New target, method and application for inducing mucosal immune response of helicobacter pylori

Abstract

The invention belongs to the technical field of biology, and particularly relates to a new target spot, a method and application for inducing a helicobacter pylori mucosal immune response reaction. Aiming at the problems of poor immune effect, safety risk and the like due to the fact that the existing helicobacter pylori mucosal immune response is induced mainly through oral administration or nasal drop treatment, the invention provides a new target spot for inducing the helicobacter pylori mucosal immune response, and the target spot is positioned at the oropharynx of a mouse. The invention further provides a novel method for inducing the helicobacter pylori mucosal immune response. The method comprises the step of performing immunostimulation on the oropharyngeal mucosa of a mouse by adopting the helicobacter pylori antigen. The invention further provides application of the novel target spot, and the novel target spot is used for developing helicobacter pylori mucosal immune vaccines. The target spot can induce humoral immunity and mucosal immunity response, can efficiently inhibit helicobacter pylori colonization in the stomach of a mouse, and reduces the infection rate. The vaccine based on the target spot has the advantages of being convenient to use and remarkable in effect, and a reliable strategy is provided for prevention and treatment of helicobacter pylori.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a new target, method and use for inducing mucosal immune response of Helicobacter pylori. Background Art

[0002] Helicobacter pylori (Hp) is a microaerophilic, Gram-negative pathogen that inhabits the gastric mucosa. Hp infection initially causes chronic gastritis, which can lead to gastric ulcers and atrophy, and in severe cases, gastric cancer. Approximately 50% of people worldwide are infected with H. pylori. The current mainstay of treatment is quadruple therapy. However, with increasing antibiotic resistance, the effectiveness of H. pylori treatment and the recurrence rate are not optimistic. Current research on mucosal vaccines targeting H. pylori focuses on oral or intranasal routes. Oral administration is convenient and fast, making it considered the most ideal route of administration. However, peptides and proteins are susceptible to degradation or denaturation in the harsh gastrointestinal environment. While oral H. pylori vaccines have shown initial success in clinical trials, they also suffer from the high oral dosage, making them unsuitable for immunization in children, necessitating consideration of alternative routes of administration. While intranasal immunization can induce potent mucosal immunity, it carries the potential risk of neurotoxicity. Therefore, developing safe and effective novel mucosal immunization strategies is of great research value.

[0003] The oropharyngeal mucosa is the intersection of the digestive tract and the respiratory tract, and its unique structure provides an ideal target for vaccine immune induction. In rodents, nasopharynx-associated lymphoid tissue (NALT) exists on both sides of the nasopharyngeal duct, located on the dorsal side of the cartilaginous soft palate. It is considered to be similar to the pharyngeal lymphoid ring (Waldeyer's ring) in humans. This area is composed of stratified squamous epithelium and is rich in microfold cells (M cells), dendritic cells, B lymphocytes, and T lymphocytes. Among them, M cells can efficiently take up and transport antigens to the underlying lymphoid tissue. Studies have shown that oropharyngeal immunization can simultaneously activate the mucosal immune response of the digestive tract and respiratory tract, forming cross-site immune protection. In addition, oropharyngeal immunization can avoid the destruction of vaccine antigens by gastric acid, and its operational convenience is significantly better than other immunization routes, and it has higher clinical applicability.

[0004] Although the oropharyngeal mucosa is involved in immune recognition in the early stages of pathogen invasion, there are currently no reports of vaccines that use the oropharyngeal immunization route to induce specific anti-Helicobacter pylori mucosal immunity, and development is urgently needed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the existing methods of inducing mucosal immune response of Helicobacter pylori are mainly through oral administration or nasal drops, which have the problems of poor immune effect when stimulating immune response and inconvenient operation of the treatment method.

[0006] The technical solution of the present invention to solve the above technical problems is: providing a new target for inducing Helicobacter pylori mucosal immune response, which is located in the oropharynx of mice.

