动物实验 z-bo 2020-08-25 475

Abstract Vaccination is an effective strategy to prevent infectious and immune-related diseases, and has made significant contributions in human history. Recently, more and more attention has shifted to mucosal vaccines due to its multiple advantages over traditional methods. Mucosal vaccine subunit or peptide antigen immunization is more reasonable than live or attenuated pathogens, but the immune response needs to increase adjuvants. For different causes, many mucosal adjuvants have developed the best immune response. Compared with adjuvants derived from pathogens, the inclusion of natural endogenous molecules into mucosal vaccines shows outstanding adjuvant effects and safety. At present, cytokines are the most widely used mucosal adjuvant to participate in immune response signal transduction, activate innate immunity and polarized adaptive immunity. Based on the specific interaction between cytokines and corresponding receptors, the ideal immune response is rapid and effective. In addition, some other natural molecules have also been identified as effective mucosal adjuvants. This article reviews natural endogenous adjuvants to clarify the development of mucosal vaccines.

　　Keywords Mucosal vaccines Adjuvants Innate endogenous molecules

　　 Introduction: Mucosal vaccination is a direct strategy to prevent infection. Before entering the body, pathogens initiate infection through the host mucosa. Compared with other methods of vaccination, mucosal vaccination is preferable for many reasons. First of all, for vaccination, the recipient and the community provide safe and free vaccines, especially in developing countries. Free cold chain distribution reduces the transportation and storage costs of vaccines. The ease of operation of mucosal immunity helps to get rid of dependence on vaccinators, which is the flexibility of vaccination. In addition, mucosal immunity increases the compliance of the recommended vaccination schedule and reduces the side effects of vaccination, especially in children. The most obvious advantage of mucosal vaccines is that the rapid delivery of mucosal sites effectively induces mucosal immune responses, especially mucosal IgA secretion. Mucosal IgA is the monitor or neutralize mucosal pathogens and activate the pathway of import complement. In addition, mucosal vaccines also induce systemic immune responses, including the production of serum antibodies and cell-mediated responses at remote sites.

　　 There are abundant immune organs on the mucosal surface, so theoretically all mucosal parts can be used as a way of vaccination. From logical and practical considerations, the sites of mucosal vaccines are concentrated in oral and nasal. Although oral vaccines have been successfully used to combat polio, cholera, and typhoid fever, nasal administration is the most effective way to induce effective and extensive mucosal immune responses in multiple mucosal sites. In addition, a large number of immunogens induce oral tolerance, and oral administration is easily digested by gastric acid, inhibiting the use of oral vaccines. The small area of the nasal mucosa requires a small amount of antigen, and the lack of an acidic environment in the nasal environment maintains the stability of the antigen.

　　Live vaccines or live attenuated vaccines are effective in controlling infectious diseases. However, the safety of live vaccines or attenuated vaccines has always been controversial, hindering its clinical application process. It is possible that the vaccinated pathogen may return to the wild type or replicate into a highly toxic organism in the host. Therefore, purified protective antigens are a new generation of mucosal vaccines that receive more attention. Direct mucosal immunization of soluble antigens causes only relatively low immunogenicity. Therefore, safe and effective mucosal adjuvants are often combined with antigens to improve the immunogenicity of non-live vaccine antigens. In addition, adjuvants are defined as immunopotentiators different from previous delivery systems.

Adjuvants or derivatives derived from pathogenic bacteria are widely used in adjuvant systems for mucosal vaccines. Although some adjuvants have potential and extensive immune responses, the toxicity threats and side effects of non-human products should be carefully evaluated. For example, a nasotoxin adjuvant influenza inactivated vaccine was withdrawn after a period of time on the market. The use of this adjuvant may cause facial paralysis. Obviously, due to the low toxicity of innate substances, innate molecules are more beneficial than other ingredients as adjuvants. The nature of the innate adjuvant to prevent infection and the development of mucosal vaccines will be discussed extensively in the following section.

　　 Cytokines: Cytokines are scattered, classified as signal proteins, which are released by a wide range of cells.

　　The cytokine network activates and regulates the development of innate immunity and adaptive immunity, and balances humoral immunity and cell-mediated immune response. Therefore, they are important in host defense and modulation of immune-related diseases. The immune response can be initiated by simply increasing these signaling molecules such as proteins or DNA fragments. Emphasize potential vaccine adjuvants. Some cytokines in the immune system have been well studied and discussed to improve the efficacy of the vaccine.

