Vaccines are one of the most successful medical inventions in history. For antigens with low immunogenicity, adjuvants may enhance immune responses. One of the mechanisms is to activate pattern recognition receptors (PRRs) of immune cells, thereby initiating a series of immune responses and promoting the production of antibodies and cytokines. A variety of sugar molecules, as important signals on the surface of microorganisms, can effectively activate PRRs, thus having the potential to be used as adjuvant materials. Elucidating the immune regulatory mechanisms of these sugar molecules will provide a theoretical basis to guide the design of future vaccine adjuvants. Professor Wang Chunming and Dr. Mu Ruoyu from the University of Macau and Professor Dong Lei from Nanjing University published a review paper in Trends in Immunology, a journal of Cell Press, summarizing the latest development trends of carbohydrate-based adjuvants, focusing on polysaccharides, oligosaccharides or How the physicochemical properties of glycopolymers—including molecular size, assembly state, monosaccharide composition, and functional group patterns—affect the process of antigen presentation and immune response.
Although adjuvants have been widely used in modern vaccine development, the mechanism of action of even the most classic aluminum-based adjuvant and the widely used Adjuvant System 04 (AS04) is still unclear. With the deepening of the understanding of the innate immune system, it has been gradually discovered that PRRs such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and NOD-like receptors (NLRs) recognize microbial pathogen-associated molecular patterns (PAMPs). ), triggering intracellular signals and initiating immune responses. Therefore, PRRs agonists can be designed by mimicking the structure of PAMPs on the surface of pathogenic microorganisms. Sugar molecules are components of PAMPs of many pathogenic microorganisms. For example, bacterial PAMPs: lipopolysaccharide (LPS) and peptidoglycan (PGN); fungal PAMPs: β-glucan and mannan, both can stimulate some PRRs, such as TLR4 and Dectin-1, showing adjuvant activity. Although there is an increasing number of studies on carbohydrate-based adjuvants, challenges such as their structural complexity, high synthesis difficulty, and lack of research technology have hindered the exploration of the molecular basis of the potential adjuvant activity of carbohydrates.
Key factors affecting the interaction between sugar molecules and PRRs
There are many decisive factors that affect the biological activity of sugar molecules and their recognition and activation of PRRs, including molecular mass, solubility, monosaccharide composition, functional group substitution degree, etc. (Figure 1).
- Molecular weight/solubility
Molecular weight is an essential characteristic of all polymers, including polysaccharides. Early studies found that only large molecular weight granular polysaccharides have immune activating effects. For example, both soluble and insoluble fungal polysaccharides can bind to Dectin receptors, but only the latter can induce efficient receptor aggregation and activation. However, the latest research shows that soluble (~20 nm) Candida albicans-derived mannan does not induce peripheral immune responses after being injected subcutaneously on the back of mice, but can directly enter draining lymph nodes (dLNs) and initiate lymph node innate immune responses. This discovery may provide a new idea for adjuvant design and vaccine development.
- Functional groups and monosaccharide composition
The functional group pattern of sugars also affects their recognition with PRRs. For example, chitosan obtained by deacetylation of chitin can activate the macrophage immune response by activating the NLRP3 inflammasome, while chitin originally does not have this activity. In addition, the recognition between pectin and TLR2-1 is not only regulated by the degree of methyl esterification modification, but also depends on the distribution of methyl esters. A clinical trial found that acetyl groups also affect the biological function of pectin: O-acetylated pectin can effectively induce IgG production in healthy volunteers, but does not cause serious vaccine-related adverse reactions.
The structure of the monosaccharides in polysaccharides also affects their precise interactions with PRRs. It was previously thought that DC-SIGN and L-SIGN, as closely related CLRs, recognize similar monosaccharide structures. However, the latest research found that compared to DC-SIGN, L-SIGN has a higher binding affinity to N-mannan.
- advanced conformation
Changes in the primary structure of sugars will affect their higher-order conformation and their recognition with PRRs. For example, acetylation can change the hydrophilic and hydrophobic properties of glucomannan. As the degree of modification increases, the structure of acetylated glucomannan can transform from linear to granular, and when the degree of modification of the acetyl group reaches 1.8, relatively uniform particles of 200-300 nanometers in size can be formed. Activates TLR2 on the surface of macrophages to initiate an immune response. This finding suggests that finely tuned three-dimensional morphologies assembled from sugar molecules contribute to biological activity.
