Polyionic vaccine adjuvants: another look at aluminum salts and polyelectrolytes

Vaccination is among humanity's greatest achievements for reducing death, disability, and inequity caused by infectious diseases. The net benefit to health and well-being afforded by vaccination has been triumphant across nearly 300 years of use. A recent analysis of public records in the United States concluded that vaccines prevented 103.1 million cases of communicable and environmental disease over the past 90 years [1]. This accomplishment supported another benefit, namely, the increase in life expectancy of 30.4 years over the same time period [2]. Today, the global mortality prevention rate is about 2.5 million lives saved worldwide each year by immunization programs. The World Health Organization (WHO) immunization program includes the original six childhood vaccines-against diphtheria, tetanus, pertussis (whooping cough), measles, polio, and tuberculosis-plus more recently introduced vaccines against hepatitis B virus, Haemophilus influenzae type b, mumps, pneumococcal disease, rotavirus and rubella (German measles) [3]. Regional, national, and local or high-risk occupational immunization programs have added vaccines for varicella (chickenpox), meningococcal, typhoid fever, herpes zoster (shingles), human papillomavirus, yellow fever, Japanese encephalitis, as well as a list of investigational status biodefense vaccines [3, 4, 5]. Due in large part to worldwide vaccination programs, the global annualized rate of under-five-year-old deaths was reduced in half, from 12.6 million in 1990 to 6.6 million in 2012 [6]. Nevertheless, such reports also reveal that low income families represented 13 times the average case rate, immunization coverage is incomplete in many regions, vaccine stocks shortages are reported, and case rates have recently been increasing for some targeted diseases [6]. Beyond the remarkable impact on mortality rates, vaccines have also helped raise life quality, work productivity, and social equity by averting untold numbers of disabling post-infection disease sequelae, most notably blindness, deafness, neurological disorders [7, 8].

Citing a list of accomplishments-the global eradication of smallpox in 1979 [9, 10, 11, 12], an active endgame strategic plan for the eradication of polio [3, 13, 14], regional initiatives to eliminate or control several remaining vaccine-preventable childhood diseases, plus robust international post-marketing surveillance programs and periodic benefit-to-cost assessments [15, 16, 17, 18, 19, 20, 21, 22, 23]-the value of prophylactic vaccines against infectious diseases remains undisputed in the scientific, medical, and public policy communities as well as the general public at large [9, 10, 24, 25, 26, 27]. However, significant advances in knowledge and technology are now helping to rapidly expand product development goals for immunization beyond the traditional and proven capabilities of vaccinology. Challenging new targets include vaccines for recent and ancient pathogens (human immunodeficiency virus [HIV], pandemic influenza, and malaria), improved capabilities to extend immunity to the very young, elderly, and immune compromised populations, for post-exposure prophylaxis indications in preparedness against biological threat agents (anthrax and Ebola virus) and emerging pandemic strains (H5N1 influenza, Middle East respiratory syndrome coronavirus, and methicillin-resistant Staphylococcus aureus), as well as immune treatments and protections against important non-infectious targets including cancers, autoimmune diseases, neurological disorders, and other medical conditions.

Adjuvants and the broader phenomenon of adjuvantation have been partner to vaccines successes throughout this history. The proportion of vaccines formulated with added adjuvant has increased over time and now comprises about half of the vaccines either licensed or in clinical testing (Fig. 1). A more detailed understanding of adjuvants has only recently appeared with the discovery and deepening characterization of immune signaling by biomolecules and physicochemical structures derived from pathogens or related to cell damage, now referred to as pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). This growth in understanding recently includes contributions from structural vaccinology and synthetic biology. Initially added to vaccines through empirical discovery, adjuvants are presently developed through more deductive ("rational") design and testing. The informed incorporation of adjuvant materials will continue to be an essential component of forthcoming vaccines, particularly as vaccines are engineered to meet rising global demand for greater potency using less antigen, wider disease and population coverage in fewer formulations, easier administration, and reduced logistical costs.

Fig. 1
Proportion of human vaccines containing adjuvant through stages of history. Circles depict periods in vaccine development with fractional amount containing adjuvant shaded in blue, and diameter proportional to the log of number of different vaccines. A, up to 1899; B, 1900 to 1949; C, 1950 to 2012; D, 2014 U.S. licensed as listed by Food and Development Administration (FDA); E, 2014 in clinical testing as listed by HuVax (http://www.violinet.org). Note that the licensed vaccines group D contains many more multiple products for similar or overlapping indications, most of which are non-adjuvanted, while the experimental group E includes all existing and new candidate adjuvants reported in clinical testing.

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Here we first look back at the origin of adjuvants, and then focus on a few classical and recent adjuvants as examples of the category of ionic polymers and ionic materials. Recent advances in immunology and materials science have improved the understanding and capability of aluminum salts which remain the most widely used adjuvant, and of synthetic polymers which represent a newer material showing wide ability and promise.

Adjuvant technology emerged from medical necessity, regulatory pressures, and scientific discovery. Recounted here are a few of the seminal events leading to the development and use of adjuvants as a medical tool that continues to evolve in capability and importance, now more rapidly than ever before. This history shows that empirical observation and testing were critical to the discovery and development of the first immunologic adjuvants.

