants need to be compared and any new adjuvant must prove it provides better protection, tolerability or safety, or preferably all, when compared to aluminum adjuvant. This has proved extremely hard to achieve, explaining aluminum’s ongoing dominance. Aluminum’s action was initially thought to be due to local antigen depot effects but the situation is now recognized as more complex with NALP3-mediated inflammasome activation, interleukin (IL)-1 production, cell necrosis, DNA release, and activation of DAMP and PAMP receptors, all proposed to contribute to it’s action [22–24]. Other metal salts including iron and beryllium [25, 26] that also induce lysosomal rupture and phagocyte cell death share alum’s adjuvant activity [27, 28] suggesting induction of cell death is a common feature of adjuvants based on metal salts [29]. The propensity to kill phagocytes may help explain alum’s inability to induce robust cellular immunity as live antigen presenting cells are required for efficient antigen cross-presentation to CD8 T cells [30]. Aluminum adjuvants suffer from a number of minor toxicities potentially explained by its mechanisms of action. For example, aluminum induces injection site pain and tenderness [31] that may reflect cell necrosis and induction of inflammasome activation and IL1 production [32]. Aluminum salts’ propensity to induce cell death and inflammasome activation could also explain why some subjects develop persistent lumps and granulomas at the injection site [31]. Aluminum adjuvants also induce contact dermatitis to aluminum in a fraction of immunized subjects [33]. Aluminum adjuvant-containing vaccines can cause post-immunization headache, arthralgia and myalgia, which could reflect alum’s propensity to induce IL-1, with IL1 administration to human subjects reproducing these symptoms [34]. On the positive side, aluminum adjuvants rarely cause severe local reactions and are not normally associated with systemic inflammatory problems such as pyrexia.

A potential issue is aluminum adjuvants’ propensity to induce Th2 immune bias with increased eosinophils and IgE production, thereby increasing risk of allergy and anaphylaxis [14, 35–37]. This phenomenon can be reproduced in a murine ovalbumin sensitization model where sensitization by repeated immunization with ovalbumin plus aluminum adjuvant induces susceptibility to allergic asthma and lethal anaphylaxis upon subsequent ovalbumin re-exposure. Aluminum adjuvant-associated allergic sensitization can be prevented in IL4 receptor knockout mice or by administration of IFN-γ [38] or CpG oligonucleotides [39], indicating that allergy sensitization is due to aluminum adjuvants’ excessive Th2 bias. Th2 immune bias may be a particular problem in children who are already genetically-biased towards excessive Th2 immune responses and allergies [40]. Excess Th2 bias is a particular problem for vaccines against viruses such as respiratory syncytial virus (RSV) or SARS coronavirus (SARS), where aluminum-adjuvanted vaccines have been shown to increase the risk of lung eosinophilic immunopathology upon virus infection [41, 42]. This mechanism is thought to have contributed to deaths of children administered an experimental formalin-inactivated aluminum-adjuvanted RSV vaccine after they became infected by RSV [43]. In a mouse model, SARS lung eosinophilic immunopathology could be prevented if animals were immunized with SARS antigen in combination with a non-Th2 polarizing delta inulin adjuvant in place of the aluminum adjuvant [42]. This suggests that adjuvants that do not share the Th2 bias of aluminum would be safer for use with vaccines against pathogens such as RSV or SARS where an excessive Th2 bias could otherwise result in detrimental immune responses in response to viral infection.

In cats, dogs and ferrets, aluminum adjuvants cause local chronic granulomatous lesions that can progress to malignant fibrosarcomas [44]. Why similar tumors are not seen in aluminum-immunized humans is not known. However, aluminum adjuvants in humans have been reported to cause chronic granulomatous inflammation, called macrophagic myofasciitis (MMF) [15, 45]. Symptoms of MMF syndrome include myalgia, arthralgia, marked asthenia, muscle weakness and fever [15, 45]. Abnormal findings in MMF patients include elevated creatine kinase and erythrocyte sedimentation rate plus a myopathic electromyograph. Muscle biopsies of MMF patients have shown infiltration by sheets of macrophages with granular periodic-acid-Schiff positive content and with aluminum deposits being demonstrated in the lesions by energy dispersive X-ray microanalysis [46]. The syndrome is hypothesized to be due to the persistence of vaccine-derived aluminum tissue deposits, resulting in a perpetual cycle of macrophage ingestion of alum, intracellular lysosomal rupture, phagocyte death, and ingestion of alum-containing dead phagocytes by newly recruited macrophages, leading to a chronic inflammatory reaction [46], although the link between the muscular MMF lesion and the described MMF symptoms remains a contentious area of debate[16]. Some MMF patients have been reported to demonstrate neurological manifestations resembling multiple sclerosis [47]. Since its original description in 1993, more than 600 cases of MMF have been diagnosed in France [21], with sporadic case reports from other countries [10]. These numbers need to be put in the perspective of the total immunized French population, which likely numbers over 64 million. Currently the only treatment is surgical resection of the aluminum at the original muscle injection site. Interestingly, the symptoms of MMF closely resemble those of Muckle-Wells syndrome, which is caused by inherited mutations that result in constitutive inflammasome activation [48]. As aluminum adjuvants are now known to also induce inflammasome activation [32] it is possible to speculate that MMF might occur in individuals who are also susceptible to chronic inflammasome activation. If so, MMF could essentially represent a low-grade acquired form of Muckle-Wells syndrome, a plausible mechanism given aluminum’s known molecular actions. While GACVS accepts that MMF is a lesion containing aluminum salts identified by histopathological examination found at the site of previous vaccination with an aluminum-containing vaccine, they have concluded “that there is no evidence to suggest a resulting clinical illness or disease” [16]. GACVS recommended that to further understand MMF, additional research studies need to be undertaken to evaluate clinical, epidemiological, immunological and basic science aspects of this disease[16].

