Comparative safety of vaccine adjuvants: a summary of current evidence and future needs

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615573/

Abstract

Improved use of highly pure antigens to improve vaccine safety has led to reduced vaccine immunogenicity and efficacy. This has led to the need to use adjuvants to improve vaccine immunogenicity. The ideal adjuvant should maximize vaccine immunogenicity without compromising tolerability or safety or posing undue risk. Unfortunately, adjuvant research has lagged behind other vaccine areas such as antigen discovery, with the consequence that only a very limited number of adjuvants based on aluminum salts, monophosphoryl lipid A and oil emulsions are currently approved for human use. Recent strategic initiatives to support adjuvant development by the National Institutes of Health should translate into greater adjuvant choices in the future. Mechanistic studies have been valuable in better understanding adjuvant action but mechanisms of adjuvant toxicity are less well understood. The inflammatory or danger-signal model of adjuvant action implies that increased vaccine reactogenicity is the inevitable price for improved immunogenicity. Hence, adjuvant reactogenicity may be avoidable only if it is possible to separate inflammation from adjuvant action. The biggest remaining challenge in the adjuvant field is to decipher the potential relationship between adjuvants and rare vaccine adverse reactions such as narcolepsy, macrophagic myofasciitis or Alzheimer’s disease. While existing adjuvants based on aluminum salts have a strong safety record, there is an ongoing need for new adjuvants and for more intensive research into adjuvants and their effects.

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 [2224]. 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, 3537]. 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 [6769] 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 [8587]. 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) [9799] 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, 123127]. 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.

Key points

  • Existing human adjuvants have a high level of safety
  • The relationship between specific adjuvants and rare adverse reactions such as narcolepsy or macrophagic myofasciitis remains to be resolved
  • More research is needed into adjuvants and how they work

Acknowledgments

FUNDING: The author is supported by research funding from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, under Contracts No. HHSN272200800039C and HHSN272201400053C. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

COMPLIANCE WITH ETHICAL STANDARDS

CONFLICTS OF INTEREST: Nikolai Petrovsky is a director of Vaxine Pty Ltd.

REFERENCES

1. Poolman JT. Shortcomings of pertussis vaccines: why we need a third generation vaccine. Expert Rev Vaccines. 2014 Oct;13(10):1159–1162. [PubMed]
2. Li-Kim-Moy J, Booy R. The manufacturing process should remain the focus for severe febrile reactions in children administered an Australian inactivated influenza vaccine during 2010. Influenza Other Respir Viruses. 2015 Aug;:10. [PubMed]
3. Aichinger G, Grohmann-Izay B, van der Velden MV, Fritsch S, Koska M, Portsmouth D, et al. Phase I/II randomized double-blind study of the safety and immunogenicity of a nonadjuvanted vero cell culture-derived whole-virus H9N2 influenza vaccine in healthy adults. Clinical and vaccine immunology : CVI. 2015 Jan;22(1):46–55. [PMC free article] [PubMed]
4. Rockman S, Dyson A, Koernig S, Becher D, Ng M, Morelli AB, et al. Evaluation of the bioactivity of influenza vaccine strains in vitro suggests that the introduction of new strains in the 2010 Southern Hemisphere trivalent influenza vaccine is associated with adverse events. Vaccine. 2014 Jun 24;32(30):3861–3868. [PubMed]
5. Petrovsky N, Heinzel S, Honda Y, Lyons AB. New-Age Vaccine Adjuvants: Friend or Foe? Biopharm International. 2007;20(8):24–33.
6. Petrovsky N. Freeing vaccine adjuvants from dangerous immunological dogma. Expert Rev Vaccines. 2008 Feb;7(1):7–10. [PubMed]
7. Petrovsky N, Aguilar JC. Vaccine adjuvants: current state and future trends. Immunol Cell Biol. 2004 Oct;82(5):488–496. [PubMed]
8. Ahmed SS, Schur PH, MacDonald NE, Steinman L. Narcolepsy, 2009 A(H1N1) pandemic influenza, and pandemic influenza vaccinations: what is known and unknown about the neurological disorder, the role for autoimmunity, and vaccine adjuvants. Journal of autoimmunity. 2014 May;50:1–11. [PubMed]
9. Vera-Lastra O, Medina G, Cruz-Dominguez Mdel P, Jara LJ, Shoenfeld Y. Autoimmune/inflammatory syndrome induced by adjuvants (Shoenfeld’s syndrome): clinical and immunological spectrum. Expert review of clinical immunology. 2013 Apr;9(4):361–373. [PubMed]
10. Gherardi RK, Authier FJ. Aluminum inclusion macrophagic myofasciitis: a recently identified condition. Immunol Allergy Clin North Am. 2003 Nov;23(4):699–712. [PubMed]
