CN102813920A - Vaccine adjuvant - Google Patents

Abstract

The invention belongs to the field of biological products and relates to a vaccine adjuvant, in particular to the use of praziquantel as a vaccine adjuvant and for preparing vaccine complexes and a preparation method thereof. In the present invention, praziquantel is used as a vaccine adjuvant, and it has been confirmed by experiments that the praziquantel can be used as a vaccine adjuvant to prepare antiviral vaccines, including the preparation of hepatitis B vaccine, AIDS vaccine or influenza vaccine, especially the preparation of DNA therein Vaccines, nucleic acid vaccines or subunit protein vaccines. The invention uses praziquantel as an adjuvant, and compared with the existing adjuvants, it can more effectively activate the body's humoral and cellular immunity levels, significantly enhance the immune effect of vaccines, and improve antigen-specific CTL responses. At the same time, the vaccine adjuvant of the present invention provides a better method for eliminating viruses, and is convenient to use, low in cost, has few side effects, and is easy to popularize.

Description

a vaccine adjuvant

technical field

The invention belongs to the field of biological products and relates to a vaccine adjuvant, in particular to the use of praziquantel as a vaccine adjuvant and for preparing vaccine complexes and a preparation method thereof. the

Background technique

Hepatitis B is an infectious disease caused by hepatitis B virus, transmitted through blood and body fluids, and mainly damages the liver. This disease poses a great threat to human health and has become a serious public health problem. Studies have shown that after infection with hepatitis B, some patients will develop chronic persistent infection, and some may develop liver cirrhosis or primary hepatocellular carcinoma. According to reports, my country is a high prevalence area of hepatitis B virus infection, and about 350,000 people die of hepatitis B-related diseases (such as liver cirrhosis, liver cancer, etc.) every year; The antigen (HBsAg) population carrying rate was 10%. It is estimated that there are 300 million HBsAg carriers in the world, and my country accounts for one-third of them. The transmission of hepatitis B has become an important issue affecting the quality of the population. The development of hepatitis B vaccine has gone through the stages of blood-derived vaccine and genetic engineering vaccine. Hepatitis B vaccine plays an important role in the prevention and control of hepatitis B. the

Studies have shown that effective immunotherapy for hepatitis B should be based on stimulating the immune system in virus carriers. The internationally recognized index for evaluating therapeutic hepatitis B vaccine is antigen-specific cellular immune response, especially to stimulate high-level activity of hepatitis B-specific CD8-positive CTL cells and secrete high-level gamma-interferon and other effective immune responses. components. Although it has been reported in the world that the S or C antigen of hepatitis B virus is used as the immunogen in combination with different immune enhancers to improve the level of activated cellular immunity, the clinical effect is still under evaluation. The prior art discloses that the vaccination of hepatitis B vaccine is the most effective measure to control hepatitis B; said hepatitis B vaccine can be divided into synthetic peptide vaccine, gene (nucleic acid) vaccine and recombinant subunit vaccine according to the composition, wherein, synthetic peptide vaccine It is a vaccine formed by imitating certain peptide chains or protein artificially synthesized antigens of specific antigens. Gene (nucleic acid) vaccines are relative to gene (nucleic acid) immunization, which is to clone the gene sequence containing the specific antigen protein into Prepare a nucleic acid expression vector on a suitable plasmid vector, introduce it into the body by intramuscular injection, etc., synthesize the antigen protein through the transcription system of the host cell, and stimulate the body's immune system to generate a specific immune response against the foreign protein. The nucleic acid expression vectors used in the above-mentioned genetic immunization process are called gene vaccines, also known as nucleic acid vaccines. The third type of hepatitis B vaccine is a recombinant subunit vaccine, which is formed by expressing the virus antigen gene in Escherichia coli or yeast system, extracting and purifying it and then mixing it with aluminum adjuvant. In the 1980s, the hepatitis B recombinant subunit vaccine developed rapidly. Since 1981, Merck successfully developed a recombinant subunit vaccine in which the hepatitis B gene S protein was expressed in yeast and compatible with aluminum adjuvant and commercialized. It plays an important role in the prevention and control of hepatitis B worldwide. the

Influenza (abbreviated as influenza) is an acute respiratory infectious disease caused by influenza virus. Influenza has brought huge disasters to human health. It has caused four influenza pandemics in human history, with tens of millions of deaths. In recent years, some countries and regions have caused annual small-scale influenza epidemics, and 9% of the world's population is infected with influenza viruses every year. Influenza virus is easy to mutate and has the characteristics of multiple serotypes; for example, the highly pathogenic H5N1 subtype and the H1N1 subtype A influenza in recent years have strong variability, and are important viral respiratory pathogens, which are very easy to infect people and cause major epidemics . Therefore, in order to prevent the occurrence of the above-mentioned potential outbreaks, effective vaccines against this novel virus urgently need to be widely promoted and produced; however, because the currently widely used inactivated vaccines can only cause antibody responses to antiviral proteins, they fail to meet this requirement. need. Studies have reported that specific cytotoxic T lymphocyte responses against viruses play an important role in controlling viral infections. Therefore, for an inactivated vaccine to control viral infection, it is more important whether it can effectively stimulate the body to produce a specific CTL response than merely inducing the corresponding antibody. the

Research reports, Acquired Immunodeficiency Syndrome (AIDS) for short, has become a serious infectious disease that endangers the whole world. AIDS is an immunodeficiency disease caused by the destruction of the body's CD4 ⁺ T cells after the HIV virus enters the body. There are three main genes encoding HIV retroviral proteins: Env, Pol, and Gag. Pol and Gag mainly encode the nuclear protein and reverse transcriptase protein of the virus, while the Env gene mainly encodes the protein on the surface of the virus membrane, and the epitopes of neutralizing antibodies are basically on the Env gene. Currently there are no effective vaccines and drugs for the prevention and elimination of HIV infection. As an emerging vaccine, DNA vaccine has many advantages such as being irreplaceable by traditional vaccines. DNA vaccines are also regarded as one of the key technologies to overcome AIDS at home and abroad. However, the biggest problem currently restricting the development of DNA vaccines is the weak immunogenicity. It is recognized that one of the important ways to evaluate and prevent HIV virus infection is how to effectively mobilize cellular immune responses, especially to stimulate high levels of HIV-specific CD8-positive CTL cell activity and secrete high levels of γ-interferon and other effective immune responses. components. Therefore, finding an effective adjuvant to enhance the effect of DNA vaccine is imminent.

Studies have confirmed that in the process of vaccine preparation, adjuvants, as substances that can enhance immunogenicity or change the type of immune response together with antigens or pre-injected into the body, are favored by researchers in the development of new and highly effective vaccine complexes. Pay attention to. Adjuvants, also known as non-specific immune proliferators, are not antigenic in themselves. They are substances that precede or act simultaneously with antigens and can non-specifically change or enhance the body's specific immune response to antigens. Together or pre-injected into the body can enhance immunogenicity or change the type of immune response. The main biological effects of immune adjuvants include the following aspects: After the antigenic substance mixed with the adjuvant is injected into the body, the physical properties of the antigen are changed, the antigenic substance can be released slowly, and the action time of the antigen is prolonged; after the adjuvant absorbs the antigen , increase the surface area of the antigen, so that the antigen is easy to be phagocytized by antigen-presenting cells; adjuvants can stimulate antigen-presenting cells to process antigens; adjuvants can promote contact between lymphocytes and enhance the role of helper T cells; can stimulate sensitization The division of lymphocytes and the production of antibodies by plasma cells. the