[0007] Furthermore, the present invention also provides a novel method for inducing a mucosal immune response to Helicobacter pylori, which comprises the steps of using Helicobacter pylori antigens to immunostimulate the oropharyngeal mucosa of mice.

[0008] Furthermore, the novel method for inducing a mucosal immune response to Helicobacter pylori comprises the following steps:

[0009] After mixing the Helicobacter pylori recombinant urease protein and the adjuvant, the mixture was slowly dripped into the oropharynx of the mice for immune induction; and on the 14th, 21st, and 28th days after immune induction, the Helicobacter pylori recombinant urease protein and adjuvant mixture was dripped again for immune boosting.

[0010] Wherein, in the above-mentioned new method for inducing gastric Helicobacter pylori mucosal immune response, the Helicobacter pylori recombinant urease proteins are UreA and UreB.

[0011] Wherein, in the above-mentioned new method for inducing gastric Helicobacter pylori mucosal immune response, the adjuvant is LTs63k.

[0012] Among them, in the above-mentioned new method for inducing Helicobacter pylori mucosal immune response, the dosage of the Helicobacter pylori recombinant urease proteins UreA and UreB is 50 μg, and the dosage of the adjuvant is 10 μg.

[0013] Wherein, in the above-mentioned novel method for inducing mucosal immune response to Helicobacter pylori, the dosage of the dripped mixture is 20 μl per mouse.

[0014] The present invention also provides a use of the above-mentioned new target for inducing Helicobacter pylori mucosal immune response, which is used for the development of Helicobacter pylori mucosal immune vaccine.

[0015] Furthermore, in the above use, the new target is located in the oropharynx of mice.

[0016] Furthermore, in the above use, the Helicobacter pylori mucosal immune vaccine is a vaccine for oropharyngeal immunization.

[0017] Furthermore, in the above use, the vaccine comprises Helicobacter pylori recombinant urease protein and an adjuvant.

[0018] Furthermore, in the above use, the dosage of the Helicobacter pylori recombinant urease protein is 50 μg, and the dosage of the adjuvant is 10 μg.

[0019] The beneficial effects of the present invention are:

[0020] The present invention experimentally discovered for the first time that the mouse oropharynx can serve as a new target for inducing a mucosal immune response to Helicobacter pylori. Stimulating the oropharynx with Helicobacter pylori antigens effectively generated a mucosal response, induced the production of higher titers of specific antibodies in mouse serum, and more effectively inhibited Helicobacter pylori colonization in the mouse stomach. Therefore, the present invention provides a new approach for the development of Helicobacter pylori mucosal immune vaccines. By using the oropharyngeal immunization route, new Helicobacter pylori mucosal immune vaccines can be developed, which not only makes immunization more convenient and efficient, but also enhances the vaccine's protective efficacy, thus having important practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are the results of quantitative plate culture determination of Helicobacter pylori colonization in the stomachs of immunized mice challenged with the virus. Each dot represents an individual mouse. Points on the x-axis indicate that no H. pylori colonies were detected or were below the limit of detection in the plate culture of gastric tissue from that mouse. n = 10, *p < 0.05, **p < 0.01, ***p < 0.001, ns indicates no significant difference. DETAILED DESCRIPTION

[0022] The present invention provides a new target for inducing Helicobacter pylori mucosal immune response, and the target is located in the oropharynx of mice.

[0023] For a long time, the industry's research on Helicobacter pylori immune vaccines has mostly focused on oral or nasal drops. The antigens of oral vaccines mainly act on the intestinal mucosa and induce immune responses through the gut-associated lymphoid tissue (GALT). However, oral vaccines also have the following defects: First, the immunogens that enter the gastrointestinal tract orally are at risk of being degraded or denatured by the strong gastric acid environment, resulting in a reduction or failure of the effective immunogens that induce the body's immune response; second, oral immunization requires a large dose of immunogens, which is not economical in terms of vaccine preparation, and oral tolerance may occur. The other type of nasal drop immunization mainly acts on the respiratory mucosa, especially the nasal mucosa, and induces immune responses through the nasopharyngeal associated lymphoid tissue (NALT). Nasal drop immunization has the disadvantages of being inconvenient to operate and easily causing the risk of neurotoxicity.