Type I interferons: Type I interferons (IFNs) have always been considered antiviral drugs and initiate immune response development. Some clinical reports clearly show that the mucosal transmission of human interferon alpha has direct prevention and treatment for cancer, autoimmune diseases and respiratory syncytial virus, measles virus, papilloma virus, HIV and hepatitis virus caused by influenza virus infection. effect. After the mucosal administration of type 1 interferon, the expression of gene-encoded chemokines, cytokines and proteases was significantly increased. It is closely related to antigen processing and lymphocyte activation, migration, apoptosis and protein degradation.

　　IFNα/β treatment increases the uptake of antigen by APCs in murine nasal mucus layer. IFN-α low-dose mucosal administration effectively activates natural killer cells, the proliferation of B cells and T cell subsets in the peripheral circulation. Type I interferons play a signaling role in some well-studied adjuvants, including Th1 cells and CTL polarization types. On the other hand, the application of type I interferon, immunosuppressant in the mucosa effectively induces suppressor T cells and reduces cytotoxic T cells and related cytokine products when encountering autoimmune and inflammatory diseases. Oral mucosal administration of mouse IFN-γ significantly reduced the production of specific allergen IgE and the production of eosinophils in mice without toxicity.

　　IFN-α/β and influenza virus subunit vaccine intranasal immunization can cause an effective humoral immune response to prevent influenza virus infection and weight loss in mice. Mice immunized with mouse type I interferon and influenza virus A can produce high levels of serum and mucosal antibodies, effectively clearing the virus in the lungs. However, human trials have shown that both serum hemagglutination inhibition and neutralizing antibody response mice have significantly increased type I interferon IFN. Nasal administration of mouse interferon IFN-β and influenza inactivated vaccines significantly reduced the lethal dose of influenza virus is a challenge to mice death. Oral administration of mouse type I interferon with lethal dose of encephalomyocarditis virus, vesicular stomatitis virus or varicella-zoster virus can significantly increase the survival rate of mice.

"Poxvirus and interferon epsilon nasal immunization increased the expression of lung-specific CD8+ CD107a + interferon-γ+. Enhance lymphocyte alveolar filtration to reduce inflammation, improve the function of functional/cytotoxic CD8 + CD4 + T cell subsets (CD3hiCCR7hiCD62Llo) in mouse lung lymph nodes, and enhance rapid lung clearance.

　　 The immune stimulation effect of type I interferon is heavily dependent on the time and the dose of mucosal administration. For example, administration of immune interferon inhibits the production of immunoglobulin and the conversion of B cells. In addition, high-dose type I interferons have immunosuppressive effects. At the same time, type I interferon is considered to be an immunosuppressant in the case of autoimmune diseases and inflammation. Some reports indicate that the use of type I interferon is harmful to the elimination of harmful pathogens. This shows that the mysterious role of type I interferon as an immunomodulator or immunosuppressant requires further research. However, it is not limited to the transportation location, the adjustment mechanism, the optimal dosage and the time of use of type I interferon, as well as diseases and environmental conditions.

　　IFN-γ FN-γ, mainly produced by T cells and NK cells, reacts to antigens or mitogens. The activation of T cells and the establishment of adaptive immunity play a key role. The lethal dose of mouse interferon-γ and influenza inactivated vaccine administers nasal cavity challenges to the survival rate of mice. Further analysis showed that IgA and IgG antibody titers were enhanced in the IFNγ comprehensive group in the early stage of infection. The HI antibody titer of the same group was significantly lower than that of mice in the late stage of infection that received only the vaccine. Oral lethal doses of encephalomyocarditis virus and interferon IFNγ can significantly increase the survival rate of mice. Systemic and mucosal antigen-specific IgG1 and IgA antibodies are enhanced by intranasal co-injection of polylactic acid microcapsules V Yersinia antigen and IFN-γ. However, to prevent systemic bacterial infections in mouse models, this formulation is harmful. Low-dose mouse IFN-γγ was provided to adult HAM/ICR mice through drinking water. The following is the challenge of Salmonella typhimurium one day later. Compared with control mice, the application of interferon IFNγ significantly prolonged the survival time of mice. Reduce Salmonella invasion of intestinal epithelial cells, Salmonella growth and reproduction. Adverse reactions were observed in the report. Like type I interferon, the dosage and IFN-γ mucosal therapy immune schedule γ should be optimized.