The adjuvant is not only an important component in the production of inactivated vaccines, but also plays a role in improving the immune effect of DNA vaccines and subunit protein vaccines. Especially in inducing a more effective antiviral immune response, the participation of T cell immunity is essential. At present, there are almost no adjuvants that improve the immune response of vaccine cells in clinical application, and the adjuvants currently used, such as aluminum salt adjuvant, oil adjuvant and SF59 adjuvant, are all adjuvants that mainly improve the antibody level. Therefore, Adjuvants to enhance cellular immune responses, especially CD8 T cell immune responses, are urgently needed clinically. Therefore, for the screening of adjuvants for enhancing the immune effect of new hepatitis B, AIDS and influenza vaccines, developing a new type of adjuvant for hepatitis B and influenza vaccines has become an urgent research focus. the

Praziquantel (PZQ) is a new type of broad-spectrum antiparasitic drug, which has obvious inhibitory effect on the glucose metabolism of worms. Currently, it is used as a broad-spectrum tapeworm drug, anti-schistosomimetic drug and anti-flukeworm drug. Studies have found that after treatment with praziquantel, it can effectively enhance the secretion of IgE, inhibit the secretion of IgG4, and at the same time enhance the expression of Th2 cytokines, which can effectively resist the recurrence of schistosomiasis. It has been reported that after praziquantel treatment, the proportion of regulatory T cells in patients will decrease, and the ability of natural regulatory T cells to express CD45O will be greatly weakened, which will enhance the body's ability to resist re-infection of schistosomiasis. For a long time, the research on praziquantel has mainly focused on the field of prevention and treatment of human or animal parasitic diseases. the

So far, there have been no reports about praziquantel as an antiviral (especially hepatitis B, AIDS and influenza) vaccine adjuvant. the

Contents of the invention

The purpose of the present invention is to provide a new vaccine adjuvant, especially to enhance cellular immune response, especially to enhance the vaccine adjuvant of CD8 T cell immune response, described vaccine adjuvant hepatitis B is vaccine adjuvant, AIDS vaccine adjuvant or influenza vaccine adjuvants. the

The present invention provides a new medicinal use of praziquantel, specifically relates to the use of praziquantel as a vaccine adjuvant and for preparing vaccine complexes and its preparation method; the present invention utilizes the praziquantel as an adjuvant It can enhance the immune effect of vaccines, significantly increase the level of antibodies and cellular immunity, and improve the CTL response of antigen specificity. the

The present invention uses praziquantel as a vaccine adjuvant, and experiments have confirmed that the praziquantel can be used as a vaccine adjuvant for preparing antiviral vaccines, especially for preparing anti-hepatitis B, AIDS or influenza vaccines; wherein, the The vaccines available are DNA vaccines, nucleic acid vaccines or subunit protein vaccines. the

In the present invention, the chemical name of the praziquantel: 2-cyclohexylformyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-α]isoquinoline -4-ketone, molecular formula: C19H24N2O2, molecular weight: 312.41, has the structure of formula (I),

In the present invention, the hepatitis B vaccine is hepatitis B DNA vaccine, hepatitis B subunit vaccine; wherein, the hepatitis B subunit vaccine is to express the virus antigen gene in Escherichia coli or yeast system to produce protein through genetic engineering technology, Made after extraction and purification with an adjuvant, especially with praziquantel adjuvant;

In the present invention, the AIDS vaccine is an AIDS DNA vaccine, which can be amplified in Escherichia coli, extracted and purified, and then formulated with an adjuvant, especially with praziquantel adjuvant;

In the present invention, the influenza vaccine is an inactivated influenza virus vaccine, which can be prepared by inoculating chick embryos, amplified, extracted and purified, and then compatible with an adjuvant, especially praziquantel adjuvant. the

Another object of the present invention is to provide a vaccine compound for preventing hepatitis B, AIDS or influenza; said vaccine compound is formulated by combining viral antigen or DNA vaccine or subunit protein vaccine with praziquantel adjuvant production. the

The invention provides a vaccine compound for treating hepatitis B or AIDS; the vaccine compound is made of virus antigen, DNA vaccine or subunit protein vaccine and praziquantel adjuvant. the

In the present invention, the vaccine for the treatment of hepatitis B is hepatitis B DNA vaccine, hepatitis B subunit vaccine; the described AIDS vaccine is AIDS DNA vaccine; The mass is 0.25-1% of the vaccine complex, wherein the preferred praziquantel adjuvant is 0.25%, 0.5%, 1%, more preferably the praziquantel adjuvant is 0.5%, and the vaccine complex It may also contain pharmaceutically acceptable carriers, solvents or other auxiliary components. the

In the present invention, the preparation method of described vaccine complex is:

Those skilled in the art can use conventional techniques to compound the praziquantel adjuvant with the vaccine to prepare a vaccine complex; for example, dissolve an appropriate amount of praziquantel in an appropriate amount of ethanol to prepare a mother liquor, and then add the mother liquor to An appropriate amount of normal saline is prepared as a standard solution (the concentration can be adjusted by adding normal saline according to the need), and finally the pre-prepared vaccine is dissolved in the standard solution to obtain a vaccine compound that can be directly used; or, the standard After the vaccine is added to the solution, it is prepared into a dry powder preparation or injection by conventional techniques (such as freeze-drying, or freeze-spray drying, etc.) for storage. the

The present invention provides the use of the praziquantel as a vaccine adjuvant and vaccine complex and its preparation method, but those skilled in the art can use any known hepatitis B vaccine in the prior art during the specific implementation process (such as hepatitis B DNA vaccine pcD-S2, hepatitis B subunit vaccine prepared by recombinant rHBsAg pure antigen, influenza vaccine of inactivated virus, etc.); in addition, conventional techniques in the art can also be used to prepare praziquantel adjuvant, For example, 0.2 grams of praziquantel is dissolved in 3 milliliters of ethanol to prepare a 6.7% mother solution, then add 15 milliliters of mother liquor to 85 milliliters of normal saline to make a 1.0% solution, and finally add normal saline to configure into 0.5% and 0.25% solutions. the

In the present invention, the praziquantel can be used to prepare hepatitis B, AIDS or influenza vaccine adjuvant or vaccine complex, and the described hepatitis B, AIDS or influenza vaccine is prepared by DNA vaccine, subunit protein, or inactivated virus as antigen The vaccines, all of which are for human use. the

In the present invention, the content of the antigen (or vaccine) in the vaccine complex belongs to common knowledge or can be determined through routine exploration. the

The present invention also proves through experiments that the effect is most obvious when the mass of the praziquantel adjuvant (that is, the praziquantel as an adjuvant) is 0.25-1% of the vaccine complex. the

Compared with the prior art, the vaccine adjuvant of the present invention has the following advantages:

(1) The present invention utilizes praziquantel as a hepatitis B vaccine adjuvant, which can effectively enhance the immune response of the hepatitis B vaccine, and provides new research directions and hotspots for the research of vaccine adjuvants;

(2) The praziquantel adjuvant of the present invention can more effectively activate the body's humoral and cellular immunity levels, significantly enhancing the immune effect;

(3) The vaccine adjuvant of the present invention is easy to use, low in cost, has little side effects, and is easy to popularize;

(4) The vaccine adjuvant of the present invention can activate the CD8 ⁺ T cell response of the body more highly, including the activation of Tc1 and Tc17, and provide a better way for clearing the virus.