[0024] To address this issue, the present invention experimentally discovered for the first time that the mouse oropharynx also has a target that induces an immune response against Helicobacter pylori. By stimulating this target, a mucosal immune response can be induced, leading to the development of a novel vaccine that acts via the oropharynx. This discovery of a target located in the oropharynx is a universal target, and in the examples of this application, we have verified the use of this target in the development of a mucosal immune vaccine against Helicobacter pylori. However, the scope of protection of this invention is not limited to mucosal immune vaccines against Helicobacter pylori but is also applicable to the development of other vaccines that induce humoral and mucosal immune responses.

[0025] The oropharyngeal immunization pathway discovered for the first time in the present invention can not only have the advantages of convenient operation of oral immunization, but also avoid the risk of neurotoxicity caused by nasal drop immunization, and can also effectively control the immunization dose to avoid the phenomenon of immune tolerance. Therefore, the oropharyngeal immunization pathway has higher clinical applicability. The reason why the oropharyngeal immunization effect of the present invention is better than the oral effect may be that: the structure and composition of the antigen in the vaccine are relatively stable when acting on the oropharynx, reducing the risk of degradation or denaturation and improving its immunogenicity; the antigen dose and immune volume are both small during immunization, which can effectively avoid the occurrence of immune tolerance, that is, the weakening or loss of the immune response; the immunogen induces an immune response through the nasopharyngeal associated lymphoid tissue (NALT), which can not only produce specific antibodies locally in the respiratory mucosa, but also through the common mucosal immune system, so that the relevant immune cells and specific antibodies produce cross-protection effects in other mucosal sites such as the gastrointestinal tract, inducing a systemic immune response.

[0026] In the subsequent development of human vaccines, stimulating immune responses through pharyngeal spray administration can be considered.

[0027] The following is a clear and complete description of the technical solutions of various embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments derived by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts belong to the present invention.

[0028] Example 1 Animal Immunization

[0029] In this example, recombinant Helicobacter pylori urease proteins UreA and UreB were used as antigens, prepared according to the methods disclosed in CN114350696A and CN116286757A, respectively. Protein LTs63k was used as an adjuvant, prepared according to the literature (Feng Qiang. Construction, Expression, and Properties of Recombinant Escherichia coli Heat-Labile Enterotoxin, Its Mutants, and Its B Subunit. [D]. Chongqing University. 2003).

[0030] The animal groups are shown in Table 1 below. The experimental animals were female Balb / c mice, 6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., with 10 mice in each group.

[0031] In this experiment, the oral immunization dose was set to twice the oropharyngeal immunization dose, mainly because the antigen would be degraded or denatured by the gastrointestinal environment when administered orally, thus increasing the dosage.

[0032] Table 1 Animal immunization groups

[0033]

[0034] The immunization method is as follows:

[0035] (1) First immunization

[0036] Oropharyngeal immunization: Mix 50 μg each of UreA and UreB antigens with 10 μg of adjuvant LTs63k and place on ice until ready for immunization. After anesthetizing the mouse, carefully pull out the tongue with forceps and open the mouth to facilitate pipetting into the oropharynx. Pipette 20 μl of immunogen and slowly drip it into the pharynx behind the tongue (avoid injuring the mouse with the pipette tip). After immunization, place the mouse in a lateral position until it wakes up.

[0037] Oral immunization: Mix 100 μg of each UreA and UreB antigens with 20 μg of adjuvant LTs63k and place in an ice box until ready for immunization. 0.5 h before immunization, administer 100 μl of 7.5% sodium bicarbonate solution by oral gavage to neutralize gastric acid. Finally, administer 200 μl of the immunization volume by oral gavage.

