Vaccine for preventing body injury caused by trauma inflammation and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a vaccine for preventing body injury caused by trauma inflammation and application. The vaccine for preventing the injury of the traumatic inflammation to the body is prepared by taking DAMPs as an immunogen and mixing and emulsifying the DAMPs and an adjuvant. The vaccine for preventing the injury of the trauma inflammation to the body can enable a human body to form immune memory on DAMPs related to the trauma and prevent the injury of the trauma inflammation to the body, so that the capability of protecting the trauma is realized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a vaccine for preventing body damage caused by traumatic inflammation and its application. Background Art

[0002] Vaccines are a type of biological preparation that can produce specific immunity in the body. Vaccination enables the recipient to gain immunity and is a classic means of preventing infectious diseases. The main active ingredients of vaccines are immunogens and adjuvants. The role of immunogens is to induce the desired specific immune response, while the role of adjuvants is to regulate the intensity and type of immune response. In traditional vaccine research, immunogens are designed and developed based on exogenous molecules such as pathogen-associated molecular patterns (PAMPs) of target pathogens. Today, with the development and maturity of vaccine technology, vaccines are gradually being applied to non-communicable diseases. Relevant progress in the design and development of cancer vaccines and hypertension vaccines based on endogenous molecules provides a blueprint for the development of trauma vaccines based on traumatic endogenous molecules.

[0004] Trauma is the destruction of tissues or organs caused by mechanical factors applied to the human body. Cells in tissues or organs die, are damaged or stressed, leading to the release of intracellular molecules that play physiological roles in cells under normal conditions. The released molecules enter the internal environment and are called damage-associated molecular patterns (DAMPs). DAMPs are recognized by the immune system as "alarms" and cause inflammatory responses to promote the recovery of trauma. At the same time, in order to prevent the inflammatory response from being too strong and damaging normal tissues and organs, the immune system will produce anti-inflammatory responses to limit the scope and intensity of inflammation. DAMPs were originally a series of intracellular molecules that were not recognized by the immune system under normal circumstances, played physiological functions in cells, and did not cause immune responses. However, in severe trauma, the massive release of DAMPs leads to an excessively strong inflammatory response, which in turn causes sterile inflammation and immune dysfunction with an imbalance between pro-inflammatory and anti-inflammatory effects, affecting the calming of the inflammatory response and ultimately causing damage to normal organ tissues. It is a common pathological process that seriously affects the prognosis of trauma. DAMPs bind to pattern recognition receptors on innate immune cells and share similar receptors and signaling pathways with PAMPs in traditional vaccines. Since they share common immunological mechanisms, vaccines, as a means of eliminating pathogens, also have the potential to eliminate excess DAMPs.

[0005] The top priority of vaccine development is to find effective immunogens, and for this purpose, it is necessary to select appropriate DAMPs. Ideal candidate DAMPs must at least meet the following conditions: ① released in large quantities after trauma; ② being the main molecule that causes immune system response and inflammatory response; ③ being the primary pathogenic factor causing trauma-related complications. DAMPs such as HMGB1, histones, myoglobin, and nucleic acids are released in large quantities from cells after trauma, play an important role in the post-traumatic pathological process, and are the main trauma-related DAMPs. Therefore, they can be used as the preferred immunogen for developing trauma vaccines.

[0006] Specifically, extracellular HMGB1 has a strong pro-inflammatory ability, transmitting tissue damage signals to macrophages and dendritic cells, thereby inducing the activation of monocytes, macrophages, endothelial cells, enterocytes, hepatocytes, etc. and secreting more HMGB1, triggering a positive feedback loop, ultimately leading to distal organ damage.

[0007] Histone subunits H3 and H4 are crucial DAMPs and are important factors leading to endothelial dysfunction and multiple organ failure. Myoglobin, as an important component of muscle cells, is released into the blood in large quantities after muscle cell damage when rhabdomyolysis or crush syndrome (CS) occurs. It accumulates in the kidneys with blood filtration, causing crush syndrome associated acute kidney injury (CS-AKI). It is a highly specific DAMP.

[0008] Free DNA participates in the formation of neutrophil extracellular traps, which can have harmful effects on surrounding tissues and lead to complications such as coagulopathy and multiple organ dysfunction syndrome.