For ease of understanding, the vaccine adjuvant of the present invention will be described in detail below with reference to the accompanying drawings and specific examples. It should be pointed out that the specific embodiments and accompanying drawings are only for illustration. Obviously, those skilled in the art can make various amendments or changes to the present invention according to the description herein, and these amendments and changes will also be included within the scope of the present invention. the

Description of drawings

Figure 1 shows the detection of IgG of praziquantel as a hepatitis B vaccine adjuvant by quantitative ELISA in the present invention. the

Fig. 2 shows the detection result of T lymphocyte expansion of the adjuvant praziquantel in the present invention to enhance the immune response of hepatitis B DNA vaccine. the

表示6只正常未免疫的C57BL/6小鼠对照；图中的数值为6只小鼠的平均值±标准差；^*表示P＜0.05。  Figure 3 shows the results of detecting the expression of IFN-γ, IL-4 in CD4 T cells and IFN-γ in CD8 T cells in the present invention; wherein, the number of CD4+ cells expressing IL-4 or IFN-γ,

Indicates 6 normal non-immunized C57BL/6 mouse controls; the values in the figure are the mean ± standard deviation of 6 mice; ^* indicates P<0.05.

Fig. 4 shows the in vitro CTL response after the adjuvant praziquantel enhances the immune response detected by flow cytometry in the present invention. the

Fig. 5 shows the IgG antibody quantitative detection results of the adjuvant praziquantel in the present invention enhancing the immune response of the hepatitis B subunit vaccine. the

Fig. 6 shows the detection result of DTH enhanced by the adjuvant praziquantel in the present invention in the immune response of hepatitis B DNA vaccine. the

Figure 7 shows the detection results of the adjuvant praziquantel in the present invention enhancing the immune response of hepatitis B DNA vaccine Tc1 and Tc17 cell activity. the

Fig. 8 shows the detection results of the Tc1 and Tc17 cell activity of the adjuvant praziquantel in the present invention to enhance the immune response of hepatitis B DNA vaccine in HBsAg transgenic mice. the

Figure 9 shows the detection results of the adjuvant praziquantel in the present invention enhancing the hepatitis B DNA vaccine's ability to clear the surface antigen-expressing hepatocytes by CD8 cells after immunizing hepatitis B surface antigen transgenic mice. the

Figure 10 has shown the detection result by quantitative ELISA method among the present invention, and wherein, A is the total IgG content that detects praziquantel as AIDS DNA vaccine adjuvant, B is the IgG2a subtype that detects praziquantel as AIDS DNA vaccine adjuvant C is the IgG1 subtype content of praziquantel as an AIDS vaccine adjuvant. the

Fig. 11 shows the results of detecting the expression of IFN-γ in CD4 T cells when the adjuvant praziquantel enhances the immune response of AIDS DNA vaccine in the present invention. the

Figure 12 shows the results of detecting the expression of IL-4 in CD4 T cells when the adjuvant praziquantel enhances the immune response of AIDS DNA vaccine in the present invention. the

Fig. 13 shows the detection results of detecting IFN-γ in CD8 T cells when the adjuvant praziquantel enhances the immune response of AIDS DNA vaccine in the present invention. the

Figure 14 shows the CTL response in vivo after the adjuvant praziquantel enhances the immune response detected by flow cytometry in the present invention. the

Figure 15 shows the levels of cytotoxicity and antibody responses in mice after immunization in the present invention; wherein, C57BL/6 mice were immunized with H5N1 inactivated vaccine, or solvent or praziquantel; specific cell lysis in A The ratio is obtained by summarizing three independent experiments; the serum in B was collected on the 14th day after immunization, and the antibody level was detected by ELISA, and the data shown is a representative data of three independent experiments; *, p<0.05.ns, p>0.05), Inactive V represents inactivated vaccine. the

Figure 16 shows the expression of CD8 ⁺ T cell antigen-specific cytokines analyzed by FACS assay in the present invention; wherein, 7 days after the initial immunization, CD8 ⁺ T cells were sorted from the spleen of C57BL/6 mice, and treated with NP peptide Cultured after stimulation for 6 hours; CD8 ⁺ T cells were stained intracellularly for IFN-γ and IL-17; percentages in A and B are from three independent experiments; **, p<0.01.ns, p>0.05.

Figure 17 shows the level of cytotoxic response in mice after wild-type and CD8K0 or IL-17K0 immunization in the present invention; wherein, A is 7 days after the initial immunization, CTL in vivo was performed on wild-type and CD8K0 mice, and specific The rate of sexual lysis is summarized; B is 7 days after the initial immunization, CTL in vivo was performed on wild-type and IL-17K0 mice, and the rate of specific lysis is summarized; *p<0.05.ns, p>0.05. K0 stands for gene knockout. the

Fig. 18 has shown the infection situation of protecting highly pathogenic avian influenza virus in the present invention; Wherein, A is the detection of body weight change from the 7th day to the 13th day after the immunized mouse is infected with the virus, and C57BL/6 mouse is immunized for the first time The virus was challenged one month later; B is the monitoring of the death of the immunized mice from the 7th day to the 17th day after the virus infection, the data came from three independent experiments, and 10 mice were analyzed each time. the

Figure 19 shows the CD8+T cell adoptive transfer of the present invention slows down the symptom of virus infection; Wherein, mouse carries out vaccine immunization 7 days after taking out CD8+T cell, and adoptive transfer in normal mouse body; A is that mouse is infected with virus Detection of body weight changes from day 7 to day 13, each mouse received 2x10 ⁶ cells, and then challenged immediately; B is the monitoring of the death of mice from day 7 to day 17 after virus infection, the data comes from Three independent experiments, each analysis of 10 mice.

Detailed ways

The experimental methods mentioned in the following examples are conventional methods unless otherwise specified; the mentioned percentages are all mass percentages unless otherwise specified. the

Embodiment 1 prepares DNA vaccine

The hepatitis B DNA vaccine pcD-S2 uses the virus in the serum of hepatitis B patients as a template, reverse transcribes it into cDNA, and then amplifies preS2 by PCR. After the PCR product is digested and recovered, it is connected to the pcDNA3. proves the expression. the

Plasmid DNA was extracted from Escherichia coli, protein was removed in phenol-chloroform solution, and double-stranded DNA was separated by ethanol precipitation. the

Above-mentioned extraction method and technique are recorded in " Molecular Cloning " (second edition 1998, Cold Spring Harbor Laboratory Press, New York) of people such as Sambrook and " biochemistry and molecular biology experimental technique " (Zhejiang University Press) edited by Li Chaolong etc. , methods and techniques known in the art. the

Embodiment 2 prepares DNA or nucleic acid subunit vaccine

The pure rHBsAg antigen of hepatitis B was purchased from North China Pharmaceutical Group, expressed by CHO cells, purified by HPLC system to obtain pure rHBsAg antigen (purity ≥ 99%), and prepared DNA or nucleic acid subunit vaccine. the

Example 3 The experiment of the hepatitis B vaccine compound immunization model animal comprising adjuvant praziquantel

Praziquantel (PZQ) bulk drug was purchased from North China Pharmaceutical Group Co., Ltd. (batch number 081008);

Prepare praziquantel mother solution: 0.2 g of praziquantel is dissolved in 3 ml of ethanol to form a 6.7% mother solution, then add 15 ml of the mother solution to 85 ml of normal saline to form a 1.0% solution, and finally add physiological Saline, configured as 0.5% and 0.25% solutions. the

6-8 weeks old female C57BL/6 mice were divided into 9 groups, with 6 mice in each group, and the experimental groups were shown in Table 1,

Table 1. Injection groups

Each group of vaccines was dissolved in normal saline, and each mouse was injected with 100 microliters. On day 0, the vaccine was injected intramuscularly for the first time, and then re-injected at an interval of 14 days after the first injection, for a total of three injections. the

Embodiment 4 adjuvant praziquantel enhances IgG, IgG1 and IgG2a antibody quantitative detection of hepatitis B DNA vaccine immune response