11. Willhite CC, Karyakina NA, Yokel RA, Yenugadhati N, Wisniewski TM, Arnold IM, et al. Systematic review of potential health risks posed by pharmaceutical, occupational and consumer exposures to metallic and nanoscale aluminum, aluminum oxides, aluminum hydroxide and its soluble salts. Critical reviews in toxicology. 2014 Oct;44(Suppl 4):1–80. [PMC free article] [PubMed]
12. Leventhal JS, Berger EM, Brauer JA, Cohen DE. Hypersensitivity reactions to vaccine constituents: a case series and review of the literature. Dermatitis : contact, atopic, occupational, drug. 2012 May-Jun;23(3):102–109. [PubMed]
13. Powell BS, Andrianov AK, Fusco PC. Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes. Clinical and experimental vaccine research. 2015 Jan;4(1):23–45. [PMC free article][PubMed]
14. Butler NR, Voyce MA, Burland WL, Hilton ML. Advantages of aluminium hydroxide adsorbed combined diphtheria, tetanus, and pertussis vaccines for the immunization of infants. British medical journal. 1969 Mar 15;1(645):663–666. [PMC free article] [PubMed]
15. Cherin P, Gherardi RK. Emergence of a new entity, the macrophagic myofasciitis. GERMMAD Study Group of the French Association Against Myopathies Study and Research Group on Acquired Dysimmunity-related Muscle Disease. Rev Rhum Engl Ed. 1998 Oct;65(10):541–542. [PubMed]
16. Safety WGACoV. Questions and Answers about macrophagic myofasciitis (MMF) 2008 [cited 2015 19 August 2015]; Available from:
17. Aghazadeh-Habashi A, Kohan MH, Asghar W, Jamali F. Glucosamine dose/concentration-effect correlation in the rat with adjuvant arthritis. Journal of pharmaceutical sciences. 2014 Feb;103(2):760–767.[PubMed]
18. Marson A, Housley WJ, Hafler DA. Genetic basis of autoimmunity. J Clin Invest. 2015 Jun;125(6):2234–2241. [PMC free article] [PubMed]
19. Nohynek H, Jokinen J, Partinen M, Vaarala O, al e. AS03 Adjuvanted AH1N1 Vaccine Associated with an Abrupt Increase in the Incidence of Childhood Narcolepsy in Finland. PLoS ONE. 2012;7(3):33536.[PMC free article] [PubMed]
20. Partinen M, Saarenpaa-Heikkila O, Ilveskloski I. Increased Incidence and Clinical Picture of Childhood Narcolepsy following the 2009 H1N1 Pandemic Vaccination Campaign in Finland. PLoS One. 2012;7(3):33723. [PMC free article] [PubMed]
21. Rigolet M, Aouizerate J, Couette M, Ragunathan-Thangarajah N, Aoun-Sebaiti M, Gherardi RK, et al. Clinical features in patients with long-lasting macrophagic myofasciitis. Frontiers in neurology. 2014;5:230. [PMC free article] [PubMed]
22. Lima H, Jr, Jacobson LS, Goldberg MF, Chandran K, Diaz-Griffero F, Lisanti MP, et al. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death. Cell Cycle. 2013 Jun 15;12(12):1868–1878. [PMC free article] [PubMed]
23. Gupta RK. Aluminum compounds as vaccine adjuvants. Adv Drug Deliv Rev. 1998 Jul 6;32(3):155–172. [PubMed]
24. Goto N, Kato H, Maeyama J, Shibano M, Saito T, Yamaguchi J, et al. Local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties. Vaccine. 1997 Aug-Sep;15(12–13):1364–1371. [PubMed]
25. Pusic K, Aguilar Z, McLoughlin J, Kobuch S, Xu H, Tsang M, et al. Iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 2013 Mar;27(3):1153–1166. [PMC free article] [PubMed]
26. Lee JY, Atochina O, King B, Taylor L, Elloso M, Scott P, et al. Beryllium, an adjuvant that promotes gamma interferon production. Infect Immun. 2000 Jul;68(7):4032–4039. [PMC free article] [PubMed]
27. Lin Y, Epstein DL, Liton PB. Intralysosomal iron induces lysosomal membrane permeabilization and cathepsin D-mediated cell death in trabecular meshwork cells exposed to oxidative stress. Investigative ophthalmology & visual science. 2010 Dec;51(12):6483–6495. [PMC free article] [PubMed]
28. Dinsdale D. Lysosomal involvement in beryllium phosphate toxicity. British journal of experimental pathology. 1982 Feb;63(1):103–108. [PMC free article] [PubMed]
29. Hall JG. Studies on the adjuvant action of beryllium IV The preparation of beryllium containing macromolecules that induce immunoblast responses in vivo. Immunology. 1988 Jun;64(2):345–351.[PMC free article] [PubMed]
30. Doherty PC, Turner SJ, Webby RG, Thomas PG. Influenza and the challenge for immunology. Nat Immunol. 2006 May;7(5):449–455. [PubMed]
31. Jensen-Jarolim E. Aluminium in Allergies and Allergen immunotherapy. The World Allergy Organization journal. 2015;8(1):7. [PMC free article] [PubMed]
32. Aimanianda V, Haensler J, Lacroix-Desmazes S, Kaveri SV, Bayry J. Novel cellular and molecular mechanisms of induction of immune responses by aluminum adjuvants. Trends Pharmacol Sci. 2009 Jun;30(6):287–295. [PubMed]
33. Netterlid E, Hindsen M, Siemund I, Bjork J, Werner S, Jacobsson H, et al. Does allergen-specific immunotherapy induce contact allergy to aluminium? Acta dermato-venereologica. 2013 Jan;93(1):50–56.[PubMed]
34. Dinarello CA, van der Meer JW. Treating inflammation by blocking interleukin-1 in humans. Semin Immunol. 2013 Dec 15;25(6):469–484. [PMC free article] [PubMed]
35. Audibert FM, Lise LD. Adjuvants: current status, clinical perspectives and future prospects. Immunology today. 1993 Jun;14(6):281–284. [PubMed]
36. Bomford R. Aluminium salts: perspectives in their use as adjuvants. In: Gregoriadis GAAG, Poste G, editors. Immunological adjuvants and vaccines. New York: Plenum Press; 1989. pp. 35–41.
