2apos; , 3apos; application of cGAMP in preparation of medicine for enhancing varicella-zoster virus vaccine immune response

Abstract

The invention belongs to the technical field of immunology, and particularly discloses application of 2 ', 3'-cGAMP in preparation of a medicine for enhancing chicken pox-herpes zoster virus vaccine immune response. The 2 ', 3'-cGAMP combined varicella-zoster virus vaccine disclosed by the invention can improve the level of a specific IgG antibody, enhance the cellular immune response intensity of a gE antigen and improve the secretion amount of IFN-gamma and IL-2, so that the immune response of the varicella-zoster virus vaccine is enhanced, the immunoprophylaxis effect is enhanced, and the 2 ', 3'-cGAMP combined varicella-zoster virus vaccine adjuvant can be used for preparing a vaccine adjuvant for treating the varicella-zoster virus. Good application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology, and specifically relates to the application of 2',3'-cGAMP in the preparation of a medicine for enhancing the immune response of a varicella-zoster virus vaccine. Background Art

[0002] Varicella-zoster virus (VZV), a double-stranded DNA virus belonging to the alphaherpesvirus subfamily of the herpesviridae family, poses a significant threat to human health worldwide.

[0003] Varicella mainly attacks children, and shows obvious seasonal epidemic characteristics in temperate regions, with winter and spring being the peak seasons. Globally, the annual incidence of varicella in children in developed countries can reach 3,000 to 5,000 per 100,000 people. The incidence is often higher in developing countries due to factors such as high population density and uneven sanitary conditions. In my country, varicella is also a very common infectious disease among children. Although its incidence has declined in recent years with the promotion and application of varicella vaccines, the prevention and control of varicella is still severe in places where children gather, such as schools. Varicella is mainly transmitted through respiratory droplets and direct contact. Children usually have typical symptoms such as fever and rash. The rash is distributed centripetally and goes through four stages: macules, papules, herpes and scabs.

[0004] Herpes zoster is more common in middle-aged and elderly people, and its incidence is positively correlated with age. From a global perspective, the annual incidence of herpes zoster in the general population is about 3-5‰, while in developed countries, due to the accelerated aging of the population, the incidence of herpes zoster in the elderly over 60 years old can rise to about 10-15 people per 1,000 people. With the increasingly prominent problem of an aging population, the incidence of herpes zoster is not optimistic. It is estimated that the incidence rate in people over 50 years old is about 2-3 people per 1,000 people. The pathogenesis of herpes zoster is the reactivation of VZV lurking in the ganglion. Patients often show clustered blisters distributed in a band-like manner along one side of the peripheral nerve, accompanied by neuralgia. The pain symptoms of some patients can last for months or even years, seriously affecting the patient's quality of life.

[0005] The existing varicella-zoster virus vaccine has played a certain role in preventing and controlling the disease. Varicella vaccine has been included in the immunization plan or recommended for vaccination in many countries around the world. For example, the two-dose vaccination rate of varicella vaccine in the United States exceeds 90%. Although varicella vaccine in my country is a Class II vaccine, the vaccination rate is also gradually increasing, and in some economically developed regions it can reach 70%-80%. The herpes zoster vaccine is also widely used in Europe and the United States for middle-aged and elderly people over 50 years old, and has achieved the effect of reducing the risk of disease and alleviating symptoms. It is also gradually recognized and accepted by the public in my country. However, the existing vaccines still have limitations.

[0006] At the immune response level, specific IgG antibodies are crucial for resisting VZV infection. They can specifically recognize and bind to the virus, thereby preventing the virus from invading and spreading. The gE antigen plays an important role in the cellular immune response, and the intensity of its related cellular immune response is directly related to the body's immune clearance ability against VZV. In addition, cytokines IFN-γ and IL-2 occupy a core position in the immune regulatory network. IFN-γ can enhance the phagocytic function of macrophages, promote T cell differentiation, and activate natural killer cells, while IL-2 is indispensable for the proliferation, activation, and formation of immune memory of T cells. However, the current varicella-zoster virus vaccine has not yet reached the ideal state in stimulating the body to produce sufficiently high levels of specific IgG antibodies, sufficiently strong gE antigen cellular immune response intensity, and sufficient IFN-γ and IL-2 secretion.

[0007] In view of this, research and development of enhanced adjuvants specifically for the immune response to varicella-zoster virus vaccine, to increase the level of specific IgG antibodies, enhance the intensity of cellular immune response to gE antigen, and increase the secretion of IFN-γ and IL-2, is of extremely important scientific value and urgent practical need for further optimizing the immune effect of varicella-zoster virus vaccine and strengthening the prevention and control of varicella and herpes zoster diseases. Summary of the invention

[0008] The purpose of the present invention is to provide the use of 2',3'-cGAMP in the preparation of a drug for enhancing the immune response of varicella-zoster virus vaccine.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides the use of 2',3'-cGAMP or a pharmaceutically acceptable salt thereof in preparing a drug for enhancing the immune response of a varicella-zoster virus vaccine.

[0011] Preferably, the drug is a varicella-zoster virus vaccine adjuvant.