The antiphospholipid syndrome is an autoimmune disorder manifested by elevated titers of antiphospholid antibodies, arterial and venous thromboembolic events, recurrent spontaneous abortions, and thrombocytopenia [49]. Tetanus toxoid hyper-immunization is able to reproduce the antiphospholipid syndrome (APS) in mice, which correlates with the induction of cross-reactive low affinity anti-β(2) glycoprotein I (β(2)GPI) antibodies [50]. In C57BL/6 mice, tetanus toxoid hyper-immunization with aluminum adjuvants but not glycerol resulted in an increase in low affinity anti-β(2)GPI IgG antibodies and a decrease in maternal fecundity consistent with the aluminum adjuvant being a critical component in this model of antiphospholipid syndrome [50]. To what extent aluminum-adjuvanted tetanus vaccines might contribute to the rare human cases of antiphospholipid syndrome is not known.

High aluminum levels in the body predominately affect the brain and bone tissues, causing fatal neurological syndrome and dialysis-associated dementia [51]. Cerebral aluminum accumulation has also been observed in Alzheimer’s disease [52]. Aluminum exposure through pediatric parenteral nutrition have been shown to impair bone mineralization and delay neurological development [11]. While low doses of aluminum are renally excreted, under conditions of reduced renal function aluminum can accumulate in the body and become toxic. Furthermore, environmental aluminum loads are greater than in the past to which the additional load of a multiplicity of alum-based vaccines must be added [53]. Research using aluminum oxyhydroxide particles labeled with fluorescent functionalized nanodiamonds confirmed that 21 days post-immunization the brains of mice contained on average 15 solid aluminum particles and parallel studies in vitro confirmed that aluminum adjuvant was toxic to neuronal cell cultures [54]. This is consistent with mouse studies showing neurotoxic effects including neural apoptosis and both motor and behavioral deficits of aluminum adjuvant [55, 56]. What contribution cumulative doses of aluminum adjuvants might make to human chronic disorders such as Alzheimer’s disease [11, 57] or chronic bone disease [58] is simply unknown and warrants more thorough investigation. In particular, parenterally-administered aluminum particles can behave very differently in the body to soluble aluminum as these particles can be transported to unusual sites such as the brain after phagocytosis[54], whereas soluble aluminum ions on which current exposure limits are set are easily excreted by the kidneys [11]. GACVS have characterized studies suggesting adverse effects of aluminum adjuvants in humans as ‘seriously flawed’ but unfortunately have failed to comment on the validity or otherwise of the animal toxicology data and its potential relevance to human immunization [59]. Any adverse finding against alum adjuvants would clearly have serious ramifications [60] in view of the current lack of adjuvant alternatives and the overwhelming public health benefit of current vaccines containing aluminum adjuvants, particularly in developing countries where deaths from vaccine-preventable infectious diseases remain high. Hence a very high standard of proof is required before any claim of aluminum adjuvant toxicity could be endorsed, and the risk-benefit of inclusion of alum adjuvant in vaccines, in the absence of a viable alternative, remains overwhelmingly positive.

It is important to note that not all forms of aluminum adjuvants are necessarily the same. For example, reports of MMF have been largely linked to use of aluminum hydroxide which may reflect the fact that than interstitial fluid containing organic acids with an alpha-hydroxy carboxylic acid able to chelate aluminum reacted more readily with aluminum phosphate than with aluminum hydroxide, with the result that three times more aluminum is excreted from rabbits vaccinated with aluminum phosphate with aluminum hydroxide having a much longer tissue residence time[61] as also suggested by a monkey study in which histopathological lesions similar to human MMF lesions were still present 12 months after aluminum hydroxide-adjuvanted vaccine administration versus 3 months for aluminum phosphate [62]. In this study, none of the 24 immunized monkeys developed clinical symptoms despite the presence of MMF-like lesions [62], although this still does not exclude the possibility that clinical symptoms are associated with MMF lesions in some human subjects genetically or otherwise predisposed to developing this rare syndrome.