37. Goto N, Kato H, Maeyama J, Eto K, Yoshihara S. Studies on the toxicities of aluminium hydroxide and calcium phosphate as immunological adjuvants for vaccines. Vaccine. 1993;11(9):914–918. [PubMed]
38. Nieuwenhuizen N, Herbert DR, Lopata AL, Brombacher F. CD4+ T cell-specific deletion of IL-4 receptor alpha prevents ovalbumin-induced anaphylaxis by an IFN-gamma-dependent mechanism. J Immunol. 2007 Sep 1;179(5):2758–2765. [PubMed]
39. Xu W, Tamura T, Takatsu K. CpG ODN mediated prevention from ovalbumin-induced anaphylaxis in mouse through B cell pathway. Int Immunopharmacol. 2008 Feb;8(2):351–361. [PubMed]
40. White OJ, McKenna KL, Bosco A, A HJvdB, Richmond P, Holt PG. A genomics-based approach to assessment of vaccine safety and immunogenicity in children. Vaccine. 2012 Feb 27;30(10):1865–1874.[PubMed]
41. De Swart RL, Kuiken T, Timmerman HH, van Amerongen G, Van Den Hoogen BG, Vos HW, et al. Immunization of macaques with formalin-inactivated respiratory syncytial virus (RSV) induces interleukin-13-associated hypersensitivity to subsequent RSV infection. Journal of virology. 2002 Nov;76(22):11561–11569. [PMC free article] [PubMed]
42. Honda-Okubo Y, Barnard D, Ong CH, Peng BH, Tseng CT, Petrovsky N. Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection while Ameliorating Lung Eosinophilic Immunopathology. Journal of virology. 2015 Mar 15;89(6):2995–3007. [PMC free article] [PubMed]
43. Openshaw PJ, Culley FJ, Olszewska W. Immunopathogenesis of vaccine-enhanced RSV disease. Vaccine. 2001 Oct 15;20(20) Suppl 1:27–31. [PubMed]
44. Vascellari M, Melchiotti E, Bozza MA, Mutinelli F. Fibrosarcomas at presumed sites of injection in dogs: characteristics and comparison with non-vaccination site fibrosarcomas and feline post-vaccinal fibrosarcomas. Journal of veterinary medicine A, Physiology, pathology, clinical medicine. 2003 Aug;50(6):286–291. [PubMed]
45. Cherin P, Gherardi RK. Macrophagic myofasciitis. Curr Rheumatol Rep. 2000 Jun;2(3):196–200.[PubMed]
46. Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, et al. Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle. Brain. 2001 Sep;124(Pt 9):1821–1831. [PubMed]
47. Authier FJ, Cherin P, Creange A, Bonnotte B, Ferrer X, Abdelmoumni A, et al. Central nervous system disease in patients with macrophagic myofasciitis. Brain. 2001 May;124(Pt 5):974–983. [PubMed]
48. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nature genetics. 2001 Nov;29(3):301–305. [PMC free article] [PubMed]
49. Bingley PJ, Hoffbrand BI. Antiphospholipid antibody syndrome: a review. Journal of the Royal Society of Medicine. 1987 Jul;80(7):445–448. [PMC free article] [PubMed]
50. Zivkovic I, Petrusic V, Stojanovic M, Inic-Kanada A, Stojicevic I, Dimitrijevic L. Induction of decreased fecundity by tetanus toxoid hyper-immunization in C57BL/6 mice depends on the applied adjuvant. Innate immunity. 2012 Apr;18(2):333–342. [PubMed]
51. Reusche E, Seydel U. Dialysis-associated encephalopathy: light and electron microscopic morphology and topography with evidence of aluminum by laser microprobe mass analysis. Acta neuropathologica. 1993;86(3):249–258. [PubMed]
52. Miu AC, Benga O. Aluminum and Alzheimer’s disease: a new look. Journal of Alzheimer’s disease : JAD. 2006 Nov;10(2–3):179–201. [PubMed]
53. Kramer MF, Heath MD. Aluminium in allergen-specific subcutaneous immunotherapy–a German perspective. Vaccine. 2014 Jul 16;32(33):4140–4148. [PubMed]
54. Eidi H, David MO, Crepeaux G, Henry L, Joshi V, Berger MH, et al. Fluorescent nanodiamonds as a relevant tag for the assessment of alum adjuvant particle biodisposition. BMC medicine. 2015;13:144.[PMC free article] [PubMed]
55. Shaw CA, Li Y, Tomljenovic L. Administration of aluminium to neonatal mice in vaccine-relevant amounts is associated with adverse long term neurological outcomes. Journal of inorganic biochemistry. 2013 Nov;128:237–244. [PubMed]
56. Shaw CA, Tomljenovic L. Aluminum in the central nervous system (CNS): toxicity in humans and animals, vaccine adjuvants, and autoimmunity. Immunol Res. 2013 Jul;56(2–3):304–316. [PubMed]
57. Shaw CA, Li D, Tomljenovic L. Are there negative CNS impacts of aluminum adjuvants used in vaccines and immunotherapy? Immunotherapy. 2014;6(10):1055–1071. [PubMed]
58. Fanni D, Ambu R, Gerosa C, Nemolato S, Iacovidou N, Van Eyken P, et al. Aluminum exposure and toxicity in neonates: a practical guide to halt aluminum overload in the prenatal and perinatal periods. World journal of pediatrics : WJP. 2014 May;10(2):101–107. [PubMed]
