Gene modified neisseria gonorrhoeae and neisseria gonorrhoeae outer membrane vesicle vaccine

Abstract

The invention discloses genetically modified neisseria gonorrhoeae and a neisseria gonorrhoeae outer membrane vesicle vaccine, and a gene modification mode is that at least one of a coding gene of PorB1a protein and a coding gene of PorB1b protein is subjected to gene mutation, so that the capability of inducing mitochondrial autophagy is reduced or weakened. According to the present invention, the induction of the mitochondrial autophagy pathway is effectively prevented by precisely mutating the key amino acids (PorB1a-K117Q / K171Q or PorB1b-K117Q / K170Q) at the 117 site and the 171 / 170th site of the PorB protein; the protective efficacy of the neisseria gonorrhoeae OMVs vaccine is improved compared with that of the neisseria gonorrhoeae OMVs vaccine prepared from wild bacteria, and a new treatment means is provided for gonorrhoeae prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, specifically relating to a genetically modified Neisseria gonorrhoeae vaccine and a Neisseria gonorrhoeae outer membrane vesicle vaccine. Background Technology

[0002] Neisseria gonorrhoeae ( Neisseria gonorrhoeae Gonorrhea is a Gram-negative diplococcus, oval or spherical in shape, about 0.6-0.8 μm in size. It is often arranged in pairs, with a flat or slightly concave contact surface, and is kidney-shaped or coffee bean-shaped. It is mainly transmitted through sexual contact and causes gonorrhea, which is mainly characterized by purulent infection of the genitourinary system. In severe cases, it can cause inflammation of the reproductive system, infertility, and systemic infection.

[0003] Antibiotics are the first-line treatment for gonorrhea. However, in recent years, the resistance of Neisseria gonorrhoeae to multiple antibiotics has increased significantly and is widely distributed globally, with the proportion of drug-resistant strains reaching as high as 48.96% in some regions. Therefore, we urgently need new treatment methods to control Neisseria gonorrhoeae infection in the long term, namely, the development of vaccines.

[0004] Due to the high variability of Neisseria gonorrhoeae (such as the diversity of fimbriae and outer membrane proteins), research on Neisseria gonorrhoeae vaccines is fraught with challenges. A retrospective case-control study in New Zealand demonstrated that the group B outer membrane vesicle (OMV) vaccine (MeNZB) against Neisseria gonorrhoeae infection has a vaccine efficacy of up to 31%, proving the biological rationale for OMV-mediated protective immunity against Neisseria gonorrhoeae (Petousis-Harris, Helen et al. “Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study”). Lancet (London, England) vol. 390,10102 (2017): 1603-1610.doi:10.1016 / S0140-6736(17)31449-6).

[0005] OMVs are spherical vesicles released by bacteria during growth or stress, rich in pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), which can activate the immune system.

[0006] Neisseria gonorrhoeae OMVs contain a variety of components, among which outer membrane porin PorB accounts for about 35% of the total protein. PorB has high immunogenicity and can be transported to host cells by OMVs, participating in the pathogenic process of bacteria. PorB is mainly divided into PorB1a and PorB1b, which are expressed at the same genetic locus and have about 60%-80% amino acid sequence homology. A single strain of Neisseria gonorrhoeae only expresses one type of PorB protein. Therefore, the immune cross-protection between PorB1a epitopes and PorB1b epitopes is variable Figure 1 ).

[0007] In host cells, PorB can target mitochondria, trigger mitochondrial membrane potential loss, activate mitochondrial autophagy, and thus destroy the normal function of host cells. These immunomodulatory properties of Neisseria gonorrhoeae OMVs and PorB affect their effectiveness as Neisseria gonorrhoeae vaccines.

[0008] Chinese invention patent application with publication number CN116096871A discloses the use of genetically modified Neisseria gonorrhoeae FA1090 and OMVs obtained therefrom in medicine. The genetic modification involves the lauroyltransferase (lpxL1) lpxL1 ) gene and the modified protein (lpxT) rmp ) gene.