7. Oil Emulsion Adjuvants

This class of adjuvants includes a wide spectrum of oil-in-water or water-in-oil emulsions. Water in oil adjuvants such as complete Freund’s adjuvant (CFA) rank as the most reactogenic of known adjuvants and hence are unsuitable for human use. Oil-in-water emulsions have lower although still significant reactogenicity and include the squalene-based adjuvants such as MF59, AS02, and AS03 [7], the various Montanide oil adjuvants, and the liposomal adjuvant CAF01 which is composed of a cationic liposome vehicle (dimethyldioctadecyl-ammonium (DDA)) stabilized with trehalose 6,6-dibehenate, a glycolipid synthetic variant of mycobacterial cord factor [63]. The mechanism of action of oil emulsions reflects their ability to induce a strong inflammatory reaction at the injection site with local cell death leading to production of DAMPs and inflammasome activation [64]. The oil component also forms a potential long-term depot that entraps the antigen and slows down its systemic release [65]. Local toxicities of oil emulsions include severe injection site pain due to local tissue damage followed by severe inflammatory reactions that in some cases may progress to formation of a sterile granuloma or ulceration at the injection site [64]. Overall, emulsion adjuvants tend to be at the high end of the local reactogenicity scale and hence are not ideal for prophylactic vaccine use, particularly in pediatric populations [66].

Oil emulsions can also cause generalized systemic symptoms including fever, headache, malaise, nausea, diarrhea, arthralgia, myalgia, and lethargy, reflecting induction of inflammation [6]. A major recurring concern is the potential association between oil emulsion adjuvants and autoimmune disease induction as seen in animal [67–69] and fish [70] models. A single intradermal injection of a range of oil emulsions, including squalene emulsions, induces adjuvant arthritis in susceptible murine and rat models [17]. Adjuvant arthritis is transferrable using T cells, inhibited by anti-T-cell antibodies and associated with increased expression of pro-inflammatory cytokines including IL1 and IFNγ in the draining lymph nodes [71], indicating that oil emulsion adjuvants activate autoreactive arthritogenic T cells. Administration of CFA or IFA alone to C57Bl/6 mice can also induce experimental autoimmune hepatitis [72]. Susceptibility to oil emulsion-induced autoimmune disease is closely linked to genetic factors [73]. There is a theoretical risk that any humans who share similar genetic susceptibility features to these models could similarly be prone to develop adjuvant arthritis, lupus, autoimmune hepatitis, uveitis or some other form of autoimmune disease after exposure to oil emulsion adjuvants alone or when combined with other potent innate immune activators such as monophosphoryl lipid A [9, 74]. This might be relevant to the AS03 adjuvant containing squalene and tocopherol contained in the narcolepsy-associated pandemic influenza vaccine [19, 20]. The causative factor(s) that triggered narcolepsy are still not known but the AS03 adjuvant could have played a major role as no increase in narcolepsy was seen in children who received alternative unadjuvanted vaccines [75]. Hence, it could be hypothesized that inflammation induced by the AS03 adjuvant could have contributed to the breaking of self-tolerance. IL17 is thought to play a major role in autoimmune disorders including multiple sclerosis, rheumatoid arthritis, psoriasis [76] and experimental allergic encephalitis (EAE) [77]. Oil emulsions are potent at inducing inflammatory cytokines including IL1 and IL17 [78]. Given the importance of IL17 for breaking self-tolerance and allowing T cells to cross the blood brain barrier, this could explain why inflammatory oil emulsion adjuvants are so important to autoimmune disease induction in animal models [76], but could also potentially explain the mechanism whereby the AS03-adjuvanted pandemic influenza caused narcolepsy in susceptible HLA DQB1*0602 (DR2+) children[19, 20].

8. Saponin Adjuvants

Saponins are tensoactive glycosides containing a hydrophobic nucleus of triterpenoid structure with carbohydrate chains linked to the nucleus. Quil A is a saponin extract derived from the bark of Quillaja saponaria [79]. Fractions purified from this extract by reverse phase chromatography, such as QS-21, induce strong humoral and T-cell responses [80]. Saponin adjuvants have been extensively utilized in experimental therapeutic cancer vaccines [81]. Through its detergent effects, saponin disrupts cell membranes resulting in moderate to severe injection site pain and muscle cell damage and death causing local redness, swelling and granuloma formation[82]. Saponin adjuvants also cause red blood cell haemolysis, reflecting the affinity of saponins for cholesterol present in erythrocyte membranes [83]. To make the saponin less toxic, QS21 can be mixed with cholesterol to form immune-stimulatory complexes (ISCOMs) [84]. ISCOM particles induce less hemolysis but still induce systemic side effects including flu-like symptoms, fever and malaise [85–87]. The potential of saponin adjuvants to trigger autoimmunity in humans is not known. Some elderly human subjects in a clinical trial of a QS21-adjuvanted experimental Alzheimer’s disease vaccine did develop meningoencephalitis [88] although the role, if any, of the QS21 adjuvant in these adverse reactions is not known [89].

