In recent years, the research on novel vaccines such as nucleic acid vaccines, genetically engineered vaccines, and synthetic peptide vaccines has made rapid progress. However, compared to traditional inactivated or live vaccines, these vaccines often have issues like poor immunogenicity, necessitating the use of adjuvants to enhance their effectiveness. Adjuvants have been proven to be crucial components of vaccines. There are many types of adjuvants, and no unified classification method exists. Currently, the most widely used adjuvants are aluminum adjuvants and Freund’s adjuvant. But with the development of new vaccines, the creation of new adjuvants is indispensable. Based on the current state of adjuvant research, they can be mainly classified into three categories: immunomodulatory molecule adjuvants, antigen delivery adjuvants, and composite adjuvants. Only seven newly developed adjuvants have been approved by the FDA. Besides aluminum adjuvants, these include MF59, AS03, AS01, AS04, CpG 1018, and Matrix-M adjuvant, which is used for emergency COVID-19 purposes.
Newly developed adjuvants at BOC Sciences
| Name | Description |
| MF59 | MF59 is a safe and effective adjuvant licensed to be included in influenza vaccines. |
| CpG 1018 | CpG 1018 is added to vaccines to enhance immune responses to protein antigens. |
| AS03 | AS03 has been shown to enhance the vaccine antigen-specific adaptive response by activating the innate immune system locally and by increasing antigen uptake and presentation in draining lymph nodes, a process that is modulated by the presence of α-tocopherol in AS03. |
Immunomodulatory Molecule Adjuvants
New vaccines like nucleic acid vaccines, peptide vaccines, and recombinant subunit vaccines are constantly being developed and applied. However, these new vaccines generally have the drawback of low immunogenicity and cannot effectively trigger the body’s immune response, thus failing to protect the body. Therefore, selecting adjuvants that have immunomodulating effects and can enhance immune responses is significant for better exerting the effects of new vaccines. Adjuvants that stimulate immune responses contain immune stimulants or enhancers that use receptor-mediated signaling pathways to modulate the immune response and enhance antigen immunogenicity, such as CpG1018.
CpG oligonucleotides (CpG ODN) refer to artificially synthesized oligodeoxynucleotide sequences with unmethylated CG dinucleotides as the core. As TLR9 receptor agonists, CpG ODN can activate the TLR9 receptor by binding to it, thereby enhancing specific antigen humoral and cellular immune responses. CpG ODN can activate natural killer cells, B cells, T cells, etc., and induce the production of various cytokines such as IFN-γ, IL-1, IL-2, IL-12. Moldoveanu et al. first studied the intranasal immunization of mice with inactivated influenza virus vaccines combined with CpG ODN. The results showed that the serum-specific antibodies induced in the experimental group increased sevenfold compared to the control group without CpG ODN. Continuous research on CpG ODN as adjuvants revealed that they could enhance immune responses to various antigens. With strong immunostimulatory activity, CpG ODN can be used as an immune enhancer with subunit vaccines or peptide vaccines to enhance vaccine immunogenicity, indicating a broad application prospect.
CpG ODN adjuvants initially derived from the Coley Pharmaceutical Group’s development of CpG DNA technology. Especially CpG2006 and CpG1018 have been extensively verified in preclinical and clinical trials, contributing to improved protective efficacy, faster onset of action, and prolonged vaccine protection time. The combination of the CpG1018 adjuvant with the Hepatitis B surface antigen has been approved, becoming a new generation of vaccines preventing HBV. These adjuvants come from chemical synthesis, offering advantages of controllable quality, large-scale production, and low cost.
CpG ODN adjuvants at BOC Sciences
| CpG-A DNA, ODN 2216 Prototype | CpG ODN BW005 |
| CpG-B DNA, Rabbit | CPG 21424 |
| CpG-B DNA, Human, Mouse, K-type | CpG-ODN 2135 |
| CpG ODN 1466 | CpG-28 |
| CpG ODN PB3 | ODN K3 |
With the advancement of biological science technology, the application scope of vaccines has extended beyond the prevention of infectious diseases to a new application field in tumor immunotherapy. With the advent of an aging society, increased lifespan, and environmental degradation, the incidence of tumors is gradually rising, creating an urgent medical need to develop highly efficient and low-toxicity anti-tumor drugs that extend the survival time of tumor-bearing patients.