Seven days after the third intramuscular injection, blood was collected to separate serum, and the levels of IgG, IgG1 and IgG2a antibodies were detected by quantitative ELISA. According to the instructions of the Hepatitis B virus surface antibody diagnostic kit (purchased from Beijing Jinhao Pharmaceutical Co., Ltd., batch number S20053020) for quantitative detection of IgG, the reference antibody against hepatitis B was 8 IU/ml (IU, international unit) (Beijing Jinhao Pharmaceutical Co., Ltd. Co., Ltd., batch number 20060701) was diluted 5 gradients by 2 times, and finally developed the color according to the instructions, placed at 450nm/620nm to measure the optical density value of each well, made a standard curve, and calculated the antibody content. For the quantitative ELISA method to detect IgG1 and IgG2a antibody levels separately, 48 wells of a 96-well microtiter plate were coated with 2ug/ml rHBsAg antigen, and 2ug/ml rabbit IgG (Sigma) was coated on the same 96-well microtiter plate In another 48 wells, overnight at 4°C, block with 3% bovine serum albumin at 37°C for 2 hours; wash 4 times with PBST (the solution obtained by dissolving Tween 20 in PBS so that the final concentration of Tween 20 is 0.05%); 1:100 times dilution, mouse anti-rabbit IgG (Sigma) was diluted 10 times from 100ng/ml in 10 gradients, incubated at 37°C for 1h; washed 4 times with PBST, 5 minutes each time; Oxidase-labeled IgG1 and IgG2a antibodies (Sigma) (both diluted 1:4000) were incubated at 37° C. for 1 h. Add substrate TMB to each well, develop color in the dark at 37°C for 5-15 minutes, add stop solution 2mol/L H ₂ SO ₄ , 50 μl per well, stop color development, place at 450nm/620nm to measure the optical density value of each well, and make a standard Curve to calculate the antibody content.

The results are shown in Figure 1 (the values in Figure 1 are the mean ± standard deviation of 6 mice; ^** represents P <0.01; mIU is milliinternational units):

空白对照组、无佐剂无抗原的pcDNA3对照组、无抗原的PZQ佐剂对照组以及无抗原的乙醇对照组，IgG水平最低，表明以上组合基本上不具备免疫效果；  (1)

In the blank control group, the pcDNA3 control group without adjuvant and antigen, the PZQ adjuvant control group without antigen, and the ethanol control group without antigen, the IgG level was the lowest, indicating that the above combinations basically have no immune effect;

(2) Both the immunized antigen group without adjuvant (pcD-S2) and the immunized antigen group with ethanol adjuvant (ethanol+pcD-S2) produced low levels of IgG, which indicated that the immunized antigen group (pcD-S2) had a certain Immune effect, and ethanol basically did not produce obvious immune-enhancing effect;

(3) Compared with the above combinations, the immune antigen group (pcD-S2) containing adjuvant PZQ (0.25%, 0.5%, 1.0%) can produce high levels of IgG, and the antigen group with 0.5% adjuvant PZQ effect is most pronounced. the

The above results show that the adjuvant PZQ can induce a high humoral immune response to the hepatitis B vaccine. the

Example 5 Adjuvant Praziquantel Enhances the Detection of T Lymphocyte Expansion of Hepatitis B DNA Vaccine Immune Response

Mice were sacrificed 7 days after the third intramuscular injection. Under aseptic conditions, the spleens of the mice were taken and crushed. Red blood cells were removed with erythrocyte lysate and B cells were passed through a nylon column to make a single cell suspension, washed with PBS. 3 times, centrifuge and count the cells, adjust the cell concentration to 1×10 ⁶ cells/ml, add 3 parts of each group of cell suspensions to the 96-well culture plate; one part is added with rHBsAg antigen to a final concentration of 5 μg/ml One part was added with ConA (concanavalin A) to a final concentration of 5 μg/ml as a positive control, and one part was added with BSA to a final concentration of 2 μg/ml as a negative control. After culturing for 48 hours, 20 μl of MTT (tetramethyl azolium salt), cultured for 4 hours, centrifuged at 2,000 rpm for 5 minutes, discarded the cell supernatant, added 100 μL DMSO (dimethyl sulfoxide), placed in a 37-degree incubator for 15 minutes, and read 490nm with a microplate reader For OD value, the stimulated index (SI) was calculated, SI=(experimental group OD-medium OD)/(cell OD-medium OD). Wherein, the OD of the experimental group refers to the OD value read by the antigen-stimulated cells, and the cell OD refers to the OD value read by the cells not stimulated by the antigen.

The results are shown in Figure 2 (in Figure 2, ConA and BSA are each the mean ± standard deviation of 6 mice, and the others are the mean ± standard deviation of 6 mice; ^* indicates P＜0.05):

(1) Since ConA has a strong effect of stimulating T cells, which usually far exceeds the stimulating effect of conventional or novel antigens, ConA as a positive control exhibits the strongest effect of stimulating T cells;

空白对照组、无佐剂无抗原的pcDNA3对照组、无抗原的佐剂PZQ对照组以及无抗原的乙醇对照组，基本上刺激T细胞的效应很低，表明以上物质如果作为佐剂而不联合抗原，其效果和阴性对照基本上相同；  (2) The negative control group of BSA,

The blank control group, the pcDNA3 control group without adjuvant and antigen-free, the adjuvant PZQ control group without antigen, and the ethanol control group without antigen basically have a very low effect on stimulating T cells, indicating that if the above substances are used as adjuvants without combining Antigen, its effect is basically the same as the negative control;

(3) Both the immunized antigen group (pcD-S2) without adjuvant and the immunized antigen group (pcD-S2) with adjuvant (ethanol or PZQ) produced a high level of T cell stimulating effect, indicating that the immune antigen group (pcD-S2) -S2) has a certain immune effect, and ethanol has basically no obvious immune-enhancing effect; in addition, the immune antigen group (pcD-S2) containing adjuvant PZQ (0.5%, 1.0%) has a more obvious effect of stimulating T cells (P<0.05), and the antigen group with 0.5% adjuvant PZQ had the most significant effect. the

Example 6 Flow cytometry detection of the expression of IL-4 and IFN-γ in CD4 cells after the adjuvant praziquantel enhances the immune response

Mice were killed 7 days after the third intramuscular injection, T lymphocytes were obtained by the above method, stimulated with 10 μg/ml rHBsAg for 12-14 hours, then acted with 2 μL of 100 μg/ml monensin for 2 hours, washed 3 times with PBS, Anti-Fcγ antibody (eBioscience, USA) was blocked at 4°C for 15min, washed three times with PBS, stained with anti-CD4-FITC antibody (eBioscience) at 4°C for 30min, washed three times with PBS, fixed with 4% paraformaldehyde at 4°C for 10min, washed with PBS 3 times, membrane rupture with 0.1% saponin for 10 minutes, washed 3 times with PBS, stained with anti-IL-4-PE or IFN-γ-FITC antibody (eBioscience Company) for 15 minutes, washed 3 times with PBS, and detected by flow cytometry. the

The expression results of IFN-γ are shown in the upper panel of Figure 3:

(1) In the blank control group, pcDNA3 control group without adjuvant and antigen, PZQ control group without antigen adjuvant and ethanol control group without antigen, the expression level of IFN-γ was very low, indicating that the above combinations basically had no immune effect;

(2) Both the immune antigen group without adjuvant (pcD-S2) and the immune antigen group with ethanol adjuvant (pcD-S2) produced low levels of IFN-γ expression, which indicated that the immune antigen group (pcD-S2) had Certain immune effect, and ethanol has basically no obvious immune-enhancing effect;

(3) Compared with the above combinations, the immunized antigen group (pcD-S2) containing adjuvant PZQ (0.25%, 0.5%, 1.0%) can produce high levels of IFN-γ, and compared with the only immunized antigen group (pcD-S2). Compared with S2), the level of IIFN-γ has been significantly increased, and the effect of the antigen group with 0.5% adjuvant PZQ is the most significant. the

