Vaccines are one of the most important inventions in human history, which has revolutionized human life and health. Typically, vaccines work by triggering an innate immune response and stimulating antigen-presenting cells, resulting in a defensive adaptive immune response against specific pathogen antigens. Adjuvants are a key ingredient that is often used as an additive to improve the efficacy and immunogenicity of vaccines. For more than 90 years, adjuvants have been an important ingredient in many human vaccines, improving their efficacy by enhancing, modulating, and prolonging the immune response.
Currently, the vast majority of vaccines approved by the European Medicines Agency and the United States Food and Drug Administration (FDA) for human use aluminum salts as adjuvants, which are the oldest adjuvant used in vaccine formulations. In order to improve the safety and effectiveness of vaccines, it is necessary to increase the type and number of novel adjuvants. First, a good adjuvant must be safe, effective, and easy to produce. It should also have good medicinal properties (pH, osmotic pressure, endotoxin levels, etc.) and durability. Finally, it is also economically affordable. Therefore, granules, emulsions, and immunostimulants all show great potential for development in vaccine production.
History of vaccine adjuvants
Significant progress has been made in the field of vaccine adjuvant research since its inception in the early 20th century. As infectious diseases become more common, immunization has become the most effective way to limit their spread and reduce the associated harms. However, early vaccines often proved ineffective because purified antigens alone failed to elicit a strong enough immune response. To overcome this challenge, adjuvants have been introduced into vaccines, which are materials that are added to vaccines to enhance their immunogenicity and thus enhance their effectiveness.
The development of modern adjuvants is a slow and challenging process. The researchers’ goal is to develop a vaccine that induces a strong immune response while remaining safe. This work includes selectively combining molecules and formulations that determine efficacy, and simultaneously exploring the discovery of more natural and synthetic compounds.
Aluminum salts were the first adjuvant to be discovered to enhance the immune response to vaccines against diseases such as hepatitis B, tetanus, diphtheria, pertussis, and human papillomavirus. The effectiveness of aluminum salts is attributed to their ability to form antibody reservoirs at the injection site, induce inflammation, and activate dendritic cells and T cells. However, although aluminum adjuvants have been used in vaccines for more than 70 years, their exact mechanism of immunity is still not fully understood.
Adjuvant developments in the late 90s of the 20th century included oil-in-water emulsions. These emulsions help the antigen to be released slowly over a longer period of time, activate the innate immune system, and induce humoral and cellular immune responses. This emulsion was first applied to Fluad, a seasonal flu vaccine for adults over 65 years of age. Other FDA-cleared adjuvants include MF59, an oil-in-water emulsion adjuvant containing squalene, a biodegradable oil, and a surfactant for use in influenza vaccines. AS03, which contains a-tocopherol, squalene, and a surfactant, is used in the H1N1 influenza vaccine and some pandemic influenza vaccines. and AS01, a mixture of liposomes and monophospholipid A, which has been used in the RTS, S/AS01 malaria vaccine, and the Shingrix shingles vaccine. In recent years, novel vaccine adjuvants, such as the TLR9 agonist CpG-1018, have also been approved by the FDA for use as an adjuvant against the hepatitis B vaccine Heplisav-B.
Although there are many other adjuvants that have shown high potency in preclinical models, most have not been licensed for human use due to safety or tolerability issues. In addition, the molecular mechanisms by which existing adjuvants, including alum, MF59, and AS0 system adjuvants, work in humans are still not fully understood.
Benefits and effects of adjuvants
Overall, vaccine adjuvants play a vital role in improving the effectiveness and accessibility of vaccines and reducing the global burden of infectious diseases. Their use is described below:
Alleviate limited vaccine supply: Adjuvants can enhance the immune response, allowing for a reduction in vaccine doses for adequate protection. This relieves pressure on the healthcare system and increases access for those who cannot receive multiple doses.
Enabling a rapid immune response: Adjuvants stimulate a stronger, longer-lasting response to vaccine antigens, providing better protection.
Expanded antibody response: Adjuvants amplify antibody responses to pathogens with significant antigenic drift or strain variation, which are essential for diseases such as influenza, human papillomavirus (HPV), and malaria parasites.
Enhance the amplitude and function of the antibody response: Adjuvants increase not only the amplitude of the antibody response, but also its function, affinity, or the number of functional antibodies produced by the immune system.
Enhance T cell responses: Many novel vaccine adjuvants can induce more efficient participation of T helper cells to optimize the quality and persistence of antibody responses, or induce effector CD4+ or CD8+ T cells that can target and eliminate intracellular pathogens. Therefore, new vaccines may include agonists of toll-like receptors (TLRs) and other innate immune receptors to promote the production of T helper cell responses. This approach is able to overcome the limitations of traditional adjuvants and create more effective vaccines that provide long-lasting protection against a wider range of pathogens.