Recognition of a need for supplemental processing or formulation to improve immunization arose with the expansion of vaccine design beyond the attenuated and inactivated or killed strain preparations used since the dawn of vaccinology [28, 29]. In the early 20th century, difficulties accompanied the initial toxoid type vaccines which comprised dilute active toxin administered with antitoxin (horse anti-serum). Discovered forty years earlier by Kitasato and Von Behring, for which Von Behring later won the first Nobel Prize in medicine [30], these toxin/serum type vaccines were marketed boldly and were desired for the protection they afforded against diphtheria and tetanus, commonly fatal childhood diseases. Unlike most other endemic and epidemic diseases awaiting effective vaccines, the bacterial exotoxin proteins associated with these diseases reproduced disease pathologies in isolation of and apart from the etiological, live infectious organisms. The activity of exotoxins could induce neutralizing antibodies, as demonstrated first in animal testing and later by passive transfer in humans, and small doses could induce immunity if co-administration with protective antiserum [29, 31, 32]. Diphtheria was a leading cause of illness and death among children then, estimated at 200,000 cases and 16,000 deaths annually, and various toxin/antitoxin products afforded prophylaxis. However, in 1901 tetanus-contaminated diphtheria antiserum caused the death of 13 children in St. Louis, and soon afterward 9 children died in New Jersey from tetanus contaminated smallpox vaccine [33, 34]. These tragedies underscored a need for regulation, standardization, and quality control and drove legislative action for public safety. The Biologics Control Act of 1902 launched federal requirements for annual licensing and inspections of biologics manufacturers, rules for product testing, purity standards and quality control, truth in labeling, and also created the Hygienic Laboratory of the U.S. Public Health Service which eventually became the National Institutes of Health [34]. Subsequent acts and amendments added and consolidated regulations, then eventually transferred oversight of biologics to the Food and Drug Administration (FDA) in 1972. Thus, the events of 1902 were groundbreaking as "the first enduring scheme of national regulation for any pharmaceutical product...the very first premarket approval statute in history," and set precedent for "shifting from retrospective post-market to prospective pre-market government review" [35]. This general period and area of research also yielded the first biological standard, established by Paul Ehrlich in working with Von Behring's antiserum to control for variations in content and temperature in proposing universal units of activity.

The genesis of immunologic adjuvants emerged within the same setting of medical and regulatory pressure surrounding toxoid vaccines and antisera, though detailed knowledge of the underlying science about how adjuvants enhance immunity would not coalesce until the 1990s. A series of discoveries which focused on reducing the toxicity of diphtheria toxin while improving its immunity as an antigen illustrate the inception of adjuvant science and methodology:

The last achievement listed, on the effectiveness of pre-formed aluminum salts compared to co-precipitation of antigen with aluminum phosphate or alum (potassium aluminum sulfate) before that, connects this line of development to modern aluminum gels which are mostly employed today. Often considered together, research has shown that the various aluminum salt gels do not exhibit universally equivalent attributes as adjuvant materials. The full chemical formula and process formulation of aluminum salt adjuvants for some licensed vaccines are not publically released. The discovery and evolution of aluminum salt adjuvants surveyed here is just one thread of several adjuvants in a deep history of vaccine development, and illustrates that empirical findings were foundational both in general and specifically to the largest class of adjuvant still in use today. A depiction of the development of several licensed vaccines and their adjuvant is shown in Fig. 2. This illustration includes timelines for the common vaccine classes (attenuated or killed, serum or toxoid, conjugate, etc.) and a few technologies supporting new development of vaccines and adjuvants, as reprinted from Rappuoli et al. [2].

Fig. 2
Timeline of vaccines and adjuvants development. Shown is an historical view of the origin and development for some early and currently licensed human vaccines, the time needed to translate a scientific discovery (shown in red) into a safe and scalable vaccine technology (orange bars), the evolution of adjuvants, and of some supporting areas of science and technology. The blue bars represent the time needed to develop and implement one or more vaccines derived from the same technology. IPV, inactivated polio virus; OPV, oral polio virus; Hib, Haemophilus influenzae type b; HBV, hepatitis B virus; HPV, human papillomavirus. Reproduced from Rappuoli et al. (2014), with permission of the author and publisher, Proceedings of the National Academy of Sciences [2].

