IL-1β directly influences the proliferation and differentiation of CD4 and CD8 T cells, particularly IL-4-producing cells, and also enhances the tissue localization and memory responses of CD8 T cells (21). The production and release of IL-1β are stimulated by pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). IL-1β as an adjuvant with a recombinant PRRSV vaccine induce a robust T-cell immune response, increase IFN-γ and IL-4 levels, and elicit higher serum antibody levels (9). This indicates that IL-1β has a dual role in enhancing both cellular and humoral immune responses. Furthermore, IL-1β serves as an effective mucosal vaccine adjuvant based on the capacity of attracting both innate and adaptive immune cells through the induction of chemokines and adhesion molecules (22), specifically inducing tissue-resident memory T cells mediated a rapid clearance of secondary IAV infections in mice, which improves heterosubtypic immunity against influenza A viruses (10).

IL-2 is a cytokine that promotes the growth of bone marrow-derived T lymphocytes and was one of the first cytokines to be characterized at the molecular level (23). IL-2 enhances the growth activity of various cells, particularly the proliferation of CD4+ and CD8+ T lymphocytes (24). Additionally, IL-2 promotes the production of cytokines by natural killer (NK) cells and synergizes with IL-12 to enhance NK cell cytotoxic activity (25). In B cells, IL-2 primarily influences antibody secretion (26). There have been numerous reports on the use of recombinant IL-2 as a vaccine adjuvant (27, 28). Recombinant IL-2 encapsulated in nano-liposomes significantly enhances the levels of foot-and-mouth disease virus (FMDV)-specific antibodies and the concentrations of IFN-γ secreted from spleen cells through Th1 immune response as well as maintain longer periods of time to stimulate T and B cell proliferation and differentiation to improve antibody secretion, which successfully solves the shortcoming of a short half-life of IL-12. Additionally, it increases the proliferative responses of antigen-specific spleen cells, demonstrating its effective adjuvant properties (11). Co-expression of the IL-2 and VP60 genes in a DNA vaccine for rabbit hemorrhagic disease induced higher levels of neutralizing antibodies and IL-4 expression, while reducing tissue damage upon challenge, confirming the effectiveness of IL-2 as an adjuvant (12).

IL-4 is a type I cytokine with a four-α-helix bundle structure that exhibits pleiotropic effects across multiple lineages. While IL-4 is produced by various immune cells, it is primarily secreted by activated CD4+ T cells (29). IL-4 mediates host sensitization and parasitic responses via IgE and induces IgG production, particularly IgG1 in B cells (30–32). In humans and mice, IL-4 acts as a T-cell growth factor and promotes Th2 cell differentiation. Studies have shown that recombinant Newcastle disease virus (NDV) expressing chicken IL-4 significantly reduced organ damage and virus shedding in vaccinated chickens compared to wild-type virus, indicating potential antiviral and protective adjuvant effects of IL-4 (13). As a genetic adjuvant, IL-4 co-expressed in a Trichinella spiralis DNA vaccine significantly elevated Th1/Th2 cytokine levels, alleviated intestinal damage, and demonstrated effective adjuvant functionality (14).

IL-6 is a multifunctional pro-inflammatory cytokine with diverse roles in inflammation, immune responses, and hematopoiesis. IL-6 synergizes with transforming growth factor-β (TGF-β) to promote the differentiation of naïve CD4+ T cells, thereby enhancing adaptive immune responses (33). Furthermore, IL-6 promotes the production of IL-21, aids in the differentiation of T follicular helper (Tfh) cells (34) and CD8+ T cells (35), and induces B cell differentiation into plasma cells (36), thereby enhancing antibody production. The co-expression of IL-6 as the molecular adjuvant with FMDV DNA vaccine, induced a higher ratio ofIgG2a/IgG1, higher levels of expression of IFN-γin CD4+ and CD8+ T cells, IL-4 in CD4+ T cells, and in vivo antigen-specific cytotoxic response, which confirm both Th1 and Th2 immune response are activated (37). Both recombinant IL-6 protein and plasmids expressing the IL-6 gene have been used as adjuvants in studies on Japanese flounder (Paralichthys olivaceus). These adjuvants increased antibody levels, enhanced immune responses, reduced bacterial infection-induced damage, and significantly decreased mortality (15). Moreover, vaccination of mice with recombinant rabies virus expressing IL-6 resulted in earlier and higher antibody titers compared to the wild-type virus, demonstrating the potential adjuvant activity of IL-6 in enhancing vaccine immunogenicity (16).