Apart from traditional cytotoxic drugs, activating tumor immunity has become a new emerging therapeutic approach. Based on the immunostimulatory activity of CpG ODNs, there are attempts to use CpG ODNs in combination with tumor antigens and as standalone therapeutic agents in anti-tumor therapy.
Research on the interaction mechanisms between various CpG ODNs and ten human Toll-like receptors (TLRs) provides the theoretical basis for designing and screening the next generation of immunostimulatory novel adjuvants. Polyinosinic-polycytidylic acid (Poly (I:C)) is a synthetic analogue of viral dsRNA that can be specifically recognized by TLR3 and MDA-5 receptors to activate the body’s innate immunity, thus being used as an immune-enhancing drug for viral inflammation. Synthetic dsRNA can target and activate TLR3 and melanoma differentiation-associated gene 5 (MDA5) on APCs, leading to the production of pro-inflammatory cytokines (such as IL-12 and type I interferon) and promoting a strong Th1 immune response and CTLs. Poly-I:C and its modified version Poly-ICLC are the most extensively studied synthetic dsRNA immunostimulants. Poly-I:C and Poly-ICLC can induce the maturation of human peripheral blood monocyte-derived dendritic cells, leading to the secretion of IFN-β and pro-inflammatory cytokines IL-6 and IL-12, allowing CD8+ T cells to achieve cross-presentation of extracellular antigens.
Preclinical and clinical studies have shown that Poly-I:C and its modified version Poly-ICLC are promising adjuvants capable of enhancing antibody production and CD8+ T cell immune responses. A human study found that dendritic cell (DC) vaccines with Poly-ICLC as an adjuvant triggered innate immune responses similar to live virus vaccines. Poly-I:C and Poly-ICLC are used in peptide vaccines, DC, and whole-cell vaccines, mainly tumor vaccines. Side effects include dose-related fever and coagulation abnormalities. Therefore, a suitable delivery system is needed to prevent its degradation, so that more Poly-I:C/Poly-ICLC acts on APCs, aiming to ensure the lowest dose activates APCs and reduces adverse reactions. Poly (I:C) enhances vaccine immunogenicity, especially for synthetic peptide vaccines and subunit vaccines, with a significant enhancement in both humoral and cellular immunity when used in combination with Poly (I:C), indicating its potential as an immune enhancer.
Cytokines are small soluble peptide proteins secreted by immune cells and some non-immune cells under certain stimulatory conditions, often playing regulatory roles intercellularly and intracellularly, making them a widely applied type of molecular adjuvant. Commonly researched cytokines include interferons (IFNs), interleukins (ILs), tumor necrosis factors (TNFs), granulocyte-macrophage colony-stimulating factor (GM-CSFs), chemokines, etc. Cytokines enhance the immunomodulatory function of natural killer cells, promote the differentiation of T lymphocytes, play a role in upregulating immune responses in the body, and protect the body against bacterial, viral, and parasitic invasions, serving as effective immune enhancers. Studies continually demonstrate that cytokines used as adjuvants are powerful tools for modulating host immune responses induced by recombinant DNA or protein vaccines, playing significant roles in specific immune responses, and revealing significant potential as vaccine adjuvants.
Bacterial flagellin, as a pathogen-associated molecular pattern (PAMP), can bind to Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4) to activate the body’s innate and adaptive immunity and effectively induce the production of cytokines and nitric oxide, which are natural immune effectors, serving as unique inflammatory stimulation molecules. Flagellin can be mixed with extraneous antigens or expressed through fusion with these antigens to produce recombinant vaccines, effectively inducing both innate and acquired immune responses and activating dendritic cells, resulting in the migration of induced cytokines to secondary lymphoid organs to enhance immune responses. Therefore, flagellin effectively enhances the immunogenicity of external antigens, showing significant potential in the development of recombinant vaccines. Flagellin has four domains: D0, D1, D2, and D3, with D0 and D1 being the conserved regions and D2 and D3 as the hypervariable regions. Its unique structural characteristics provide effective and flexible adjuvant activity, allowing for the design of various vaccine types to prevent different diseases.