Development of new vaccines: Adjuvants are essential for the development of vaccines against diseases that have not been effective with conventional vaccines, to enhance immune responses, and to target multiple strains of pathogens. They can enhance the immune response to vaccines and help target a wide range of pathogens.
Improved safety: By enhancing the immune response, adjuvants can reduce the risk of vaccine-related adverse events.
Types of adjuvants and their mechanism of action
Aluminum adjuvant
Aluminum adjuvant was the first adjuvant to be used in authorized human vaccines and remains the most widely used. They are made up of aluminum hydroxide, aluminum phosphate, or a combination of the two and can form an antigen reservoir at the injection site, slowly releasing vaccine antigens over time, resulting in a stronger and prolonged immune response. They have been shown to enhance immune responses to a variety of antigens, including those found in hepatitis B, HPV, and pneumococcal vaccines.
Inorganic nanoparticle adjuvant
Inorganic nanoparticle-based vaccine adjuvants, such as Nanoaluminum, Layered Dimetallic Hydroxide (LDH), and Nano/Mesoporous silica, Nanodiamond, and Quantum dots, have been shown to have the potential to enhance vaccine immune responses. The mechanism of action of inorganic nanoparticle-based vaccine adjuvants involves their interaction with the immune system to enhance and modulate the immune response to antigens present in the vaccine. Inorganic nanoparticles act as carriers of antigens, ensuring efficient delivery to antigen-presenting cells (APCs). Some inorganic nanoparticle adjuvants, such as aluminum-based adjuvants, induce local inflammation at the injection site, attract immune cells to this site, and promote APC recruitment. Inorganic nanoparticle adjuvants help develop long-lasting immune memory. This ensures that the immune system remembers the antigens encountered, providing protection in the event of subsequent exposure to the pathogen.
Emulsion adjuvant
Emulsion is formed when two liquids that are unable to form a homogeneous mixture come together. The emulsion consists of two immiscible liquids, usually the oil phase and the aqueous phase, which are stabilized by surfactants. Emulsions work by forming small droplets of antigens in the oil phase, which are then dispersed throughout the aqueous phase. The antigen is contained in the water droplets, and the oil acts as a reservoir, releasing the antigen slowly and enhancing the immune response by reducing the time of clearance and prolonging the time of antigen exposure. In addition, emulsion adjuvants also activate the innate immune system, inducing the ability of humoral and cellular immune responses.
One of the first oil-in-water adjuvants approved for use in human vaccines was MF59, which contains squalene, polysorbate 80, sorbitan trioleate, and trisodium citrate dehydrate. MF59 enhances antigen-specific immunity at the vaccination site by inducing ATP release and upregulation of cytokines and chemokines. Another oil-in-water emulsion adjuvant is AS03, which contains the surfactant polysorbate 80 and two biodegradable oils, squalene and DL-a-tocopherol. In 2009, the European Union approved the marketing authorization of the AS03 adjuvanted Pandemrix, and in 2013, the AS03 adjuvanted influenza A (H5N1) monovalent vaccine was approved by the FDA.
TLR agonists
TLRs are pattern recognition receptors that play a crucial role in the innate immune response to invading pathogens. These receptors are divided into two groups: cell surface TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) and intracellular TLRs (TLR3, TLR7, TLR8, and TLR9), which are expressed on the endosomal membrane.
CpG is a synthetic DNA molecule with a phosphorothioate backbone containing an unmethylated CpG motif. Activation of TLR9 by CpG enhances specific humoral and cellular immune responses to antigens. CpG is a potent adjuvant that induces a type 1 helper T (Th1) response and promotes cytotoxic T lymphocyte (CTL) production and IFN-γ secretion. In addition, CpG can promote the immunostimulatory effect of antigens, activate antigen-presenting cells, and accelerate immune responses. CpG also promotes the expression of major histocompatibility complexes (MHCs), CD40, and CD86 on plasmacytoid dendritic cells (pDCs) and enhances antigen processing, leading to robust and sustained immune responses.
Liposomes adjuvants
Liposomes are small, spherical structures made of phospholipids and cholesterol that contain antigens and other immune-stimulating molecules. They are biodegradable, biocompatible, and allow for multifunctional structural modifications that allow for tunable properties and toxicity. Liposomes can be used to deliver antigens to antigen-presenting cells, such as dendritic cells, which activate the immune system. In addition, liposomes are able to enhance humoral and cellular immune responses, inducing Th1-biased responses, which are important for the removal of intracellular pathogens and enhance antibody responses, making them attractive adjuvants for vaccine development. A unique feature of liposomes is their ability to be modified to target specific cell types. They can be modified with antibodies or peptides that target specific receptors on dendritic cells to increase the uptake of liposome-encapsulated antigens. Liposomes can also be used to co-deliver multiple antigens or immunostimulatory molecules, resulting in a more robust immune response, especially for complex pathogens that may require multiple antigens to induce a protective immune response.
