Vaccination is undoubtedly one of the most remarkable health achievements in human history. In just over two centuries, vaccines have enabled us to achieve extraordinary goals, such as eradicating smallpox, eradicating polio from most parts of the world, and dramatically reducing mortality and morbidity from many infectious diseases.
Vaccination policies are the cornerstone of public health, and there is a strong emphasis on ensuring safe and effective vaccines. The effectiveness of the vaccine depends not only on the antigenic composition, but also on the adjuvants that are often used, stimulating the immune system in a more effective way. Adjuvants are components that are added to vaccines to improve the immune response to antigens. In addition, adjuvants have several benefits, such as reducing the amount of antigen and the number of vaccinations per dose of the vaccine, and in some cases, they also increase the stability of the antigenic component, extend its half-life, and indirectly improve its immunogenicity.
Antigens are associated with adjuvants, and they are able to induce a local pro-inflammatory response by activating the innate immune system, resulting in the recruitment of immune cells to the injection site. The antigen-adjuvant complex activates the pattern recognition receptor (PRR) pathway through pathogen-associated molecular patterns (PAMPs). This leads to the activation of innate immune cells, which produce cytokines and chemokines.
Currently, the vast majority of vaccines approved by the European Medicines Agency and the U.S. Food and Drug Administration for human use aluminum salts as adjuvants, which are the oldest adjuvants used in vaccine formulations. In order to improve the safety and efficacy of vaccines, it is necessary to increase the variety and number of new adjuvants. Advances in modern technologies, such as nanotechnology and molecular biology, have strongly facilitated the development process of adjuvant components, thereby increasing the efficacy of vaccines. A good adjuvant must be safe, effective, easy to produce, have good medicinal properties (pH, osmolality, endotoxin levels, etc.) and durability and, finally, be economically affordable. Particles, emulsions, and immunostimulants show great potential in vaccine production.
Fig. 1 Mechanism of Action of Adjuvants.1
Delivery System Adjuvant
Adjuvants can be classified according to different criteria, such as their physicochemical properties, origin, and mechanism of action. One of the most talked about classification systems is based on their mechanism of action, which divides them into two broad categories: delivery system adjuvants and immune-enhancing adjuvants.
Creative Biolabs’ Adjuvant Products for Vaccine Development:
| Aluminum | Oil Adjuvant | Saponin |
| Lipopolysaccharide | CpG | PRRs Agonist |
The adjuvant properties of aluminum salts were discovered in the twenties of the twentieth century, and these compounds have been used as vaccine adjuvants since 1926. As the longest-used and most frequently used adjuvant in vaccines, about one-third of currently licensed vaccines contain aluminum.
In vaccines, aluminum exists in the form of a composite polymer of crystalline aluminum hydroxide (AlH) or amorphous aluminum hydroxyl phosphate (AlP) to form aggregated nanoparticles. AlH has the appearance of needle-like nanoparticles, whereas when viewed under transmission electron microscopy, AlP appears as a reticulated. Both forms of aluminum adjuvants are generally soluble in citrate, but AlP is more soluble than AlH. The antigen is adsorbed on the surface of the adjuvant particle through electrostatic interaction and ligand exchange. Aluminum salt/antigen binding enhances antigen uptake and presentation in antigen-presenting cells (APCs). In addition, aluminum salts stimulate the activation of the NLRP3 inflammasome, leading to the production of IL-1β and IL-18, which cause local inflammation and APC recruitment.
Many vaccines use aluminum adjuvants, such as those against diphtheria and tetanus, pertussis, hepatitis B, and pneumococcal and meningococcal bacteria. In Europe, the European Pharmacopoeia sets the aluminium content in vaccines at a maximum of 1.25 mg per dose. In the United States, the Code of Federal Regulations sets the aluminum content in biological products, including vaccines, at 0.85 mg/dose. Unlike aluminum, which is predominantly in the form of soluble citrate or chloride salts in food, inorganic aluminium compounds used as adjuvants are less soluble as part of their auxiliary mode of action. Therefore, due to this poor solubility at physiological pH, the absorption rate of aluminum contained in the vaccine after intramuscular or subcutaneous injection will be very slow.