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The only other adjuvant classes used in currently licensed vaccines include emulsions systems, liposomes and toll-like receptor (TLR)-based compounds. As with aluminum salts, their discoveries have been critical to the general development of adjuvant science and technology. Their introduction as modern version adjuvants is also noted in the adjuvants timeline of Fig. 2. Currently licensed emulsion systems and liposome type adjuvant formulations used in vaccines for seasonal flu, pandemic influenza, and hepatitis A [42] trace back to Freund's complete adjuvant (1951) [43]. Complete Freund's adjuvant (CFA) is an emulsion system of soluble antigen mixed into mineral oil (water-in-oil [w/o]) with surfactant and inactivated dried mycobacteria. Though CFA became popular because of its potency [43], it was deemed too toxic for human use as evidenced by severe inflammatory effects and granulomatous reactions [44]. Reversed emulsion formulations containing an oil-in-water (o/w) mixture without mycobacterium, including incomplete Freund's adjuvant (IFA), were less viscous so easier to prepare and inject as well as less reactogenic [28, 45]. Squalene, a biodegradable oil that is still extracted from shark liver, emerged as part of effective o/w emulsion adjuvants used in few licensed vaccine formulations today including MF59 (squalene, Tween-80, and sorbitan trioleate) [46], AS03 (squalene, Tween-80, and α-tocopherol) [47], as well as AF03, a candidate pandemic flu vaccine not yet licensed [48]. Several candidate emulsion and liposome adjuvants are also being evaluated in preclinical and clinical testing and are not yet licensed, including the GlaxoSmithKline "adjuvant system" formulations AS01 (liposome, 3-O-desacyl-4'-monophosphoryl lipid A [MPL], and saponin) and AS02 (o/w, MPL, and saponin). No TLR or other pattern recognition receptor (PRR) mechanism of action has been indicated for squalene [49] or other emulsion and liposome type adjuvants though these are being investigated as vehicles to combine with known TLR ligands.

The synthetic compound MPL is the only TLR class of adjuvant presently used in a US licensed vaccine. This is a detoxified version of the gram-negative bacterial endotoxin lipopolysaccharide (LPS) which signals innate immunity via the TLR4 complex. LPS was the first TLR ligand out of many PAMPs now intensively investigated for development as potential adjuvants with new capabilities to improve vaccines. Discovery of bacterial endotoxin in the late 1890s by Richard Pfieffer, and subsequent demonstrations that this heat stable substance could evoke neutralizing antibody which suppressed the poisonous effect [50] initiated a long line of research that underlies current knowledge of PAMPs and TLR-directed innate immune response. Among several other discoveries that also spurred the advance of immunologic adjuvants were medical reports of "Coley's toxins," extracts from infectious bacteria that successfully treated cancer for almost 50 years (1895-1944) [51]. The wide study of immune-stimulating bacterial components converged in the 1990s with discoveries and understanding that certain microbial substructures embody PAMPS which serve as ligands for immune cell PRRs (aka PRRs) that signal the innate immune system [52, 53]. The identification of TLRs (10 in human), nod-like receptors (NLRs, 3 classes), and other PRRs has revolutionized the science and practice of vaccine adjuvant characterization and development [54, 55, 56, 57, 58, 59, 60, 61, 62].

It is evident from this synopsis that the early period of vaccine development was determinative both for the science of adjuvantation and for the standards of biologics regulation. Since then the variety of vaccine types has expanded, and adjuvants have co-developed with each vaccines type licensed or under investigation today with a few exceptions, principally whole organism vaccines and carbohydrate vaccines.

With a large body of cumulative knowledge, the nature of adjuvant development and testing has changed from one of empirical, trial-and-error testing toward one more akin to rational design [55, 63], though much is left to be understood about adjuvant mechanism of action, and particularly for DAMP-type class of substances. Several fields of science intersect as drivers for the co-evolution of vaccine and adjuvant for improved immunization capability [54, 55]. The largest single driver of innovation and improvements in adjuvant technology is now scientific knowledge and information, for the rapid growth in primary data and the improved, more accessible means of connecting and cross-referencing information. Due to the high interdependency and rapid pace of development, this shared endeavor will benefit from comprehensive, descriptive reporting and networking of data and results from testing that is designed and performed with standards then annotated and communicated with universal scientific meaning.

Adjuvants improve vaccine effectiveness by modulating the immunogenicity of antigen delivered in a vaccine product. In addition to the original goal of increasing antibody titer, immunologic adjuvants are now being engineered to alter the natural adaptive immune response to an antigen for increased potency and duration, shortened time to immunity, altered immune polarization, increased scope and breadth for disease target and patient population (Table 1).

Table 1
Immunological roles for adjuvants in vaccine development

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These goals are sought while also aligning with efforts to improve vaccine tolerability and safety, product manufacture and stability, as well as to aid the cost and global logistics of transport, distribution, stockpiling and administration of new vaccines, including to underdeveloped and challenging environments.

A few types of vaccine do not require adjuvant, though their total product number for licensed indications exceeds those of vaccines containing adjuvant (Fig. 1). These include the live attenuated vaccines (adenovirus, Bacillus Calmette-Guerin [BCG], flu, mumps and rubella vaccine, oral polio virus polio, rotavirus, smallpox, Salmonella Typhi Ty21a, varicella, and yellow fever), split virion vaccines (flu), some inactivated vaccines (rabies and inactivated poliovirus virus [IPV] polio), and carbohydrate vaccines (pneumococcal, meningococcal, and Typhium Vi). While the live attenuated, inactivated and split virion vaccines do not require adjuvant because they include sufficient PAMPs for self-adjuvantation, the carbohydrate vaccines do not require adjuvant because they are T-cell independent. Carbohydrate antigens such as capsular polysaccharide directly bind B-cells to elicit activation and proliferation without MHC-restricted T-cell help [64]. This response generally is of short duration with no affinity maturation and fails to elicit immunologic memory. For this reason, the newer carbohydrate-based vaccines consist of carbohydrates conjugated to protein which provide T-cell epitopes for a T-cell dependent response capable of antibody class switching, affinity maturation, immunologic memory, and longer duration. Although adjuvants are often added to conjugate vaccines to enhance the T-cell dependent response, some candidate conjugate proteins also appear to have the capacity to function simultaneously as true adjuvants through TLR interaction. For example, neisserial porins signal via a TLR1/TLR2 complex [65, 66]. In addition, other proteins may provide TLR-based adjuvant effects when fused with a target protein antigen. As an example, salmonella flagellin interacts with TLR5 when genetically fused with influenza hemagglutinin [67].