IL-12 is a member of the interleukin-12 (IL-12) family cytokines with an integral effect in activating cellular immune responses in mammals (38). When pathogens infect the host, IL-12 stimulates Th1 cell to release IFN-γ, promoting the Th1 cellular immune response and enhance the host’s property to clear the pathogens. For intracellular pathogens, IL-12 induces macrophages or cytotoxic T lymphocytes (CTLs) to destroy infected cells (39, 40). Macrophages exhibit strengthened activation activities based on regulation of IL-12 and upregulate the production and release of nitric oxide (NO) to further enhance the ability for antigen clearance (41). Multiple functional studies have highlighted IL-12 as a potential vaccine adjuvant with immunomodulatory properties. Co-delivery of an IL-12-expressing plasmid with an NDV F gene DNA vaccine using electroporation has been shown to significantly enhance immune responses in chickens, resulting in higher neutralizing antibody levels, increased lymphocyte proliferation, reduced viral shedding, and complete protection compared to the DNA vaccine alone (17). In addition, co-immunization with an IL-12 eukaryotic expression plasmid and a Toxoplasma gondii multi-epitope vaccine (pcROP8) enhanced the Th1 response and IFN-γ secretion, thereby providing heightened vaccine protection (18).

IL-15 is a critical factor for the development, proliferation, and activation of effector NK cells and CD8+ memory T cells. It plays important roles in NK cell proliferation, cytotoxicity, cytokine production, NK cell-macrophage interactions, and the maintenance of CD4+/CD8+ memory T cell homeostasis (42). IL-15 supports the long-term persistence of CD8+ T cells and effectively extends immune duration, making it a preferred adjuvant for improving immune responses and vaccine longevity (43). The inactivated vaccine is short activities to the immune response, when bovine-derived IL-15 has been used as an adjuvant in guinea pigs immunized with an inactivated FMDV vaccine, the IL-15 adjuvanted vaccine maintained neutralizing antibody levels for up to six months in animals receiving. As well as Compared to animals immunized with the inactivated vaccine alone, those vaccinated with IL-15 adjuvants exhibited stronger Th1 and Th2 immune responses (19).

IL-18, initially identified as an interferon-γ-inducing factor (44), synergizes with IL-12, mitogens, or microbial agents to promote IFN-γ production by T cells and NK cells (45–47). IL-18 also induces the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in peripheral blood mononuclear cells (PBMCs) (44, 48–50) and stimulates IL-13 production (51). When IL-18 plasmids encapsulated in PLGA nanoparticles were used as adjuvants in combination with a foot-and-mouth disease virus DNA vaccine, they induced higher antibody titers and stronger CTL responses in guinea pigs, compensating for the limited cellular immunity often observed with inactivated FMDV vaccines (20).

Type I IFNs possess immunomodulatory properties and can regulate the activity of other cytokines (62). They enhance the maturation and activation of dendritic cells (56, 63), promote Th1-type immune responses, and activate B cells to facilitate antibody production (64, 65). A combination of recombinant porcine IFN-α protein and inactivated influenza vaccine has been shown to significantly upregulate the expression of immunomodulatory cytokines such as IL-2, IL-6, IL-10, IL-18, and IFN-γ. This combination also significantly increases the transcription of homing factors CCR9 and CCR10, induces a strong mucosal innate immune response, and enhances antibody levels (53). In another study, immunization with a Venezuelan equine encephalitis virus (VEEV) vaccine containing an IFN-α plasmid adjuvant in mice resulted in the robust expression of antiviral proteins and induced specific immunity against VEEV (57).

Type II IFN is primarily produced by activated Th cells and NK cells (66). It is a multifunctional homodimeric cytokine, with IFN-γ being its sole member (58, 59). The main biological function of IFN-γ is to induce the expression of various immune factors, thereby enhancing the body’s immune response. Several studies have demonstrated that IFN-γ is an effective adjuvant for veterinary vaccines. For instance, immunization of mice with a recombinant Hyalomma asiaticum rHasCPL protein subunit vaccine combined with an IFN-γ adjuvant increased the production of IFN-γ and IL-4, enhanced antibody levels, and improved the protective efficacy of the vaccine in mice (67). In vitro experiments have shown that the expression of porcine IFN-γ can significantly enhance the pro-inflammatory immune response in cells infected with PRRSV (68). Additionally, in Japanese flounder, the use of an IFN-γ adjuvant with an Edwardsiella tarda subunit vaccine effectively increased survival rates, upregulated the expression of immune-related genes, and enhanced antibody production (69). Furthermore, a study assessing the immunoadjuvant effects of a recombinant poIFN-γ-poGM-CSF fusion protein in an inactivated PRRSV vaccine administered to piglets found that the coadministration of poIFN-γ-linker-poGM-CSF and PRRSV KV significantly increased neutralizing antibody titers, accelerated viral clearance, reduced clinical symptoms, and prevented highly pathogenic PRRSV infection (70). This reinforces the critical role of IFN-γ and its fusion proteins in enhancing vaccine efficacy and providing protection against viral infections in veterinary medicine.