[0009] So far, there is no effective treatment for post-traumatic sterile inflammation and the traumatic complications caused by it. Current trauma emergency treatment and follow-up treatment mainly include traditional treatment methods such as respiratory support, circulatory support, wound management, and drug therapy, that is, maintaining the stability of vital signs and supplementing symptomatic treatment, and the effect of trauma treatment is not ideal. The reason is that the excessive release of DAMPs causes post-traumatic sterile inflammation and immune dysfunction, which is an important cause of the high morbidity and mortality of traumatic complications. Reducing the intensity of inflammation and controlling the development of inflammation can significantly improve the prognosis of trauma patients. The main clinical treatment methods currently include hemodialysis, anti-inflammatory treatment, etc., but these treatment methods are difficult to meet the needs of all patients in a timely and sufficient manner when traumatic events, especially large-scale disasters, occur. Summary of the invention

[0010] The purpose of the present invention is to provide a vaccine for preventing traumatic inflammation from damaging the body, so as to solve the technical problem of high morbidity and mortality of traumatic complications caused by excessive release of damage-related molecular patterns leading to post-traumatic sterile inflammation and immune dysfunction in severe trauma.

[0011] The second object of the present invention is to provide a use of a vaccine for preventing damage to the body caused by traumatic inflammation.

[0012] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0013] A vaccine for preventing damage to the body caused by traumatic inflammation, characterized in that the vaccine for preventing damage to the body caused by traumatic inflammation uses damage-associated molecular patterns as immunogens and is prepared by mixing and emulsifying the damage-associated molecular patterns with adjuvants.

[0014] Furthermore, the damage-associated molecular pattern includes one or more of myoglobin, HMGB1, histone, nucleic acid, calcium-binding protein, and heat shock protein.

[0015] Furthermore, the adjuvant is one or more of Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, AS03 adjuvant, and AS04 adjuvant.

[0016] Furthermore, the preparation method of the vaccine for preventing damage to the body caused by traumatic inflammation is as follows: dissolving the damage-associated molecular pattern in physiological saline or phosphate buffer solution, and then mixing and emulsifying with an adjuvant.

[0017] Furthermore, the concentration of the damage-associated molecular pattern solution is 0.2-20 μg / μL; the volume of the adjuvant accounts for 10-50% of the total volume of the vaccine for preventing traumatic inflammation from damaging the body; the concentration of the vaccine for preventing traumatic inflammation from damaging the body is 0.1-10 μg / μL.

[0018] A use of the above vaccine in a medicine for preventing damage to the body caused by traumatic inflammation.

[0019] Beneficial effects of the present invention:

[0020] The vaccine for preventing damage to the body caused by traumatic inflammation of the present invention can enable the human body to form immune memory to the damage-related molecular patterns related to trauma, prevent damage to the body caused by traumatic inflammation, and thus realize the protective ability against trauma.

[0021] The vaccine for preventing body damage caused by traumatic inflammation of the present invention can be used as a preventive vaccine to first form protection for people at high risk of trauma, thereby reducing the harm to people in dangerous situations.

[0022] The vaccine for preventing damage to the body caused by traumatic inflammation of the present invention forms immune memory in advance, can quickly activate immune response after trauma occurs, and produces antibodies to clear damage-related molecular patterns more promptly, thereby preventing damage to the body caused by traumatic inflammation. It is a new treatment plan for preventing damage to the body caused by traumatic inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A is a schematic diagram of the indirect ELISA experimental process in Example 1;

[0024] Figure 1 B is a graph showing the results of the indirect ELISA method for measuring the anti-myoglobin antibody level in mouse serum in Example 1;

[0025] Figure 2 A is the BUN content in the serum of mice in the NC, CS, V1+CS, V2+CS, and V3+CS groups after squeezing in Example 1;

[0026] Figure 2 B is the SCr content in the serum of mice in the NC, CS, V1+CS, V2+CS, and V3+CS groups after squeezing in Example 1;

[0027] Figure 2 C is a graph showing the WB results of the renal tissues of mice in the NC, CS, V1+CS, V2+CS, and V3+CS groups after compression in Example 1;

[0028] Figure 2 D is the result diagram of the relative optical density of NGAL protein in the kidney tissue of mice in the NC, CS, V1+CS, V2+CS, and V3+CS groups after compression in Example 1;

[0029] Figure 2 E is the result graph of the number of damaged renal tubules in mice in the NC, CS, V1+CS, V2+CS, and V3+CS groups after compression in Example 1. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the embodiments of the present invention and the accompanying drawings.

[0031] Example 1

[0032] The vaccines used in this embodiment to prevent damage to the body caused by traumatic inflammation are divided into primary immunization vaccines and booster immunization vaccines.

[0033] The preparation method of the first immunization vaccine is to dissolve three groups of myoglobin in phosphate buffer solution (PBS solution), the concentrations of the three groups of myoglobin solutions are 16μg / μL, 8μg / μL, and 4μg / μL respectively, and the three groups of myoglobin solutions are mixed with equal volumes of Freund's complete adjuvant respectively, and the myoglobin solution and Freund's complete adjuvant are fully mixed and emulsified by the syringe push-pull method. The concentrations of the three groups of first immunization vaccines prepared are 8μg / μL, 4μg / μL, and 2μg / μL respectively. Freund's complete adjuvant formula: Each milliliter of Freund's complete adjuvant contains 1mg of heat-killed dried Mycobacterium tuberculosis, 0.85mL of paraffin oil and 0.15mL of dimannol monooleate.