[0038] (2) Second immunization: The second immunization is carried out on the 14th day, and the immunization dose and method are the same as above;

[0039] (3) The third immunization was carried out on day 21, and the immunization dose and method were the same as above;

[0040] (4) The fourth immunization was carried out on the 28th day, and the immunization dosage and immunization method were the same as above.

[0041] Samples were taken from mice after different immunization treatments to measure relevant immune indicators.

[0042] 1. Serum specific antibody IgG and fecal antibody sIgA detection

[0043] (1) Elisa detection of specific antibodies in serum of immune mice

[0044] Six days after the fourth immunization, orbital venous blood was collected from Balb / c mice, kept at 4°C for 3 h, and then centrifuged at 3000 rpm for 5 min to separate serum. ELISA was used to detect changes in the levels of recombinant urease UreA and UreB protein-specific IgG.

[0045] a. Antigen coating: Dilute Helicobacter pylori urease proteins UreA and UreB to 4 μg / mL in coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight.

[0046] b. Blocking: Add 300 μL of blocking solution per well, incubate at 37°C for 1 h, wash the plate with PBST, and store at 4°C until use.

[0047] c. Specimen dilution: Serum dilution starts from 1:512 and is serially diluted to 1:8192.

[0048] d. Sample addition: Take the coated ELISA plate and add diluted serum in sequence, 100 μL / well, with duplicates for each sample, incubate at 37°C for 1 hour, and wash 4 times with PBST;

[0049] e. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgG in antibody diluent 1:10,000, 100 μL / well, incubate at 37°C for 30 min, and wash four times with PBST;

[0050] f. Color development: Add 100 μL / well of substrate color development solution, incubate at 37°C for 10 min, then add 50 μL / well of stop solution, and measure the OD value at a wavelength of 450 nm on a microplate reader;

[0051] g. Result judgment: A sample / A negative ≥ 2.1 is positive.

[0052] The coating buffer in a is 0.05 mM carbonate / bicarbonate buffer, pH 9.6 (15 mM Na₂CO₃, 35 mM NaHCO₃). The blocking buffer in b is 10 mM PBS (pH 7.4) + 1% BSA. The PBST wash buffer in d is 10 mM PBS (pH 7.4) + 0.05% Tween-20. The antibody dilution buffer in e is 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.5% BSA. The developing buffer in f is a 10:90:1 ratio of TMB stock solution: substrate buffer: 3% hydrogen peroxide; the TMB stock solution is 1 mg / mL TMB dissolved in DMSO; the substrate buffer is 0.53 mM citric acid (pH 5.0) and 100 mM Na₂HPO₄. The stop buffer in f is 2 M H₂SO₄.

[0053] The results showed that the sera of mice immunized oropharyngeally had 100% and 80% positive rates of specific antibodies to recombinant urease UreA and UreB, respectively, with geometric mean titers of 1:4096 and 1:2195, respectively. After oral gavage, the sera of mice immunized orally had 10% positive rates of specific antibodies to recombinant urease UreA and UreB, with geometric mean titers of 1:27.8 and 1:21.1, respectively. The results are shown in Table 2, indicating that oropharyngeal immunization can induce higher titers of specific antibodies in mouse serum than oral immunization.

[0054] Table 2 Statistics of serum specific IgG antibodies to Helicobacter pylori urease proteins UreA and UreB

[0055]

[0056] (2) Detection of specific antibodies sIgA in feces of immunized mice by Elisa

[0057] Six days after the fourth immunization, feces were collected and weighed from each group of mice. PBS buffer was added based on fecal weight and placed on ice at a rate of 5 ml / g / mouse. After collection, the mixture was vortexed for 10 minutes and then centrifuged at 12,000 g for 5 minutes. The supernatant was used to analyze specific sIgA levels using an ELISA.