Several studies have evaluated the kinetics of aluminum after intramuscular injection. An experiment was studied based on the intramuscular injection of AlH and AlP labeled with 26Al with a total dose of 0.85 mg of aluminum. The results showed that the absorption rates of AlH and AlP were 17% and 51%, respectively, during the 28 days of the experiment. The maximum serum concentration (Cmax) of 26Al is 2 μg/L. In addition, the neurotoxicity of aluminum has been studied in vitro, ex vivo, in animal models, and in humans. Some in vitro studies of bacteria have shown no mutagenicity. However, some in vivo studies are often inconsistent and contradictory, and there may be methodological shortcomings. Therefore, to date, it has not been possible to determine whether aluminum salts used as adjuvants (at recommended doses) have toxic effects, despite the fact that they produce more or less intense oxidative stress.
- Freund’s Adjuvant
Freund’s adjuvants include both complete and incomplete Freund’s adjuvants. Both adjuvants are water-in-oil emulsions that are capable of carrying antigens and stimulating the innate immune system. Complete Freund’s adjuvant (CFA) includes in its structure heat-killed mycobacteria, which enhance the stimulation of the immune response. However, CFA is able to induce intense, long-lasting local inflammation, which can be significantly painful and may ulcerate at the injection site. Incomplete Freund’s adjuvant (IFA) does not contain mycobacteria and was used as an adjuvant in human influenza vaccines in the 1950s, where it can induce a stronger, longer-lasting antibody response than the same vaccine without adjuvant.
The adjuvant activity of IFA is based on its characteristic of being a deposit of oily antigens, from which antigens are continuously released at the injection site. This simultaneously results in an increase in the antigen half-life as well as strong local innate immune stimulation through phagocytosis, leukocyte recruitment and infiltration, and cytokines. However, the routine use of IFA in human vaccine formulations has triggered strong side effects. According to a survey conducted by the World Health Organization in 2005, 40,000 people who received immunizations from about 1 million IFA subjects experienced serious side effects (e.g., sterile abscesses).
- MF59
MF59 is a water-in-oil emulsion consisting of squalene, Span 85, and Tween 80 in 10 mM sodium citrate buffer at pH 6.5 with an average particle size of approximately 165 nm. It was the first oil-in-water emulsion approved in Italy for use in a human vaccine in 1997. Currently, it is used in the trivalent and quadrivalent (TIV and QIV) influenza vaccine, Fluad (Seqirus). Studies have shown that the presence of MF59 increases the effectiveness of the influenza vaccine in children under 2 years of age. MF59 has also been used as an adjuvant in HBV vaccines, which is able to elicit a strong immune response that is better than aluminum-induced immune responses.
Regarding the mechanism of action, MF59 has a similar effect to aluminum salts. The reservoir activity at the injection site is negligible, and studies have shown that it has a half-life of 42 hours. In contrast, MF59 has a potent ability to induce cellular and humoral immune responses, including the production of high-titer functional antibodies. The presence of MF59 stimulates local innate immune cells to secrete chemokines such as CCL4, CCL2, CCL5, and CXCL8, which in turn drive leukocyte recruitment, antigen uptake, and migration to lymph nodes by triggering adaptive immune responses. MF59 is safe and well tolerated, with millions of doses administered in more than 35 countries.
- AS03
AS03 is an oil-in-water adjuvant emulsion consisting of the surfactant polysorbate 80 and two biodegradable oils, namely squalene and DL-α-tocopherol, in phosphate buffer. This adjuvant has been used in influenza vaccines, eliciting an immune response similar to MF59, and is also used in malaria vaccines. The European Union approved the marketing of the AS03 adjuvanted vaccine Pandemrix in 2009, while the AS03 adjuvanted influenza A (H5N1) monovalent vaccine was approved by the FDA in 2013.
