Vaccination is the primary measure for preventing infectious diseases. Traditional vaccines, prepared through the inactivation or attenuation of pathogens, pose concerns over safety due to their complex composition and potential for virulence reversion. With the continuous and rapid advancements in immunology and genetic engineering technologies, novel vaccines such as nucleic acid vaccines, recombinant subunit vaccines, and synthetic peptide vaccines have been continuously developed and applied. The new-generation vaccines prepared using genetic engineering and molecular cloning techniques exhibit characteristics of relatively small molecular weight, high antigen purity, and enhanced biosafety. However, compared to traditional vaccines, these novel vaccines also have drawbacks, including weaker immunogenicity, difficulty in crossing cell membranes, and rapid degradation in the cellular microenvironment.

To enhance the immune response of these antigens within the body, prolong their presence, and improve their immunological efficacy, adjuvants must be administered concurrently with or prior to the antigens. Adjuvants function as immune potentiators, boosting the immune response to antigens, and as carriers, delivering antigens to the appropriate immune cells while reducing their degradation, thereby strengthening the immune protection of the body.

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 the antigens or alter the type of immune reaction. Adjuvants are non-specific immune enhancers that do not possess antigenicity themselves. An ideal adjuvant not only enhances the immune response but also enables the body to achieve optimal protective immunity. Currently, there is no universally established classification standard for adjuvants internationally. Based on their chemical compositions, adjuvants can be categorized into several types, including aluminum salt adjuvants, protein-based adjuvants, nucleic acid-based adjuvants, lipid-containing adjuvants, novel oil-in-water emulsion adjuvants, and composite adjuvants.

1. Aluminum Salt Adjuvants and Their Mechanism of Action

Aluminum salts have been used clinically for over 80 years and are a classic adjuvant approved by the US FDA for human use. Aluminum-containing adjuvants have been widely employed in vaccines for diseases such as diphtheria, tetanus, and hepatitis B. Currently, among the human vaccines approved by the FDA, 25 contain aluminum. Many vaccines, including diphtheria-tetanus-pertussis (DTP) vaccines and Haemophilus influenzae type b (Hib) vaccines, contain aluminum salts as part of their composition.

Vaccines using aluminum salts as adjuvants can be classified into two types based on their preparation process: aluminum-adsorbed vaccines and aluminum-precipitated vaccines. In aluminum-adsorbed vaccines, the antigen is added to a solution of aluminum hydroxide or aluminum phosphate, whereas in aluminum-precipitated vaccines, a suspension of aluminum salt is added to the antigen solution. Aluminum hydroxide and aluminum phosphate are the commonly used aluminum adjuvants. Studies have found that aluminum-adjuvanted vaccines can reduce the amount of antigen required and enhance the intensity and durability of the immune response in the body.

The exact mechanism of aluminum salts is not fully understood at present, but it is generally believed to encompass several aspects: ① The antigen adsorbs onto aluminum salt particles to form a gel-like state, which, when injected into animals, creates an antigen depot. These insoluble particles can sequester antigenic materials, enhancing the surface area of the antigen. ② Adjuvants facilitate the formation of granulomas rich in macrophages at the injection site, delaying antigen absorption and subsequently prolonging antigen stimulation from days to weeks under normal conditions. This also enhances the antigen uptake capability at the injection site. ③ As an adjuvant, aluminum hydroxide activates Th2 cells to secrete IL-4, inducing the expression of CD83, CD86, and MHC-II molecules, thereby eliciting a Th2-type humoral immune response. ④ Aluminum salts can induce the activation of the NLRP3 inflammasome, stimulating the production of IL-1b and IL-18. This, in turn, triggers local inflammation, the recruitment of antigen-presenting cells (APCs), dendritic cell maturation, enhanced antigen uptake, and T-cell stimulation. ⑤ Aluminum salts, acting as adjuvants, promote complement activation, thereby enhancing immune responses through B cells and dendritic cells.

2.Protein Adjuvants and Their Mechanism of Action

Protein adjuvants are primarily bioactive substances synthesized and secreted by immune and non-immune cells upon stimulation. They belong to the category of cytokines, typically small molecular peptides or glycoproteins, encompassing interferons (IFNs), interleukins (ILs), tumor necrosis factors (TNFs), granulocyte-macrophage colony-stimulating factors (GM-CSFs), and chemokines, among others. Cytokines are potent immune modulators that enhance the immunoregulatory functions of natural killer cells, promote the differentiation of T lymphocytes, and broadly upregulate the immune response of the body. They also protect the body against bacterial, viral, and parasitic infections, making them effective immune enhancers.