[0058] a. Antigen coating: Dilute Helicobacter pylori urease UreA and UreB proteins to 4 μg / mL in coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight.

[0059] b. Blocking: Add 300 μL of blocking solution per well, incubate at 37°C for 1 h, wash the plate with PBST, and store at 4°C until use.

[0060] c. Specimen dilution: Stool samples were serially diluted starting from 1:2 to 1:64.

[0061] d. Sample addition: Take the coated ELISA plate and add the diluted lavage fluid samples in sequence, 100 μL / well, with duplicates for each sample, incubate at 37°C for 1 hour, and wash 4 times with PBST;

[0062] e. Add secondary antibody: dilute HRP-labeled goat anti-mouse IgA in antibody diluent 1:10,000, 100 μL / well, incubate at 37°C for 30 min, and wash four times with PBST;

[0063] f. Color development: Add 100 μL / well of substrate color development solution, incubate at 37°C for 10 min, then add 50 μL / well of stop solution, and measure the OD value at a wavelength of 450 nm on a microplate reader;

[0064] g. Result judgment: A sample / A negative ≥ 2.1 is positive.

[0065] The coating buffer in a is 0.05 mM carbonate / bicarbonate buffer, pH 9.6 (15 mM Na₂CO₃, 35 mM NaHCO₃). The blocking buffer in b is 10 mM PBS (pH 7.4) + 1% BSA. The PBST wash buffer in d is 10 mM PBS (pH 7.4) + 0.05% Tween-20. The antibody dilution buffer in e is 10 mM PBS (pH 7.4) + 0.05% Tween-20 + 0.5% BSA. The developing buffer in f is a 10:90:1 ratio of TMB stock solution: substrate buffer: 3% hydrogen peroxide; the TMB stock solution is 1 mg / mL TMB dissolved in DMSO; the substrate buffer is 0.53 mM citric acid (pH 5.0) and 100 mM Na₂HPO₄. The stop buffer in f is 2 M H₂SO₄.

[0066] The experimental results showed that the fecal conversion rate of specific antibodies to the recombinant urease proteins UreA and UreB in mice immunized orally was 90-100%, with the highest antibody titers reaching 1:32 and 1:16. The fecal conversion rate of specific antibodies to the recombinant urease proteins UreA and UreB in mice immunized orally by gavage was 30% and 10%, with the highest antibody titers reaching 1:4 and 1:2, respectively. The results are shown in Table 3. This indicates that oropharyngeal immunization induces a stronger mucosal immune response in mice than oral immunization.

[0067] Table 3 Statistics of fecal specific sIgA antibodies to Helicobacter pylori urease protein subunit A and B

[0068]

[0069] 2. Evaluation of protection against virus attack after immunization

[0070] The specific steps are as follows:

[0071] (1) Oral gavage of mice: 10 days after the last immunization, Helicobacter pylori (purchased from ATCC, No. 700824) was orally gavaged for the challenge experiment. The mice were fasted for 24 hours and deprived of water for 17 hours before gavage. The infection dose for each mouse was 2.0×10 7 CFU, and water and food were restored 2 h after gavage.

[0072] (2) Plate culture: One week after oral gavage, the mice were slaughtered and their gastric tissues were weighed and minced, placed in PBS buffer, and vortexed for 3 minutes. The washed stock solution and 10-fold dilution were then spread on Skirrow plates (pH 7.4) containing 5% defibrinated sheep blood and 0.5% compound antibiotics (vancomycin 1.67 mg / mL, polymyxin 0.0694 mg / mL, trimethoprim 0.5 mg / mL, amphotericin B 0.2 mg / mL). The plates were cultured at 37°C under microaerophilic conditions (5% O2, 10% CO2, 85% N2) for 3 days and then observed.