Saponins are natural glycosides of steroidal or triterpenoid structures, exhibiting numerous biological and pharmacological activities. Saponins activate the immune system of mammals. The most widely used saponin-based adjuvants are Quil-A and its derivative QS-21, which can modify T cells and antigen-presenting cells, inducing pro-inflammatory Th1/Th2 or anti-inflammatory Th2 immune responses. Saponin adjuvants were initially used in cancer vaccines (e.g., melanoma, breast cancer, and prostate cancer) and later applied to vaccines for Alzheimer’s disease and infectious diseases, including AIDS, influenza, simple herpes, malaria, and hepatitis B, etc. Saponins enhance immune stimulation as vaccine adjuvants. For novel vaccines such as antiviral and antitumor vaccines, various types of saponins and their derivatives exhibit good adjuvant effects. Therefore, research into novel saponin adjuvants is crucial for the development of modern vaccines.
Saponins adjuvants at BOC Sciences
| Ginsenoside Rb1 | Ginsenoside Re |
| Ginsenoside Rg1 | QS-21 |
| Saponin | Ginsenoside Rg3 |
| Platycodin D | Platycodin D2 |
| QS-17 | QS-18 |
| QS-7 | Quil A |
| AJS75 | IA-05 |
| QS-7-Api | GPI-0100 |
| VSA-1 |
Lipopolysaccharides (LPS) naturally occur in the outer membrane of Gram-negative bacteria, strongly activating the innate immune system and are key factors in triggering adaptive immune responses after bacterial infection. LPS and their derivatives can be added to antigen vaccines to enhance immunity, but natural LPS often increases vaccine reactogenicity by exhibiting endotoxin activity. Thus, modifying the structure of LPS reduces its toxicity while triggering appropriate immune responses needed against specific pathogens. LPS generally consists of three parts: polysaccharide O-antigen, core oligosaccharide, and hydrophobic lipid A. Mammalian immune cells recognize lipid A through the Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2) pattern recognition receptor complex, activating immune cells and releasing inflammatory cytokines. Lipid A is an effective inducer of innate immunity and an effective enhancer of immune responses. LPS is one of the most active pathogen-associated molecular patterns known; chemically or genetically modifying LPS to reduce its toxicity is crucial in its application as an adjuvant. For example, monophosphoryl lipid A (MPLA) is modified from lipid A, reducing its endotoxin activity while retaining its adjuvant effects. Due to its low toxicity and good immune stimulation, MPLA effectively enhances immune responses to recombinant proteins or peptide antigens with low immunogenicity. The potential of LPS modifications for adjuvant preparation in the future is immense.
Antigen Delivery Adjuvants
Regarding novel safe subunit vaccines characterized by weak immunogenicity, poor stability, and easy degradation in the cellular microenvironment, employing carriers to deliver vaccines offers a viable solution. Carriers can control the spatial and temporal presentation of antigens within the immune system, promoting sustained release and targeting of vaccines, even allowing low-dose weak immunogens to effectively stimulate immunity. Various antigen delivery carriers like liposomes, nanoparticles, microspheres, and micelle systems are continually being developed and applied in the market, with approved examples like aluminum adjuvants, MF59, AS03, etc.