59. Safety WGACoV. GACVS Report of meeting Jun 2012. WHO Weekly Epidemiological Record. 2012;87(30):282–283.
60. von Biela LM. A disclosure dilemma: what you don’t know can kill you, but so can what you do know. Food and drug law journal. 2010;65(2):317–346. ii. [PubMed]
61. Lindblad EB. Aluminium adjuvants–in retrospect and prospect. Vaccine. 2004 Sep 9;22(27–28):3658–3668. [PubMed]
62. Verdier F, Burnett R, Michelet-Habchi C, Moretto P, Fievet-Groyne F, Sauzeat E. Aluminium assay and evaluation of the local reaction at several time points after intramuscular administration of aluminium containing vaccines in the Cynomolgus monkey. Vaccine. 2005 Feb 3;23(11):1359–1367. [PubMed]
63. van Dissel JT, Joosten SA, Hoff ST, Soonawala D, Prins C, Hokey DA, et al. A novel liposomal adjuvant system, CAF01, promotes long-lived Mycobacterium tuberculosis-specific T-cell responses in human. Vaccine. 2014 Dec 12;32(52):7098–7107. [PubMed]
64. Stills HF., Jr Adjuvants and antibody production: dispelling the myths associated with Freund’s complete and other adjuvants. Ilar J. 2005;46(3):280–293. [PubMed]
65. Herbert WJ. The mode of action of mineral-oil emulsion adjuvants on antibody production in mice. Immunology. 1968 Mar;14(3):301–318. [PMC free article] [PubMed]
66. McCarthy JS, Marjason J, Elliott S, Fahey P, Bang G, Malkin E, et al. A phase 1 trial of MSP2-C1, a blood-stage malaria vaccine containing 2 isoforms of MSP2 formulated with Montanide(R) ISA 720. PLoS ONE. 2011;6(9):24413. [PMC free article] [PubMed]
67. Vera-Lastra O, Medina G, Cruz-Dominguez Mdel P, Ramirez P, Gayosso-Rivera JA, Anduaga-Dominguez H, et al. Human adjuvant disease induced by foreign substances: a new model of ASIA (Shoenfeld’s syndrome) Lupus. 2012 Feb;21(2):128–135. [PubMed]
68. Whitehouse M. Oily adjuvants and autoimmunity: now time for reconsideration? Lupus. 2012 Feb;21(2):217–222. [PubMed]
69. Kuroda Y, Akaogi J, Nacionales DC, Wasdo SC, Szabo NJ, Reeves WH, et al. Distinctive patterns of autoimmune response induced by different types of mineral oil. Toxicological sciences : an official journal of the Society of Toxicology. 2004 Apr;78(2):222–228. [PubMed]
70. Koppang EO, Bjerkas I, Haugarvoll E, Chan EK, Szabo NJ, Ono N, et al. Vaccination-induced systemic autoimmunity in farmed Atlantic salmon. J Immunol. 2008 Oct 1;181(7):4807–4814. [PubMed]
71. Holm BC, Lorentzen JC, Bucht A. Adjuvant oil induces waves of arthritogenic lymph node cells prior to arthritis onset. Clin Exp Immunol. 2004 Jul;137(1):59–64. [PMC free article] [PubMed]
72. Howell CD, Yoder TD. Murine experimental autoimmune hepatitis: nonspecific inflammation due to adjuvant oil. Clinical immunology and immunopathology. 1994 Jul;72(1):76–82. [PubMed]
73. Lorentzen JC, Klareskog L. Susceptibility of DA rats to arthritis induced with adjuvant oil or rat collagen is determined by genes both within and outside the major histocompatibility complex. Scand J Immunol. 1996 Dec;44(6):592–598. [PubMed]
74. Svelander L, Erlandsson Harris H, Lorentzen JC, Trollmo C, Klareskog L, Bucht A. Oligodeoxynucleotides containing CpG motifs can induce T cell-dependent arthritis in rats. Arthritis and rheumatism. 2004 Jan;50(1):297–304. [PubMed]
75. Tsai TF, Crucitti A, Nacci P, Nicolay U, Della Cioppa G, Ferguson J, et al. Explorations of clinical trials and pharmacovigilance databases of MF59(R)-adjuvanted influenza vaccines for associated cases of narcolepsy. Scand J Infect Dis. 2011 Sep;43(9):702–706. [PubMed]
76. Cosmi L, Liotta F, Maggi E, Romagnani S, Annunziato F. Th17 and non-classic Th1 cells in chronic inflammatory disorders: two sides of the same coin. International archives of allergy and immunology. 2014;164(3):171–177. [PubMed]
77. Sutton C, Brereton C, Keogh B, Mills KH, Lavelle EC. A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J Exp Med. 2006 Jul 10;203(7):1685–1691. [PMC free article] [PubMed]
78. Vitoriano-Souza J, Moreira N, Teixeira-Carvalho A, Carneiro CM, Siqueira FA, Vieira PM, et al. Cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete Freund’s adjuvant, and monophosphoryl lipid A. PLoS ONE. 2012;7(7):40745. [PMC free article] [PubMed]