How adjuvants promote immune stimulating effects is an active and growing area of research in which ongoing discoveries are helping to build areas of understanding in vaccine adjuvant immunology, as is briefly discussed next.

Today, information about the mechanisms of immune potentiation is accumulating quite rapidly, and mechanisms are frequently challenged or supported by experimental evidence. Consensus exits for several pathways involved in adjuvant mechanisms of action, the most detailed of which are for PRRs and the TRL class of signal receptors that helped revolutionize understandings of immune phenomenology [68, 69]. In summary, TLRs1-6 located on the plasma membrane of immune cells (macrophages, dendritic cells [DC], and neutrophils, etc.) recognize PAMPs from the surface of bacterial and fungal pathogens such as LPS and flagellin, while endosomally located TLR3 and TLRs7-9 recognize viral and microbial RNA and DNA [70]. Several other PRRs are described, such as NLRs, RIG-1 like helicases, extracellular C-type lectin receptors, all of which function via adaptor proteins (such as MyD88, TIRAP, TRIF, and ASC, etc.) to coordinate signaling between and among PAMP pathways. PRRs including some TLRs (e.g., TLR2/4) also recognize endogenous DAMP ligands like heat shock proteins 60 and 70, fibrinogen, and fibronectin produced in cell damage, injury, stress, or metabolic imbalance [71, 72]. On activation, TLRs in neutrophils can activate NADPH oxidase to produce reactive oxygen species (ROS) which mediate release of other DAMPs driving an inflammatory response [71, 73, 74]. In this way TLRs, NLRs and other PRRs transmit and help coordinate immunomodulatory responses signaled by pathogens and endogenous stressors [73, 75]. The net immediate effect of responses involving the inflammasome is to activate the inflammatory process by promoting the processing and secretion of the proinflammatory cytokines interleukin (IL)-1β and IL-18. This initiates processes driving the recruitment, activation, and maturation of immune cells to direct innate and adaptive immune functions, processes at the heart of functional mechanisms of immunologic adjuvants. Excellent reviews have been published with great detail about mechanisms for TLR-dependent and TLR-independent adjuvants [44, 54, 55, 61, 64, 68, 69, 70, 75, 76, 77]. Many of these reviews provide generalized summary illustrations which are instructive in developing, challenging, and refining models toward an understanding for immune regulation and adjuvant function. Naturally, concepts and models occasionally require adjustment to adapt as knowledge evolves.

Current evidence does not yet connect the functioning of aluminum salt gels and polyelectrolyte adjuvants, topics of this review, to known TLR pathways. However, the activity of some TLR-independent adjuvant materials such as aluminum and oil emulsions are linked to DAMP pathways, including activation of the inflammasome by ROS [78] and deformation or re-structuring of the plasma membrane [79, 80, 81]. The NLRP3-inflammasome plays an important role, though it has also been shown not to be universally essential in DAMP-signaling by adjuvants. Interestingly, an NLRP3-oligemeric complex also acts as an extracellular DAMP particle itself that disperses to neighboring macrophages to amplify the inflammatory response [82]. Generalized roles for cells, pathways, and the NLRP3-inflammasome are depicted in Fig. 3, reproduced and modified slightly from De Gregorio et al. [68]. This illustration shows how some TLR and non-TLR adjuvant activities focus on the DC as the central controller of adaptive immunity.

Fig. 3
A model of multiple mechanisms of adjuvanticity that converge toward dendritic cell (DC) activation. Adjuvants activate DCs either through direct interactions or through cellular intermediaries. toll-like receptor (TLR)-dependent adjuvants (in black) act directly on DCs; however, they can also activate other cells expressing the responding TLR. TLR-independent adjuvants (in red) can act directly either on DCs or on accessory cells. Aluminum salts, PLG, MSU, and Quil-A activate the NLRP3 inflammasome. Aluminum salts and MF59 act on monocytes, macrophages, or granulocytes to induce cytokines that generate a local immunostimulatory environment eventually leading to DC activation. In addition they also promote monocyte differentiation into DCs. MF59 can also activate muscle cells at injection site. It has been suggested that aluminum salts causes local necrosis of stromal cells leading to the release of uric acid, an endogenous danger signal that activates the inflammasome. Mast cell activators such as c48/80 can also act as adjuvants in a DC-dependent mechanism. iNKT activation by a-GalCer presented on CD1d leads to DC activation. Beta glucans activate DC through dectin-1 and ISCOMATRIX directly promotes cross-presentation in DCs [68]. So far, a role by TRLs has not been shown for the adjuvant action of polyelectrolytes. Reproduced from De Greggorio et al. (2009) with slight update and with permission of the author and publisher, Elsevier, Current Opinion in Immunology [68].

