Adjuvants, also known as immunomodulators or immune enhancers, are additives used in vaccines. When injected into the body before or mixed with antigens, adjuvants can enhance the immune response of the body to antigens or alter the type of immune reaction. Adjuvants belong to non-specific immune enhancers and do not possess antigenicity themselves. An ideal adjuvant can not only strengthen the immune response but also enable the body to obtain the best protective immunity. In addition to the aluminum salt adjuvants, protein adjuvants, and nucleic acid adjuvants introduced above, this article will continue to introduce lipid-containing adjuvants, new oil-in-water emulsions adjuvants, other adjuvants, and compound adjuvants.

Related Reading: "An Introduction to the Research Progress of Vaccine Adjuvants (Part 1)"

4. Lipid-Containing Adjuvants and Their Mechanisms of Action

Lipid-containing adjuvants encompass two main categories: lipopolysaccharides (LPS) and liposomes. LPS naturally occur in the outer membranes of Gram-negative bacteria and serve as potent activators of the innate immune system, playing a pivotal role in initiating adaptive immune responses following bacterial infections. LPS typically consists of three components: the polysaccharide O-antigen, the core oligosaccharide, and the hydrophobic lipid A. In mammals, immune cells recognize lipid A through the pattern recognition receptor complex Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), which activates immune cells and triggers the release of inflammatory cytokines. Lipid A is an effective inducer of innate immunity and an enhancer of immune responses.

Researchers have employed LPS as an adjuvant in pertussis vaccines to immunize mice. Experimental results show that LPS not only enhances the immunological efficacy of the vaccine but also reduces the occurrence of type I hypersensitivity reactions. However, due to its high toxicity, lipid A-based adjuvants are considered unsuitable for use in human vaccines. LPS is one of the most potent pathogen-associated molecular patterns (PAMPs) known, and chemical or genetic modifications to LPS to reduce its toxicity are crucial for its application as an adjuvant.

GLA-SE and GLA-AF are lipid A analogues that have demonstrated exceptional efficacy in enhancing protection against H5N1 with low toxicity and fewer side effects. GLA-AF has also been explored as a replacement for emulsions in various vaccines, most notably pandemic influenza vaccines. Adjuvant systems containing deacylated monophosphoryl lipid A, when combined with hepatitis B surface antigen, can induce higher antibody titers more rapidly and provide longer-lasting protection.

Liposomes are spherical structures composed of non-immunogenic, non-toxic, and biodegradable phospholipids derived from natural products that can encapsulate antigens. They serve as antigen delivery adjuvants, acting as a tool for vaccine delivery. A key advantage of liposomes as a vaccine carrier system lies in their multifunctionality and versatility. By harnessing their chemical properties, water-soluble antigens (such as proteins, peptides, nucleic acids, carbohydrates, and haptens) can be entrapped within the aqueous interior space of the liposomes, while lipophilic compounds (lipopeptides, antigens, adjuvants, and linker molecules) are embedded within the lipid bilayer. Additionally, antigens or other adjuvants can be attached to the liposome surface through adsorption or stable chemical linkages.

When DNA vaccines are encapsulated within liposomes, they can more efficiently traverse cell membranes to enter cells for expression, with the liposomes providing protection and sustained release during this process. Liposomes can be modified with immunostimulatory molecules and targeting moieties, functioning as a multifunctional vaccine adjuvant delivery system involved in the immune process. They are capable of targeting immune cells or even subcellular organelles, enabling lysosomal escape and promoting cross-presentation of antigens, thereby significantly enhancing the immune effect of vaccines. Studies have shown that liposomes can enhance both humoral and cellular immune responses in the body.

When used in combination with Freund's adjuvant or aluminum hydroxide, liposomes exhibit synergistic effects. Liposomes are widely utilized in clinical trials for vaccines against COVID-19, tumors, and influenza, among others. However, as adjuvants, liposomes also have limitations. For instance, during storage, the unsaturated fatty acids in the phospholipids can gradually oxidize. Liposomes are prone to fusion, which can lead to the release of encapsulated antigens during the fusion process. Additionally, the production of liposomes requires advanced technology and can be costly. Through continuous research on liposomes, several novel types have been designed for vaccine delivery, including ultradeformable vesicles (Transfersomes), multilamellar vesicles with interlamellar cross-linking, and solid-core liposomes.

