疫苗 z-bo 2020-09-22 532

　　1. Vaccines

Since Edward Jenne, vaccines have completely changed global public health, successfully removing infectious diseases such as diphtheria, Haemophilus influenzae type B (Hib), hepatitis B virus infection, tetanus, measles, mumps, Tens of thousands of lives have been saved in neonatal tetanus, whooping cough, streptococcus pneumoniae infection, rubella, and group C meningococcal infection. It has been about 30 years since the World Health Organization (WHO) announced the complete control and elimination of smallpox. With the increase in the vaccination rate, the eradication of polio is almost complete. With the exception of Nigeria, Pakistan and Afghanistan, the polio-endemic countries have seen a 99% reduction in the number of polio cases since 1988. Therefore, the discovery of vaccines has become one of the greatest achievements and safest interventions in biomedical scientific research.

　　 Although vaccines are one of the most successful scientific breakthroughs, basic immunology needs further research. The success of the vaccine depends on the quality, quantity and duration of the acquired immune response after vaccination. To initiate an adaptive immune response, many signals need to pass through immature T cells. Among these signals, signal 1 is vaccine-derived, and signal 1 is the major histocompatibility complex (MHC) class II and class I of the vaccine, polypeptide antigen (Ag) bound to the surface of antigen presenting cells (APCS). Signal 2 is also called "co-stimulation", and more importantly, together with signal 1, induces an immune response. Signal 2 includes cross-linking CD28 and other T cell receptors through costimulatory molecules B7-1 (CD80), B7-2 (CD86) and other ligands expressed through APC. Signal 3 is provided by cytokines, transported from APC to T cells, and determines the differentiation of T cells into effector cells. Signal 2, signal 3 is presented to T cells by activated and mature APCS such as dendritic cells (DCs). Mature dendritic cells can induce clonal expansion and primary immune response of cells, so the understanding of vaccines is very important.

　　 Dendritic cells will mature when they receive specific stimuli from the environment. Such as exposure to Toll-like receptor (TLR) ligands, necrosis, inflammatory factors (cytokines), T cell ligands (such as CD40 ligands), and contact between homodendritic cells are interrupted. The maturation of dendritic cells involves changes in their phenotype and location, transforming them from a monitor cell into a potentially activating naive T cell. The characteristic of DC maturation is the appearance of dendrites, increased expression of MHC class II molecules, costimulatory molecules, chemokine receptor 7 (CCR7) and cytokines. Under such circumstances, MHC class II molecules present antigens and costimulatory molecules help activated T cells, and CCR7-expressed chemokine receptors mediated cell migration to draining lymph nodes. Cytokines are involved in a variety of functions, such as the transport of local lymph node cells at the vaccine injection site, the activation of T cells and the differentiation of T cells.

　　 In the lymph nodes, dendritic cells are located at the T cell entry site of the Epstein-Barr virus induced by the receptor ligand chemokine. Chemokine ligand 9 (chemokine) and secondary lymphoid tissue chemokine (SLC) or CCL21 can enhance the attractiveness of DC-T cells. In the paracortical area of the lymph node, B cells use surface immunoglobulin to bind antigens and become activated. As a result, they rapidly differentiated in plasma cells, and produced low-affinity antibodies (Ab) in the paracortical area that caused the activation, proliferation, and differentiation of antigen-specific CD4 + T cells into T helper cells ( Th) 1, 2, and 17, and follicular helper T cells (TFH). Th2 and Tfh cells start the germinal center response, and the cells and follicular DCS (FDCS) provide powerful activation signals to B cells. Active B cells produce plasma cells that secrete antigen-specific high affinity.

　　 The frontier field of biomedical sciences has increased the practical and theoretical understanding of pathogenic biology, molecular biology, biochemistry, immunology and biotechnology. Therefore, vaccine development has shifted from trial and error-based empirical research to a more rational and reductionist approach. However, these methods have had limited success in developing effective vaccines for emerging diseases, such as human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and re-emerging diseases such as tuberculosis and malaria. This may be due to factors such as rapid elimination by the body, lack of recognition by the immune system, and insufficient stimulation of appropriate immune cells. Therefore, the discovery of suitable and safe antigens has become the main goal of vaccination. In recent years, the development of adjuvants has shared the immunogenicity of antigens and has also received equal attention. The use of adjuvants reduces the dose of the vaccine and induces a specific protective response (CD4 or CD8, Th1 or Th2). In order to strengthen the wide-ranging immune response, the design and development of adjuvant vaccines are very necessary. Therefore, designing an effective adjuvant is the key to vaccine development.