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For many years, particulate and colloidal adjuvants such as aluminum salts and polymers were considered to function mainly as inert delivery vehicles, assisting in antigen stability, uptake and delivery. The original depot model for slow-release of antigen by aluminum adjuvants has been challenged [83] with experimental evidence that some antigens desorb from some aluminum adjuvants soon after injection [84]. Nevertheless, the carrier or transport role is not debunked. Antigen stability in both structure and potency can be greatly enhanced as bound to aluminum salt gels compared to their free, soluble form [85, 86, 87]. Importantly, recent work demonstrated that the simple inclusion of 4 mM phosphate with Alhydrogel (aluminum oxyhydroxide) saline formulation resulted in hydrated antigen and re-established soluble-like structure compared to formulation with plain Alhydrogel [85]. There have also been contrasting conclusions about whether aluminum salts adjuvants are internalized by antigen presenting cells [88, 89, 90]. Recent reports provide convincing evidence of internalization [89, 91]. In particular, the fluorescent molecular probe for aluminum, lumogallion, was used to confirm intracellular location of aluminum oxyhydroxide adjuvants taken up by a monocytic T helper 1 cell line [91]. Thus, immune activation is supported both with and without internalization of aluminum adjuvant particles.

What began as a handful of materials to boost antigen titer 90 years ago has now become a short list of licensed vaccine adjuvants and a very long list of potential adjuvant materials under preclinical and clinical testing. Adjuvants are commonly divided into two broad functional categories as delivery vehicles or immunopoteniators, though many show both functions including the mineral salts and polyelectrolytes discussed below. Subcategories of licensed vaccine adjuvants include mineral salts (alum and aluminum hydroxide, etc.), oil-in-water emulsions (MF59 and AS03), liposomes (virosome), and a combination adjuvant (AS04) [42]. On the other hand, there are very many adjuvants and combination adjuvant formulations under exploration or development, including many in clinical testing. The Vaxjo web-based database tool for vaccine adjuvant informatics (http://www.violinet.org/vaxjo/adjuvant) lists 103 different vaccine adjuvant compounds [92, 93]. The Huvax database, another vaccine informatics tool within the VIOLONET suite, lists 101 human vaccines or vaccine candidates containing adjuvant. The ClinicalTrials.gov registry database [94, 95] lists 869 clinical trials globally that involve vaccine adjuvant. The number and proposed indications for vaccine adjuvants continues to expand as increasing knowledge and information enables rational justification for new combination products or testing a new potential ligand, receptor, or pathway [96]. The remainder of this review focusses on polyionic classes of adjuvants.

It has been generally known for at least few decades that polyelectrolytes-ionic macromolecules of either synthetic or natural origin-serve as immunostimulants when introduced as mixtures with typical antigens, thereby enhancing immune responses by several fold [113]. Moreover, this effect is increased by orders of magnitude if the antigen is chemically bound to synthetic polyelectrolytes [113]. It was suggested that membrane activity and immunostimulation are inherent to polyelectrolytes with different chemical structures of the repeating units as a class and depend critically on molecular size, self-assembly, and other characteristics of the system. Although, there are examples of licensed products and clinical use of such materials [113, 114], this family of polymers is generally reviewed less frequently in immunoadjuvants literature. This may have been partly due to challenges related to isolation, purification, and characterization of natural and some synthetic polyelectrolytes In addition, their ability to self-assemble in aqueous solutions under certain conditions can often lead to poor reproducibility, conflicting results, and difficulties in interpretation. Nevertheless, the synthetic and purification approaches to this class of polymers are greatly improving and the body of literature related to these compounds is rapidly growing. Natural and synthetic polyelectrolytes possess a number of common features and parameters affecting their biological function in solution. A few important parameters are: length and conformation of the polymer chains, only a few types of repeating units, flexibility of the backbone, ionic density, tacticity (regularity of repeating units), solvent interaction parameters, and hydrodynamic behavior. Thus, in summarizing reports related to immunoadjuvant properties of these polymers, it was important to review literature related to both natural and synthetic types of these long-chain, polyionic macromolecules.

Chitin and its derivative, chitosan, are linear polysaccharides which having increased attention as biomaterials, drug delivery carriers and immunomodulators [115, 116, 117]. Chitin is a long-chain polymer of a N-acetyl-D-glucosamine. While it is one of the most abundant polysaccharides in nature, its life sciences applications are somewhat limited due to insolubility in water. In contrast, chitosan, which is produced by partial deacetylation of chitin, is a copolymer consisting of randomly distributed ionizable D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) (Fig. 4), and consequently is more amendable for water-soluble and hydrogel nanoparticulate formulations. Moreover, cationic derivatives of chitosan provide improved water-solubility and have been also widely tested [118].

Fig. 4
Schematic chemical structure of chitosan.