Liposomes, composed of nontoxic, non-immunogenic, and biodegradable phospholipids derived from natural products, can encapsulate antigens, serving as antigen delivery adjuvants and tools for vaccine delivery. A key advantage of liposome vaccine carrier systems is their versatility and adaptability. Utilizing their chemical properties, hydrophilic antigens (proteins, peptides, nucleic acids, carbohydrates, haptens) can be encapsulated within the aqueous internal spaces of liposomes, while lipophilic compounds (lipopeptides, antigens, adjuvants, linker molecules) are embedded in the lipid bilayer. Additionally, antigens or other adjuvants can attach to the liposome surface through adsorption or stable chemical linkage. DNA vaccines encapsulated in liposomes can more effectively cross cell membranes for intracellular expression, with liposomes playing protective and sustained-release roles. Liposomes can be modified with immune stimulatory molecules and targeting molecules, participating in the immune process as a multifunctional vaccine adjuvant delivery system, capable of targeting immune cells or even organelles, inducing lysosomal escape, promoting antibody cross-presentation, and thereby greatly improving vaccine immunogenicity. However, as adjuvants, liposomes have some drawbacks: During storage, unsaturated fatty acids in phospholipids gradually oxidize; liposomes tend to fuse, allowing encapsulated antigens to release during fusion; liposome preparation techniques are complex and costly.
Nanoparticles (NPs) are ultra-fine particles typically less than 100 nm in diameter. Their larger specific surface area offers more surface active centers, better adsorbing and catalyzing antigens, and good biocompatibility, allowing sustained antigen release, maintaining antigen at effective concentrations. Currently, both organic and inorganic nanoparticles are widely applied in adjuvant delivery system research, such as calcium phosphate, chitosan, silica nanoparticles, etc.
Composite Adjuvants
Composite adjuvants are mainly divided into two categories: carrier-based forms or non-carrier adjuvants. Composite adjuvants combining the first two types of adjuvants include GlaxoSmithKline’s AS04 and AS01 adjuvants. AS04 comprises aluminum adjuvant and MPL, with MPL adsorbing onto the aluminum adjuvant and activating TLR4 responses.
Carrier-based composite antigen delivery adjuvants often exhibit easily modifiable characteristics, allowing their combination with immune enhancers or targeting molecules, raising antigen targeting and extending antigen existence, inducing effective and long-lasting immune effects and memory antibodies. The combination of immune stimulatory molecules comprises the AS01 adjuvant, a vaccine adjuvant system based on liposomes containing two immune stimulatory agents: 3-o-deacyl-4’-monophosphoryl lipid A (MPL) and saponin QS-21. It not only delivers antigens but also enhances humoral and cellular immunity to antigens. The immune-stimulating complex (ISCOM) is a hollow cage-like structure formed from a mixture of saponin, cholesterol, and phospholipids, with particle sizes between 30-40 nm. Antigens can be encapsulated within the hollow cage for delivery, while saponins, acting as immunomodulatory molecules, also induce stronger immune responses. Carrier-based adjuvant composites allow for simultaneous presentation of immune stimulatory molecules and vaccines through antigen delivery systems to immune cells, maximizing the effectiveness of adjuvants and vaccines.
Non-carrier adjuvant composites typically use single immune stimulants formulated in water-in-oil emulsions. Recently, composite adjuvants comprising multiple immune stimulants have shown promising potential. The immune system can activate innate defense systems by recognizing different pathogen components, inducing multiple immune responses referred to as pathogen-associated molecular patterns (PAMPs). Various PAMPs are often utilized as adjuvants to activate specific pattern recognition receptors (Pattern PRRs), thereby enhancing vaccine immunogenicity. Naturally or synthetically optimized PAMP derivatives serving as ligands for TLRs can elicit strong Th1 responses, which many vaccines lack. Furthermore, other types of PAMPs like the stimulator of interferon genes (STING), retinoic acid-inducible gene I (RIG-I), and C-type lectin (CLR) agonists are beginning to be explored as potential adjuvants. Different immune stimulatory molecules target specific PRR categories, providing broad immune responses; activating different receptors induces distinct signaling pathways, thereby affecting adaptive immune responses to generate determined cellular and antibody responses.
References
- Zhao T, Cai Y, Jiang Y, et al. Vaccine adjuvants: mechanisms and platforms. Signal Transduct Target Ther. 2023;8(1):283.
- Zeng Z, Wang HN, Zhang ZF, et al. Research progress of new vaccine adjuvants. Chinese Journal of Biotechnology, 2021, 37(1): 78-87.