Other adjuvants
Cytokines are a group of multiple signaling molecules that play an important role in the immune response. They are secreted by various cells of the immune system and act on specific receptors that mediate a range of biological functions such as inflammation, cell proliferation, and differentiation. Cytokines can be divided into different classes based on their structure and function, including interleukin, interferon, tumor necrosis factor, and chemokines.
Polymers are compounds made up of repeating units that are covalently bonded together. They can be natural or synthetic. In vaccine applications, polymers can be used as carriers to create sustained-release libraries that carry antigens and other immunomodulators with their large cross-linked structures. This results in sustained antigen presentation and activation of the immune system, resulting in a stronger, longer-lasting immune response.
Saponins are naturally occurring amphiphilic compounds that can enhance the immune response by stimulating antigen-presenting cells such as dendritic cells and promoting the production of cytokines and chemokines. An example of a saponin-based adjuvant is QS-21, which is used in combination with MPL (Monophosphoryl Lipid A) in the AS01 adjuvant system and has been used to develop vaccines against diseases such as malaria, tuberculosis, and herpes zoster.
Virions are made up of phospholipids and viral envelope proteins, which are usually derived from viruses that do not cause disease in humans. Viral envelope proteins provide an immunogenic surface that can activate the immune system, while phospholipids help stabilize virion structure and increase its uptake by antigen-presenting cells.
The role of various vaccines and adjuvants
Adjuvants have been widely used in various vaccines. This includes whole-virus vaccines (e.g., live attenuated vaccines, inactivated vaccines, and viral vector vaccines), subunit vaccines, and nucleic acid vaccines (including DNA and mRNA).
Inactivated vaccines
Inactivated vaccines are usually produced and inactivated by virus-infected cells, which ensures that the virus is no longer infectious, but retains its antigenic properties, allowing it to contribute to the production of an immune response. Adjuvants used in inactivated vaccines include aluminum, MF59, AS03, and AS04. These adjuvants have been used in vaccines such as the H1N1 influenza vaccine and the HPV vaccine.
Live attenuated vaccines
A live-attenuated virus vaccine is a weakened virus that replicates in the body but does not cause disease. As a result, they can elicit a strong and long-lasting immune response. Adjuvants are not usually used in live attenuated vaccines because the vaccine itself is already a powerful stimulant of the immune system. However, several studies have explored the use of adjuvants with live attenuated virus vaccines to potentially increase their effectiveness. For example, MF59 has been shown to improve the immune response in older adults as an adjuvant to live attenuated influenza vaccine.
Viral vector vaccines
Replication-capable and non-replication-competent adenoviral vectors are typically used for antigens expressing viral proteins. Adjuvants can be used in combination with viral vector vaccines to enhance the immune response generated by the vaccine. For example, the Oxford-Astra Zeneca COVID-19 vaccine is a viral vector vaccine that uses a weakened version of the chimpanzee adenovirus to deliver the genetic material of the SARS-CoV-2 spike protein. This vaccine contains an adjuvant called AS03, which is a combination of squalene, polysorbate 80 and vitamin E.
Virus-like particle vaccines
In this strategy, structural viral proteins are co-expressed to form non-infectious particles as vaccine immunogens. They resemble true virions but lack a viral genome. Adjuvants can be used in virus-like particle vaccines to enhance the immune response and increase vaccine efficacy. Commonly used adjuvants in virus-like particle vaccines include aluminum salts, MF59, and CpG. These adjuvants stimulate the immune system to produce a stronger, longer-lasting response to the VLP vaccine.
DNA vaccines
DNA-based vaccines utilize DNA plasmids as vectors to transfer antigen-encoding genes into host cells, specifically antigen-presenting cells. However, DNA vaccines tend to have limited immunogenicity compared to other vaccine types, which may limit their effectiveness. Adjuvants can help overcome this limitation by enhancing the immune response to the encoded antigen. Various adjuvants have been tested in DNA vaccines, including aluminum, CpG, and liposomes.
mRNA vaccines
mRNA vaccines inject mRNA molecules directly into host cells and then convert them into target proteins in the cytoplasm. mRNA vaccines offer many advantages over traditional vaccines, such as safety, flexibility, scalability, and cost-effectiveness. mRNA vaccines usually do not require adjuvants because the mRNA molecule itself stimulates the immune system. However, some research is also underway to explore the use of adjuvants in mRNA vaccines to increase their effectiveness, especially for certain populations that may have a weaker immune response to the vaccine, like older people.