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There are multiple reports that chitin stimulates macrophages to produce cytokines that confer anti-tumor activity and non-specific host resistance against bacterial and viral infections [119]. It is conceivable that chitin could be a potent innate immune stimulator of macrophages and other innate immune cells. There are reports that it stimulates macrophages by interacting with different cell surface receptors such as macrophage mannose receptor, TLR-2, C-type lectin receptor Dectin-1, and leukotriene B4 receptor (BLT1) [120]. It must be emphasized that that isolated chitin presents two difficulties, both related to its water-insolubility. Due to this insolubility, the chemical structure of chitin cannot be defined under physiological conditions, and may involve concurrent destruction. Formulations have always been evaluated as particulates and the above mentioned host responses vary dramatically depending on its size and possibly the nature of the chitin employed as well as the methods of preparation, timing, and route of administration [121].

Contrary to chitin, chitosan formulations are prepared by hydrolysis and are either water-soluble mixed polymers or hydrogel nanoparticulates, therefore more amenable to characterization than chitin. Unless the polymer was additionally modified, the water-solubility of chitosan is typically limited to slightly acidic to acidic conditions. Unfortunately, upon review of the literature, it is not always possible to elucidate which physical or even chemical form of chitosan was evaluated. The assessment of activity then become further complicated as various routes of administrations are used and chitosan was shown to be mucoadhesive, can promote prolonged retention times following mucosal vaccination, and additionally acts as a permeation enhancer by opening tight junctions [116]. However, there have been a number of studies reported recently focusing on the evaluation of well-characterized original materials, but not necessarily detailing the formulation itself.

However, one well defined chitosan, when tested for parenteral immunization in C57BL/6 mice using β-galactosidase as a model antigen, was shown to enhance antigen specific antibody titers over five-fold and antigen-specific splenic CD4+ proliferation over six-fold [122]. According to the authors, strong increases in antibody titers together with robust delayed-type hypersensitivity responses revealed that chitosan induced a mixed Th1/Th2 humoral and cell-mediated immune responses. By this study, chitosan was demonstrated to be equipotent to IFA and superior to Imject Alum (aluminum hydroxide + aluminum magnesium hydroxide). Mechanistic studies revealed that this viscous chitosan solution created an antigen depot when injected subcutaneously and it also induced a transient 67% cellular expansion in draining lymph nodes [122]. It was also shown that chitosan can synergistically enhance the immunoadjuvant properties of granulocyte-macrophage colony-stimulating factor, a pleiotropic, proinflammatory cytokine that was initially discovered as a growth factor capable of generating granulocyte and macrophage colonies from bone marrow precursor cells [123].

In a separate study, evaluation of three different well-defined types of chitosan for parenteral administration of model antigen, ovalbumin, in mice showed adjuvant activity for all of them, in some cases exceeding that of "alum" (Alu-S-Gel aluminum hydroxide sensitive to phosphate) [124]. However, the adjuvant activity varied significantly depending on the formulation. Authors conclude that this was probably a result of interplay between molecular weight, particle size, preparation technique, solubility and viscosity, as well as degree of deacetylation, whereas impurities were found to play a minor role. Nevertheless, authors also note that potential activation of PRR on cells of the innate immune system, which was reported for some chitin derivatives, can be potentially explained by the presence of bacterial and fungal contaminants and endotoxins since these materials are of natural origin and their ionic nature can complicate the purification efforts [124], as already noted.

These observations are mirrored in a recently published review, which emphasizes that in the majority of publications on the adjuvant activity of chitosan to date, the molecular weight, viscosity, deacetylation degree and purity level, especially endotoxins, are not provided for the chitosan under investigation and final formulation and preparation procedures are not sufficiently detailed. Due to this, evaluation of adjuvant properties is challenging, especially as it concerns elucidation of the mechanism of action or exclusion of potential interferences due to impurities [125].

Alginic acid is a linear copolymer containing (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, covalently linked together in different sequences or blocks (Fig. 5). Although alginates are widely used for the preparation of hydrogel microspheres and nanospheres for encapsulation of biological agents [126], there have been also reports on the adjuvant activity of soluble salts of alginic acid.

Fig. 5
Schematic chemical structure of alginic acid.

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Adjuvant effect of sterile sodium alginate was investigated in a subcutaneous immunization with BCG vaccine in BALB/c mice [127]. Proliferative and delayed-type hypersensitivity responses, interferon (IFN)-g, specific anti-mycobacterium total IgG, IgG1, and IgG2a production were found all significantly higher in mice immunized with adjuvanted formulation. In addition, following systemic infection with BCG, mice vaccinated with BCG plus alginate had lower mean bacterial count compared to those vaccinated with BCG alone [127].

The effect of sodium alginate was also evaluated in combination with microparticle carriers fabricated using biodegradable polymer-poly(lactic-co-glycolic acid) (PLGA) with two malaria synthetic peptides, SPf66 and S3 [128]. Despite the fact that the formulations were practically indistinguishable in terms of size and morphology, the addition of alginate improved both encapsulation efficiency and immune responses. Immunization studies in BALB/c mice by intradermal route demonstrated that incorporation of alginate elicited higher humoral and cellular immune responses leading to more balanced Th1/Th2 responses. Furthermore, administration of microparticles containing RGD-modified alginate showed evidence of cell targeting by enhancing immunogenicity of microparticles, in particular with regard to cellular responses such as IFN-c secretion and lymphoproliferation [128].