9. TLR agonist adjuvants

The TLR adjuvant category covers an extremely broad spectrum of pathogen-derived compounds including nucleic acids, proteins, lipopeptides and glycolipids, and synthetic analogues thereof [7]. Each of these types of compounds is likely to have very different toxicities. All TLR agonists activate the inflammatory transcription factor, NFkB, through the TLR adaptor proteins, MYD88 and TRIF [90]. A consequence of NFkB activation in monocytes is production of pyrogens and inflammatory cytokines thereby resulting in potential for dose-limiting inflammation and pyrexia [91]. Attempts to detoxify TLR agonists inevitably lead to some loss of adjuvant activity. This is exemplified by conversion of highly toxic TLR4 ligand, lipopolysaccharide, to the less toxic monophosphoryl lipid A (MPL) [92]. Given its modest potency MPL needs to be combined with aluminum or other adjuvants for best effect [92]. AS04 is an example of a combination adjuvant of MPL and aluminum adjuvant and is included in an approved prophylactic hepatitis B virus (HBV) vaccine for low responder renal dialysis patients [93] and a prophylactic human papilloma virus vaccine [94]. HBV-AS04 vaccine was more locally reactogenic than a standard aluminum adjuvanted vaccine with pain at the injection site occurring with 41% of HBV-AS04, versus 19% of standard vaccine, doses, consistent with increased vaccine reactogenicity due to the MPL component [93]. In animal models, TLR4 adjuvants have been shown to cause aberrant immune responses associated with toxicity [95]. For example, inclusion of a TLR4 agonist with an intranasal influenza vaccine in mice caused exacerbated illness and death when immunized animals were challenged with influenza, with the exacerbated lung pathology subsequently found to be due to the TLR4 agonist inducing an excessive IL17 response [95]. TLR4 agonists have also been shown to be able to break tolerance and induce autoimmunity in susceptible animal models[96]. For example, TLR4 agonists, just like the inflammatory agents, trehalose dimycolate, β-glucan, pristane and squalene oil, are potent inducers of inflammatory arthritis in susceptible strains [96]. However, the potential significance of these findings to human safety is not known and relative doses used in human adjuvants are likely to be much lower than those used in animal models.

TLR9 agonists based on unmethylated CpG-containing oligonucleotides (CpG) [97–99] are also under development as human vaccine adjuvants. Binding of CpG to TLR9 leads to activation of NFkB and release of inflammatory cytokines [100], thereby stimulating Th1 immune responses [101]. CpG can also bind directly to B-cell expressed TLR9 leading to B-cell proliferation and antibody secretion [102]. Initially developed for anti-cancer use, CpG was shown to be well tolerated when injected intravenously in high doses to cancer subjects [103]. In general, the phosphodiester linkages in native CpG sequences are considered unsuitable for in vivo use because they are rapidly degraded by DNases [104]. Hence, the synthetic phosphorothioate-backbone is almost exclusively used for current CpG adjuvants in human development. However, the phosphorothioate-backbone has been shown to cause increased adverse effects in murine models including splenomegaly, lymphoid follicle destruction and immunosuppression [103, 105]. In the last 10 years, phosphorothioate-backbone CpG adjuvants have been used in human clinical trials for a broad range of vaccine applications in infectious disease (hepatitis B, influenza, malaria, anthrax, HIV), cancer (melanoma, non-small cell lung cancer) and allergy rhinitis [106]. When CpG7909 (0.5 or 1mg) was added to aluminum-adjuvanted hepatitis B vaccine seroprotection after just a single dose was seen in ~50% of subjects versus none that received the aluminum-adjuvanted vaccine alone [107]. Adverse events including injection site reactions, flu-like symptoms and headache were more frequent in CPG 7909 groups but were predominantly of mild to moderate intensity [107]. 1018 ISS is a synthetic TLR9 agonist oligonucleotide used as an adjuvant in Heplisav®, a vaccine in development for hepatitis B prophylaxis. Vaccine containing 1018 ISS (3mg) promoted faster seroprotection than the comparator Engerix B vaccine [108]. Symptoms of local or systemic reactogenicity in the first 7 days post-immunization were not significantly different to an aluminum-adjuvanted control vaccine, although other studies reporting a higher rate of injection site reactions in the HBsAg-1018 group. [109, 110]. Due to the occurrence of a case of autoimmune Wegener’s granulomatosis in a subject receiving HBsAg-1018 in a trial [109], potential autoimmune events were monitored for in subsequent trials where 3 new-onset autoimmune events, 2 cases of hypothyroidism and 1 of vitiligo all occurred in the HBsAg-1018 group and none in the comparator group, although due to small numbers and 4:1 randomization ratio this difference was not significant [111]. Nevertheless, in 2013 the FDA Vaccines and Related Biological Products Advisory Committee reviewing the Biologic License Application for Heplisav deemed there was still insufficient data to adequately support the safety of Heplisav [112].