79. Kensil CR, Kammer R. QS-21: a water-soluble triterpene glycoside adjuvant. Expert Opin Investig Drugs. 1998 Sep;7(9):1475–1482. [PubMed]
80. Wu JY, Gardner BH, Murphy CI, Seals JR, Kensil CR, Recchia J, et al. Saponin adjuvant enhancement of antigen-specific immune responses to an experimental HIV-1 vaccine. J Immunol. 1992 Mar 1;148(5):1519–1525. [PubMed]
81. Stewart TJ, Drane D, Malliaros J, Elmer H, Malcolm KM, Cox JC, et al. ISCOMATRIX adjuvant: an adjuvant suitable for use in anticancer vaccines. Vaccine. 2004 Sep 9;22(27–28):3738–3743. [PubMed]
82. Waite DC, Jacobson EW, Ennis FA, Edelman R, White B, Kammer R, et al. Three double-blind, randomized trials evaluating the safety and tolerance of different formulations of the saponin adjuvant QS-21. Vaccine. 2001 Jul 16;19(28–29):3957–3967. [PubMed]
83. Lorent JH, Quetin-Leclercq J, Mingeot-Leclercq MP. The amphiphilic nature of saponins and their effects on artificial and biological membranes and potential consequences for red blood and cancer cells. Organic & biomolecular chemistry. 2014 Nov 28;12(44):8803–8822. [PubMed]
84. Morein B, Sundquist B, Hoglund S, Dalsgaard K, Osterhaus A. Iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature. 1984;308(5958):457–460. Mar 29-Apr 4. [PubMed]
85. Claassen I, Osterhaus A. The iscom structure as an immune-enhancing moiety: experience with viral systems. Res Immunol. 1992 Jun;143(5):531–541. [PubMed]
86. Ronnberg B, Fekadu M, Morein B. Adjuvant activity of non-toxic Quillaja saponaria Molina components for use in ISCOM matrix. Vaccine. 1995 Oct;13(14):1375–1382. [PubMed]
87. Davis ID, Chen W, Jackson H, Parente P, Shackleton M, Hopkins W, et al. Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans. Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10697–10702. [PMC free article][PubMed]
88. Orgogozo JM, Gilman S, Dartigues JF, Laurent B, Puel M, Kirby LC, et al. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology. 2003 Jul 8;61(1):46–54. [PubMed]
89. Asuni AA, Boutajangout A, Scholtzova H, Knudsen E, Li YS, Quartermain D, et al. Vaccination of Alzheimer’s model mice with Abeta derivative in alum adjuvant reduces Abeta burden without microhemorrhages. The European journal of neuroscience. 2006 Nov;24(9):2530–2542. [PMC free article][PubMed]
90. Verstak B, Hertzog P, Mansell A. Toll-like receptor signalling and the clinical benefits that lie within. Inflammation research : official journal of the European Histamine Research Society [et al] 2007 Jan;56(1):1–10. [PubMed]
91. Collins SE, Mossman KL. Danger, diversity and priming in innate antiviral immunity. Cytokine & growth factor reviews. 2014 Oct;25(5):525–531. [PubMed]
92. Alving CR, Peachman KK, Rao M, Reed SG. Adjuvants for human vaccines. Current opinion in immunology. 2012 Jun;24(3):310–315. [PMC free article] [PubMed]
93. Tong NK, Beran J, Kee SA, Miguel JL, Sanchez C, Bayas JM, et al. Immunogenicity and safety of an adjuvanted hepatitis B vaccine in pre-hemodialysis and hemodialysis patients. Kidney international. 2005 Nov;68(5):2298–2303. [PubMed]
94. Didierlaurent AM, Morel S, Lockman L, Giannini SL, Bisteau M, Carlsen H, et al. AS04, an aluminum salt- and TLR4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity. Journal of immunology. 2009 Nov 15;183(10):6186–6197.[PubMed]
95. Maroof A, Yorgensen YM, Li Y, Evans JT. Intranasal vaccination promotes detrimental Th17-mediated immunity against influenza infection. PLoS Pathog. 2014 Jan;10(1):1003875. [PMC free article] [PubMed]
96. Naik SR, Wala SM. Arthritis, a complex connective and synovial joint destructive autoimmune disease: animal models of arthritis with varied etiopathology and their significance. Journal of postgraduate medicine. 2014 Jul-Sep;60(3):309–317. [PubMed]
97. Hartmann G, Weeratna RD, Ballas ZK, Payette P, Blackwell S, Suparto I, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol. 2000 Feb 1;164(3):1617–1624. [PubMed]
98. Klinman DM, Yi AK, Beaucage SL, Conover J, Krieg AM. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2879–2883. [PMC free article] [PubMed]