Type III IFNs (IFN-λ1, λ2, and λ3) are structurally related to Type I IFNs and the IL-10 family (60), and are also known as IL-29, IL-28a, and IL-28b (61, 71). Their receptor, IL-28Rα, is expressed on a limited range of cells such as macrophages, peripheral blood lymphocytes, conventional dendritic cells, epithelial cells, and plasmacytoid dendritic cells (61, 72). IFN-λ primarily acts on these cell types to regulate antiviral immunity, thus possessing potential as an adjuvant to enhance immune responses. In studies where a Porcine Reproductive and Respiratory Syndrome (PRRS) DNA vaccine expressing IFNλ1 was used to immunize mice, there was an upregulation of serum antibodies and activation of the STAT signaling pathway. This suggests that IFNλ1 can enhance the immune protective effect of PRRSV DNA vaccines (73, 74).

CCL4, also known as Macrophage Inflammatory Protein-1β (MIP-1β) (79), is effective chemoattractant for CD4+CD25+ T cell populations and is a phenotypic characteristic of regulatory T cells (80). CCL35.2 in crucian carp has the highest identity with mammalian CCL4. Using CCL35.2 plasmid adjuvant in combination with a DNA vaccine to immunize crucian carp can effectively upregulate the mRNA expression of key immune genes IL-1β, IL-2, IFN-γ2, and viperin in Carassius auratus gibelio. It also increases the levels of complement C3, lysozyme, and total superoxide dismutase, significantly enhancing the resistance of crucian carp to Cyprinid herpesvirus 2 (81).

CCL28, also known as Mucosa-Associated Epithelial Chemokine (MEC), has unique immunoregulatory properties in various mucosal areas, attracting IgA and directing their migration to different mucosal sites (82, 83). Many chemokines are effective immune adjuvants in various model systems, enhancing protection induced by viral, bacterial, and parasitic vaccines, and regulating the direction and magnitude of induced immune responses produced by DNA, protein, subunit, or peptide vaccines (77). When used as a virus-like particle (VLP) vaccine adjuvant for influenza vaccines, CCL28 can act as an immune stimulant in a membrane-bound form, eliciting a systemic mucosal immune response and significantly enhancing the host’s cross-protective efficacy against heterologous viruses (84). Furthermore, studies have shown that the addition of a CCL28 adjuvant in H3N2 influenza vaccines can induce a significant increase in IgA levels and hemagglutination inhibition (HI) titers, enhancing long-term cross-protection against H3N2 influenza virus (85). A vaccination strategy that employs the intramuscular co-delivery of CCL27 or CCL28 has been shown to generate strong systemic and local immune responses, leading to the production of long-lived antibodies that neutralize influenza (86).

GM-CSF is a hematopoietic growth factor produced by various immune cells (87). It stimulates the proliferation and differentiation of bone marrow progenitor cells into granulocytes and macrophages (88), as well as activating and maintaining mature bone marrow cells (89). GM-CSF responds to immune cell survival, differentiation, and proliferation by inducing various signaling pathways, which is a key step in helping the immune system fight infections (90). GM-CSF levels significantly increase during inflammatory responses (90, 91), and multiple studies have demonstrated that T cell-derived GM-CSF plays an essential role in immune responses against a variety of pathogens, such as Mycobacterium tuberculosis, Epstein-Barr virus, and Human Immunodeficiency Virus and the rabies virus (92–96). Based on its pro-inflammatory effects induced by recruiting and activating bone marrow cells (90), recombinant pGM-CSF has been tested as a co-adjuvant with pFLIC protein in conjunction with a Porcine Circovirus (PCV) vaccine to immunize pigs. This approach significantly elevated PCV-specific antibody levels, stimulated CD4+ and CD8+ T cell proliferation, and upregulated the transcription of IL-1, IL-8, and IL-17, indicating a robust enhancement of both humoral and cellular immune responses (97). Similarly, the combination of GM-CSF and APS as a complex immunostimulant in a PRV vaccine model resulted in higher levels of PRV-specific gB and neutralizing antibodies, and concurrently increased the production of cytokines including IL-4, IL-10, IL-2, and IFN-γ (98). These findings indicate that GM-CSF-based adjuvant strategies have promising application prospects.