　　GM-CSF: Granulocyte-macrophage colony stimulating factor (GM-CSF) improves the recruitment and activation of antigen presenting cells (APCs). Nasal administration of human GM-CSF enhances rabbit serum GM-CSF levels and increases total white blood cell count. The levels of lung antigen presenting cells (APCs) and cytokines, including IFN-γ and IL-12p40, were significantly increased after intranasal administration of GM-CSF in recombinant mice expressing RSV with respiratory syncytial virus. In addition, GM-CSF encodes virus virus-specific Th2 transfer to Th1 type. Intranasal injection of HIV DNA vaccine and GM-CSF expressed by mouse plasmids induces high levels of systemic and mucosal antigen-specific antibodies and enhances delayed-type hypersensitivity in mice. Adenovirus vector-mediated intranasal immune white dominance antigen-specific IgG1 and IgG2b responses of mouse GM-CSF and β-amyloid egg mice, suggesting that GM-CSF polarizes Th2 immune response. Expressing mouse GM-CSF recombinant vesicular stomatitis virus is highly inactivated in terms of virus transmission and pathogenesis. Further analysis showed that genetic GM-CSF enhanced the recruitment of macrophages, CD8 T cell memory and mouse immune recall response.

"Compared with the parental virus, mouse oral immunization with recombinant rabies virus expressing GM-CSF can increase the number of DCS and immune B cells in the periphery, a higher level of adaptive immune response and increase the resistance of the virus. Intranasal administration of GM-CSF expressing deactivated HSV induces a protective immune response to the virus to reduce the lethal dose used in mice. Nasal administration of BCG vaccine expressing GM-CSF in mice significantly increased the number of lung DCs and the secretion of immunomodulatory cytokine IL-12. Correspondingly, antigen-specific CD4 + T cells were increased in mediastinal lymph nodes and lungs. More importantly, compared with mice immunized with BCG alone, the use of BCG:GM-CSF can significantly reduce the dose of Mycobacterium tuberculosis infection. On the basis of the above evidence, the co-administration of GM-CSF is conducive to the recruitment of APCs to antigens, thereby initiating immune response.

　　 Tumor necrosis factor (TNF) family: The tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily is extremely critical in maintaining the homeostasis of the immune system. In inflammation and host defense, it has a good protective effect. Tumor necrosis factor-α is used intranasally with the antigen to induce antigen-specific IgG and mucosal IgA antibodies in mice. Through cytokine analysis, mucosal application of TNF-α can induce Th2-type immune response. In mouse experiments, it is proved that the adjuvant effect of TNF-α is effective and safe, and the immune adjuvant of TNF-α is at least partially related to the increase of epithelial permeability. The HIV mutant and TNF-α immune adjuvant are used as mucosal vaccines to induce antigen-specific IgG. When administered through the nasal cavity of mice, it caused a local or distant mucosal IgA antibody response. Other members of the TNF family have also been tested for mucosal adjuvants in mice. Nasal administration of antigen with TL1A or APRIL induces strong antigen-specific mucosal immunity and humoral immunity. Interestingly, the immune response induced by tumor necrosis factor and CTB is comparable in size, indicating that these adjuvant candidates are expected to replace toxin adjuvants.

IL-1: IL-1 secreted by various types of cells is a pro-inflammatory cytokine that has a wide range of host immune system effects, including up-regulation and down-regulation of many other cytokines related chemokines, adhesion, cell differentiation and migration gene. The soluble antigen combined with human IL-1α or IL-1β mouse nasal immunization produces humoral and cellular immunity. It has the same effect as antigen and CT adjuvant. After antigen and IL-1α or IL-1β are used together, antigen-specific serum IgG, vaginal IgG and IgA, systemic delayed hypersensitivity and lymphocyte proliferation response are significantly increased. Nasal administration of IL-1 mice with α induces effective systemic and mucosal immune responses, which depend on the expression of IL-1R1 in stromal cells. In addition, IL-1α induced adaptive immunity is dependent on CD11c+ cells. After nasal immunization in mice, IL-1α showed significant adjuvant activity in inducing HIV-specific serum IgG. In addition, after nasal immunization with IL-1α and HIV antigens in mice, significant CTL responses and antigen-specific IFN-γ secreting cells were induced. In addition, nasal administration of human IL-1α has been shown to be safe and tolerable in primate models. Streptococcal protein antigens and human IL-1 cause tonsils to use higher levels of mucosal IgA and serum IgG antibodies than antigens alone. Delayed-type hypersensitivity to streptococci, confirmed by ear swelling and increased levels of IFN-γ, can only be found in rabbits immunized with IL-1. Nasal administration with influenza virus hemagglutinin (HA) and mouse IL-1α/β caused an increase in Th1 and Th2 cytokines and systemic IgG and mucosal IgA antibodies. In addition, mouse IL-1α/β and HA are co-administered to protect against lethal infection by influenza virus. After a lethal dose of Streptococcus pneumoniae in mice, the nasal administration of human IL-1β adjuvant is as effective as the subcutaneous immune effect of alum adjuvant, producing equivalent antibody levels and protection. Like subcutaneous immune ISA-51 (water-in-oil emulsion adjuvant) and nasal CTX adjuvant, IL-1β is an effective nasal vaccine adjuvant to induce the body to produce protective immunity against systemic tetanus toxin. Although IL-1 adjuvant activity is very effective, the pleiotropic characteristics of IL-1 can easily cause adverse immune reactions, and a clear optimization procedure is required for the mucosal adjuvant of IL-1.