99. Krug A, Rothenfusser S, Selinger S, Bock C, Kerkmann M, Battiany J, et al. CpG-A Oligonucleotides Induce a Monocyte-Derived Dendritic Cell-Like Phenotype That Preferentially Activates CD8 T Cells. J Immunol. 2003;170(7):3468–3477. [PubMed]
100. Klinman DM, Barnhart KM, Conover J. CpG motifs as immune adjuvants. Vaccine. 1999 Jan;17(1):19–25. [PubMed]
101. Krieg AM, Efler SM, Wittpoth M, Al Adhami MJ, Davis HL. Induction of systemic TH1-like innate immunity in normal volunteers following subcutaneous but not intravenous administration of CPG 7909, a synthetic B-class CpG oligodeoxynucleotide TLR9 agonist. J Immunother (1997) 2004 Nov-Dec;27(6):460–471. [PubMed]
102. Jegerlehner A, Maurer P, Bessa J, Hinton HJ, Kopf M, Bachmann MF. TLR9 signaling in B cells determines class switch recombination to IgG2a. J Immunol. 2007 Feb 15;178(4):2415–2420. [PubMed]
103. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nature reviews Drug discovery. 2006 Jun;5(6):471–484. [PubMed]
104. Agrawal S, Temsamani J, Tang JY. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7595–7599. [PMC free article] [PubMed]
105. Heikenwalder M, Polymenidou M, Junt T, Sigurdson C, Wagner H, Akira S, et al. Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration. Nat Med. 2004 Feb;10(2):187–192. [PubMed]
106. Scheiermann J, Klinman DM. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine. 2014 Nov 12;32(48):6377–6389. [PMC free article][PubMed]
107. Cooper CL, Davis HL, Morris ML, Efler SM, Adhami MA, Krieg AM, et al. CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in healthy adults: a double-blind phase I/II study. J Clin Immunol. 2004 Nov;24(6):693–701. [PubMed]
108. Halperin SA, Dobson S, McNeil S, Langley JM, Smith B, McCall-Sani R, et al. Comparison of the safety and immunogenicity of hepatitis B virus surface antigen co-administered with an immunostimulatory phosphorothioate oligonucleotide and a licensed hepatitis B vaccine in healthy young adults. Vaccine. 2006 Jan 9;24(1):20–26. [PubMed]
109. Halperin SA, Ward B, Cooper C, Predy G, Diaz-Mitoma F, Dionne M, et al. Comparison of safety and immunogenicity of two doses of investigational hepatitis B virus surface antigen co-administered with an immunostimulatory phosphorothioate oligodeoxyribonucleotide and three doses of a licensed hepatitis B vaccine in healthy adults 18–55 years of age. Vaccine. 2012 Mar 28;30(15):2556–2563. [PubMed]
110. Sablan BP, Kim DJ, Barzaga NG, Chow WC, Cho M, Ahn SH, et al. Demonstration of safety and enhanced seroprotection against hepatitis B with investigational HBsAg-1018 ISS vaccine compared to a licensed hepatitis B vaccine. Vaccine. 2012 Mar 30;30(16):2689–2696. [PubMed]
111. Heyward WL, Kyle M, Blumenau J, Davis M, Reisinger K, Kabongo ML, et al. Immunogenicity and safety of an investigational hepatitis B vaccine with a Toll-like receptor 9 agonist adjuvant (HBsAg-1018) compared to a licensed hepatitis B vaccine in healthy adults 40–70 years of age. Vaccine. 2013 Nov 4;31(46):5300–5305. [PubMed]
113. Mizel SB, Bates JT. Flagellin as an adjuvant: cellular mechanisms and potential. J Immunol. 2010 Nov 15;185(10):5677–5682. [PMC free article] [PubMed]
114. Treanor JJ, Taylor DN, Tussey L, Hay C, Nolan C, Fitzgerald T, et al. Safety and immunogenicity of a recombinant hemagglutinin influenza-flagellin fusion vaccine (VAX125) in healthy young adults. Vaccine. 2010 Dec 6;28(52):8268–8274. [PubMed]
115. Turley CB, Rupp RE, Johnson C, Taylor DN, Wolfson J, Tussey L, et al. Safety and immunogenicity of a recombinant M2e–flagellin influenza vaccine (STF2.4xM2e) in healthy adults. Vaccine. 2011 Jul 18;29(32):5145–5152. [PubMed]
116. Larena M, Holmgren J, Lebens M, Terrinoni M, Lundgren A. Cholera Toxin, and the Related Nontoxic Adjuvants mmCT and dmLT, Promote Human Th17 Responses via Cyclic AMP-Protein Kinase A and Inflammasome-Dependent IL-1 Signaling. J Immunol. 2015 Mar;:18. [PubMed]
117. Holmgren J, Lycke N, Czerkinsky C. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine. 1993 Sep;11(12):1179–1184. [PubMed]
118. Freytag LC, Clements JD. Mucosal adjuvants. Vaccine. 2005 Mar 7;23(15):1804–1813. [PubMed]