[0009] Currently, there is no public report or patent disclosure on the application of OMVs targeting the immunogenicity optimization of the mitochondrial autophagy pathway in modified Neisseria gonorrhoeae strains in the prevention of gonorrhea. SUMMARY

[0010] The present application aims to develop a Neisseria gonorrhoeae OMVs vaccine with enhanced immunogenicity by site-directed mutagenesis of the key amino acids at positions 117 and 171 / 170 of the PorB protein (PorB1a-K117Q / K171Q or PorB1b-K117Q / K170Q) to reduce or eliminate its mitochondrial autophagy activity. Studies have confirmed that the vaccine has significant application value in the prevention and treatment of gonorrhea and exhibits better immune protection effect against Neisseria gonorrhoeae infection.

[0011] A genetically modified Neisseria gonorrhoeae, the genetic modification is to mutate the coding gene of the PorB protein, thereby reducing or weakening the ability to induce mitochondrial autophagy. The PorB protein is one of PorB1a and PorB1b.

[0012] Preferably, the genetic modification is any of the following: The coding gene of the PorB1a protein is mutated to cause K117Q / K171Q double mutation of the PorB1a protein; The gene encoding the PorB1b protein is mutated to cause double mutation of K117Q / K170Q of the PorB1b protein.

[0013] Preferably, the starting strain of Neisseria gonorrhoeae is Neisseria gonorrhoeae ATCC 49226 strain.

[0014] The application further provides the use of the genetically modified Neisseria gonorrhoeae in the preparation of a Neisseria gonorrhoeae outer membrane vesicle vaccine.

[0015] The application further provides a Neisseria gonorrhoeae outer membrane vesicle vaccine, which is prepared by culturing the genetically modified Neisseria gonorrhoeae and extracting outer membrane vesicles.

[0016] Preferably, the Neisseria gonorrhoeae outer membrane vesicle vaccine further comprises a pharmaceutically acceptable carrier, which comprises one or more of an adjuvant, a buffer, and a freeze-drying protective agent.

[0017] The application further provides a preparation method of the Neisseria gonorrhoeae outer membrane vesicle vaccine, which comprises the following steps: culturing the genetically modified Neisseria gonorrhoeae, extracting outer membrane vesicles, and preparing the extracted outer membrane vesicles into the Neisseria gonorrhoeae outer membrane vesicle vaccine.

[0018] Preferably, when the extracellular vesicles are extracted, the supernatant after culture is separated by ultracentrifugation.

[0019] Compared with the prior art, the application has the following beneficial effects: The application effectively prevents the induction of the mitochondrial autophagy pathway by precisely mutating the K117Q / K171Q site of the PorB1a protein or the K117Q / K170Q site of the PorB1b protein. The immunogenicity is significantly enhanced, and the protection efficacy of the Neisseria gonorrhoeae OMVs vaccine is improved compared with that of the wild-type strain, thereby providing a new treatment method for gonorrhea prevention and treatment. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Sequence alignment of PorB1a from Neisseria gonorrhoeae ATCC 49226 and PorB1b from ZJXSH86 strain. Loop 3 is represented in green, Loop 4 is represented in blue; lysine 117 (PorB1a and PorB1b), lysine 171 (PorB1a) and lysine 170 (PorB1b) are marked in red.

[0021] Figure 2PorB1a WT and PorB1a-K117Q / K171Q mutant proteins. The differences of TOM20, TIM23 and LC3 protein levels were detected by Western Blot.

[0022] Figure 3 To evaluate the effect of N. gonorrhoeae OMVs expressing PorB1a WT and PorB1a-K117Q / K171Q mutant proteins as prophylactic vaccines in a mouse model of vaginal gonococcal infection. Among them, Figure 3 A in FIG. 1 is the infection of mice, Figure 3 B in FIG. 1 is the bacterial load of the colonized mice, Figure 3 C in FIG. 1 is the area under the curve. DETAILED DESCRIPTION

[0023] In the following examples, specific embodiments of the present application are described in detail. These are illustrative of the present application and are not limiting of the scope of the application. Unless otherwise indicated, all reagents were purchased from the market.