　　Aluminum preparation

　　Although there are a large number of new adjuvants developed, aluminum adjuvants dominate all currently approved and licensed adjuvants in the world. This adjuvant was first used by Alexander T. Glenny to prepare potassium aluminum sulfate or aluminum preparation adjuvant and diphtheria toxoid (DT) vaccine co-precipitated and dissolved in carbonate buffer. Due to the reproducibility of production, the technology of aluminum preparation precipitation has been replaced by the technology of prefabricated alumina or aluminum gel, aluminum phosphate (AP) or adjusted phosphate gel. Aluminum compounds, such as aluminum chloride, aluminum silicate, algammulin, cesium alum (CA), were replaced by aluminum hydroxide adjuvant in the experiment. Although these compounds are often collectively referred to as "aluminum preparations, aluminum preparations are actually a unique chemical composition. More importantly, these compounds have been used to emphasize their comparative role in the immune response induced in the body."

　　 Although these adjuvant vaccines have been used in humans for about 90 years, their mechanism of action is still elusive. The effects of some aluminum preparations may help improve the immunogenicity of vaccines. However, in many cases, these effects are only partially described or lack clear causal links and auxiliary functions.

　　2 Mechanism of action: in vivo and in vitro experiments

　　Adjuvant biologists hypothesized that adjuvants work in accordance with the formation of depots, targeted antigens, and inflammation. These hypotheses are based on in vitro studies and a small number of in vivo validation tests. Although we have constantly updated knowledge and understanding of the immunization process, our research on vaccine adjuvants still relies mostly on experience. Reductionist methods, such as the analysis of the adjuvant effects of major immune system cells in vitro, will help to determine the characteristics of adjuvants that are critical to determining their functions, and greatly improve our understanding of the mechanisms involved. However, in the body, the adjuvant and its environment have complex interactions on the immunological, physiological and anatomical levels. In vitro, under different experimental conditions, a single cell can exhibit different behaviors. Therefore, understanding how cells behave in the body and their interaction with the environment will be the key to our understanding of the mode of action of adjuvants. The published comments are about the mechanism of action of aluminum preparations. However, in vitro and in vivo data have never been completely compared and evaluated. Therefore, this review compares the mode of action of adjuvants in vitro and in vivo.

　　The challenge of warehouse model theory

　　 Warehouse model hypothesis was first proposed by Glenny, Buttle and Stevens in 1931 after co-precipitation of diphtheria and aluminum preparations. They excised part of the injection site (diphtheria-aluminum preparation precipitate or soluble diphtheria toxin) from the guinea pig 3 days after administration. They ground the skin into a lotion and injected it into unimmunized guinea pigs. The diphtheria-aluminum preparation precipitated animals were successfully immunized but the control group that received diphtheria toxin was not immunized, and the antitoxin titer was measured. This experiment led them to hypothesize that the precipitation of antigens from a single injection site of aluminum preparations will slowly eliminate for a long period of time, which can increase the primary and secondary stimulation of the antigen, leading to an increase in the relevant antibody titer. Similarly, Harrison proved this hypothesis by transferring aluminum nodules from one guinea pig to another. White and his colleagues believe that the persistent inflammation caused by the adjuvant's warehouse model can stimulate immune cells in the regional lymph nodes and induce granulomas to produce antibodies in plasma cells. In subsequent studies, 2-3 antigens were monitored in granulomas induced by alumina gel. It has been confirmed that the adjuvant consists of a powerful adsorbent, which can ensure a high local concentration of the antigen for a period of time. This is also necessary for antigen uptake and activation of APCs-like dendritic cell DCs.

　　 Warehouse hypothesis theory can make a reasonable explanation based on the high combination of aluminum preparation and Ag. This will result in the retention of the antigen at the injection site and its slow release in the body. Aluminum adjuvants have been proven to pass electrostatic interactions, coordination exchanges, and hydrophilic-hydrophobic interactions and interactions for each combination. Mainly depends on the characteristics of the antigen, pH, ionic strength, and the nature of the surfactant. As a result of adsorption, the soluble antigen becomes granular.