The antioxidant and immunostimulatory properties of α-tocopherol appear to be stronger compared to MF59. In addition, studies have shown that AS03 is able to stimulate the immune system by activating NF-κB, thereby inducing cytokine and chemokine secretion in muscles and lymph nodes and promoting the migration of innate immune cells. AS03 can also stimulate CD4+ T cell-specific immune responses, which can lead to long-lasting neutralizing antibody production and higher levels of memory B cells. AS02 is further supplemented with two powerful immunostimulants, QS-21 (a saponin extracted from Astragalus membranaceus) and 3-O-deacyl-4′-monophospholipid A (MPL), on top of AS03 to enhance its immunogenicity.
- Virus-like particles
Virus-like particles (VLPs) are icosahedral or rod-shaped nanoparticles (Å20–200 nm) composed of the outer shell of a self-assembling capsid protein, which have been used in long-term research for vaccine development. They are non-infectious particles and do not include any genetic material. VLPs are formed from an external viral shell with repetitive epitopes that are immediately recognized by the immune system as non-self, resulting in a robust immune response. In addition to these repeated structural motifs, VLPs are similar in size to viruses (typically between 20–800 nm) and are processed quickly and efficiently to produce a rapid and durable immune response, even in the absence of adjuvants.
Currently, there are two important vaccines that use virus-like particle adjuvants to induce immunity: hepatitis B and papillomavirus (HPV) vaccines. The currently used hepatitis B vaccine, a recombinant DNA vaccine containing hepatitis B surface antigen (HBsAg) in the form of VLP, is used to prevent hepatitis B infection. It is administered to infants, children, and adolescents under 15 years of age, or to people at high risk of hepatitis B infection, and has also shown good immunogenicity (95–99% efficacy) in newborns born to mothers of hepatitis B carriers.
The HPV vaccine is also a vaccine based on the VLP platform. The current 9-valent HPV vaccine protects against nine different viral genotypes, which can cause 90% of cervical cancers and 80–95% of anogenital cancers. The 9-valent HPV vaccine contains the L1 protein of nine different genotypes of HPV (6, 11, 16, 18, 31, 45, 53, 58) to form VLP and synthesize it through recombinant DNA technology.
- Virions
Virions are a vaccine platform that is structurally very similar to native viruses. Structurally, they are VLPs formed by recombinant influenza virus envelopes composed of hemagglutinin (HA), neuraminidase (NA), and phospholipids (phosphatidylethanolamine and phosphatidylcholine), which lack viral genetic material. The first use of virions in 1975 to make influenza vaccines, an adjuvanted influenza vaccine for all age groups, is effective in healthy and immunocompromised children, adults, and the elderly. It is capable of inducing B cell responses and producing specific antibodies. Virions retain the receptor-binding capacity and membrane fusion activity of viral HA, but due to the lack of viral RNA, they are unable to induce infection in cells after binding.
Virions are a perfect delivery system capable of transferring antigens into the cytoplasm of antigen-presenting cells and inducing cytotoxic T lymphocyte (CTL) responses. However, due to their weak adjuvant properties, virions are not very effective in activating APCs and facilitating cross-presentation. This intrinsic limitation can be eliminated by the addition of stronger adjuvants. For example, a novel influenza vaccine based on a virion has recently been developed, supplemented with the Toll-like receptor 4 (TLR4) ligand monophosphoryllipid a (MPLA) and the metal ion chelating lipid DOGS NTA-Ni adsorbed into the membrane. In vivo immunization of mice with virions with these MPLA adjuvants can induce specific CTL.
The significant advantage of virion delivery system adjuvants is their ability to adsorb antigens to their surface and lumen through hydrophobic lipid interactions. Adsorption of antigens to the surface of the fluid phospholipid bilayer of the virion stimulates interaction with host cell receptors. The FDA has approved virions as nanocarriers for human use, and they are very well tolerated and safe. In contrast to subunit vaccines, which do not respond well to viral invasion, virions are able to induce robust humoral and cellular immunity in a manner very similar to natural infection and other potent adjuvants.