Polyoxidonium-a water-soluble cationic polymer is a polyelectrolyte, which was specifically designed as an immunomodulator for use in vaccines [113, 129]. It is a ternary copolymer of 1,4-ethylenepiperazine, 1,4-ethylenepiperazine-N-oxide, and (N-carboxymethylene)-1,4-ethylenepiperazinium bromide with a molecular weight of 60-100 kDa (Fig. 6). A derivative of poly(1,4-ethylenepiperazine), it is synthesized by a partial oxidation of the parent polymer with hydrogen peroxide to introduce N-oxide groups followed by the quaternization of some of the nonoxidized aminogroups with bromoacetic acid. The introduction of N-oxide groups is critically important as the optimal composition was selected in order to minimize toxicity, typically inherent to polyamines, and to maintain an appropriate level of immunostimulation [113]. In addition, the N-oxide units in the backbone are capable of degradation at elevated temperatures rearranging to oximes and then to amine and aldehyde groups. As a result, the copolymer chains are cleaved to shorter fragments, which then can be released from the body [113].

Fig. 6
Schematic chemical structure of polyoxidonium.

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The analysis of immunomodulating effect induced by polyoxidonium proved its stimulating activity on proinflammatory cytokines production in vitro, such as IL-1H, tumor necrosis factor (TNF)-α and IL-6 [129]. A dose-dependent increase in the intracellular killing by blood phagocytes was also observed for this polymer. In another study, it was established that polyoxidonium could affect the bactericidal activity of leukocytes [130]. Many chronic infectious inflammatory diseases are characterized by a sluggish, recurrent course, resistant to adequate therapy and requiring additional immunostimulation. It was established that a one-hour incubation of human peripheral blood leukocytes with polyoxidonium increased the ability of leukocytes to kill the ingested Staphylococcus aureus in a dose-dependent manner [130]. This increase was observed with leukocytes obtained both from healthy persons and from patients with chronic granulomatous disease. Polyoxidonium was also shown to have antioxidant activity at all dose range of 100 to 500 µg/mL [130]. Polyoxidonium displayed ability to enhance immune responses to the live brucellosis vaccine, Brucella abortus strain 82-PS (penicillin sensitive) in a guinea pig model [131].

One of the first clinical applications of polyoxidonium was with the commercial influenza vaccine, in which polyoxidonium was covalently conjugated to antigenic components of the vaccine-hemagglutinin and neuraminidase [113]. Reportedly, about 50 million recipients were vaccinated, and extensive data indicating the efficacy and safety character of the preparation was obtained [113]. In a clinical study polyoxidonium was also evaluated with trivalent live attenuated measles, mumps and rubella vaccine [132]. Although findings indicate that healthy children needed no fortification of their immune responses on the vaccination as they can produce a high level of specific antibodies, children with previous exposure to harmful factors affecting normal T cell content (viral and other diseases) may benefit from the use of polyoxidonium. However, authors also note that the detected increase in TNF-β level and skewing the dominant immune responses from Th1 to Th2 type could not be appreciated as positive effect of polyoxidonium in this environment [132].

The mechanism of action of polyphosphazene immunoadjuvants is not yet fully understood. It has been demonstrated, that on the molecular level, the parameters of interactions between polyphosphazene and the antigen play an important role. Similarly to other polyelectrolyte adjuvants [113], biological activity of PCPP strongly depends on its association with the antigen, however, contrary to their conventional counterparts, PCPP can form stable water-soluble, non-covalent complexes with antigenic molecules spontaneously and thus do not require chemical conjugation [133, 150]. The molecular size and antigen loading of the complex have been shown to correlate with its activity in vivo.

Recent study explored the effect of PCPP and its multimeric formulation with recombinant Gag-HIV antigen on the maturation, activation and antigen presentation by human adult and newborn DCs in vitro [150]. PCPP treatment induced DCs activation and maturation as assessed by upregulation of co-stimulatory molecules and cytokine production. The effect of PCPP was found to be more robust than that of "alum" (Adju-Phos) and resulted in the release of mixed Th1/Th2 cytokine responses, promoting both cellular and potentially humoral responses to the formulated antigen. PCPP also induced an innate immune transcriptome in human adult and newborn DCs [150]. Authors note that the ability of PCPP to activate neonatal monocyte derived DCs to levels similar to their adult counterparts is particularly noteworthy and suggests that this adjuvantation platform may have additional applicability for early life immunization, a key point of healthcare contact at which impaired immune responses are noted to many current vaccine formulations.

Much attention has been also focused on the 'next generation' polyphosphazene adjuvants. It has been clearly elucidated that structure and composition of polyphosphazenes play an important role in their immunopotentiation effect and novel molecules can differentiate significantly from PCPP in their activity and mechanism of action. Although about two dozen "new generation" polyphosphazenes, have been synthesized, only few have been explored for their immunoadjuvants properties, namely or PCPP copolymers containing oxyethelyne side groups (Fig. 7B) [151] poly[di(carboxylatoethylphenoxy)phosphazene], and PCEP (Fig. 7C) [152]. It was found that depending on the type of polyphosphazene the IgG isotype profiles of the immune response can vary indicating potential differences in the mechanism of action. PCPP primarily enhances IgG1 antibody responses, which are typically associated with Th2-type response, whereas its sister polymers have been shown to also enhance IgG2a [141, 151], which can be associated with Th1-type immune responses providing protection against intracellular pathogens [153].