　　 Therefore, compared with soluble antigens, particles effectively interact with APCs to enhance cell phagocytosis. . And through internalization, soluble antigens may be trapped in large irregular aggregates formed by fiber primary particles, the size (1-10μm). With the continuous release of antigen in the tissue culture medium in vitro, these main particles are loose in aggregates and are easily degraded. A similar release is also observed in the body, because aluminum-containing adjuvants are rapidly chelated and solubilized by α-hydroxy carboxylic acids such as citric acid, lactic acid, and malic acid in the interstitial fluid, which can be absorbed into the tissues and finally passed Excretion in urine. As time changes, as antigen release, aluminum preparation elution or interstitial fluid. The antigen is first taken up by phagocytes, encapsulated and finally swallowed. Immediately, the soluble protein released from the aluminum preparation aggregates is pinocytosed by phagocytes. The elution of antigen from the adjuvant surface in vivo is critical, because APCS-like dendritic cells residing in the lymph nodes can capture the antigen from the injection site. Although dendritic cells with lymph nodes pinocytose and present antigens, they are not enough to cause an immune response in the body. The second round of antigen presentation by dendritic cells from the injection site interacts with immature T cells in lymph nodes to induce an effective immune response. Therefore, the uptake of antigen by dendritic cells at the injection site, the endocytosis of sustained release of antigen at the lymph node site, and the presentation of antigen at the injection site are all critical. However, the retention of antigen and its slow release have proven that aluminum adjuvants are dispensable to increase the antigen response in vivo. In this case, through the mixed use of ovalbumin and aluminum hydroxide and phosphate-treated aluminum hydroxide, the rate of antigen adsorption and elution has been quantified (adsorption: 91%, completely dissolved in interstitial fluid: 4 hour). Dephosphorylated α-casein (DPAC) mixed with aluminum hydroxide (adsorption: 100%; completely eluted in interstitial fluid: <6 hours) or phosphate-treated aluminum hydroxide (PTAH-B) (adsorption: 40%) , Completely soluble in interstitial fluid: 1 hour). Taking into account these in vitro data, the author injected these vaccine preparations into mice subcutaneously, and observed enhanced independent antibody titers and antigen adsorption properties in vivo. Therefore, it shows that strong adsorption may damage the physical and chemical properties of the vaccine. However, a slight interaction between the aluminum preparation and the antigen is necessary, for example, a good immune response can be observed after the interaction between the aluminum preparation and the anthrax antigen or hepatitis B virus. The interaction between the aluminum preparation and the antigen is necessary and has been proved in other experiments. The injection of the antigen or aluminum preparation alone did not produce an immune response. Although a higher quantitative antibody titer was obtained in a similar experiment. Therefore, WHO recommends that more than 80% of diphtheria toxin DT and tetanus toxoid (TT) should be adsorbed. The minimum requirement in the United States is that> 75% of DT and TT antigens should be adsorbed in aluminum adjuvants. There is no other correct candidate adjuvant. However, these are all in vitro experiments, so it is not clear what is happening in the body.

　　The antigen retention hypothesis has not been confirmed by subcutaneously immunizing mice with radioimmune mice (14C) labeled TT antigen adsorption adjuvant. Subsequently, studies confirmed the retention of antigen at the injection site and the increase in antibody titers in rat serum after 5 weeks of immunization and the increase in antibodies after 2 weeks. In this study, rats were injected with 111In-labeled α-casein (IDCAS) antigen adsorbed to aluminum hydroxide (AH) or IDCAS antigen adsorbed to phosphate-treated aluminum hydroxide (AP), or non-adsorbed IDCAS antigen (Constituted by phosphate-treated AP) Inject by subcutaneous route. Observe that the antigen retention is in the following order: IDCAS?+?AH?>?IDCAS?+?AP?>?PTAP?=?IDCAS. The order of antibody titer is PTAP?=?IDCAS?+?AP?>?IDCAS?+?AH>?>?IDCAS. It reveals that there is an inverse correlation between antigen retention and antibody titer. In this study, de Veer and his colleagues concluded that aluminum preparations can reduce the number of soluble antigens entering blood vessels. Although their adjuvant activity is not related to the slow release of antigen. Therefore, in vivo, aluminum preparations exert strong adsorption and dispersion, antigen retention, and slow release in antibody reactions, indicating that aluminum preparations adjuvants may have other mechanisms of action.