To date, in addition to the two virion-based vaccines against influenza and hepatitis A mentioned above, several virion-based vaccines are being studied, including those against HIV, human papillomavirus, respiratory syncytial virus, and malaria.
Immune Enhancer Adjuvant
- TLR1/2 agonists
Among TLR1/2 agonists, L-pampo is a potent adjuvant system consisting of Pam3Csk4 (Pam3) and polyinosinyl:polycytidylate (polyI:C). In one study, L-pampo induced the production of stronger anti-HBV antibodies than the aluminum adjuvant, and also involved cell-mediated immune responses such as increased multifunctional CD4+ T cells.
In addition, bacterial lipoproteins are the most potent ligands for TLR2 recognition. Studies have shown that synthetic lipopeptides derived from bacterial lipoproteins are strong activators of B cells and macrophages and can be used as vaccine adjuvants. Macrophage-activated lipoprotein-2 (MALP-2) from Mycoplasma fermentum is shown to activate immune cells via TLR2- and MyD88-dependent signaling pathways. In addition to MALP-2, Pam2CSK4 and Pam3CSK4 are also recognized TLR2 agonists, and they have been evaluated as therapeutics against infectious diseases such as Leishmania, malaria, and influenza.
- TLR3 agonists
TLR3 is an endosomal receptor that detects viral dsRNA, which recognizes poly(I:C) because it structurally mimics viral RNA, thereby inducing the production of type I and type III IFNs and eliciting Th1 cytokine responses. Type I IFNs produced following TLR3-poly(I:C) interactions are particularly important for the efficient activation of CD8+ T cell responses by traditional dendritic cells (cDCs). In addition, poly(I:C)-generated type I IFNs stimulate clonal expansion of T cells, increasing the ratio of effector T cells and the number of antigen-specific B cells.
Poly(I:C) has been extensively studied as a potential adjuvant. However, poly(I:C) has a toxic effect on humans. Therefore, scientists’ attention has focused on derivatives of poly(I:C), such as poly(ICLC) and poly(IC12U), as well as other synthetic TLR3 agonists, such as ARNAX, IPH3102, and RGC100.
To date, several studies have used poly (ICLC) as a vaccine candidate for infectious diseases, such as Plasmodium falciparum and HIV, as well as cancer. Studies have shown that poly (ICLC) is able to elicit a stronger Th1 immune response compared to other TLR agonists such as LPS and CpG. A new TLR3 agonist with adjuvant potential is ARNAX, a TLR3-specific ligand with lower toxicity than poly(I:C). Two of the most important areas of ARNAX research are cancer immunotherapy and influenza vaccines.
- TLR4 agonists
TLR4 agonists studied as vaccine adjuvants include AS01, AS02, and AS04, all of which contain the ligand MPLA for endosomal TLR4. AS01 has been used to develop vaccines against malaria, HIV, and tuberculosis. AS01 is a combined adjuvant system consisting of two different immunostimulatory molecules, MPLA and QS-21, encapsulated in a liposomal structure. These two compounds use liposomes as carriers to reach intracellular levels through cholesterol-dependent endocytosis. Intracellularly, QS-21 causes lysosomal instability and promotes the activation of the protein kinase SYK. MPLA acts on endosome TLR4 to induce TRIF-dependent signaling pathways. AS01 activates caspase-1, thereby promoting the activation of the NLRP3 inflammasome and the release of IL-1β and IL-18 from APCs. The release of IL-18 leads to the rapid production of IFN-γ, especially in the lymph nodes, which promotes the maturation of DCs and the induction of Th1-type immune responses.
