Current Status and Future Opportunities in Vaccine Adjuvant Research

Introduction

Vaccines represent one of the most pivotal inventions in human history, fundamentally revolutionizing global health and safety. Typically, vaccines function by triggering innate immune responses and stimulating antigen-presenting cells, thereby generating a defensive adaptive immune response against specific pathogen antigens. Adjuvants are crucial components, often used as additives, to enhance the potency and immunogenicity of vaccines. For over 90 years, adjuvants have been essential elements in many human vaccines, improving their efficacy by enhancing, modulating, and prolonging immune responses.

Currently, the vast majority of human vaccines approved by the European Medicines Agency and the US Food and Drug Administration (FDA) utilize aluminum salts as adjuvants, the oldest class of adjuvants used in vaccine formulations. To improve vaccine safety and efficacy, expanding the variety and number of new adjuvants is highly necessary. Advances in modern technologies like nanotechnology and molecular biology have significantly facilitated the development of adjuvant components, consequently boosting vaccine effectiveness. An ideal adjuvant must be safe, effective, easy to produce; possess good pharmaceutical properties (pH, osmolarity, endotoxin levels, etc.) and durability; and finally, be economically viable. Particles, emulsions, and immunostimulants show immense potential in vaccine production.

History of Vaccine Adjuvant Development

The field of vaccine adjuvant research has seen remarkable progress since its inception in the early 20th century. As infectious diseases became increasingly prevalent, immunization emerged as the most effective method to curb their spread and reduce associated harm. However, early vaccines often proved ineffective because purified antigens alone could not elicit a sufficiently strong immune response. To overcome this challenge, adjuvants were introduced into vaccines. Adjuvants are materials added to vaccines to enhance their immunogenicity, thereby improving their effectiveness.

The development of modern adjuvants has been a slow and challenging process. Researchers aim to create vaccines that induce robust immune responses while maintaining safety. This work involves selectively combining molecules and formulations with proven efficacy while also exploring and discovering more natural and synthetic compounds.

Aluminum salts were the first adjuvants discovered to enhance immune responses 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 an antigen depot at the injection site, induce inflammation, and activate dendritic cells and T cells. However, despite over 70 years of use in vaccines, the exact immunological mechanisms of aluminum adjuvants remain incompletely understood.

Adjuvant development in the late 1990s included oil-in-water emulsions. These emulsions facilitate the slow release of antigen over an extended period, activate the innate immune system, and induce both humoral and cellular immune responses. Such an emulsion was first used in Fluad, a seasonal influenza vaccine for adults aged 65 and over. Other FDA-licensed adjuvants include MF59, an oil-in-water emulsion adjuvant containing squalene, a biodegradable oil, and a surfactant, used in influenza vaccines; AS03, containing α-tocopherol, squalene, and a surfactant, used in H1N1 influenza vaccines and some pandemic influenza vaccines; and AS01, a mixture of liposomes and monophosphoryl lipid A, used in the RTS,S/AS01 malaria vaccine and the Shingrix herpes zoster vaccine. In recent years, novel vaccine adjuvants, such as the TLR9 agonist CpG-1018, have also received FDA approval for use as an adjuvant in the hepatitis B vaccine Heplisav-B.

Although many other adjuvants have shown high efficacy in preclinical models, most have not been licensed for human use due to safety or tolerability concerns. Furthermore, the molecular mechanisms of how existing adjuvants, including alum, MF59, and the AS0x systems, work in humans remain incompletely understood.

Benefits and Effects of Adjuvants

In summary, vaccine adjuvants play a vital role in enhancing vaccine effectiveness and accessibility, thereby helping to alleviate the global burden of infectious diseases. Their uses and other practical applications are detailed below:

(1) Alleviating Limited Vaccine Supply: Adjuvants can enhance immune responses, allowing for dose-sparing and achieving full protection with fewer doses. This reduces strain on healthcare systems and increases accessibility for those who cannot receive multiple doses.
(2) Enabling Rapid Immune Responses: Adjuvants stimulate stronger and more durable responses to vaccine antigens, providing better protection.
(3) Broadening Antibody Responses: Adjuvants broaden antibody responses against pathogens with significant antigenic drift or strain variation, which is crucial for diseases like influenza, human papillomavirus (HPV), and malaria.
(4) Enhancing Antibody Response Magnitude and Functionality: Adjuvants not only increase the magnitude of the antibody response but also its functionality, affinity, or the amount of functional antibodies produced by the immune system.
(5) Enhancing T-cell Responses: Many new vaccine adjuvants can induce more effective engagement of T-helper cells to optimize the quality and durability of antibody responses, or induce effector CD4+ or CD8+ T cells that can target and eliminate intracellular pathogens. Consequently, new vaccines may include agonists for Toll-like receptors (TLRs) and other innate immune receptors to promote T-helper cell responses. This approach can overcome the limitations of traditional adjuvants, creating more effective vaccines that provide durable protection against a wider range of pathogens.
(6) Developing New Vaccines: Adjuvants are crucial for developing vaccines against diseases where traditional vaccines are ineffective, enhancing immune responses, and targeting multiple pathogen strains. They can augment immune responses to vaccines and aid in targeting multiple pathogens.
(7) Improving Safety: By enhancing immune responses, adjuvants can potentially reduce the risk of vaccine-related adverse events.