　　Although, the main effect of aluminum preparations is the storage effect, leading to the sustained release of antigens. Since the 1950s, this role has been questioned. Holt challenged this storage effect for the first time. It was observed that the antibody titer caused by DT did not change 7 days or more after the excision of the injection site of the aluminum preparation precipitate.

　　 Recently, using the Eα-green fluorescent protein (EαGFP)/YAe system, Hutchison and his colleagues failed to observe the transfer of antigen-specific T cells from mice to the receptor after 5 days of immunization with OVA?+? aluminum preparations. When the antigen is internalized by dendritic cells, EαGFP is degraded and Eα peptides are presented by MHC class II epitopes on the surface of type I antibody cells. MHC class II molecular complexes can be effectively detected by YaeAb to stain cells because these antibodies of this company can effectively bind Eα(52-68) and I-Ab MHC class II epitopes. Therefore, the YAeAb recognizes the T cell receptor of the same MHC epitope. This system uses in situ hybridization to combine the antigen and Y-Ae antibody to evaluate the uptake and consumption of the antigen. It is worth noting that Hutchison’s research further confirmed the absence of antigen in draining lymph nodes and the presentation of phagocytes. It is worth noting that: B cells are a type of phagocytic cell that presents Eα; MHC class II molecular complexes will present these complexes within 6-12h after immunization, and cDCs will present these complexes within 12-24h after immunization with aluminum preparations?+?EαGFP. The dendritic cells present antigen within 48-72 hours of the use of aluminum preparations in the draining lymph nodes. In monkeys 2 hours after injection site ablation, no difference was observed between B cells, cDCs, and pDCs in antigen uptake and expression. With the use of EαGFP, it is proved that the aluminum adjuvant has no effect on the storage of antigen.

　　 Alternative Theory of Antigen Targeting

　　 Landsteiner explained the mechanism of the slow absorption and delayed removal of the aluminum preparation in the antigen. This indicates that the "particle" nature of these adjuvants will facilitate the subsequent phagocytosis of macrophage activators. The antigen targeting theory is further proved by APCs by observing the phagocytic cells at the injection site of injected antigen and phosphate-treated aluminum hydroxide. This is a groundbreaking research that led to the hypothesis of antigen targeting by aluminum adjuvants. Antigen targeting is divided into three different stages: the first stage includes the accumulation of cells at the injection site; the second stage includes the induction of signal 1 and the maturation of APCs to generate signals. The third process involves the transfer of antigen-loaded phagocytes to draining lymph nodes.

　　3.2.1 Aluminum preparation can promote the recruitment of APC at the injection site and produce chemokines and cytokines

　　 Aluminum preparations can produce immune recruitment of various subgroups of white blood cells, such as neutrophils, eosinophils, macrophages, monocytes, and dendritic cells, in a time-dependent manner at the injection site. The recruitment of cytokines related to the enhancement of mRNA expression, chemokines and cell adhesion molecules, their secretions are related and can enhance the complement cascade. Various spatial and temporal inflammatory cell recruitment data have been obtained. The effect on the recruitment of immune cells may be related to the formulation and delivery route of aluminum preparations and antigens. It has been found that macrophages, epithelioid cells, lymphoid tissue, and collagen fibers are produced in lymphatic tissues after immunization with aluminum preparations or aluminum preparations +?TT. It is worth noting that neutrophils have been shown to migrate to the injection site at 6, 24, and 72 hours after immunization with aluminum preparations. Neutrophil transport is related to increased expression of macrophage inflammatory protein 2 (MIP2) or chemokine (chemokine) master ligand 2 (CXCL2) and keratinocyte chemokine (KC) expression. Increase expression by enhancing proteolytically lysed extracellular matrix. Subcutaneous immunization with AH+lysozyme can increase the consumption of neutrophils, the activation of antigen-specific T cells and the activity of antibody reactivity. This therefore indicates that neutrophils compete with dendritic cells and macrophages for antigen, thereby interfering with antigen presentation. On the contrary, treatment of neutrophils with anti-Ly6G antibody cannot affect the typing and degree of antibodies after AH?+?OVA muscle immunization. These different results may be related to the formulation of the vaccine and different immunization routes.