The above results indicated that PZQ can well induce hepatitis B vaccine to produce IFN-γ. the

IL-4 expression results are shown in the middle panel of Figure 3:

空白对照组、无佐剂无抗原的pcDNA3对照组、无抗原的佐剂PZQ对照组以及无抗原的乙醇对照组，IFN-γ表达水平很低，表明以上组合基本上不具备免疫效果；  (1)

In the blank control group, pcDNA3 control group without adjuvant and antigen, PZQ control group without antigen adjuvant and ethanol control group without antigen, the expression level of IFN-γ was very low, indicating that the above combinations basically had no immune effect;

(2) Antigen-containing pcD-S2 groups (such as pcD-S2, pcD-S2+ethanol, pcD-S2+PZQ) all showed better expression levels of IL-4I than the above combinations, although antigen+0.5%PZQ had no obvious IL-4I expression The expression of -4 increased, but antigen + ethanol also did not significantly increase the expression of IL-4, indicating that PZQ has the ability to promote the immune response of hepatitis B vaccine in the direction of Th1. the

Example 7 Flow cytometry detection of the expression of IFN-γ in CD8 cells after the adjuvant praziquantel enhances the immune response

CTL response is closely related to the expression of IFN-γ in the immune system, and it is an important cytokine that reflects CTL response. Mice were sacrificed 7 days after the third intramuscular injection, T lymphocytes were obtained by the method in step 2, and 10 ^-6 mol of hepatitis B S antigen short peptide (aa208-215) was added to 1×10 ⁶ cells (Shanghai Jier Biochemical Co., Ltd. Co., Ltd., product number is 52633), after stimulation for 12-14 hours, 2 μL of 100 μg/ml monensin was applied for 2 hours, washed 3 times with PBS, blocked with anti-Fcγ antibody (eBioscience, USA) for 15 minutes at 4°C, washed 3 times with PBS, Anti-CD8-PE antibody (eBioscience, USA) was stained at 4°C for 30 minutes, washed three times with PBS, fixed with 4% paraformaldehyde at 4°C for 10 minutes, washed three times with PBS, ruptured with 0.1% saponin for 10 minutes, washed three times with anti- IFN-γ-FITC antibody (eBioscience, USA) was stained for 15 minutes, washed 3 times with PBS, and detected by flow cytometry.

表示6只正常未免疫的C57BL/6小鼠对照；图中的数值为6只小鼠的平均值±标准差；^*表示P＜0.05)：  The results are shown in the lower figure of Figure 3 (the ordinate is the number of CD8+ cells expressing IFN-γ;

Indicates 6 normal unimmunized C57BL/6 mouse controls; the values in the figure are the mean ± standard deviation of 6 mice; ^* indicates P<0.05):

空白对照组、无佐剂无抗原的pcDNA3对照组、以及无抗原的乙醇对照组，IFN-γ表达水平很低并且水平基本相同，表明以上组合基本上不具备免疫效果；  (1)

In the blank control group, the pcDNA3 control group without adjuvant and antigen, and the ethanol control group without antigen, the expression level of IFN-γ is very low and the level is basically the same, indicating that the above combinations basically have no immune effect;

(2) The adjuvanted PZQ control without antigen, the immune antigen group without adjuvant (pcD-S2) and the immune antigen group with ethanol adjuvant (pcD-S2) all produced higher levels of IFN-γ expression, among which, The PZQ control group has the highest level, indicating that the effect of the adjuvant PZQ group has been better than that of the immune antigen group (pcD-S2) without adjuvant PZQ and the immune antigen group (pcD-S2) with ethanol adjuvant, while ethanol did not produce any Effect;

(3) Compared with the above combinations, the immunized antigen group (pcD-S2) containing adjuvant PZQ (0.25%, 0.5%, 1.0%) can produce higher levels of IFN-γ, and compared with the only immune antigen group (pcD-S2) Compared with -S2), the level of IIFN-γ has been significantly improved, and the effect of the antigen group with 0.5% adjuvant PZQ is the most significant. the

Example 8 Flow cytometry detection of in vitro CTL response after the adjuvant praziquantel enhances the immune response

Normal unimmunized 5-7 week-old C57BL/6 mice were sacrificed, T lymphocytes were obtained by the above method, and two equal parts of the isolated T lymphocytes were obtained; one part was incubated with 10 ^-6 M hepatitis B S antigen short Peptide (aa208～215) (Shanghai Jill Biochemical Co., Ltd., product number is 52633), and another part was incubated with an equal amount of irrelevant peptide OVA (aa323～339) (Shanghai Jill Biochemical Co., Ltd., product number is P01662), the volume of each plate 1-2 mL, 37 ° C, 5% CO ₂ culture for 4 hours (this step can appropriately increase the number of target cells according to the number of experimental groups); after 4 hours, transfer the cells to a 15 mL cell tube, centrifuge at 2000rmp for 5 minutes, and The target cells incubated with OVA (aa 257-264) were stained with a low concentration of CFSE (0.5uM), the target cells incubated with a short peptide of hepatitis B S antigen were stained with a high concentration of CFSE (5uM), and stained at 37°C for 15 minutes in the dark. After staining, stop the reaction with an equal volume of fetal bovine serum, centrifuge at 2000rmp for 5 minutes, discard the supernatant, and wash 3 times with 10 mL of PBS; mix equal volumes of low-concentration and high-concentration stained target cells, and use 2 per mouse. ×10 ⁷ cells were injected into the mice of the above-mentioned immune groups and normal mice through the tail vein to carry out the cytotoxic reaction in vivo; 4 hours after the injection, the experimental mice were killed, and the splenocytes were obtained by separating them in the dark, filtered with copper mesh, and then transferred into In a FACS dedicated tube, ready for instrument detection and analysis. Killing rate (%)=[1-(percentage of specific lysis/percentage of non-specific killing)]×100.

表示6只正常未免疫的C57BL/6小鼠对照，数值为6只小鼠的平均值)：  The result is as shown in Figure 4 (indicates the percentage of killing in the figure,

Represents 6 normal unimmunized C57BL/6 mouse controls, and the value is the average value of 6 mice):

(1) The blank control group, the pcDNA3 control group without adjuvant and antigen-free, the adjuvant PZQ control group without antigen and the ethanol control group without antigen had a very low level of CTL response. Among them, The blank control group and the ethanol control group without antigen are close to 0, indicating that the above combinations basically do not have the effect of stimulating CTL response;

(2) The experimental groups containing antigen pcD-S2 (such as pcD-S2, pcD-S2+ethanol, pcD-S2+PZQ) all showed significantly better levels of stimulated CTL responses than the above combinations, indicating that the antigen pcD-S2 played a role in stimulating play a major role in the level of CTL response;

(3) The antigen pcD-S2 plays a major role in stimulating the level of CTL response, but in the test group added with adjuvant PZQ, its ability to stimulate the level of CTL response is still significantly stronger than that of pcD-S2, pcD-S2+ethanol group, Among them, 0.5% PZQ can produce the highest level of CTL response. the

The above results show that the adjuvant PZQ can promote the immune antigen group (pcD-S2) to produce a higher level of CTL response, wherein, antigen plus 0.5% PZQ can produce a higher level of CTL response, and PZQ can stimulate the hepatitis B nucleic acid vaccine to produce more Consistent with more CD8+IFN-γ. the

Example 9 The experiment of the hepatitis B subunit vaccine complex immunization model animal comprising adjuvant praziquantel

6-8 weeks old female C57BL/6 mice were divided into 6 groups, 6 mice in each group, and the experimental groups were shown in Table 2,