[0034] Preparation method of booster vaccine: dissolve three groups of myoglobin in PBS solution, so that the concentration of myoglobin solution is 8μg / μL, 4μg / μL, and 2μg / μL respectively, mix the three groups of myoglobin solution with equal volume of Freund's incomplete adjuvant respectively, and use the syringe push-pull method to fully mix and emulsify the myoglobin solution and Freund's incomplete adjuvant to obtain the booster vaccine. The concentration of the prepared booster vaccine is 4μg / μL, 2μg / μL, and 1μg / μL. Freund's incomplete adjuvant formula: each milliliter of Freund's incomplete adjuvant contains 0.85mL paraffin oil and 0.15mL mannide monooleate.

[0035] Vaccine Immunization Process

[0036] Fifteen 8-week-old male BALB / c mice were divided into three groups: high-dose V1, medium-dose V2, and low-dose V3, with 5 mice in each group. The three groups of mice were first injected with the first immunization vaccine. The 5 mice in the high-dose V1 group were injected with 100uL of the first immunization vaccine with a concentration of 8μg / μL, the 5 mice in the medium-dose V2 group were injected with 100uL of the first immunization vaccine with a concentration of 4μg / μL, and the 5 mice in the low-dose V3 group were injected with 100uL of the first immunization vaccine with a concentration of 2μg / μL. 14 days after vaccination, the three groups of mice were injected with booster immunization vaccines. The 5 mice in the high-dose V1 group were injected with 100uL of the booster immunization vaccine with a concentration of 4μg / μL, the 5 mice in the medium-dose V2 group were injected with 100uL of the booster immunization vaccine with a concentration of 2μg / μL, and the 5 mice in the low-dose V3 group were injected with 100uL of the booster immunization vaccine with a concentration of 1μg / μL. The booster vaccine requires a total of three injections, with one booster injection every two weeks.

[0037] Before each vaccination, mice were weighed and their fur color was recorded. Vaccination was performed by subcutaneous multi-point injection, with injections made at five points: the back, both armpits, and both waists.

[0038] The DAMPs inoculation amount of the vaccine for preventing body damage caused by traumatic inflammation is 1 to 1000 μg per mouse each time.

[0039] Immune effect evaluation

[0040] ①Serum separation: Before the first immunization experiment of mice and on the 7th day after the first immunization and each booster immunization, about 150 μL of blood was collected from each mouse by the inner canthus blood collection method, left to stand at room temperature for 1 hour, and centrifuged at 3000g for 20 minutes. The serum was separated and stored at -80℃ for later use. Depending on whether the mice were vaccinated with vaccines to prevent traumatic inflammation from damaging the body when the serum was separated, the serum before the first immunization was called negative serum, and the serum after the first immunization was called antiserum.

[0041] ② Plate coating: dilute myoglobin to 2μg / mL with coating solution, take 50μL of the diluted myoglobin to the ELISA plate, and then put the ELISA plate into a 37℃ constant temperature incubator for 2h. The coating solution is 100mM carbonate buffer with a pH of 9.6.

[0042] ③ Blocking: Discard the coating solution in the ELISA plate, then add 60 μL of blocking solution to the corresponding wells of the ELISA plate with incubated myoglobin to block non-specific binding sites, and incubate the ELISA plate with the blocking solution in a 37°C constant temperature incubator for 2 hours. The blocking solution is a PBS solution containing 1% bovine serum albumin solution.

[0043] ④ Multiple dilution: Thaw the negative serum and antiserum obtained after the last booster vaccine injection on ice, and dilute them with blocking solution. The dilution ratios are 1:250, 1:1000, 1:4000, and 1:16000.

[0044] Discard the blocking solution of the ELISA plate in step ③, wash with washing solution for 3 times, and spin dry the ELISA plate. Add 50 μL of negative serum and the gradient dilution of antiserum taken after the last booster vaccine injection to the corresponding wells of the ELISA plate, and incubate in a 37°C constant temperature incubator for 2 hours. The washing solution is TBST, TBS containing 0.5% Tween-20.