A further TLR-based adjuvant approach that has been tested in preliminary human trials is the TLR5 ligand, flagellin [113]. Since flagellin is a protein it can be conveniently expressed as a fusion protein with the antigen itself, and this has been successfully applied to its use in an influenza hemagglutinin-based vaccine [114]. The globular head of the HA1 domain of A/Solomon Islands/3/2006 (H1N1) influenza virus fused to flagellin induced a functional antibody response with the most common local adverse event being pain at the injection site of mild or moderate intensity. Systemic symptoms include fatigue and headache and 2 subjects, who received higher antigen doses had moderately severe systemic symptoms accompanied by substantial increases in serum C-reactive protein (CRP) consistent with a marked inflammatory response[114]. Clinical trials were also conducted with a fusion protein comprising 4 copies of the ectodomain of influenza matrix protein 2 fused to flagellin[115]. Following the first injection at higher doses (3 and 10 µg), self-limited but severe symptoms were noted in some subjects and were associated with elevated CRP levels believed to be mediated by TLR5-stimulated cytokine release [115]. Hence, the major challenge posed by flagellin-based adjuvant approaches, and also mirrored with TLR4 ligand adjuvants, is whether it is possible to titrate the dose to achieve sufficient vaccine immunogenicity on the one hand, while avoiding excess reactogenicity and inflammation, on the other.

10. Enterotoxin adjuvants

A major category of mucosal adjuvants includes cholera toxin (CT) and Escherichia coli heat-labile toxin (LT), and mutated variants thereof [116]. These mucosal adjuvants that are thought to work via their ability to bind distinct ganglioside cell surface receptors and stimulate ADP-ribosylating activity thereby activating adenylate cyclase and increasing intracellular cAMP [117, 118]. CT has a complex range of adjuvant activities, promoting CD40, CD80 and CD86 costimulatory molecule expression, and IL-4 expression thereby enhancing Th2 responses and a B-cell isotype-switch to IgA and IgG production, while suppressing interferon regulatory factor-8, IL-12 production and T cell CD40 ligand expression, thereby suppressing Th1 responses [119]. In gut epithelial cells, cAMP elevation leads to secretion of electrolytes and water into the gut lumen with severe diarrhea being the major dose-limiting toxicity of unmodified CT adjuvant. Whilst detoxified versions of CT and LT have been developed[116], human development of enterotoxin-based mucosal adjuvants was severely set back following a clinical influenza vaccine trial in which the use of a detoxified LT-based adjuvant with an intranasal inactivated vaccine caused facial nerve palsy in a small number of subjects [120].

11. Polysaccharide Adjuvants

The polysaccharides including the polyglucans, polyfructans, and mannans share the benefit of biocompatibility and biodegradability while having potentially useful immunological activities [121]. Polysaccharide adjuvants can be separated into two classes based on whether they activate NFkB and hence are pro-inflammatory (dextran, zymosan, β-glucan, mannan) or don’t activate NFkB and are non-inflammatory (delta inulin) [121]. The polysaccharide adjuvants that activate NFkB and inflammation behave like emulsion adjuvants and are able to induce adjuvant arthritis in susceptible animal models [96]. The polysaccharide adjuvant known as delta inulin or Advax™ [122] enhances humoral and cellular immune responses to a wide variety of viral and bacterial antigens but without evidence of inflammatory side effects [42, 123–127]. Delta inulin adjuvant has been safely administered to pregnant dams [128] and 7-day-old mouse pups [129], where it was able to induce protection with a single influenza vaccine dose. By contrast, MF59, a squalene emulsion adjuvant, failed to protect pups even after two vaccine doses [130]. Delta inulin adjuvant enhanced vaccine immunogenicity and was well tolerated in human clinical trials of hepatitis B [131], pandemic influenza [132] and bee sting allergy [133] vaccines. If inflammation is the key mechanism behind adjuvant-associated toxicity including autoimmune disease induction, then a non-inflammatory adjuvant such as delta inulin may help avoid such toxicity and safety issues. This possibility warrants further exploration as it could provide a route to the development of safer and better-tolerated adjuvants. With respect to safety, polysaccharides, particularly when in particulate form, activate complement causing anaphylatoxin (C5a and C3a) release and basophil and mast cell activation, and potentially symptoms of anaphylactoid shock. In general, however complement activation sufficient to induce anaphylactoid shock is only seen after intravenous, but not intramuscular or subcutaneous, injection. Furthermore, many polysaccharides including dextran and delta inulin bind plasma lipoproteins and may thereby providing negative feedback to downstream complement activation [134].