5. Novel Oil-in-Water Emulsion Adjuvants and Their Mechanisms of Action

Currently, the representative novel oil-in-water emulsion adjuvants approved by the FDA for market use are MF59 and AS03. MF59 is a uniformly emulsified droplet formed under high-pressure conditions by blending squalene, Tween-80, and sorbitan trioleate. It is capable of inducing robust humoral immune responses in the body. MF59 functions by inducing a chemokine cascade, which modifies immune cells in mice, stimulating immune responses, and subsequently directing monocytes towards differentiation into dendritic cells (DCs), thereby providing an effective immune environment for the body. Furthermore, it enhances IgG antibody levels post-immunization.

Primarily used in influenza vaccines, MF59 enhances immune responses and improves cross-reactivity against influenza strains. Studies have shown that when MF59 is incorporated as an adjuvant, even with a reduced antigen dose of one-quarter to one-half, the split vaccine adjuvanted with MF59 remains effective against H7N9 avian influenza virus infections, during large outbreaks of the disease. Additionally, MF59 aids in enhancing the efficacy of seasonal or pandemic influenza vaccines for young children and the elderly. In trivalent vaccines for older adults, MF59 induces a more potent humoral immune response compared to aluminum adjuvants. It has also been explored as an adjuvant for subunit vaccines against Coronavirus Disease 2019 (COVID-19), enhancing the immunogenicity of subunit vaccines based on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) proteins through intramuscular injection.

AS03: Another Oil-in-Water Emulsion-Based Adjuvant.AS03 is another adjuvant formulated as an oil-in-water emulsion. In 2009, the European Commission granted marketing authorization for Pandemrix, an influenza vaccine containing the AS03 adjuvant. Subsequently, in 2013, the US Food and Drug Administration (FDA) approved an influenza A (H5N1) monovalent vaccine formulated with the AS03 adjuvant. Since then, AS03 has played an increasingly important role in influenza vaccines and malaria vaccines.

AS03 comprises the surfactant polysorbate 80 and two biodegradable oils: squalane and DL-α-tocopherol. DL-α-tocopherol is the most bioavailable form of vitamin E, and studies have shown that the addition of DL-α-tocopherol can modify the characteristics of innate immune responses, enhancing antibody responses. AS03 induces the production of cytokines and chemokines in muscles and draining lymph nodes (dLNs), triggering the migration of monocytes, dendritic cells, and granulocytes to the dLNs. This, in turn, stimulates a CD4+ T cell-specific immune response.

6. Other Adjuvants: Viral-Like Particles (VLPs)

Viral-like particles (VLPs) are polymeric particles self-assembled from viral capsid proteins. They possess suitable sizes and geometries, making them effective platforms for vaccine antigen delivery. Typically ranging in size from 20 to 100 nanometers, VLPs facilitate entry into the lymphatic vasculature and targeting of lymph nodes, where they are efficiently taken up by specialized antigen-presenting cells (APCs). Due to their highly repetitive and rigid structure, VLPs can display multivalent antigenic epitopes on their surfaces, effectively engaging B cell receptors (BCRs), activating B cells, and inducing strong and long-lasting antibody responses.

VLPs have been successfully employed in the development of vaccines against human papillomavirus (HPV) and hepatitis B virus, among others, and are widely available in the market. Furthermore, VLPs can deliver both endogenous and heterologous antigens, with the capability of undergoing chemical conjugation or genetic fusion modifications on their surfaces. Studies have demonstrated the successful use of VLPs in clinical trials for vaccines against Zika virus, influenza, cancer, and encephalitis.

Saponins are natural glycosides of spirostanol compounds or triterpenes that can activate the immune system of mammals. Saponins extracted from natural plants form complexes with cholesterol on cell membranes, which jointly promote the proliferation of T and B lymphocytes, induce the production of specific MHC-I antigen-restricted CTLs (cytotoxic T lymphocytes), and stimulate the secretion of various cytokines. As such, saponins can be used as adjuvants.