　　 At the peak of immune cell infiltration seven days after immunization with aluminum preparations, the transfer of macrophages to the injection site has been studied for many years. The transfer may be related to the high expression of CCL2 and carbon tetrachloride at the injection site. A similar pattern of chemokines expressed by human monocytes can react with aluminum preparations in vitro and has also been verified in vivo. However, several studies have also reported the disappearance of macrophages at the injection site of aluminum preparations. Different data may be related to different immunization routes and adjuvant antigen formulations. Importantly, the role of adjuvants in aluminum preparations for activating macrophages has been questioned. Because of intraperitoneal (ip) injection of aluminum preparations, macrophage system depletion does not affect the antibody response. Even after subcutaneous injection of aluminum preparations can increase the antibody response.

　　Similarly, eosinophils increased within 24 hours, and inflammatory cells increased to 25% on the sixth day after immunization with aluminum preparations. The recruitment of eosinophils may be related to interleukin (IL) 5 and histamine released by mast cells, unknown factors secreted by macrophages, and secretion of eosinophil chemotactic protein (CCL11 and CCL24 molecules) after the second immunization . After inoculation, the number of mast cells at the injection site decreased, possibly due to mast cell granulation and cell death. Despite the fact that mast cells are IL-5, IL-16, granulocyte colony stimulating factor (G-CSF), KC, and MIP2, mast cells and macrophages induce secretion of IL-1β and IL-1 receptors. The main source of drug (Ra), IL-6, and eosinophil chemokine. Studies of cell depletion have shown that these cells respond in vivo to unwanted aluminum agents. Compared with its receptor, CCR3, and expression on mouse eosinophils, the role of CCL11 is controversial. It has been confirmed that eosinophils are dispensable after aluminum immunization. Although eosinophils are the main source of IL-4 factors and may cause the initiation of B cells and the production of antibodies, their absence in the body does not affect the quality of the aluminum adjuvant vaccine antibody response. The transfer of monocytes and DC at the injection site is very important because the interaction of APC is the interface between innate immunity and adaptive immune response. The transfer of monocytes is related to various signals of neutrophils. The ability of inflammatory monocytes to differentiate into dendritic cells indicates that an immune response to specific antigens followed by vaccination. It has been confirmed that dendritic cells DCS are actively recruiting to the injection site from 1 day to 7 days after inoculation. The depletion of dendritic cells almost completely abolishes the immune response of T cells and the production of antibodies, which means that dendritic cells mediated by aluminum preparations in vivo are very critical. In this case, the lack of CD8α+ DCs, and other unknown dendritic cell subgroups, have been confirmed to appear in the CD4+ helper T cell initiation process.

　　3.2.2 Aluminum preparations can improve antigen uptake and presentation

　　 reported that aluminum adjuvants can enhance the antigen uptake ability of macrophages and human peripheral blood monocytes and dendritic cells in vivo, and can enhance the antigen uptake ability of dendritic cells in vitro. The use of EαGFP/Yae system method in vitro can show that aluminum preparations enhance the antigen targeting function of dendritic cells. This system shows that aluminum hydroxide has this effect on intracellular antigen storage, and can provide a continuous antigen release to MHCII molecules and prolong the antigen presentation process. Although these data have not been confirmed in vivo experiments. Other factors such as the potential function of dendritic cells uptake of aluminum particles and further results still need to be evaluated. In in vitro experiments, macrophages swallowed aluminum particles and further cell rupture. Flach and his colleagues also confirmed through similar experiments that aluminum preparations can bind to the lipid membrane of dendritic cells (depending on cholesterol and cell movement). Subsequently, the lipid ordering changes, resulting in increased antigen transport without the need for internalization of aluminum formulations. In the same study, aluminum particles were found to exist in dendritic cells, indicating that dendritic cells are phagocytic cells that efficiently take up particles. In vitro experiments have verified in vivo observations, such as histochemistry, electron microscopy, X-ray microanalysis, and atomic absorption spectrophotometry in the subcutaneous injection site granuloma test can find aluminum preparation crystals. They have been confirmed to exist in macrophages. Multinucleated giant cells and MHCII+ monocytes, most likely dendritic cells, T helper cell 1 cell line (THP-1) indicate that these immune helper cells actively engulf aluminum preparations in the body.

"In vivo and in vitro, the uptake of aluminum preparations by MHCII molecule antigen-presenting cells has been observed, but very little is known about the number of MHCII molecule antigen-presenting cells.

　　 3.2.3 Aluminum preparations can enhance the activation and maturation of APC

　　 3.2.4 In vivo aluminum preparations help APCs cells to take up antigen and transport it to lymphatic tissues

　　 3.2.5 Aluminum preparation can produce the default Th2 response