[0024] Example 1

[0025] N. gonorrhoeae ATCC 49226 strain (purchased from American Type Culture Collection). The PorB1a-K117Q / K171Q ATCC 49226 mutant strain was constructed as follows: Construction of plasmid template: the kanamycin resistance gene kan was inserted into the lower end sequence of PorB1a and cloned into the pUC57 vector. The inserted sequence is shown in SEQ ID No. 1, wherein 1-984 bp is the PorB1a-K117Q / K171Q sequence; 985-1041 bp is the PorB1a downstream sequence 1; 1042-2208 bp is the kanamycin sequence; 2209-2913 bp is the PorB1a downstream sequence 2. The recombinant plasmid was transformed into E. coli DH5α competent cells and verified by PCR.

[0026] Plasmid extraction: Axygen plasmid extraction kit was used for operation. First, 2 ml of E. coli bacterial solution was placed in a centrifuge tube, centrifuged at 12000 x g for 1 min, and the supernatant was discarded; the precipitate was resuspended with 500 μl of S1 buffer, and 500 μl of S2 buffer was added after uniform mixing, and the mixture was mixed uniformly by gently shaking up and down; 500 μl of S3 buffer was added, and after mixing thoroughly, it was centrifuged at 12000 x g at 4°C for 10 min; the supernatant was transferred to a preparation tube, centrifuged at 12000 x g at 4°C for 1 min, and the filtrate was discarded; 500 μl of washing buffer was added to the preparation tube, and centrifuged at 12000 x g at 4°C for 1 min, and the filtrate was discarded, and the operation was repeated once; the preparation tube was placed back into the centrifuge tube, and centrifuged at 12000 x g at 4°C for 2 min, and after the alcohol evaporated completely, the preparation tube was placed into a 1.5 ml EP tube, 60 μl of ultrapure water was added, and centrifuged at 12000 x g at 4°C for 2 min, and the DNA was eluted.

[0027] Construction of point mutation plasmid: The Hieff MutTM Site-Directed Mutagenesis Kit (Yeasen) was used for site-directed mutagenesis of the plasmid template, and glutamine was used to replace the 117th lysine and the 171st lysine.

[0028] Transformation: Many bacteria have the ability to horizontally transfer some drug-resistant or suitable genes through plasmid or chromosomal DNA, so as to cause the bacteria to obtain drug resistance function or have better adaptability for further spread, and this ability is called natural transformation. The main gene exchange way of Neisseria gonorrhoeae is natural transformation (Marri, Pradeep Reddy et al. “Genome sequencing reveals widespread virulence gene exchange among human Neisseria species.” PloS One vol. 5,7 e11835. 28 Jul. 2010, doi:10.1371 / journal.pone.0011835). The activated gonococci ATCC 49226 were resuspended in GCB medium, 10 μl was dropped on the surface of GC solid medium, and after the liquid was dried, 10 μl of purified PCR product was added, and after being cultured at 37°C for 7 h, all the bacteria were scraped and spread on the resistant GC agar plate. After being cultured at 37°C for 48 h, single colonies were picked and amplified for PCR identification.

[0029] Example 2

[0030] The present embodiment discloses a method for extracting bacterial outer membrane vesicles, in particular, a method for extracting OMVs of Neisseria gonorrhoeae ATCC 49226 strain and PorB1a-K117Q / K171Q ATCC 49226 mutant strain, comprising the following steps: A1: inoculate Neisseria gonorrhoeae ATCC 49226 strain and PorB1a-K117Q / K171Q ATCC 49226 mutant strain onto the surface of GC solid plates, and place them in a 37°C incubator for overnight growth.

[0031] A2: use a inoculating loop to scrape the overnight activated Neisseria gonorrhoeae into GC liquid medium, and vortex to resuspend the bacteria. Then transfer them to 1L GC liquid medium containing 1% Vitox, and incubate at 37°C with 220 rpm shaking until the OD600 is 0.8. Centrifuge the bacterial solution at 12000 rpm and 4°C for 10 min, and collect the supernatant.