- Synergizes with other PRRs agonists
Sugars can not only act as immune agonists alone, but can also act synergistically with other PRRs agonists. For example, a new adjuvant p(Man-TLR7) is a copolymer composed of mannose and TLR7 agonist. It promotes uptake by dendritic cells (DCs) by recognizing mannose receptor (MR) and activating TLR7 in cells to promote DC activation, thereby achieving synergy between MR and TLR7. In addition, polysaccharides themselves can also induce synergistic cooperation between different PRRs. Studies have found that glucomannan can activate TLR4 and MR at the same time. The above findings suggest that we can consider the synergistic effect of sugars on PRRs in adjuvant design.
Carbohydrate-based PRRs agonists as vaccine adjuvants modulate immune responses
Sugar-based PRRs agonists participate in regulating immune responses mainly through the following three aspects: (1) Antigen transport pathway (Figure 2); (2) Antigen uptake and presentation (Figure 3); (3) Adaptive immune response (Figure 4).
1. Regulation of antigen transport pathways
Generally, vaccines are injected subcutaneously or intramuscularly to produce an adaptive immune response in secondary lymphoid organs. Therefore, the role of the adjuvant is to induce the local immune environment at the injection site to produce chemokines, recruit antigen-presenting cells (APCs), initiate cell-mediated antigen transport, and transport the antigen to dLNs. However, certain viruses, nanoparticles or soluble proteins can directly enter lymph nodes through lymphatic vessels and activate macrophages, DCs or B cells in lymph nodes. Some studies have found that nanoparticles with a diameter of 5 ~ 100 nm can be transported to lymph nodes through lymphatic vessels. Particles larger than 100 nm may be trapped in the extracellular matrix at the injection site and need to be phagocytized by APCs before they can migrate to LNs. Particles smaller than 5 nm may be trapped in the extracellular matrix at the injection site. Particles of nm will diffuse through the blood endothelial basement membrane and directly enter the circulation. Therefore, the size of the vaccine delivery system can be adjusted to adjust the antigen transport path to obtain different immune effects.
2. How to regulate antigen uptake and antigen presentation
The initiation of T cell immune responses depends on the uptake and presentation of antigens by APCs. After the antigen is taken up by APCs, it is degraded into peptides in lysosomes, loaded on MHC II molecules and transported to the cell surface to activate CD4+ T helper cells. In addition, the ingested exogenous antigen may escape from lysosomes and be presented by MHC I molecules, inducing a CD8+ T cell response, which is called cross-presentation. Some carbohydrate-based adjuvants can modulate the way antigen is presented. For example, saponin-based adjuvants (SBAs) can form liposomes (LBs) within cells and enable cross-presentation of exogenous antigens through the PKR-like endoplasmic reticulum kinase (PERK) pathway. In addition, an interesting study found that not only peptides can be presented to T cells by APCs, but the polysaccharide component of glycoconjugated vaccines can also be presented by MHC II and activate carbohydrate-specific CD4+ T cells (Tcarbs).
3. Regulate adaptive immune response
Carbohydrate-based adjuvants can also participate in the regulation of adaptive immune responses. For example, an inulin-based polysaccharide adjuvant effectively induces both Th1 and Th17 immune responses. Similarly, chitosan is also effective in inducing Th1 immune responses. It was also found that polysaccharide structure also affects the type of adaptive immune response. For example, polysaccharides containing β-1,3-glucan on the surface of yeast cells can induce DCs to secrete IFN-γ and promote Th1 cell differentiation. Another polysaccharide component, mannan/β-1,6-glucan (MGCP), induces the differentiation of mouse naive CD4+ T cells into regulatory T cells (Treg). In addition, carbohydrate-based agonists can also act directly on B cells (which also express PRRs) and trigger humoral immunity. Studies have found that chitosan oligosaccharide (COS) can directly induce B cell proliferation and differentiation by activating the MR and integrin of IgM+ B cells in the spleen of grass carp.