Table 2 Injection groups

Each group of vaccines was dissolved in normal saline, and each mouse was injected with 100 microliters. On day 0, the vaccine was injected intramuscularly for the first time, and after the first injection, the vaccine was injected again at an interval of 14 days, for a total of two injections. the

Example 10 Adjuvant praziquantel enhances IgG antibody quantitative detection of hepatitis B subunit vaccine immune response

Seven days after the third intramuscular injection, blood was collected to separate serum, and IgG antibody levels were detected by quantitative ELISA. Quantitatively detect IgG according to the instructions of the Hepatitis B virus surface antibody diagnostic kit (Beijing Jinhao Pharmaceutical Co., Ltd., batch number S20053020). , Batch No. 20060701) were diluted 5 gradients by 2 times, and finally developed the color according to the instructions, placed at 450nm/620nm to measure the optical density value of each well, made a standard curve, and calculated the antibody content. the

The results are shown in Figure 5 (values are the mean ± standard deviation of 6 mice; ^* indicates P＜0.05):

空白对照组、无抗原的乙醇对照组、无抗原的佐剂PZQ对照组，IgG抗体反应水平相当低，表明单一的佐剂(乙醇或PZQ)基本上不具备刺激IgG抗体反应的效果；  (1)

In the blank control group, ethanol control group without antigen, and adjuvant PZQ control group without antigen, the level of IgG antibody response is quite low, indicating that a single adjuvant (ethanol or PZQ) basically does not have the effect of stimulating IgG antibody response;

(2) The experimental groups containing antigen rHBsAG (such as rHBsAG, rHBsAG+ethanol, rHBsAG+PZQ) all showed significantly better levels of stimulated CTL responses than the above combinations, indicating that the antigen rHBsAG played a major role in stimulating the level of IgG responses;

(3) Antigen rHBsAG plays a major role in stimulating the level of IgG response, but in the test group added with adjuvant PZQ, its ability to stimulate IgG response level is significantly stronger than that of rHBsAG, rHBsAG+ethanol group, indicating that ethanol does not promote immunity. effect. the

The above results indicated that the immunized antigen group (rHBsAg) produced a low level of IgG, and ethanol did not have any effect of promoting immunity, while the antigen plus 0.5% PZQ could produce a higher level of IgG (P<0.05=.

Embodiment 11 adjuvant praziquantel enhances the animal experiment of enhancing the effect of AIDS DNA vaccine

The adjuvant praziquantel was prepared and purified in the laboratory; 100ug interleukin 17A was dissolved in 10ml of 10mM pH7.0 PBS buffer to obtain a 10ug/ml solution. AIDS Env protein-specific neutralizing antibody epitope peptide mB1 (ENFDMWKNDM), mB2 (QKVYALFYRLD), mB3 (PNNNTRKSIRIGPGQTFYAT), specific CTL epitope peptide mCTL1 (WYIKIFIMI), mCTL2 (RYLKDQQLL), mCTL3/Th (TSAITQACPKVSFDPIPIHYCAPAG) epitope All peptides were synthesized by Shanghai Sangon Biochemical Co., Ltd. the

Source and preparation of AIDS nucleic acid vaccine pGX-Envc:

(空白对照组)；第2组免疫30μg pGX-Envc；第3组免疫30μg pGX-Env+EP(电击仪法)；第4组免疫30ug pGX-Env+PZQ+EP；第5组免疫30ug pGX-Env+CIM佐剂+EP；第6组免疫30ugpGX-Env+LMS+EP。免疫采用肌肉注射加电击仪70伏于小鼠后腿股四头肌的方法进行免疫；各组小鼠均免疫3次，每次间隔14天，第三次免疫后的7天处死小鼠检测各项免疫学 指标，  Preparation of DNA vaccine and plasmid DNA: The DNA vaccine pGX-Env plasmid was donated by Professor Dr. David Weiner of the University of Pennsylvania; the plasmid DNA was transferred to DH5α for fermentation and culture, extracted by large-scale alkaline lysis, and purified by Qiagen purification column , and adjust the mass concentration to 1mg/ml with physiological saline and store at -20°C. Animal grouping and immunization methods: Balb/c mice were randomly divided into 6 groups, 10 in each group, the first group

(blank control group); the 2nd group was immunized with 30 μg pGX-Envc; the 3rd group was immunized with 30 μg pGX-Env+EP (electric shock method); the 4th group was immunized with 30ug pGX-Env+PZQ+EP; the 5th group was immunized with 30ug pGX -Env+CIM adjuvant+EP; Group 6 was immunized with 30ugpGX-Env+LMS+EP. Immunization was carried out by intramuscular injection and electric shock at 70 volts on the quadriceps of the mouse hind legs; mice in each group were immunized 3 times with an interval of 14 days, and the mice were killed 7 days after the third immunization. Various immunological indicators,

(1)

(2) pGX-ENVC;

(3) pGX-ENVC+E.P;

(4) pGX-ENVC+PZQ+E.P;

(5) pGX-ENVC+CIM+E.P;

(6) pGX-ENVC+LMS+E.P. the

Embodiment 12 Adjuvant praziquantel enhances IgG, IgG1 and IgG2a antibody quantitative detection of AIDS DNA vaccine immune response

Blood was collected 7 days after the third intramuscular injection to separate the serum, diluted 5 times in 3 gradients, and the antibody levels of IgG, IgG1 and IgG2a were detected by ELISA method; then according to the final color development, the optical density value of each well was measured at 450nm/620nm , calculate the antibody titer curve, specifically, 2ug/ml HIV-1 ENV polypeptide is used as the antigen-coated 96-well ELISA plate at 4°C overnight, and 3% bovine serum albumin is blocked at 37°C for 2h; PBST (dissolved in Tween 20 PBS, the solution obtained by making the final concentration of Tween 20 0.05%) was washed 4 times; the mouse serum was diluted 1:10, 1:50, 1:250 times, and incubated at 37°C for 1h; washed 4 times with PBST, each 5 minutes; add goat anti-mouse horseradish peroxidase-labeled total IgG, IgG1 and IgG2a antibodies (Sigma) (diluted at 1:4000), incubate at 37°C for 1h; add substrate TMB to each well, protect from light at 37°C After developing color for 5-15 minutes, add stop solution 2mol/L H ₂ SO ₄ , 50 μl per well, stop color development, measure the optical density of each well at 450nm/620nm, make a standard curve, and calculate the antibody titer curve.

The result is shown in Figure 10 (the numerical value in the figure is the average value of 6 mice):

空白对照组抗体水平最低，表明上述组合基本上不具备免疫效果；  (1)

The antibody level of the blank control group was the lowest, indicating that the above combination basically has no immune effect;

(2) DNA vaccine immunization group without adjuvant (pGX-ENVC), DNA vaccine immunization without adjuvant plus electric shock method group (pGX-ENVC+EP), and DNA vaccine with CIM adjuvant plus electric shock method immunization Antigen group (pGX-ENVC+CIM+EP) and DNA vaccine with LMS adjuvant plus electroshock method (pGX-ENVC+LMS+EP) all produced low levels of IgG, IgG2a and IgG1 showed that immune DNA vaccine had Certain immune effect, but other adjuvant groups basically did not produce obvious immune-enhancing effect;

(3) Compared with the above combinations, the immunized antigen group containing adjuvant PZQ (pGX-ENVC+LMS+EP) can produce high levels of IgG, and the effect of the antigen group with 0.5% adjuvant PZQ is the most significant. the

The above results show that the adjuvant PZQ can induce the body to produce a high humoral immune response to the AIDS DNA vaccine. the

Cytokine expression levels detected by flow cytometry

Seven days after the third immunization, isolate splenocytes under aseptic conditions, remove B cells through a nylon column to make a single cell suspension, adjust the cell concentration to 5×10 ⁶ cells/mL, add 100 μL of cell suspension to each well to 96 On the well cell culture plate, add HIV-1 type antigen epitope peptide to each well at a final concentration of 5 μg/mL, and set 3 replicate wells for each group of cells. 37°C, 5% CO ₂ , incubate for 8 hours, add monensin and incubate for another 2 hours, collect cells by centrifugation, block with anti-Fcγ antibody, fix with 4% paraformaldehyde for 12 minutes, rupture membrane with 0.1% saponin for 7 minutes, wash twice with PBS , stained with 10 μL fluorescent monoclonal antibody for 30 min at 4°C in the dark, and took 300 μL for flow cytometry detection. FlowJo software for analysis.