[0045] ⑤ Incubate the secondary antibody: After discarding the serum from the negative serum and antiserum ELISA plate in ④ incubation, wash the ELISA plate 3 times with washing solution and spin dry. Add 50 μL of alkaline phosphatase-linked anti-mouse secondary antibody to the ELISA plate at a dilution concentration of 1:3000, that is, the volume ratio of the secondary antibody to the blocking solution is 1:3000. Incubate in a 37°C constant temperature incubator for 2 hours. Wash the ELISA plate 3 times with washing solution, spin dry, add alkaline phosphatase substrate, incubate at 37°C in the dark for 20 minutes, then add the stop solution to terminate the reaction, and immediately use an ELISA instrument to measure the absorbance (OD) value at 405 nm. The OD value is not less than 2.1 times that of the blank control group as the standard for the positive sample to be tested, and then the maximum positive dilution of the corresponding sample is calculated, which is the titer of the antibody produced after this animal is immunized. The results are as follows Figure 1 As shown, the serum titer of myoglobin antibody in mouse antiserum can reach up to 1:16000, and the serum positive reaction rate is 100%. Among them, P / N represents the absorbance value of antiserum and negative serum at 405nm, and P / N>2.1 represents that the antiserum titer can reach this dilution. The stop solution is 3N NaOH, the pH of the alkaline phosphatase substrate is 9.8, and the alkaline phosphatase substrate contains 1mg / mL pNPP, 1mol / L diethanolamine, 0.5mM CaCl 2 .

[0046] Vaccine protection effect testing

[0047] The mice that were not vaccinated were randomly divided into a control group (NC) and a compression group (CS), and the mice that were vaccinated with a vaccine for preventing traumatic inflammation from damaging the body were divided into V1+CS, V2+CS, and V3+CS groups. After the mice were anesthetized, the hind limbs of the mice in the CS, V1+CS, V2+CS, and V3+CS groups were continuously squeezed for 16 hours under a pressure of 1 kg using a self-developed digital animal compression simulation platform with an invention patent number of 201610428085.2 to prepare a CS-AKI mouse model. The mice in the NC group were not treated otherwise. All squeezed mice were killed 6 hours after decompression, and blood and kidney tissues were collected for subsequent testing.

[0048] Blood biochemical test results such as Figure 2 A, Figure 2 B. Figure 2 C. Figure 2 D and Figure 2 As shown in E, Figure 2 A and Figure 2 The blood biochemical results of B showed that compared with the CS group, the blood urea nitrogen (BUN) and serum creatinine (SCr) concentrations of mice in the V1+CS, V2+CS, and V3+CS groups were significantly lower. Figure 2As can be seen in C, compared with the CS group, the expression of neutrophil gelatinase-associated lipocalin (NGAL protein), a molecular marker of renal injury, was reduced in the renal tissues of mice in the V1+CS group and the V3+CS group. The amount of myoglobin deposition in the kidney was also significantly reduced. Figure 2 As can be seen in D, the immunohistochemical results of renal tissue sections also showed that the in situ expression of NGAL protein in the vaccination group was significantly reduced. Figure 2 As can be seen in E, the HE staining results of the renal tissue sections showed that the number of damaged renal tubules in the V1+CS, V2+CS, and V3+CS groups was significantly reduced compared with the CS group. In summary, vaccination with the vaccine for preventing traumatic inflammation from damaging the body can reduce kidney damage caused by squeezing.

Claims

1. A vaccine for preventing traumatic inflammation from damaging the body, characterized in that: The vaccine for preventing body damage caused by traumatic inflammation uses damage-associated molecular patterns as immunogens, and is prepared by mixing and emulsifying the damage-associated molecular patterns with adjuvants.

2. The vaccine for preventing damage to the body caused by traumatic inflammation according to claim 1, characterized in that: The damage-associated molecular pattern includes one or more of myoglobin, HMGB1, histone, nucleic acid, calcium-binding protein, and heat shock protein.

3. The vaccine for preventing damage to the body caused by traumatic inflammation according to claim 1, characterized in that: The adjuvant is one or more of Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, AS03 adjuvant, and AS04 adjuvant.

4. The vaccine for preventing damage to the body caused by traumatic inflammation according to claim 1, characterized in that: The preparation method of the vaccine for preventing traumatic inflammation from damaging the body is as follows: dissolving the damage-related molecular pattern in physiological saline or phosphate buffer solution, and then mixing and emulsifying with an adjuvant.

5. The vaccine for preventing damage to the body caused by traumatic inflammation according to claim 4, characterized in that: The concentration of the damage-associated molecular pattern solution is 0.2-20 μg / μL; the volume of the adjuvant accounts for 10-50% of the total volume of the vaccine for preventing traumatic inflammation from damaging the body; the concentration of the vaccine for preventing traumatic inflammation from damaging the body is 0.1-10 μg / μL.

6. Use of the vaccine according to claim 1 in a medicine for preventing damage to the body caused by traumatic inflammation.

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