Quil A and its derivative QS-21 are the most widely used saponin adjuvants with potent adjuvant activity. They can be employed to modify T cells and antigen-presenting cells (APCs). Quil A is capable of not only stimulating Th1 immune responses to exogenous antigens but also inducing CTL responses. Currently, Quil A has become an ideal adjuvant for subunit vaccines and cancer vaccines.

Composite Adjuvants

With the development of novel vaccines, single-function adjuvants often fail to meet the needs of the public. As a result, it is necessary to harness the synergistic effects between different components of adjuvants. Composite adjuvants are designed to maximize the adjuvant effect by combining two or more adjuvants with distinct immunological effects. This combination of adjuvants can further modulate or enhance the immune response, providing stronger immunological protection.

The AS adjuvant system was developed by GlaxoSmithKline in the late 1980s, primarily for enhancing the immunogenicity and immune response of vaccines. The primary component of the AS adjuvant system is monophosphoryl lipid A (MPL), which, in combination with other auxiliary components, forms a formulation capable of effectively activating the immune response. The AS adjuvant system has been widely utilized in the development and production of various vaccines, including pertussis vaccines, influenza vaccines, pneumococcal vaccines, and more. By incorporating the AS adjuvant system, these vaccines are able to elicit stronger immune responses post-vaccination, thereby enhancing the level of immune protection. Furthermore, the AS adjuvant system aids in prolonging the immunological effects of vaccines, providing more durable immune protection. Currently, the FDA has approved the marketing of AS01, AS03, and AS04 adjuvant systems.

Covaxin (BBV152), an inactivated COVID-19 vaccine utilizing an aluminum-TLR7/8 agonist as a composite adjuvant, received emergency use authorization from the World Health Organization in 2021. The neutralization antibody levels induced by Covaxin are 1.5 times higher than those induced by aluminum adjuvant-based inactivated vaccines. The TLR7/8 agonist contributes to stimulating a Th1 cell-biased immune response, enhancing Covaxin's ability to induce CD8+ T-cell responses.

The SCB-2019 COVID-19 vaccine, which was approved for emergency use in China in 2022, employs a composite adjuvant of CpG1018-alum. In Phase I clinical trials, the CpG1018-alum vaccine achieved a serum conversion rate of 100% in both adults and elderly individuals 36 days after the initial immunization, outperforming the unadjuvanted vaccine group and inducing high levels of neutralizing antibodies. Phase II/III clinical trials of the SCB-2019 COVID-19 vaccine have also demonstrated that two doses of the vaccine can provide effective protection against COVID-19 in humans, with protective efficacies of 100%, 83.7%, and 67.2% against critical illness, moderate-to-severe infection, and symptomatic infection caused by any strain of the novel coronavirus, respectively.

AS37 is a novel composite adjuvant that consists of a synthetic TLR7a (TLR7 agonist) adsorbed onto aluminum hydroxide, capable of enhancing Th1 cell-biased immune responses. In preclinical mouse experiments with meningococcal group C conjugate vaccines, the AS37 composite adjuvant demonstrated favorable adjuvant effects. All three dose groups of the AS37 adjuvant vaccine significantly increased the serum bactericidal activity (SBA) titers against Neisseria meningitidis serogroup C after the primary immunization, surpassing the aluminum adjuvant vaccine group. Furthermore, eight months after the second dose, the SBA titers against N. meningitidis serogroup C remained at high levels in all three dose groups of the AS37 adjuvant vaccine.

PIKACA is a unique vaccine adjuvant used in humans in China, which is a complex composed of double-stranded polyinosinic acid, kanamycin, and calcium chloride. In 2018, the National Medical Products Administration of China approved the clinical trial of PIKACA rabies vaccine for human use. The PIKACA rabies vaccine for human use can produce high titers of IFN within 2 hours after vaccination, while enhancing humoral immunity and improving its immunological efficacy, which is even better than the combination of the vaccine and antiserum. Currently, Phase I and II clinical trials of PIKACA rabies vaccine for human use have been completed abroad, and China is also conducting Phase I clinical trials.

References

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About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.