As shown in Figures 11-13, the results of CD4 ⁺ and CD8 ⁺ T cells secreting IL-4 and IFN-γ showed that there were statistically significant differences between the PZQ as adjuvant group and other adjuvant groups and no adjuvant groups (P <0.05), indicating that PZQ, as an adjuvant of DNA vaccine, can significantly promote the secretion of IL-4 and IFN-γ by CD4 and CD8 ⁺ T cells, and promote the activation of T cells, thereby enhancing the level of cellular immunity.

In vivo CTL detection

Eight days after the third immunization, the unimmunized blank mice were sacrificed, splenocytes were isolated and made into a single splenocyte suspension, and divided into two equal parts as target cells, one part was added with 10mol/L Env-specific CTL The epitope polypeptide pool (mCTL1+mCTL2+mCTL3) was stimulated, one part was not stimulated, 37°C, 5% CO ₂ , cultured for 4 hours, centrifuged to discard the supernatant, washed once with PBS, unstimulated target cells were treated with 0.5 μmol/L CFSE Stain in the dark for 10 min, and stain the stimulated target cells with 5 μmol/LCFSE in the dark for 10 min, add an equal volume of calf serum to stop the staining for 2 min, discard the supernatant by centrifugation, wash 5 times with PBS, and finally mix the two target cells in equal volumes, and 1×10 ⁷ target cells were injected into the tail vein of immunized mice to carry out cytotoxic killing reaction in vivo. After 4 hours of killing, the mice were sacrificed, and splenocytes were isolated in the dark, and 1 mL of flow cytometer was used for detection and analysis.

The test results are shown in Figure 14. There are differences between the PZQ adjuvant group and other adjuvant and no adjuvant groups, indicating that PZQ as an AIDS DNA vaccine adjuvant can induce a strong CTL killing response in the body. the

Example 13 Influenza vaccine testing results

1. Materials and methods

(1) Reagents and experimental animals

Praziquantel (Huabei Pharmaceutical, Hebei, China) was initially dissolved in ethanol to make a 6.7% solution, and then diluted to 0.5% with normal saline. The solvent group was 7.5% ethanol dissolved in normal saline. All anti-mouse fluorescently labeled monoclonal antibodies used in flow cytometry were purchased from BD Pharmingen (San Diego, California, USA). The NP366-374 peptide of influenza virus nucleoprotein (NP) as a representative CD8+ T cell epitope (ASNENMETM) was synthesized by Jill Biochemical Co., Ltd. C57BL/6 female mice, 8-10 weeks old, were purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences (Beijing, China). All experimental animals were fed with pathogen-free feed and drinking water under the condition of 12-hour photoperiod. the

(2) Vaccines

Highly pathogenic avian influenza virus (HPAIV, H5N1, A/Chicken/Henan/1/04) was stored in a biosafety level 3 laboratory (China Agricultural University, Beijing, China); chicken embryos propagated the virus, and were sterilized with formaldehyde. A live whole virus vaccine was produced; the protein content of the vaccine was determined by the Pierce BCA protein assay kit (Rockford, Illinois, USA), and the H1N1 vaccine was provided by Guangdong Dahuanong. the

(3) Immunization method

C57BL/6 mice were randomly divided into 5 groups, 10 mice in each group. The mice were immunized with 0.1 μg inactivated vaccine alone, and an equal-dose inactivated vaccine and adjuvant combined immunization group was established at the same time, and injected intramuscularly; the volume of each injection was 100 μl.

(4) attack poison

After 4 weeks of single-dose immunization, mice were infected with H5N1 influenza virus by nasal drops. The challenge dose was a lethal dose (10LD50) of mouse-adapted strain A/Chicken/Henan/1/04 (H5N1), and the dose was 20 μl of virus suspension; observe the death of all naive mice within 7-12 days of this infection. the

(5) In vivo CTL detection

Seven days after the third immunization, the C57BL/6 blank mice that had not been immunized were sacrificed, the splenocytes were isolated and made into a single splenocyte suspension, divided into two equal parts, one part was stimulated with 10 ^-6 M NP antigen peptide And stained with a high concentration of CFSE (15 μM, CFSE ^high cells) in the dark; the other was not stimulated with antigen peptides, and stained with a low concentration of CFSE (0.5 μM, CFSE ^low cells) as a non-specific target cell control group. Mix the two kinds of target cells in equal volume, and inject 2× ¹⁰⁷ cells per mouse into the experimental mice through the tail vein; kill the mice after 4 hours, separate the lymph nodes and splenocytes, pass through the target cells and the control group The difference in the fluorescence intensity of CFSE in cells can be analyzed using a flow cytometer FACSCalibur (BD Company, USA).

The formula for calculating the level of specific lysis intensity is as follows: ratio = CFSE ^low %/CFSE ^high %;

Percentage of specific lysis=[1-(ratio of group not stimulated by antigen peptide/ratio of group stimulated by antigen peptide)×100]. the

(6) Detection of anti-H5N1 avian influenza virus antibody level

Antibody levels against H5N1 avian influenza virus in serum were detected by ELISA. the

14 days after the initial immunization, the serum was collected; the ELISA was carried out on a 96-well polystyrene microwell plate, and the reagents included the inactivated vaccine of the H5N1 virus; after blocking with 3% bovine serum albumin for 1 hour After that, add diluted mouse serum to each well on the plate for incubation; then, add a 1000-fold diluted goat anti-mouse IgG antibody (Sigma, St. Louis, Missouri); dissolve with 0.025M phosphate-citrate buffer Add 10 mg of TMB (Sigma Company, St. Louis, Missouri) to each well for color development; use 2M _H2SO4 for color development, and measure the optical density at 450nm/620nm; the OD value of _the experimental well is the control well twice as positive.

(7) Analysis and detection of flow cytometry

On the 7th day after the third immunization, CD8+ T cells derived from mouse splenocytes were sorted out by separating magnetic beads (R&D Company, Huntingdon Valley, Pennsylvania, USA); on a 96-well plate, each well CD8+ T cells with a concentration of 0.5×10 ⁶ cells/20 μl were stimulated with NP antigen peptide (5 μg/ml) and anti-CD28 (5 μg/ml) monoclonal antibody and cultured for 6 hours, 37°C, 5% CO ₂ ; the last 4 hours , add monensin (2 μg/ml) to treat, and then wash the cells three times with PBS/10% FCS. Block cells with Fc receptor antibody (BD, San Diego, USA) for 30 minutes in PBS at 4°C, then add 4% paraformaldehyde and permeabilize cells with saponin, use immunohistochemistry for isotype control, or perform double Staining—such as staining with anti-CD8-FITC and anti-IFN-γ-PE, or anti-CD8-FITC and anti-IL-17-PE at 4°C for 1 hour.