Types of Adjuvants and Their Mechanisms of Action

Aluminum Adjuvants
Aluminum adjuvants were the first adjuvants used in licensed human vaccines and remain the most widely used. They consist of aluminum hydroxide, aluminum phosphate, or a combination of both. They form an antigen depot at the injection site, slowly releasing the vaccine antigen over time, leading to a stronger and prolonged immune response. They have been shown to enhance immune responses to various antigens, including those in hepatitis B, HPV, and pneumococcal vaccines.

Inorganic Nanoparticle Adjuvants
Inorganic nanoparticle-based vaccine adjuvants, such as nano-aluminum, layered double hydroxides (LDH), nano/mesoporous silica, nanodiamonds, and quantum dots, have demonstrated potential for enhancing vaccine immune responses. The mechanism of action involves their interaction with the immune system to enhance and modulate immune responses to co-administered antigens. Inorganic nanoparticles act as carriers for antigens, ensuring their efficient delivery to antigen-presenting cells (APCs). Some, like aluminum-based adjuvants, induce local inflammation at the injection site, attracting immune cells and promoting APC recruitment. They can also contribute to the development of long-lasting immune memory, ensuring the immune system 'remembers' encountered antigens for protection upon subsequent pathogen exposure.

Emulsion Adjuvants
Emulsions form when two immiscible liquids are combined, typically an oil phase and a water phase, stabilized by surfactants. Emulsion adjuvants work by forming small droplets of antigen within the oil phase, which are then dispersed throughout the water phase. The antigen is contained within the water droplets, and the oil acts as a reservoir, slowly releasing the antigen and enhancing the immune response by reducing clearance time and prolonging antigen exposure. Additionally, emulsion adjuvants can activate the innate immune system and induce both humoral and cellular immune responses.

One of the first approved oil-in-water adjuvants for human vaccines was MF59, which contains squalene, polysorbate 80, sorbitan trioleate, and sodium citrate dihydrate. MF59 induces ATP release and upregulates cytokines and chemokines to enhance antigen-specific immune responses at the vaccination site. Another oil-in-water emulsion adjuvant is AS03, containing the surfactant polysorbate 80 and two biodegradable oils, squalene and DL-α-tocopherol. AS03-adjuvanted Pandemrix was authorized in the EU in 2009, and an AS03-adjuvanted Influenza A (H5N1) monovalent vaccine received FDA approval in 2013.

TLR Agonists
TLRs are pattern recognition receptors that play a crucial role in innate immune responses to invading pathogens. These receptors are divided into two groups: cell surface TLRs (TLR1, TLR2, TLR4, TLR5, TLR6) and intracellular TLRs (TLR3, TLR7, TLR8, TLR9) expressed on endosomal membranes.

CpG is a synthetic DNA molecule with a phosphorothioate backbone containing unmethylated CpG motifs. Activation of TLR9 by CpG enhances specific humoral and cellular immune responses against the antigen. CpG is a potent adjuvant that induces a T-helper 1 (Th1) response and promotes the production of cytotoxic T lymphocytes (CTLs) and secretion of IFN-γ. Furthermore, CpG can promote the immunostimulatory effects of antigens, activate antigen-presenting cells, and accelerate immune responses. CpG also promotes the expression of Major Histocompatibility Complex (MHC), CD40, and CD86 on plasmacytoid dendritic cells (pDCs) and enhances antigen processing, leading to robust and sustained immune responses.

Liposomes
Liposomes are vaccine adjuvants composed of phospholipids and cholesterol, forming small spherical structures that can encapsulate antigens and other immunostimulatory molecules. They are biodegradable, biocompatible, and allow for multifunctional structural modifications, enabling tunable properties and toxicity. Liposomes can be used to deliver antigens to APCs like dendritic cells, which activate the immune system. Additionally, liposomes can enhance both humoral and cellular immune responses, inducing a Th1-biased response, which is important for clearing intracellular pathogens and enhancing 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 decorated with antibodies or peptides targeting specific receptors on dendritic cells, increasing the uptake of liposome-encapsulated antigen. Liposomes can also be used to co-deliver multiple antigens or immunostimulatory molecules, resulting in more potent immune responses, especially for complex pathogens that may require multiple antigens to induce protective immunity.