[0032] A3: after filtering the collected supernatant through a 0.45 µm filter, perform ultracentrifugation at 210000×g and 4°C for 3 h. After the end of the ultracentrifugation, collect the precipitate, which is the bacterial OMVs. The bacterial OMVs extracted from the wild-type strain and the mutant strain are named as OMVPorB1aWT and OMV PorB1a-K117Q / K171Q, respectively. After washing the OMVs with PBS for 3 times, dissolve them in an appropriate amount of PBS buffer, and store them in a -80°C ultra-low temperature freezer.

[0033] Example 3

[0034] Stimulate the dendritic cells isolated from the bone marrow of mice with Neisseria gonorrhoeae OMV PorB1a WT and OMV PorB1a-K117Q / K171Q, and analyze the mitochondrial marker proteins TOM20 and TIM23 and the autophagy marker protein LC3 by Western blot to study the mitochondrial autophagy.

[0035] A1: euthanize 8-10 week old C57BL / 6 mice using CO2, and soak them in 75% ethanol for 5-10 min. Use a scalpel and forceps to remove the tissue, isolate the tibia and femur, cut the two ends of the tibia and femur, and rinse the bone marrow contents into the culture medium.

[0036] A2: centrifuge the cell suspension at 300×g for 5 min. Discard the supernatant, resuspend the cells with 1 ml of red blood cell lysis solution, lyse for 5 min, and remove the red blood cells.

[0037] A3: add 9 ml of PBS, centrifuge at 300×g for 5 min. Discard the supernatant, resuspend with 10 ml of complete culture medium. Perform semi-replacement of the medium on the 3rd and 5th days of culture.

[0038] A4: On day 7 of culture, plate 6-well plates with 1 x 10 6 BMDCs (bone marrow-derived dendritic cells) were collected, total protein was extracted with RIPA lysis buffer and incubated on ice for 30 min. Protein concentration was determined by BCA method and adjusted to the same concentration (e.g., 2 pg / pL).

[0039] A5: Install the prepared polyacrylamide gel on the electrophoresis tank, add 1 x Tris-glycine electrophoresis buffer, and add 5-15 pl of treated sample to each sample well. First, concentrate the sample at 80 V, then separate the protein at 120 V until the bromphenol blue band moves to the bottom of the gel, and stop electrophoresis.

[0040] A6: Activate the PVDF membrane with methanol, and after washing with ultrapure water, soak in Western Blot transfer buffer working solution for 5 min. Wrap the gel, PVDF membrane and transfer filter paper with a transfer clamp to form a sandwich transfer structure, and pay attention not to generate bubbles during the process. Place the clamp in the transfer tank, add 1 x transfer buffer working solution and a cooling ice box, and transfer at 300 mA constant current for 90 min.

[0041] A7: After the transfer is completed, soak the PVDF membrane in Western Blot blocking buffer and incubate at room temperature for 2 h. After blocking, wash the membrane with TBST buffer three times. Incubate the PVDF membrane with the corresponding primary antibody at 4°C overnight. The next day, after washing the primary antibody with TBST buffer, add the corresponding secondary antibody and incubate at room temperature for 2 h. After washing with TBST buffer three times, develop and detect.

[0042] As shown in Figure 2 , mitochondrial autophagy occurred in dendritic cells stimulated with OMVs expressing PorB1a WT, but not in the OMV treatment group of the PorB1a-K117Q / K171Q mutant. This indicates that precise mutation of the K117Q / K171Q site of the PorB1a protein of Neisseria gonorrhoeae can effectively block the mitochondrial autophagy pathway.

[0043] Example 4

[0044] Determination of the protective efficacy of OMVs expressing wild-type PorB1a and PorB1a-K117Q / K171Q mutants on a mouse vaginal Neisseria gonorrhoeae ATCC 49226 infection model.