119. Liang S, Hajishengallis G. Heat-labile enterotoxins as adjuvants or anti-inflammatory agents. Immunological investigations. 2010;39(4–5):449–467. [PMC free article] [PubMed]
120. Lewis DJ, Huo Z, Barnett S, Kromann I, Giemza R, Galiza E, et al. Transient facial nerve paralysis (Bell’s palsy) following intranasal delivery of a genetically detoxified mutant of Escherichia coli heat labile toxin. PLoS ONE. 2009;4(9):6999. [PMC free article] [PubMed]
121. Petrovsky N, Cooper PD. Carbohydrate-based immune adjuvants. Expert Rev Vaccines. 2011 Apr;10(4):523–537. [PMC free article] [PubMed]
122. Cooper PD, Petrovsky N. Delta inulin: a novel, immunologically active, stable packing structure comprising beta-D-[2 -> 1] poly(fructo-furanosyl) alpha-D-glucose polymers. Glycobiology. 2011 May;21(5):595–606. [PMC free article] [PubMed]
123. Honda-Okubo Y, Saade F, Petrovsky N. Advax, a polysaccharide adjuvant derived from delta inulin, provides improved influenza vaccine protection through broad-based enhancement of adaptive immune responses. Vaccine. 2012 Aug 3;30(36):5373–5381. [PMC free article] [PubMed]
124. Lobigs M, Pavy M, Hall RA, Lobigs P, Cooper P, Komiya T, et al. An inactivated Vero cell-grown Japanese encephalitis vaccine formulated with Advax, a novel inulin-based adjuvant, induces protective neutralizing antibody against homologous and heterologous flaviviruses. J Gen Virol. 2010 Jun;91(Pt 6):1407–1417. [PMC free article] [PubMed]
125. Rodriguez-Del Rio E, Marradi M, Calderon-Gonzalez R, Frande-Cabanes E, Penades S, Petrovsky N, et al. A gold glyco-nanoparticle carrying a listeriolysin O peptide and formulated with Advax delta inulin adjuvant induces robust T-cell protection against listeria infection. Vaccine. 2015 Mar 17;33(12):1465–1473. [PubMed]
126. Petrovsky N, Larena M, Siddharthan V, Prow NA, Hall RA, Lobigs M, et al. An inactivated cell culture Japanese encephalitis vaccine (JE-ADVAX) formulated with delta inulin adjuvant provides robust heterologous protection against West Nile encephalitis via cross-protective memory B cells and neutralizing antibody. Journal of virology. 2013 Sep;87(18):10324–10333. [PMC free article] [PubMed]
127. Feinen B, Petrovsky N, Verma A, Merkel TJ. Advax-Adjuvanted Recombinant Protective Antigen Provides Protection against Inhalational Anthrax That Is Further Enhanced by Addition of Murabutide Adjuvant. Clinical and vaccine immunology : CVI. 2014 Apr;21(4):580–586. [PMC free article] [PubMed]
128. Honda-Okubo Y, Kolpe A, Li L, Petrovsky N. A single immunization with inactivated H1N1 influenza vaccine formulated with delta inulin adjuvant (Advax) overcomes pregnancy-associated immune suppression and enhances passive neonatal protection. Vaccine. 2014 Aug 6;32(36):4651–4659.[PMC free article] [PubMed]
129. Honda-Okubo Y, Ong CH, Petrovsky N. Advax delta inulin adjuvant overcomes immune immaturity in neonatal mice thereby allowing single-dose influenza vaccine protection. Vaccine. 2015 Jul;:29.[PMC free article] [PubMed]
130. Mastelic Gavillet B, Eberhardt CS, Auderset F, Castellino F, Seubert A, Tregoning JS, et al. MF59 Mediates Its B Cell Adjuvanticity by Promoting T Follicular Helper Cells and Thus Germinal Center Responses in Adult and Early Life. J Immunol. 2015 May 15;194(10):4836–4845. [PubMed]
131. Gordon D, Kelley P, Heinzel S, Cooper P, Petrovsky N. Immunogenicity and safety of Advax, a novel polysaccharide adjuvant based on delta inulin, when formulated with hepatitis B surface antigen: A randomized controlled Phase 1 study. Vaccine. 2014 Sep 27; Sep 27. pii: S0264-410X(14)01293-6. [PMC free article] [PubMed]
132. Gordon DL, Sajkov D, Woodman RJ, Honda-Okubo Y, Cox MM, Heinzel S, et al. Randomized clinical trial of immunogenicity and safety of a recombinant H1N1/2009 pandemic influenza vaccine containing Advax polysaccharide adjuvant. Vaccine. 2012 Aug 3;30(36):5407–5416. [PMC free article][PubMed]
133. Heddle R, Russo P, Petrovsky N, Hanna R, Smith A. Immunotherapy – 2076. A controlled study of delta inulin-adjuvanted honey bee venom immunotherapy. The World Allergy Organization journal. 2013;6(Suppl 1):158.