[0073] (3) Combined with H. pylori colony characteristics, rapid urease reagents, and microscopic examination, the presence of H. pylori on the plate was detected to determine whether the mice were successfully infected with H. pylori. The H. pylori infection-positive rate and protection rate were calculated. Wherein, the vaccine protection rate = (control group infection-positive rate - immunized group infection-positive rate) / control group infection-positive rate × 100%.

[0074] Experimental results: The mice in each group were slaughtered after the virus attack and the results of the plate culture test were shown in Table 4 and Figure 1 Groups 2 and 4 served as the experimental control groups, with a 100% positive rate for Hp infection, indicating that the post-immunization challenge evaluation was effective. Among the experimental groups, only 30% of the mice in Group 1, which were immunized orally via the pharynx, became infected with Hp after immunization, and this group of mice achieved a 70% immune protection effect. 80% of the mice in Group 3, which were immunized orally via gavage, became infected with Hp after the challenge, and this group only achieved a 20% protection rate, indicating that the oropharyngeal immunization route can achieve a higher immune protection effect.

[0075] Table 4 Statistics of the positive rate of Hp infection in mice after immunization

[0076] Mouse number 1 group 2 groups 3 groups 4 groups 1 + + + + 2 — + + + 3 — + + + 4 — + — + 5 + + + + 6 — + + + 7 — + — + 8 + + + + 9 — + + + 10 — + + + HP infection positive rate 30％ 100％ 80％ 100％ Vaccine protection rate 70％ — 20％ —

[0077] Note: “+” indicates that both the rapid urease test and the microscopic examination were positive, and “-” indicates that both the rapid urease test and the microscopic examination were negative.

[0078] In summary, the Helicobacter pylori vaccine of the present invention induced not only a humoral immune response but also a stronger mucosal immune response when administered orally to mice. Furthermore, it was more effective in inhibiting H. pylori colonization in the mouse stomach compared to oral administration. Therefore, this vaccine, administered orally, can further enhance the protective efficacy of H. pylori mucosal vaccines.

Claims

1. A new target for inducing mucosal immune response to Helicobacter pylori, characterized by: The target is located in the mouse oropharynx.

2. A novel method for inducing a mucosal immune response to Helicobacter pylori, characterized by: The method comprises the steps of using Helicobacter pylori antigen to immunostimulate the oropharyngeal mucosa of mice; specifically comprising the following steps: After mixing the Helicobacter pylori recombinant urease protein and the adjuvant, the mixture was slowly dripped into the oropharynx of the mice for immune induction; and on the 14th, 21st, and 28th days after immune induction, the Helicobacter pylori recombinant urease protein and adjuvant mixture was dripped again for immune boosting.

3. The novel method for inducing a mucosal immune response to Helicobacter pylori according to claim 2, characterized in that: The Helicobacter pylori recombinant urease proteins are UreA and UreB, and the adjuvant is LTs63k.

4. The novel method for inducing a mucosal immune response to Helicobacter pylori according to claim 2, characterized in that: The dosage of the Helicobacter pylori recombinant urease protein is 50 μg, and the dosage of the adjuvant is 10 μg.

5. The novel method for inducing mucosal immune response to Helicobacter pylori according to claim 2, characterized in that: The dose of the dripped mixture was 20 μl per mouse.

6. The use of the new target for inducing mucosal immune response to Helicobacter pylori according to claim 1, characterized in that: Used for the development of Helicobacter pylori mucosal immune vaccine.

7. The use according to claim 6, characterized in that: The novel target is located in the mouse oropharynx.

8. The use according to claim 6, characterized in that: The Helicobacter pylori mucosal immune vaccine is a vaccine for oropharyngeal immunization.

9. The use according to claim 6, characterized in that: The vaccine comprises Helicobacter pylori recombinant urease protein and an adjuvant.

10. The use according to claim 6, characterized in that: The dosage of the Helicobacter pylori recombinant urease protein is 50 μg, and the dosage of the adjuvant is 10 μg.

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