[0045] A1: 4-6 weeks old female BALB / c mice were intraperitoneally immunized with 50 pg of N. gonorrhoeae OMVs (OMV PorB1a WT group used OMV PorB1a WT, OMV PorB1a-K117Q / K171Q group used OMV PorB1a-K117Q / K171Q) or PBS control (Mock control group) at days 0, 14 and 28.

[0046] A2: Two days before infection, on the day of infection and the day after infection, each mouse was subcutaneously injected with 0.1 mg of b-estradiol to put the mice in the right estrous cycle.

[0047] A3: N. gonorrhoeae ATCC 49226 was prepared in a buffer containing 0.5 mM CaCl2, 1 mM MgCl2and 1% (w / v) gelatin to a concentration of 1 x 108CFU / mL and inoculated in the mouse vagina twice at 10 mI_ each. 9

[0048] A4: Daily swab samples were taken from the vagina of each mouse of each group and plated on culture plates containing selective antibiotics. The bacterial load was determined by counting the number of colonies formed at different dilutions. In this experiment, 4 Colony Forming Units (CFU) were set as the lowest limit of detection to ensure accuracy and reliability of the results.

[0049] Results are shown in Figure 3 Figure 4. All mice of the OMV PorB1a WT group were cleared at day 8 and all mice of the OMV PorB1a-K117Q / K171Q group were cleared at day 6. When comparing the bacterial load of the colonized mice, we observed similar results. The Mock control mice always had higher CFU counts and therefore a significantly reduced area under the curve compared to the OMV PorB1a WT and OMV PorB1a-K117Q / K171Q immunized mice. Thus, these data indicate that OMV PorB1a-K117Q / K171Q is a better vaccine antigen than OMV PorB1a WT, suggesting that reducing PorB-induced mitophagy contributes to enhance the immunoprotection of N. gonorrhoeae OMVs vaccines.​

Claims

1. A genetically modified Neisseria gonorrhoeae ( Neisseria gonorrhoeae ), characterized in that, The gene modification method involves mutating the gene encoding the PorB protein, thereby reducing or weakening its ability to induce mitophagy.

2. The genetically modified Neisseria gonorrhoeae according to claim 1, characterized in that, PorB protein is one of PorB1a and PorB1b.

3. The genetically modified Neisseria gonorrhoeae according to claim 2, characterized in that, The gene modification method is any of the following: The gene encoding the PorB1a protein was mutated to induce a double mutation of K117Q / K171Q in the PorB1a protein. The gene encoding the PorB1b protein was mutated to induce a double mutation of K117Q / K170Q in the PorB1b protein.

4. The genetically modified Neisseria gonorrhoeae according to claim 1, characterized in that, The original strain of Neisseria gonorrhoeae was Neisseria gonorrhoeae ATCC 49226.

5. The use of the genetically modified Neisseria gonorrhoeae according to any one of claims 1 to 4 in the preparation of Neisseria gonorrhoeae outer membrane vesicle vaccines.

6. A Neisseria gonorrhoeae outer membrane vesicle vaccine, characterized in that, The *Neisseria gonorrhoeae* strain modified according to any one of claims 1 to 4 is cultured, and outer membrane vesicles are extracted to prepare the *Neisseria gonorrhoeae* outer membrane vesicle vaccine.

7. The Neisseria gonorrhoeae outer membrane vesicle vaccine according to claim 6, characterized in that, It also includes pharmaceutically acceptable carriers, which include one or more of adjuvants, buffers, and lyophilization protectants.

8. The method for preparing the Neisseria gonorrhoeae outer membrane vesicle vaccine according to claim 6 or 7, characterized in that, The process includes the following steps: culturing genetically modified Neisseria gonorrhoeae, extracting outer membrane vesicles, and preparing the extracted outer membrane vesicles into the Neisseria gonorrhoeae outer membrane vesicle vaccine.

9. The method for preparing the Neisseria gonorrhoeae outer membrane vesicle vaccine according to claim 8, characterized in that, Extracellular vesicles were extracted by separating the supernatant after culture using ultracentrifugation.

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