12. Glycolipid adjuvants

A new class of adjuvants is based on glycolipids that bind the immune receptor CD1d and thereby activate NKT cells, leading to cytokine production and enhanced vaccine responses. Whilst, the most characterized NKT cell agonist galactosyl ceramide has been extensively tested in humans as a potential anti-cancer therapy, no human data on its use as a vaccine adjuvant is yet available, despite promising data on its adjuvant potency in animal studies. However, ABX196, a synthetic analogue of galactosyl ceramide, was tested in a Phase 1/II human trial at doses of 0.2, 0.4, and 2.0µg for its ability to enhance antibody responses to a hepatitis B vaccine [135]. There is known toxicity that can arise from activating NKT cells in the liver [136]. At high doses of ABX196, liver toxicity was seen in mice with a moderate elevation of hepatic and one in four monkeys developed elevated transaminases at the highest dose tested [135]. A clinical trial was then undertaken in healthy adult subjects. Peripheral blood NKT cell activation and increased circulatory IFN-γ was seen 24h post-immunization and increased antibody titers were seen at day 43 when compared to antigen alone, consistent with an adjuvant effect. However, 3 of 29 subjects that received ABX196 had serious treatment-emergent adverse events with major increases in hepatic transaminases (AST and ALT) lasting for several weeks post-immunization and had to be withdrawn from the study. It was concluded that the ABX196 as formulated was not safe for human use due to NKT cell activation resulting in hepatotoxicity [135].

13. Animal models for adjuvant safety assessment

Both aluminum and squalene oil emulsion adjuvants already in broad human use can be shown to induce major adverse effects in animal models, although relevance of such findings to humans remains unknown. Hence, data from such models is largely ignored when safety determinations are made on new vaccines containing these ‘grandfathered’ adjuvants. Regulators instead focus on vaccine safety data collected in rabbits or guinea pigs together with data from human clinical trials to assess vaccine safety [137]. Notably, there remains a need for better scientific explanation as to why specific animal model data showing adjuvant toxicity is not relevant to human use. For example, it has been know for many years that squalene oil emulsions either alone or when formulated with relevant antigens can induce autoimmune conditions, e.g. adjuvant arthritis [138], in genetically-susceptible animals. Hence, a consumer might reasonably ask why this animal toxicity data does not predict the possibility of the adjuvant causing autoimmune disease in human subjects who are also genetically susceptible. There is not currently any good answer to this question. Given the narcolepsy cases associated with use of a pandemic influenza vaccine containing AS03 squalene oil emulsion adjuvant[19, 20], is it reasonable to ask whether the AS03 adjuvant was tested for its propensity to induce autoimmune disease in genetically-susceptible animal models? Even if the influenza antigen in this vaccine turned out to be responsible for inducing narcolepsy, e.g. through a process of antigen mimicry, it is still plausible that the AS03 adjuvant played a role in breaking self-tolerance just as inflammatory adjuvants are critical to disease induction in models such as experimental allergic encephalomyelitis [139]. One possible mechanism worth investigating is whether the AS03 adjuvant induced an excessive Th17 response leading to opening of the blood-brain gate to autoreactive T cells induced by influenza antigen mimicry [140].

Hence, any toxicity may depend on the adjuvant and antigen and other ingredients with which they are combined, together with the genetic background and the age of the population being immunized. This highlights the problem of trying to assess adjuvant safety using traditional testing methods designed for assessment of small molecule drugs for organ toxicity rather than for potential immunological toxicity. In the absence of agreement of appropriate assays to screen for potential immunological toxicity, existing adjuvants most notably aluminum and squalene oil emulsions continue to be approved on a grandfathering basis, leaving extremely high barriers of entry to any new adjuvants. To remove obstacles to introduction of new adjuvants, there is a need for more adjuvant research, including research into mechanisms of adjuvant toxicity thereby hopefully allowing development of better in vivo and in vitro models for adjuvant safety assessment. Whilst the preceding sections have discussed adjuvant safety assessment generally, the sections below focus on safety aspects of specific adjuvants.