IL-2 and GM-CSF have been tested in foot-and-mouth disease vaccines. Recently, GM-CSF has been evaluated for use in hepatitis B and HIV vaccines, where its adjuvant effect is attributed to stimulating macrophage differentiation and proliferation, as well as activating antigen-presenting cells. Furthermore, IL-12, produced by monocytes and B cells, possesses multiple biological activities. It notably reduces bacterial invasion and elevates the expression levels of IgG2a and IgA in mucosal and immune systems. As a cytokine adjuvant, IL-12 holds promising applications, capable of inducing Th1-type immune responses and is currently in clinical trials for cancer and AIDS treatments.

Additionally, IL-15 has been studied as an adjuvant in Phase 1 HIV vaccine trials, exhibiting the ability to stimulate NK and T-cell proliferation. Research using GM-CSF and IL-5 as adjuvants for mZP3 DNA vaccines has revealed that co-immunization with GM-CSF and DNA vaccines effectively stimulates the maturation of antigen-presenting cells (APCs) and promotes their efficient accumulation at antigen delivery sites, thereby enhancing humoral immune responses.

3.Nucleic Acid Adjuvants and Their Mechanism of Action

In the course of vaccine research, investigators have discovered that certain nucleic acid substances possess adjuvant properties, with CpG-DNA being a representative example. CpG oligodeoxynucleotides (CpG ODNs) are synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine dinucleotides. They are highly valuable biological adjuvants, with their initial research stemming from tumor diagnosis. As a potent adjuvant, CpG ODNs can induce Th1 immune responses, stimulate the production of cytotoxic T lymphocytes (CTLs) and interferon-gamma (IFN-γ). CpG ODNs enhance humoral and cellular immune responses to specific antigens by activating TLR9. Given these unique properties, CpG ODNs can be administered as vaccine adjuvants through intramuscular, subcutaneous, oral, and intranasal routes.

As an efficient vaccine adjuvant, CpG ODNs promote the immunostimulatory effects of antigens, activate antigen-presenting cells (APCs), and accelerate immune responses. They enhance the expression of MHC, CD40, and CD86 on peripheral blood cells (pDCs), improving antigen processing and presentation. Studies have confirmed that CpG ODNs can serve as adjuvants for Newcastle disease virus vaccines and rabies vaccines in chickens, both of which are currently in clinical research. When administered intramuscularly or intranasally in conjunction with porcine reproductive and respiratory syndrome modified live vaccines (PRRS-MLV) in pigs, CpG ODNs can induce specific T-lymphocyte responses against porcine reproductive and respiratory syndrome virus (PRRSV).

As a vaccine adjuvant, CpG ODNs can also improve the immunogenicity of aluminum-containing vaccines such as hepatitis B, anthrax, and influenza vaccines. The first CpG-containing vaccine to complete Phase III trials was HEPLISAV for hepatitis B virus, and CpG-based adjuvants have also entered Phase II and III clinical trials for other human vaccines. In September 2017, a CpG adjuvant-enhanced hepatitis B preventive vaccine was approved for market. Research has shown that when CpG ODNs are combined with lactic-glycolic acid to form nanoadjuvants, they can protect against H6N1 subtype avian influenza virus infections. When coupled with subunit antigens from infectious bronchitis virus, CpG ODNs elicit a more robust humoral immune response.

In addition to CpG ODNs, other nucleic acid adjuvants include dsRNA, IL-12 DNA, interleukin-2/immunoglobulin (IL-2/Ig) DNA, and Poly-ICLC, which have been used in clinical trials for vaccines against influenza, HIV, and other diseases. Polyinosinic-polycytidylic acid (Poly (I:C)) is a synthetic viral dsRNA analogue that can be specifically recognized by TLR3 and MDA-5 receptors to activate innate immunity, making it a potential immunopotentiator for viral inflammation. Studies have demonstrated that Poly (I:C) enhances the immunogenicity of vaccines, particularly for synthetic peptide vaccines and subunit vaccines. When combined with Poly (I:C), there is a significant boost in both humoral and cellular immune responses.

To be continued...

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.