[0012] Preferably, 2',3'-cGAMP or a pharmaceutically acceptable salt thereof increases the level of specific IgG antibodies, enhances the intensity of cellular immune response to gE antigen, and increases the secretion of IFN-γ and IL-2 in the immune response to varicella-zoster virus vaccine.

[0013] Preferably, the varicella-zoster virus vaccine in the varicella-zoster virus vaccine immune response reaction is a gE protein vaccine. In some specific embodiments, the weight ratio of 2',3'-cGAMP or a pharmaceutically acceptable salt thereof to the varicella-zoster virus vaccine is 1:1.

[0014] The present invention also provides a varicella-zoster virus vaccine adjuvant, wherein the adjuvant contains 2',3'-cGAMP or a pharmaceutically acceptable salt thereof at an effective dosage concentration.

[0015] The present invention also provides the use of the varicella-zoster virus vaccine adjuvant in preparing a drug for enhancing the immune response of the varicella-zoster virus vaccine.

[0016] The present invention also provides a varicella-zoster virus vaccine composition, which comprises a varicella-zoster virus antigen, 2',3'-cGAMP or a pharmaceutically acceptable salt thereof.

[0017] Preferably, the varicella-zoster virus antigen is gE protein. In some embodiments, the mass ratio of the varicella-zoster virus antigen to 2',3'-cGAMP or a pharmaceutically acceptable salt thereof is 1:1.

[0018] The beneficial effects of the present invention are:

[0019] The present invention discloses for the first time that 2',3'-cGAMP combined with varicella-zoster virus vaccine can increase the level of specific IgG antibodies, enhance the cellular immune response intensity of gE antigen, and increase the secretion of IFN-γ and IL-2, thereby enhancing the immune response of varicella-zoster virus vaccine and the effect of immune prevention. It can be used as a varicella-zoster virus vaccine adjuvant and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the graph of IgG content in mouse Day-1 serum;

[0021] Figure 2 This is a graph of IgG content in mouse serum on Day-8;

[0022] Figure 3 This is a graph of IgG content in mouse serum on Day-15;

[0023] Figure 4 This is a graph of IgG content in mouse serum on Day-22;

[0024] Figure 5 is the graph of IgG content in mouse serum on Day-31;

[0025] Figure 6 The results of the changes in IFN-γ content in the spleen of mice after immunization; A is the photo taken under a microscope, and B is the statistical result of the changes in IFN-γ content;

[0026] Figure 7The results of the changes in IL-2 content in the spleen of mice after immunization; A is the photo taken under a microscope, and B is the statistical result of the changes in IL-2 content;

[0027] Figure 8 The results of flow cytometry CD4 cell population in spleen of immunized mice;

[0028] Fig. 9 The results are flow cytometry CD8 cell population in the spleen of immunized mice;

[0029] Fig.10 This is the result of weighing the spleen of mice after immunization;

[0030] Fig.11 H&E-stained sections of the spleen of mice after the in vivo immunization strategy;

[0031] Fig.12 The results show the weight changes of mice during immunization. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above contents of the present invention are covered within the scope that the present invention is intended to protect. The experimental methods in the following embodiments, unless otherwise specified, are conventional methods; the instruments and equipment used in the following embodiments, unless otherwise specified, are conventional laboratory instruments and equipment; the test materials used in the following embodiments, unless otherwise specified, are all conventional biochemical reagents and can be purchased commercially. The structural formula of 2',3'-cGAMP in the present invention is as follows:

[0033]

[0034] Example 1: Preparation and immunization of varicella-zoster virus vaccine with 2',3'-cGAMP as adjuvant

[0035] gE protein and AS01 B Adjuvants were all from GSK. Varicella-zoster virus vaccine and 2',3'-cGAMP prepared in advance were mixed and dissolved in PBS buffer. Female BALB / c mice aged 4-6 weeks were selected as vaccine immunization objects, 5 mice per group. Each mouse was injected with each group of samples by intramuscular injection, and PBS was used as a blank control. The treatment groups were:

[0036] AS01 B Processing:AS01 B Adjuvant 50 μL / mouse;

[0037] gE treatment: gE protein 5 μg / mouse;

[0038] AS01 B +gE treatment: AS01 B Adjuvant 50 μL / mouse, gE protein 5 μg / mouse;

[0039] 2',3'-cGAMP treatment + gE treatment: 2',3'-cGAMP 5μg / mouse, gE protein 5μg / mouse;

[0040] During the immunization period, the body weight of the mice was measured. Fig.12 Blood was collected from the retro-orbital venous plexus of mice on the 1st, 8th, 15th, 22nd and 31st day after the first immunization, and serum was separated by centrifugation.

[0041] The immunization process was performed twice in total, with one injection four weeks apart. Mice were treated one week after all immunizations were completed to collect spleen and serum samples.