Other Adjuvants

• Cytokines: A diverse group of signaling molecules that play important roles in immune responses. They are secreted by various cells of the immune system and act on specific receptors to mediate a range of biological functions such as inflammation, cell proliferation, and differentiation. Cytokines can be classified into different categories based on structure and function, including interleukins, interferons, tumor necrosis factors, and chemokines.

• Polymers: Compounds consisting of repeating units connected by covalent bonds, which can be natural or synthetic. In vaccine applications, polymers can be used as carriers to create slow-release depots, carrying antigens and other immunomodulators within their large cross-linked structures. This leads to sustained antigen presentation and activation of the immune system, resulting in stronger and longer-lasting immune responses.

• Saponins: Naturally occurring amphiphilic compounds. Saponins can enhance immune responses by stimulating APCs like dendritic cells and promoting the production of cytokines and chemokines. An example of a saponin-based adjuvant is QS-21, which is used in the AS01 adjuvant system combined with MPL (Monophosphoryl Lipid A) and has been used in vaccines against diseases like malaria, tuberculosis, and shingles.

• Virosomes: Composed of phospholipids and viral envelope proteins, typically derived from viruses that do not cause disease in humans. The viral envelope proteins provide an immunogenic surface that can activate the immune system, while the phospholipids help stabilize the virosome structure and increase its uptake by APCs.

Role of Adjuvants in Various Vaccines

Adjuvants have widespread applications in various vaccines, including whole-virus vaccines (e.g., live attenuated, inactivated, viral vector vaccines), subunit vaccines, and nucleic acid vaccines (including DNA and mRNA).

Inactivated Vaccines
Inactivated vaccines are typically produced from virus-grown cells that are then inactivated, ensuring the virus is no longer infectious but retains its antigenic properties, aiding immune response generation. Adjuvants used in inactivated vaccines include aluminum adjuvants, MF59, AS03, and AS04. These adjuvants have been used in vaccines such as H1N1 influenza vaccines and HPV vaccines.

Live Attenuated Vaccines
Live attenuated viral vaccines consist of a weakened virus capable of replicating in the body but not causing disease. They thus elicit a strong and durable immune response. Adjuvants are generally not used with live attenuated vaccines because the vaccine itself is a potent stimulator of the immune system. However, some studies have explored using adjuvants with live attenuated viral vaccines to potentially improve their efficacy, such as MF59 with live attenuated influenza vaccine, which has been shown to improve immune responses in the elderly.

Viral Vector Vaccines
Replication-competent and incompetent adenovirus vectors are commonly used to express antigenic viral proteins. Adjuvants can be combined with viral vector vaccines to enhance the immune response generated by the vaccine. For example, the Oxford-AstraZeneca COVID-19 vaccine is a viral vector vaccine that uses a weakened version of a chimpanzee adenovirus to deliver the genetic material for the SARS-CoV-2 spike protein. This vaccine contains the AS03 adjuvant, a combination of squalene, polysorbate 80, and vitamin E.

Virus-Like Particle (VLP) Vaccines
In this strategy, structural viral proteins are co-expressed to form non-infectious particles serving as vaccine immunogens. They resemble authentic virus particles but lack the viral genome. Adjuvants can be used in VLP vaccines to enhance the immune response and improve vaccine efficacy. Common adjuvants for VLP vaccines include aluminum salts, MF59, and CpG. These adjuvants stimulate the immune system to generate a stronger and more durable response to the VLP vaccine.

DNA Vaccines
DNA-based vaccines use DNA plasmids as vectors to transfer genes encoding antigens into host cells, particularly APCs. However, DNA vaccines often have limited immunogenicity compared to other vaccine types, which can restrict 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 adjuvants, CpG, and liposomes.

mRNA Vaccines
mRNA vaccines involve the direct injection of mRNA molecules into host cells, where they are translated into the target protein in the cytoplasm. mRNA vaccines offer many advantages over traditional vaccines, such as safety, flexibility, scalability, and cost-effectiveness. mRNA vaccines typically do not require adjuvants because the mRNA molecules themselves can stimulate the immune system. However, research is ongoing to explore the use of adjuvants in mRNA vaccines to improve their effectiveness, particularly in populations that may have weaker immune responses to vaccines, such as the elderly.

Protein Subunit Vaccines
Protein subunit vaccines contain only one or more specific proteins of a pathogen rather than the whole pathogen. Adjuvants are commonly used with protein subunit vaccines to enhance their effectiveness by increasing the immune response to the protein antigen. Aluminum adjuvants are the most commonly used adjuvants in protein subunit-based vaccines and have been shown to enhance antibody responses to the antigen. Other adjuvants, such as MF59, AS03, and AS04, have also been used in protein subunit-based vaccines to increase their immunogenicity. Furthermore, novel adjuvants like CpG are being investigated for protein subunit vaccines to further enhance their immune responses.