14. Approaches To Adjuvant Safety Testing

It is currently not clear what types of preclinical testing might be undertaken to prove an adjuvant is immunologically safe or not. In this respect it is important to distinguish ‘immunological safety’, i.e. risk of inducing, triggering or exacerbating immune disease in a susceptible individual, from ‘toxicological safety’ as assessed by current good laboratory practice (GLP) safety tests using healthy animals [141]. GLP safety tests are designed to measure systemic safety in the context of potential direct organ damage by a substance, a method of testing that is most relevant to small molecule drugs. With adjuvanted vaccines, the components themselves are likely to be non-toxic, but the immune responses they generate may have short or long-term adverse effects, either spontaneously or upon exposure to a relevant pathogen. New vaccines, including those containing new adjuvants, need to pass standard toxicology tests with the issue of potential immunological toxicity in the too-hard basket [141]. Hence there is no agreement on what might be an appropriate predictive test of ‘immunological toxicity’ for an adjuvanted vaccine [142]. The situation is made more complex because most tests of immunological toxicity would need to be undertaken in susceptible animals, which may require the substitution of the antigen and/or the adjuvant for the purposes of assessing the safety of each component separately. Current regulatory guidelines indicate a vaccine adjuvant cannot be assessed or approved in its own right, independently of the vaccine antigen [142]. Notably, narcolepsy after influenza immunization only affected HLA DR2 positive children [8], and similarly most other autoimmune diseases only affect very specific human subpopulations, e.g. ankylosing spondylitis in HLA B27+ individuals, multiple sclerosis in HLA DR2+ individuals, type 1 diabetes in HLA DR3/4+ individuals [143]. Hence, immunological safety cannot be easily assessed in animal strains not genetically susceptible to a particular autoimmune disease. With rare exceptions, e.g. adjuvant arthritis, testing for immunological toxicity also requires adjuvants to be tested in combination with one or more self-antigens. Thus, for example, EAE can only be induced by administering neuronal self-antigen, e.g. myelin basic protein (MBP) together with a pro-inflammatory adjuvant, e.g. CFA, to genetically prone animals [139]. Hence, the EAE model could be used to assess the immunological safety of a particular adjuvant if it were combined with MBP and administered to a susceptible animal. If EAE is not induced by a particular adjuvant then this might provide reassurance that the adjuvant is unlikely to break self-tolerance and induce autoimmune disease, even if inadvertently formulated with a self-antigen mimic. In our hands, for example, neither aluminum and delta inulin adjuvant were able to induce EAE, even when formulated with MBP and use to immune susceptible animals (unpublished data). The bigger problem is if the candidate adjuvant does induce EAE in this model. What is the risk if such an adjuvant is inadvertently formulated with a vaccine antigen that turns out later to be a self-antigen mimic, such as might have happened with the narcolepsy-associated pandemic influenza vaccine [144]. It would seem preferable not to include in prophylactic vaccines adjuvants that can be demonstrated to easily break self-tolerance. Nevertheless, such adjuvants may be ideal for use in cancer vaccines where the ability to break self-tolerance might be a virtue. The EAE and adjuvant arthritis models teach us that induction of autoimmune disease is dependent on exposure of a genetically susceptible individual to the relevant self-antigen together with an inflammatory adjuvant able to break self-tolerance. By simply avoiding inclusion in prophylactic vaccines of an inflammatory adjuvant able to break self-tolerance, the risk of autoimmune disease should thereby be reduced, even if the vaccine includes a self-antigen mimic. In addition to EAE, there are many other well-established animal models of vaccine-inducible autoimmune diseases, including thyroiditis, arthritis, and uveitis that could be used to screen candidate adjuvant formulations for potential immunological toxicity due to ability to break self-tolerance. Predictably, highly pro-inflammatory adjuvants, such as oil emulsions, would fail these tests as just like CFA they can be shown to induce autoimmune disease in relevant models. Similarly, although regulatory bodies do not currently require testing of new adjuvants for potential for IgE induction or allergy-exacerbation it would seem sensible to require testing of all new adjuvants in a relevant allergy induction model where they are assessed against aluminum for their propensity to induce IgE-mediated anaphylaxis [40].

Another issue for adjuvant safety testing for adjuvants such as TLR ligands, is there may be species differences in the relevant receptor, downstream pathways and/or tissue distribution [145]. This may make it difficult to fully assess their safety in the absence of humanized animal models. In this situation it would be useful to identify in vitro surrogates of adjuvant toxicity using human cell lines or primary cells, with readouts such as potency of cytokine induction [146]. Unfortunately, such in vitro approaches may have limited value as they cannot recapitulate the complexity of adjuvant action in vivo. For example, many adjuvants including aluminum have little effect on cytokine production in vitro and yet have potent adjuvant effects in vivo. Furthermore, toxicity may occur in distant and unexpected tissue compartments such as the hepatotoxicity seen with injection of NKT cell agonists[135]. Hence, assessment of adjuvant potency, tolerability and safety will continue to require in vivo testing. Given that vaccine adverse effects may only affect rare individuals in a stochastic manner or because of underlying genetic susceptibilities, predictive animal models need to be able to recapitulate such factors. This necessitates research into the nature of human susceptibilities to adjuvant toxicity, with tools including whole genome sequencing, gene expression arrays and deep sequencing approaches now readily available to start addressing such questions.