- TLR5 Agonists
TLR5 is a receptor that recognizes bacterial flagellar proteins and is expressed by several immune cells. Ligation with ligands leads to the activation of inflammatory pathways and the release of many inflammatory mediators, such as TNF-α, IL-1β, IL-6, and nitric oxide. In addition, flagellin is able to elicit Th1 and Th2 responses, whereas unlike other TLR ligands, they can only induce Th1 responses. Flagellin induces the production and release of IL-1β by activating the NLRC4 inflammasome. To date, at least three vaccines using flagellin as adjuvants are in clinical trials: two against influenza viruses and one against Yersinia pestis.
- TLR7/8 agonists
Some studies have shown that TLR7/8 agonists are able to strongly induce Th1 immune responses. Ligand binding to TLR7/8 yields high levels of type I IFN, IL-12, TNF-α, and IL-1β. In addition, TLR7/8 and TLR9 agonists are the only agonist molecules capable of activating and promoting clonal expansion of cDCs and plasmacytoid dendritic cells (pDCs), as well as mobilizing CD14+CD16+ inflammatory monocytes and CD14dimCD16+ patrol monocytes.
The most representative TLR7/8 agonists are some synthetic small molecules, such as imiquimod (R837) and requimod (R848), which belong to the class of imidazoquinolines. Imiquimod is currently approved for the treatment of genital warts, superficial basal cell carcinoma, and actinic keratosis, while raquimod has been studied for antiviral and anticancer treatments.
However, these small molecules have some inherent limitations. In particular, they can spread away from the site of administration and thus away from antigens, leading to reduced efficacy and inducing systemic side effects. The direct binding of these molecules to aluminum adjuvants has been shown to increase vaccine efficacy. In addition, the combination of synthetic polymer scaffolds, lipid polymer amphiphiles, polyethylene glycol (PEG), nanogels, alum, and various other synthetic polymers significantly increased imidazoquinoline delivery and improved DC and antigen-specific T cell maturation. In addition, previous studies using imidazoquinoline with one or more other TLR agonists (e.g., MPLA and MPLA+CpG ODN) have shown that this combination increases the innate immune response, significantly produces antigen-specific neutralizing antibodies, and improves the Th1 response. All of these innovations highlight the outstanding potential of TLR7/8 agonists as candidate adjuvants.
- TLR9 Agonists
TLR9 naturally recognizes bacterial DNA motifs represented by the unmethylated cytosine guanine phosphate (CpG) dinucleotide and drives the activation of the innate immune system through the MyD88-dependent pathway. CpG-ODNs elicit potent chemokine, cytokine, and antibody production in NK cells, B cells, and pDCs, thereby driving a strong Th1-type immune response. So far, three classes (A-C) of different classes of CpG-ODN have been developed, but only Class B molecules have been used as adjuvants in clinical trials. Type B CpG-ODN can induce the maturation of pDCs and interact directly with B cells to enhance antibody production.
The recently licensed CpG 1018, an oligonucleotide with high chemical stability and the ability to elicit an adjuvant to a Th1 immune response, is used as an adjuvant to the hepatitis B vaccine Heplisav-B. CpG 1018 in Heplisav-B increases the efficacy of the vaccine and requires only two doses of the vaccine compared to the traditional hepatitis B vaccine, which requires three doses for optimal protection. Another CpG-ODN, CpG 7909, is also being clinically evaluated and has shown encouraging results in HBV and malaria vaccination.
In addition, other next-generation TLR9 agonists are in development. MGN1703 is a small DNA molecule that includes a CG motif, but is structurally different from CPG-ODN. The MGN1703 consists of a segment of reverse complementary DNA that is double-stranded in the middle, and a single-stranded loop at both ends includes three unmethylated CG motifs, forming a dumbbell-shaped structure. MGN1703 has been tested as an adjuvant in cancer vaccines, and it has been found to be able to activate both innate and adaptive immune responses with only mild or temporary side effects.
Reference:
- Facciolà, Alessio, et al. “An overview of vaccine adjuvants: current evidence and future perspectives.” Vaccines 10.5 (2022): 819.