　　IL-2: IL-2 is mainly produced by T cells after encountering antigens, and helps T cells grow, proliferate and differentiate during the immune response process. Using live Lactococcus lactis as a nasal delivery carrier, the bacterial expression of mouse IL-2 enhances the specific immune response of the antigen (tetanus toxin C fragment). In addition, DNA vaccines and plasmids express IL-2 to induce Th1 immune responses. The intranasal delivery plasmid encoding IL-2 transfers the Th1 type response induced by TT or CT to the Th2 type immune response. After oral administration of the plasmid-expressed immunoglobulin IL-2/Ig, the production of antigen-specific IFN-γ, IL-2 and IL-4 was enhanced. In addition, IL-2/Ig gene method can cause high CTL activity and high levels of mucosal/serum antibodies. After a chicken is immunized with a nasal drip, the combined use of inactivated avian influenza virus and IL-2 can enhance the local immune response.

"IL-4 and IL-5: In a positive feedback loop, the cytokine IL-4 induces T cells to differentiate from naive Th0 cells to Th2 cells, thereby producing additional IL-4. In addition, IL-4 is involved in activating the proliferation of B cells and T cells, and the differentiation of B cells into plasma cells. Mouse immunogenetic HIV antigen and mouse IL-4 nasal drops can induce high levels of systemic and mucosal antigen-specific antibodies. In addition, IL-4 expression plasmids mainly induce serum IgG1 subtypes, but significantly inhibit delayed-type hypersensitivity and CTL responses. Systemic and mucosal antigen-specific IgG and IgA induced by intranasal immunization with poly-L-lactic acid microcapsules V Yersinia antigen and IL-4. However, this combined vaccine can reduce the survival rate of mice after bacterial injection.

　　 IL-5 is produced by Th2 cells, mast cells stimulate the growth of B cells, increase the secretion of immunoglobulin, and mediate the activation of eosinophils. Intranasal inoculation of mice with adenovirus expressed by IL-5 increases the virus-specific IgA antibody titers in lung lavage fluid.

　　IL-6: IL-6 is a pleiotropic cytokine that regulates T cells and B cells. Adenovirus-mediated intranasal inoculation of IL-6 in mice increases virus-specific IgA/G titers in mouse lung lavage fluid. In lung lymphocytes IL-6 expressing adenovirus immunized mice, virus-specific antibody secreting cells appeared frequently. Histological analysis showed that a large number of monocytes accumulated at the pIL-6 inoculation site.

"IL-12" is naturally produced by dendritic cells, macrophages and human B lymphoblasts. It usually induces Th1 response and stimulates NK cells, T cells and B cells to produce IFN-γ and TNF-α. In mice immunized with IL-12 through the nose, the mRNA transcription of IFN-γ and IL-10 continued to increase. The use of HIV gene antigens and plasmids to express IL-12 in the nose of mice induces Th1 and Th2 type responses. In addition, after mice were immunized with IL-12 and HIV antigen, high levels of HIV-specific CTL and delayed-type hypersensitivity were enhanced. After nasal administration of IL-12, serum ovalbumin-specific antibody levels IgG2a, IgG2b, and IgG3 increased significantly, while serum IgG1 was inhibited. Intranasal immunization of mice with TT and CT combined with IL-12 increased the secretion of IFN-γ from TT-specific CD4 + T cells and decreased the secretion of Th2 cytokines. This means that the CT-induced immune response shifts to Th1 type.

　　IL-15 IL-15, mainly secreted by mononuclear macrophages, induces the innate immunity and adaptive immune system, such as the proliferation of natural killer cells and T cells.

IL-18 IL-18 induces the activation of Th1 cells, NK and CTL cells, the production of IFN-γ, and regulates the synthesis of inflammatory cytokines, including IL-2 and IL-12. Mouse IL-18 and HIV antigen intranasal drops Significant CTL response and antigen-specific IFN-γ secreting cells induced after immunization.

　　 FLT3L FMS-related tyrosine kinase-3 ligand (Flt3L) stimulates the proliferation and differentiation of different blood cell progenitor cells, including T cells, B cells, NK cells and DCs. OVA-specific mucosal and plasma immune responses were induced by nasal administration of OVA. Compared with OVA alone, the plasmid-encoded mouse Flt3L tends to have a Th2-type immune response.