Recent Advances in Novel Vaccine Adjuvants

Design Principles for Novel Adjuvants
To develop more effective adjuvant vaccines, it is important to understand how adjuvants work and follow some design principles. A problem-oriented approach involves considering four main questions: (1) The type of immune response required to prevent disease from a specific pathogen, (2) The relevant innate immune cells that can induce the desired immune response, (3) The localization of these innate cell subsets within the body, and (4) The expression of pattern recognition receptors on these cells.

Based on these questions, four steps should be considered when designing vaccine adjuvants. First, the type of immune effector elements required for the vaccine to be effective in the host should be identified. This should consider antigen type, target cell subsets and phenotypes, and immune pathways to guide the selection of delivery systems and immunostimulants. Second, the identification of suitable vaccine antigens should be based on an understanding of the molecular mechanisms of immune recognition and protection. The vaccine must deliver a sufficient amount of the correct antigen in the right conformation to the appropriate cell population to induce a protective immune response, while considering the tolerability and safety of the induced inflammatory response. Third, an adjuvant often needs a delivery system, whether synthetic or natural. This involves designing a suitable mode of antigen delivery combined with relevant immunostimulants. Fourth, the preparation process for both the vaccine and adjuvant should be relatively straightforward.

New Vaccine Adjuvant Formulations
The correct selection of immunostimulants and formulation components for an adjuvant is crucial for inducing an appropriate immune response to the target antigen. Different formulations of the same immunomodulatory molecule can induce significantly different immune responses. For example, the RTS,S candidate vaccine formulated with AS02 protected 6/7 vaccines from infection, while the same antigen formulated with AS03 or AS04 protected only 2/7 and 1/8, respectively.

To create improved adjuvants, it must be ensured that each component is necessary and adds significant value without introducing substantial undue burden. AS01 is currently the most successful adjuvant in licensed products, demonstrating 97% efficacy against shingles. The development of the AS01 adjuvant was based on the synergy between its two key components, MPL and QS-21, which together lead to synergistic innate activation not achievable with either molecule alone.

Utilizing Systems Vaccinology
Systems vaccinology is an interdisciplinary approach involving the early use of human studies to generate 'omics' data, which can be used to formulate new hypotheses about the mechanisms by which candidate adjuvants stimulate robust and durable antigen-specific T and B cell responses. These hypotheses can then be re-tested in animal models, and subsequent mechanistic insights can be used to design new adjuvant concepts. Furthermore, the systems vaccinology approach can be applied not only to the mechanism of action of adjuvants but also to the underlying mechanisms of how formulation plays a role, the mechanisms underlying adverse events occurring shortly after vaccination, and the rational design of optimal formulations for vaccine delivery. Overall, this interdisciplinary approach based on systems vaccinology has the potential to transform adjuvant science and accelerate the development of novel adjuvants for vaccines.

Non-Invasive Vaccine Delivery
Research into non-invasive vaccine delivery is a key focus with potential major implications for global mass vaccination. Priority is given to developing safe, effective, and low-cost adjuvant vaccine formulations that generate the desired immune response and long-term immunity. As our understanding of the molecular mechanisms of immune protection improves and new methods in synthetic chemistry develop, breakthroughs in vaccine development are anticipated. New technologies, such as novel glycol-conjugation methods, reverse vaccinology, and next-generation sequencing, are likely to lead to new vaccine strategies for diseases like HBV, pertussis toxin, Lyme disease, and HPV, with or without adjuvants.

In conclusion, the research goal for novel vaccines is to ensure high levels of broad protection by introducing new immunostimulants and new delivery systems that combine efficacy with long-term memory immune responsiveness and safety.

Summary

For nearly a century, adjuvants have played a crucial role in vaccine development by enhancing immunogenicity. Understanding the impact of various adjuvants on immune responses, their synergy with different antigen types and vaccine platforms, and a comprehensive analysis of these adjuvants will aid in selecting adjuvants that provide the necessary immune protection. With the ongoing challenges of emerging diseases like COVID-19 and the search for more definitive treatments, developing safe and effective vaccines is imperative. More informed selection of adjuvants and antigens can not only strengthen protection for populations that respond poorly to traditional vaccines but also open new avenues beyond prophylactic applications. This approach is essential for addressing current and future global health challenges.

References:

1. Vaccine adjuvants: current status, research and development, licensing, and future opportunities. J Mater Chem B. 2024 Apr 9.