134. Szebeni J. Complement activation-related pseudoallergy caused by amphiphilic drug carriers: the role of lipoproteins. Current drug delivery. 2005 Oct;2(4):443–449. [PubMed]
135. Tefit JN, Crabe S, Orlandini B, Nell H, Bendelac A, Deng S, et al. Efficacy of ABX196, a new NKT agonist, in prophylactic human vaccination. Vaccine. 2014 Oct 21;32(46):6138–6145. [PMC free article][PubMed]
136. Takeda K, Hayakawa Y, Van Kaer L, Matsuda H, Yagita H, Okumura K. Critical contribution of liver natural killer T cells to a murine model of hepatitis. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5498–5503. [PMC free article] [PubMed]
137. Sesardic D. Regulatory considerations on new adjuvants and delivery systems. Vaccine. 2006 Apr 12;24(Suppl 2) S2-86-7. [PubMed]
138. Beck FW, Whitehouse MW, Pearson CM. Improvements for consistently inducing experimental allergic encephalomyelitis (EAE) in rats: I. without using mycobacterium II inoculating encephalitogen into the ear. Proc Soc Exp Biol Med. 1976 Mar;151(3):615–622. [PubMed]
139. Panitch H, Ciccone C. Induction of recurrent experimental allergic encephalomyelitis with myelin basic protein. Ann Neurol. 1981 May;9(5):433–438. [PubMed]
140. Barkhordarian A, Thames AD, Du AM, Jan AL, Nahcivan M, Nguyen MT, et al. Viral immune surveillance: Toward a TH17/TH9 gate to the central nervous system. Bioinformation. 2015;11(1):47–54.[PMC free article] [PubMed]
141. Mastelic B, Garcon N, Del Giudice G, Golding H, Gruber M, Neels P, et al. Predictive markers of safety and immunogenicity of adjuvanted vaccines. Biologicals. 2013 Nov;41(6):458–468. [PubMed]
142. Sun Y, Gruber M, Matsumoto M. Overview of global regulatory toxicology requirements for vaccines and adjuvants. Journal of pharmacological and toxicological methods. 2012 Mar;65(2):49–57. [PubMed]
143. Tiwari JL, Terasaki PI. HLA-DR and disease associations. Progress in clinical and biological research. 1981;58:151–163. [PubMed]
144. Vaarala O, Vuorela A, Partinen M, Baumann M, Freitag TL, Meri S, et al. Antigenic differences between AS03 adjuvanted influenza A (H1N1) pandemic vaccines: implications for pandemrix-associated narcolepsy risk. PLoS ONE. 2014;9(12):114361. [PMC free article] [PubMed]
145. Schroder K, Irvine KM, Taylor MS, Bokil NJ, Le Cao KA, Masterman KA, et al. Conservation and divergence in Toll-like receptor 4-regulated gene expression in primary human versus mouse macrophages. Proc Natl Acad Sci U S A. 2012 Apr 17;109(16):944–953. [PMC free article] [PubMed]
146. Zaitseva M, Romantseva T, Blinova K, Beren J, Sirota L, Drane D, et al. Use of human MonoMac6 cells for development of in vitro assay predictive of adjuvant safety in vivo. Vaccine. 2012 Jul 6;30(32):4859–4865. [PubMed]
147. Pignatti F, Jonsson B, Blumenthal G, Justice R. Assessment of benefits and risks in development of targeted therapies for cancer–The view of regulatory authorities. Molecular oncology. 2015 May;9(5):1034–1041. [PubMed]
148. Thompson A, Komparic A, Smith MJ. Ethical considerations in post-market-approval monitoring and regulation of vaccines. Vaccine. 2014 Dec 12;32(52):7171–7174. [PubMed]
149. Bults M, Beaujean DJ, Richardus JH, van Steenbergen JE, Voeten HA. Pandemic influenza A (H1N1) vaccination in The Netherlands: parental reasoning underlying child vaccination choices. Vaccine. 2011 Aug 26;29(37):6226–6235. [PubMed]
150. Ropeik D. How society should respond to the risk of vaccine rejection. Human vaccines & immunotherapeutics. 2013 Aug;9(8):1815–1818. [PMC free article] [PubMed]
151. Leask J, Braunack-Mayer A, Kerridge I. Consent and public engagement in an era of expanded childhood immunisation. Journal of paediatrics and child health. 2011 Sep;47(9):603–607. [PubMed]
152. Spadea A, Unim B, Colamesta V, Meneghini A, D’Amici AM, Giudiceandrea B, et al. Is the adjuvanted influenza vaccine more effective than the trivalent inactivated vaccine in the elderly population? Results of a case-control study. Vaccine. 2014 Sep 15;32(41):5290–5294. [PubMed]
153. Begue P. Consequences of opposition to vaccination in France, Europe How to maintain effective vaccine coverage in 2010? Bulletin de l’Academie nationale de medecine. 2010 Apr-May;194(4–5):719–732. discussion 32. [PubMed]
154. Butler D. Regulators face tough flu-jab choices. Nature. 2009;460(446) [PubMed]
155. How to win trust over flu. Nature. 2009;461(7265):698. [PubMed]
156. Opel DJ, Marcuse EK. Rethinking vaccine policy making in an era of vaccine hesitancy: time to rebuild, not remodel? Human vaccines & immunotherapeutics. 2013 Dec;9(12):2672–2673.[PMC free article] [PubMed]
157. Glanz JM, McClure DL, Magid DJ, Daley MF, France EK, Salmon DA, et al. Parental refusal of pertussis vaccination is associated with an increased risk of pertussis infection in children. Pediatrics. 2009 Jun;123(6):1446–1451. [PubMed]
158. Buttenheim AM, Cherng ST, Asch DA. Provider dismissal policies and clustering of vaccine-hesitant families: an agent-based modeling approach. Human vaccines & immunotherapeutics. 2013 Aug;9(8):1819–1824. [PMC free article] [PubMed]
159. Folb PI, Bernatowska E, Chen R, Clemens J, Dodoo AN, Ellenberg SS, et al. A global perspective on vaccine safety and public health: the Global Advisory Committee on Vaccine Safety. American journal of public health. 2004 Nov;94(11):1926–1931. [PMC free article] [PubMed]
160. Elmgren L, Li X, Wilson C, Ball R, Wang J, Cichutek K, et al. A global regulatory science agenda for vaccines. Vaccine. 2013 Apr 18;31(Suppl 2):163–175. [PubMed]

 

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