Cells were detected with a flow cytometer FACScalibur and analyzed using software CellQuest Pro Software (BD Biosciences). the

(8) Statistical analysis

The results were expressed as ±S.E.M statistics, and the data were analyzed by the t test method; P<0.05 was considered statistically significant. the

2. Experimental results

(1) Praziquantel induces a high level of cytotoxicity in the inactivated vaccine

Since virus-specific CTL responses are essential for the control of virus infection, it was first tested whether combined immunization with inactivated vaccines and praziquantel could induce high levels of nucleoprotein-specific cytotoxic killing. the

Seven days after the first immunization, the mice were re-immunized and tested for cytotoxicity in vivo. Compared with the immunization with inactivated vaccine and solvent group alone, the experimental group immunized with inactivated vaccine and praziquantel showed a higher level of antigen-specific cell lysis reaction (as shown in Figure 15), the results showed that praziquantel Quinoquinones can enhance the cytotoxicity induced by inactivated vaccines. the

On the other hand, the level of antibodies induced by the inactivated vaccine is also crucial for the mice to resist the virus. 14 days after immunization, the antibody titer of the serum was detected to determine whether praziquantel can also affect the humoral immune response induced by the inactivated vaccine. (As shown in FIG. 15B ), compared with the immunization with inactivated vaccine alone and the solvent experiment group, the antibody level of the inactivated vaccine and praziquantel combined immunization group had no significant difference. the

(2) Combined immunization with praziquantel and H5N1 can enhance the activity of Tc17 cells

Since both IFN-γ-secreting CD8 ⁺ T cells (Tc1) and IL-17-producing CD8 ⁺ T cells (Tc17) have cytotoxic activity, it was necessary to determine which T cell subset plays a major role. Seven days after the initial immunization, CD8+ T cells were sorted, and intracellular staining for IFN-γ and IL-17 was performed after in vitro stimulation.

As shown in Figure 16, in the combination immunization group of praziquantel and inactivated vaccine, the ability of antigen-induced CD8 ⁺ T cells to secrete IL-17 was significantly increased, while the level of secreted IFN-γ was not significantly increased, indicating that Tc17 is The key to inducing the body to produce high levels of cytotoxicity.

(3) Knockout of CD8 or IL-17 can weaken the enhanced level of cytotoxic response

To further verify that Tc17 is crucial in the process of inducing cytotoxicity; first, CD8 knockout (KO) mice and wild-type C57BL/6 mice were immunized with inactivated vaccine and praziquantel to detect nucleoprotein-specific cell Toxic solution. As shown in Figure 17A, loss of the CD8 gene attenuated cytotoxicity, suggesting that cytotoxicity in this system is primarily mediated by CD8+ T cells. the

Then, interleukin-17 knockout (KO) mice and wild-type C57BL/6 mice were immunized with inactivated vaccine and praziquantel to detect CTL in vivo; compared with wild-type mice, after immunization, IL-17KO The ability of cytotoxicity in mice (as shown in Figure 17B) was significantly decreased, indicating that the effective expression of IL-17 in CD8+ T cells is necessary to enhance the cytotoxicity of the body. the

(4) Protection against highly pathogenic avian influenza virus infection

To determine whether co-immunization with an inactivated vaccine and praziquantel enhances resistance to lethal infection by highly pathogenic avian influenza. Four weeks after the initial immunization, the mice were challenged; within 7 days after the challenge, the body weight changes of the mice were observed, and the results were shown in Figure 18B. In the negative control group and the praziquantel experimental group, all mice The body weight of the mice all decreased by nearly 30%. Compared with the inactivated vaccine alone immunization or solvent experimental group, the degree of weight loss of the mice in the inactivated vaccine combined with praziquantel immunization group was significantly controlled. the

As shown in Figure 18A, the survival of the experimental mice inoculated with the inactivated vaccine and praziquantel was significantly longer, indicating that the high level of cytotoxicity effectively prevented the infection of the H5N1 virus. the

The mice in the negative control group and the praziquantel group all died within 12 days after challenge. the

(5) Tc17 cells participate in the body's prevention of H5N1 virus infection

IL-17KO and wild-type C57BL/6 mice were immunized with inactivated vaccine and praziquantel, and CD8+ T cells were sorted 7 days after the initial immunization. Cells were purified to a purity of 96-98%, and 2 x ¹⁰⁶ cells per recipient mouse were adoptively transferred intravenously prior to challenge.

As shown in Figure 19A, if the experimental group adoptively transferred CD8+ T cells from immunized wild-type mice, the degree of weight loss of the recipient mice was significantly lower; CD8+ T cells of IFN-γKO mice attenuated the weight loss of mice to a certain extent, but compared to CD8+ T cells of wild-type mice, the degree of weakening was limited. the

As shown in Figure 19B, after challenge, in the experimental group adoptively transferred CD8+ T cells from immunized wild-type mice, 40% of the recipient mice finally survived and received CD8+ T cells from immunized IL-17KO mice. All the experimental mice of the cells eventually died, while the CD8+ T cells of IFN-γKO mice had a certain protective effect, indicating that the Tc17-mediated cytotoxic response is crucial for the prevention and control of H5N1 virus infection. the

The above test results show that the praziquantel of the present invention can effectively enhance the immune response of hepatitis B vaccine as an adjuvant for hepatitis B vaccine, and it can more effectively activate the body's humoral and cellular immunity compared with existing adjuvants. level, significantly enhanced the immune effect; at the same time, the vaccine adjuvant of the present invention can activate the CD8 ⁺ T cell response of the body higher, including the activation of Tc1 and Tc17, providing a better way to clear the virus, and it is easy to use, The cost is low, the side effect is small, and it is easy to popularize.

Claims (11)

1. a vaccine adjuvant, is characterized in that, described vaccine adjuvant is hepatitis B vaccine adjuvant, AIDS vaccine adjuvant or influenza vaccine adjuvant, and described vaccine adjuvant adopts the praziquantel of formula (I) structure Preparation, its chemical name: 2-cyclohexylformyl-1,2,3,6,7,11b-hexahydro-4H-pyrazino[2,1-α]isoquinolin-4-one, molecular formula: C19H24N2O2, molecular weight: 312.41,

2. the purposes of the vaccine adjuvant of claim 1 in the preparation antiviral vaccine. 3. The use according to claim 2, characterized in that said antiviral vaccine is hepatitis B vaccine, AIDS vaccine or influenza vaccine. 4. The use according to claim 2, characterized in that the vaccine is a DNA vaccine, a nucleic acid vaccine or a subunit protein vaccine. 5. according to the described purposes of claim 3, it is characterized in that, described hepatitis B vaccine is hepatitis B DNA vaccine, hepatitis B subunit vaccine; Described AIDS vaccine is AIDS DNA vaccine; Described influenza vaccine is influenza virus inactivated vaccine. 6. The use according to any one of claims 3-5, characterized in that said vaccine is a human vaccine. 7. The use of the vaccine adjuvant of claim 1 in the preparation of vaccine complexes for preventing hepatitis B, AIDS or influenza. 8. The use of the vaccine adjuvant according to claim 1 in the preparation of vaccine complexes for the treatment of hepatitis B or AIDS. 9. The use according to claim 7 or 8, characterized in that, in the vaccine compound, the mass of the adjuvant praziquantel is 0.25-1% of the vaccine compound. 10. The use according to claim 7 or 8, characterized in that, in the vaccine compound, the mass of the adjuvant praziquantel is 0.5% of the vaccine compound. 11. The preparation method of the vaccine adjuvant of claim 1, is characterized in that, it comprises the step: the praziquantel of 0.2 gram is dissolved in the ethanol of 3 milliliters, is configured into the mother liquor of 6.7%, then the mother liquor of 15 milliliters is added 85 ml of normal saline, configured as 1.0% solution, and finally added with physiological saline, configured as 0.5% and 0.25% solutions.

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