It was reported recently that PCEP can induce time-dependent changes in the gene expression of many "adjuvant core response genes" including cytokines, chemokines, innate immune receptors, IFN-induced genes, adhesion molecules and antigen-presentation genes, upregulate the gene expression of the inflammasome receptor, NLRP3, and induce the production of pro-inflammatory cytokines IL-1β, and IL-18 at the site of injection [154]. Since the secretion of these cytokines is predominantly a result of activation of the inflammasome, which leads to the processing and secretion of proinflammatory cytokines, authors suggest that PCEP may modulate antigen-specific immune responses by activating early innate immune responses and promoting a strong immuno-stimulatory environment at the site of injection [154]. Furthermore, intramuscular injection of mice with PCEP induced significant recruitment of neutrophils, macrophages, monocytes, DCs, and lymphocytes at the site of injection as well as in the draining lymph nodes [155]. The results of the confocal analysis revealed intracytoplasmic lysosomal localization of PCEP in recruited immune cells [155].

Although recent publications, especially on a newer generation polyphosphazenes, clearly shed light on their mechanism of action, it should be noted that very few of these papers detail synthesis, characterization and formulation studies. This certainly needs to be addressed to allow the exclusion of the impact of the secondary factors, such as molecular size of the polymer, potential self-assembly in solution, and even the effect of any structural irregularities in the polymer, on the immunological effects.

Solubility of polyphosphazene immunoadjuvants in neutral and basic aqueous solutions provide for their straightforward formulation, however conformation-activity and molecular weight-activity relationships [133, 139], as well as ionic sensitivity [136], may play an important roles in biological activity. Therefore, formulations must be characterized, tested for compatibility with a specific antigen, and optimized accordingly to achieve superior results. Importantly, due to their water-solubility and well-defined structures, polyphosphazenes provide an attractive basis for the development of combination adjuvants. In fact, a synergistic effect between PCPP, PCEP and some important adjuvants, such as CpG has been well established [148].

Although the majority of results on polyphosphazene adjuvants have been reported for water-soluble formulations, the versatility of these polymers allows for their use as nanoparticulate delivery systems or microneedles for intradermal immunization. In fact, PCPP and some of its copolymers can be easily conformed into hydrogel nano- or microparticles in aqueous solutions using ionic complexation processes with benign agents, such as calcium chloride or spermine. Contrary to nanoparticles comprised from hydrophobic biodegradable nanoparticles like PLGA, these aqueous technologies are highly protein compatible and don't require the use of organic solvents, elevated temperatures, or complex manufacturing equipment. Although the resulting systems are well characterized, allow control of size and surface characteristics, and provide for a slow release of both antigen and immunoadjuvants, their immunological behavior and antigen delivery capabilities are yet to be studied systematically.

Another attractive feature of polyphosphazene adjuvants is in its compatibility and with various intradermal immunization techniques [156, 157]. The concept of fabricating microneedles using PCPP as a construction material stems out of the macromolecular nature of this adjuvant, which determines the required set of mechanical properties, such as strength, film forming characteristics, and adhesion. These features, combined with excellent water-solubility, make polyphosphazene adjuvant an attractive platform for vaccine transdermal patch technology. Polyphosphazenes enable straightforward production of coated or dissolvable microneedles with excellent skin penetration capabilities and fast formulation dissolution profiles once microneedles are applied to the skin. Immunization with microneedles containing recombinant HBsAg and PCPP in pigs resulted in an increase in antibody titers of at least 10-fold higher than intramuscular group with similar formulation, but injected intramuscularly as a solution, and three orders of magnitude higher than non-adjuvanted group. In these studies PCPP microneedles also showed a substantial potential for antigen dose sparing.

Overall, well-defined nature of this synthetic class of adjuvants, its biodegradable nature, ability to fine-tune the structure to achieve optimal immune response and modulate its quality, compatibility with other adjuvants, and versatility of formulations, makes this class of adjuvants especially attractive for challenging applications.

Adjuvants have established value for vaccinology and the vaccines industry and emerged as a sub-discipline of knowledge and technology, at the crossroads of immunology, cell biology, bioinformatics, nanotechnology and materials sciences, public health, and several other areas of scientific knowledge and practice. This review has updated just a few aspects of adjuvants and adjuvantation and traced the current state of knowledge back to the origins of adjuvant use and science. We focused on particles and colloids of polyionic composition-aluminum salt gels and polyelectrolyte polymers-which are historical and remain important to vaccine product development. Evidence is now showing that nanoparticulate structure and surface chemistry of aluminum salt compounds are critical determinants of adjuvant activity. Therefore, measurement and control of such attributes can improve the comparability, capability, quality and further product development for this oldest of vaccine adjuvants. Definition and control of surface chemistry and molecular structure are also important to polyelectrolyte polymer adjuvants. Recent information has improved the understanding of their molecular structure and interactions with immune cells, and their mechanism of action continues to be the subject of intensive study in the quest to expand applicability and utility for vaccine technology. Adjuvants including polyionic materials will likely gain greater importance in the development of newer vaccines of greater capability and safety.