15. Consumer perceptions of adjuvant safety

No medical intervention is completely without risk and hence all human medicines including vaccines are approved by regulators based on risk-benefit principles [147]. The interests of the public are protected by regulators such as the Food and Drug Administration whose role is to approve vaccines only if the proven benefits outweigh any measurable risks [147]. Assessment of vaccine risk-benefit is more complex than for therapeutic interventions, as benefits of vaccination can accrue to the population through herd immunity, while the risks of any adverse reactions are suffered by individuals, potentially raising complex ethical issues [148]. Hence, perceptions of risk-benefit at the individual level, i.e. “I do not want immunization because any benefits do not justify the risk of a vaccine reaction” [149], can sometimes be difficult to reconcile with risk-benefit assessments at the public health level, i.e. “if we allow too many individuals not to be immunized, herd immunity will be lost and serious infectious disease outbreaks may eventuate” [150]. Hence, policy makers and vaccine recipients might have very different perceptions of immunization risk-benefits [151]. This is also likely to shape the publics’ view of adjuvant risk-benefits, particularly in situations where both adjuvanted and unadjuvanted vaccines are available for the same indication. For example, an approved seasonal influenza vaccine in Europe contains MF59 squalene emulsion adjuvant, but the vast majority of influenza vaccines used in Europe are not adjuvanted [152]. Factors influencing consumer and practitioner utilization of adjuvanted versus unadjuvanted influenza vaccines could thereby be a useful area of study. No adjuvanted seasonal influenza vaccines are currently available in the US, which could potentially reflect differences in regulatory and consumer views across continents [141]. During the 2009 influenza pandemic both adjuvanted and unadjuvanted pandemic vaccines were utilized in Europe, with consumers not always given a choice regarding which vaccine was used [153]. By contrast, only unadjuvanted pandemic vaccines were used in the US [154]. This imposed lack of consumer choice in some European countries may have acted to reinforce negative perceptions of adjuvanted vaccines, particularly when it was subsequently revealed that a squalene-adjuvanted vaccine used during the pandemic was associated with an increased risk of childhood narcolepsy[8]. Given potential public apprehension around the term ‘adjuvant’, there is a need for more research to identify the source of such fears and develop strategies to alleviate them [155]. The origins of consumer apprehension surrounding adjuvants is likely to be multifactorial, with potential contributors being general mistrust in governments and public health policy, perceptions of lack of choice, media coverage of rare adverse reactions, confusion between issues of aluminum, thiomersal and other vaccine excipients, and citing of papers on the role of adjuvants such as oil emulsions in autoimmune disease in animal models. In respect of the latter, scientists know animal model findings may not always translate to humans, as reflected in the saying ‘mice lie’. However, in the absence of adequate education many consumers are unlikely to appreciate this point and may place undue emphasis on such data when making risk-benefit assessments in respect of adjuvanted vaccines. It is also important that more research is undertaken to better understand such adverse effects in animal models and how they relate to the human context.

16. Public health views on adjuvant safety

Even if adjuvant causality is confirmed for a rare vaccine adverse events this can create a disclosure dilemma, namely “should risks of a rare vaccine-associated adverse events be publicized given that consumers are likely to over react to them in the interests of public health should any possibility of such rare adverse events be downplayed to avoid damage to immunization campaigns”[60]. This is not an easy question to answer. To assist successful introduction of new adjuvants without risk of consumer backlash, it would be beneficial to have better understanding of public perceptions regarding adjuvants. This could then allow consumer education campaigns to be designed to address any misunderstandings or concerns [156]. Hence, alongside greater research into the mechanisms underlying potential adjuvant-associated adverse reactions, research is also needed into consumer perceptions of adjuvants [157], as policies to improve immunization rates could easily backfire if not carefully researched [158]. Whilst it can be argued that ‘society has the right and responsibility to establish laws, regulations, and choice frameworks that discourage vaccine refusal’ [150], any mandatory action that reduces consumer choice needs to be considered very carefully. Another question is the role of bodies such as the WHO Global Advisory Committee on Vaccine Safety in adjudicating on vaccine or adjuvant safety [159]. Arguably, the primary role of such bodies is to defend vaccine use in developing countries where the risk-benefits of immunization is vastly different to that in developed countries where infectious diseases are far less prevalent and old age and chronic diseases far more prevalent. Notably in most cases considered by the committee, reference is made to a lack of data and inadequate well conducted controlled studies to confirm possible vaccine risks. This highlights the remarkable lack of research into vaccine and adjuvant safety issues despite the fact that such research should fit within the framework for a ‘global regulatory science agenda for vaccines’ [160].

15. Conclusions

This paper highlights the inherent difficulties of assessing adjuvant safety and the poor state of knowledge of the mechanisms underlying potential adjuvant toxicity. Even, aluminum, an adjuvant in widespread human use for almost a century and given to billions of subjects, still has unanswered questions regarding its mechanism of action and its potential connection to conditions such as macrophagic myofasciitis or Alzheimer’s disease. While there can be no such thing as a 100% risk-free vaccine, any risks of immediate severe adverse reactions are extremely low for modern vaccines and consumers should have high confidence in the safety of available vaccines. To facilitate the introduction of new adjuvants it will be important that consumers are educated better regarding vaccine risk-benefit assessment. Given the vital importance of adjuvants to modern vaccines, additional resources are needed to support research to better understand adjuvant action and how this might relate to adjuvant toxicity. New adjuvants continue to be needed that improve vaccine potency without compromising tolerability or safety. An attractive hypothesis warranting further exploration is the concept of a non-inflammatory adjuvant able to enhance vaccine potency while avoiding any reactogenicity or safety issues.

Acknowledgments

Footnotes

REFERENCES