Protein subunit-based vaccines
Protein subunit-based vaccines contain only one or more specific proteins of the pathogen, not the entire pathogen. Adjuvants are often used with protein subunit vaccines to enhance their effectiveness by increasing the immune response to protein antigens. Aluminum adjuvants are the most commonly used adjuvants in protein subunit-based vaccines, and they have been shown to enhance antibody responses to antigens. Other adjuvants, such as MF59, AS03, and AS04, have also been used in protein subunit-based vaccines to improve their immunogenicity. In addition, new novel adjuvants, such as CpG, are being studied for protein subunit vaccines to further enhance their immune responses.
New advances in novel vaccine adjuvants
Design principles for new adjuvants
In order to develop more effective adjuvanted vaccines, it is important to understand how adjuvants work and follow some design principles. The problem-oriented principle includes consideration of four main questions: (1) the immune response required to prevent the disease of a particular pathogen, (2) the relevant innate immune cells that can induce the desired immune response, (3) the localization of these innate cell subsets in vivo, and (4) the expression of pattern recognition receptors on these cells.
Based on these issues, there are four steps to consider when designing vaccine adjuvants. First, the type of immune effector element required for the vaccine to exert efficacy in the host should be determined. This should take into account antigen type, target cell subsets and phenotypes, and immune pathways to guide the delivery system and choice of immunostimulant. Second, the identification of appropriate vaccine antigens should be based on an understanding of the molecular mechanisms of immune recognition and protection. Vaccines must deliver sufficient amounts of the correct antigen to the appropriate cell population in the correct conformation to induce immune response protection, taking into account the tolerability and safety of the induced inflammatory response. Third, adjuvants require a delivery system, whether synthetic or natural. This requires the design of appropriate antigen delivery modalities and the incorporation of relevant immunostimulants. Fourth, both the vaccine and the adjuvant should be relatively simple in their preparation.
Adjuvant formulations for new vaccines
The correct selection of the immunostimulant of the adjuvant and the composition of the formulation is essential to induce an appropriate immune response to the target antigen. Different formulations of the same immunomodulatory molecule may induce significantly different immune responses. For example, the RTS, S vaccine candidate formulated with AS02 protected 6/7 of vaccinated people from infection, while the same antigen formulated with AS03 or AS04 protected only 2/7 and 1/8, respectively.
In order to manufacture an improved adjuvant, it is essential to ensure that each ingredient is necessary and adds significant value, while not introducing a significant unreasonable burden. AS01 is currently the most successful adjuvant in licensed products, with a 97% efficacy against herpes zoster. The AS01 adjuvant was developed based on the synergistic effect of two key components, MPL and QS-21, which when used together, leads to innate activation, which cannot be achieved with either molecule alone.
Utilizing systemic vaccinology
Systematic vaccinology is an interdisciplinary discipline that involves the early use of human studies to generate omics data that can be used to formulate new hypotheses about the mechanisms of antigen-specific immune responses that are durable with candidate adjuvants. These hypotheses can then be retested in animal models, and subsequent mechanistic insights can be used to design new adjuvant concepts. In addition, a systematic vaccinological approach can be used not only for the mechanism of action of adjuvants, but also for the potential mechanisms by which the formulation works, the mechanisms by which adverse reactions occur shortly after vaccination, and the design of the optimal formulation for vaccine delivery. Overall, this interdisciplinary approach based on systems vaccinology has the potential to transform the science of adjuvants and accelerate the development of novel adjuvants for vaccines.
Non-invasive vaccine delivery
Research on non-invasive vaccine delivery is a key focus that could have a significant impact on mass vaccination worldwide. Prioritize the development of safe, effective, low-cost adjuvanted vaccine formulations to generate the desired immune response and long-term immunity. With an improved understanding of the molecular mechanisms of immune protection and the development of new methods of synthetic chemistry, breakthroughs in vaccine development are expected. New technologies, such as new ethylene glycol conjugation methods, reverse vaccinology, and next-generation sequencing technologies, may lead to new vaccine strategies for diseases such as HBV, pertussis toxin, Lyme disease, and HPV, with or without adjuvants.
In conclusion, the research goal of the novel vaccine is to ensure a high level of broad protection through the introduction of new immunostimulants and new delivery systems that combine efficacy with long-term memory immunoreactivity and safety.
Summary
Adjuvants have played a vital role in vaccine development for nearly a century, enhancing immunogenicity. Understanding the effects of various adjuvants on the immune response, which can work synergistically with different antigen types and vaccines, will help in selecting adjuvants that provide the necessary immune protection. As emerging diseases such as COVID-19 continue to be challenged and more definitive treatments are found, it is imperative to develop safe and effective vaccines. A more informed choice of adjuvants and antigens can not only enhance protection for people who have not responded well to traditional vaccines, but also open up new avenues beyond prophylactic applications. This approach is essential to address current and future global health challenges.
References
Cui, Ying, et al., Vaccine adjuvants: current status, research and development, licensing, and future opportunities. Journal of Materials Chemistry B 12.17 (2024): 4118-4137.