[0042] Example 2: Heterosexual IgG antibody titers in immunized mice

[0043] The gE protein was dissolved in carbonate buffer CBS (pH = 9.6) to prepare a 1 μg / mL solution, and added to a 96-well ELISA plate at a volume of 100 μL per well, and incubated at 4°C overnight. After washing with PBST solution (0.05% Tween in PBS solution), 5% BSA was added to block at 37°C for 2h. After blocking, PBST was used to wash several times and control dry. The serum of each group of gradient dilutions in Example 1 was added to the plate at a volume of 100 μL per well, and incubated at 37°C for 2h. PBST was used to wash several times and control dry, and 100 μL of goat anti-mouse IgG antibody modified with horseradish peroxidase (HRP) (1 / 2000 dilution) was added, and incubated at 37°C for 1h. After washing and controlling dry again, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well, and 50 μL of 2M sulfuric acid was added to stop the color development after standing for 20min away from light. The absorbance value of the wells was measured using an ELISA instrument (λ = 450 nm). The definition of the vaccine antibody titer in this article is the maximum dilution factor at which the OD450 value is at least 2 times higher than that of the blank group. The titer data showed that the 2',3'-cGAMP adjuvant system can significantly increase the level of gE antigen-specific IgG antibodies. Figure 1-5 As shown, in the system of 2',3'-cGAMP combined with varicella-zoster virus vaccine gE protein, specific IgG antibodies were significantly increased, and the specific IgG antibodies reached the maximum after the second immunization, indicating that 2',3'-cGAMP as an adjuvant can significantly increase the level of specific IgG antibodies.

[0044] Example 3: Detection of the effect of immunotherapy on IFN-γ and IL-2 secretion in spleen cells after mouse immunization

[0045] After the mice were killed, the spleen was taken out and weighed. Fig.10 As shown, the spleen weight of mice increased significantly after spleen immunization. Take the spleen tissue of the immunized mouse, first grind and disperse it, and then use a 40μm cell strainer to filter out spleen cells. Use red blood cell lysis buffer to lyse the red blood cells in the spleen cells, and then count 250,000 cells / well and add them to the ELISpot plate. Antigen stimulation concentration: gE protein 25μg / mL. After incubation for 24 hours, add the primary antibody 5H4 (100μL / well), incubate for 2 hours, add the secondary antibody HRP (100μL / well), and incubate for 1 hour. After washing with sterile PBS 5 times, add TMB (100μL / well) until obvious spots appear, and deionized water stops the color development. Dry and take pictures under a microscope. As Figure 6-7 As shown, in the system of 2',3'-cGAMP combined with varicella-zoster virus vaccine gE protein, the number of IFN-γ and IL-2 color spots increased significantly, indicating that 2',3'-cGAMP as an adjuvant can significantly increase the secretion of IFN-γ and IL-2.

[0046] Example 4: Analysis of spleen cells by flow cytometry after mouse immunization

[0047] The mouse spleen cells in Example 3 were added with antibodies CD4-FITC and CD8-PE and incubated at room temperature in the dark for 30 minutes. After the incubation, the cells were centrifuged and the supernatant was removed. The cells were suspended in PBS containing 1% sodium azide. The staining (H&E staining section results are shown in Figure 2) was performed by flow cytometry. Fig.11 The spleen cells of mice after the operation were sorted by CD4 and CD8. Figure 8-9 As shown, flow cytometry results demonstrated that 2',3'-cGAMP as an adjuvant could significantly enhance the intensity of cellular immune response to gE antigen.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of 2',3'-cGAMP or a pharmaceutically acceptable salt thereof in the preparation of a drug for enhancing the immune response of a varicella-zoster virus vaccine.

2. The use according to claim 1, wherein the drug is a varicella-zoster virus vaccine adjuvant.

3. The use according to claim 2, characterized in that: 2',3'-cGAMP or a pharmaceutically acceptable salt thereof can improve the level of specific IgG antibodies in the immune response of varicella-zoster virus vaccine, enhance the intensity of cellular immune response of gE antigen, and increase the secretion of IFN-γ and IL-2.

4. The use according to claim 2, characterized in that: The varicella-zoster virus vaccine in the varicella-zoster virus vaccine immune response reaction is a gE protein vaccine.

5. The use according to claim 4, characterized in that: The weight ratio of 2',3'-cGAMP or a pharmaceutically acceptable salt thereof to the varicella-zoster virus vaccine is 1:

1.

6. A varicella-zoster virus vaccine adjuvant, characterized in that: The adjuvant contains 2',3'-cGAMP or a pharmaceutically acceptable salt thereof at an effective dosage concentration.

7. Use of the varicella-zoster virus vaccine adjuvant according to claim 6 in the preparation of a medicament for enhancing the immune response of the varicella-zoster virus vaccine.

8. A varicella-zoster virus vaccine composition, characterized in that: The varicella-zoster virus vaccine composition comprises a varicella-zoster virus antigen, 2',3'-cGAMP or a pharmaceutically acceptable salt thereof.

9. The varicella-zoster virus vaccine composition according to claim 9, characterized in that: The varicella-zoster virus antigen is gE protein. 10 . The varicella-zoster virus vaccine composition according to claim 9 , wherein the mass ratio of the varicella-zoster virus antigen to 2′,3′-cGAMP or a pharmaceutically acceptable salt thereof is 1:1.

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