An antigen combination, a porcine contagious pleuropneumonia - Klebsiella pneumoniae pneumonia bivalent vaccine and its application

Abstract

The present invention is applicable to the technical field of veterinary vaccines, and provides an antigen combination, an Actinobacillus pleuropneumoniae - Klebsiella pneumoniae suis bivalent vaccine and its application. The present invention has developed a bivalent vaccine (APP-KP bivalent vaccine) that can simultaneously prevent and treat Actinobacillus pleuropneumoniae and Klebsiella pneumoniae suis. The vaccine has shown significant effects in reducing the incidence rate, lowering the body temperature of piglets, reducing the frequency of asthma, alleviating the pathological damage of the lungs, and effectively clearing Actinobacillus pleuropneumoniae (APP) and Klebsiella pneumoniae suis (KP) in the body. By combining the TbpB-C and AdhE protein antigens of Actinobacillus pleuropneumoniae and the inactivated bacteria antigens JLKP3 and JLKP9 of Klebsiella pneumoniae suis, the present invention provides strong support for the healthy development of the pig farming industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary vaccines, and particularly relates to an antigen combination, a porcine contagious pleuropneumonia-Klebsiella pneumoniae suis bivalent vaccine and its application. Background Art

[0002] Porcine contagious pleuropneumonia (PCP), also known as necrotic pleuropneumonia, is a highly contagious and lethal respiratory infectious disease caused by Actinobacillus pleuropneumoniae (APP). Pigs of all ages are susceptible to this disease, which is commonly found in growing pigs and adult pigs. It is mainly characterized by acute hemorrhagic fibrinous pneumonia and chronic fibrinous necrotic pleurisy. The mortality rate of the most acute form can reach 80-100%, posing a serious threat to the breeding industry. In recent years, the clinical isolation rate of APP has been increasing year by year, and it often occurs in mixed infections with other pathogens such as Haemophilus parasuis, Pasteurella multocida, Klebsiella pneumoniae, PCV2 and PRRSV, causing porcine respiratory syndrome, weakening the pulmonary defense function of pigs and increasing the difficulty of prevention and treatment.

[0003] Actinobacillus pleuropneumoniae belongs to the family Pasteurellaceae and the genus Actinobacillus. It can be divided into 19 serotypes according to its capsular polysaccharide, and there is no cross-protection between different serotypes. The serotypes of APP prevalent in different regions of China are different. The latest epidemiological survey shows that serotypes 1, 3, 5, 7 and 15 are the dominant serotypes in China. Bioinformatics research has revealed the high conservation of TbpB (transferrin protease) and AdhE (belonging to acetaldehyde / ethanol dehydrogenase) among different serotypes of APP, which are important virulence factors of APP. TbpB-C and AdhE are also important antigens of PCP vaccines. Vaccines prepared with these two antigens can provide complete protection against APP type 1 and APP type 3 infections in mice (Chinese patent with publication number CN202411212848.0).

[0004] Klebsiella pneumoniae is one of the important opportunistic pathogens, which has a serious impact on the health of humans and animals. In recent years, Klebsiella pneumoniae has gradually become the second most important opportunistic pathogen after Escherichia coli, widely distributed in the natural environment, and can be secondary infected in various parts of the animal body, causing pneumonia, mastitis, septicemia and other suppurative inflammations.

[0005] In view of the great harm of porcine contagious pleuropneumonia, the inability of existing vaccines to provide good cross-protection effects and the lack of commercial vaccines for Klebsiella pneumoniae suis, the present invention provides an antigen combination, a porcine contagious pleuropneumonia-Klebsiella pneumoniae suis bivalent vaccine and its application. Summary of the Invention

[0006] The object of the present invention is to provide an antigen combination, a porcine contagious pleuropneumonia - Klebsiella pneumoniae disease bivalent vaccine and its application, aiming to solve the problems raised in the above - mentioned background technology.

[0007] The object of the present invention is achieved through the following technical solutions:

[0008] An antigen combination of Actinobacillus pleuropneumoniae and Klebsiella pneumoniae, comprising AdhE and TbpB - C proteins of Actinobacillus pleuropneumoniae and inactivated bacteria JLKP3 and JLKP9 of Klebsiella pneumoniae; The JLKP3 strain was deposited at the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025116; The JLKP9 strain was deposited at the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025117.

[0009] An application of the antigen combination of Actinobacillus pleuropneumoniae and Klebsiella pneumoniae as described above in the preparation of a porcine contagious pleuropneumonia - Klebsiella pneumoniae disease bivalent vaccine.

[0010] A porcine contagious pleuropneumonia - Klebsiella pneumoniae disease bivalent vaccine, comprising the antigen combination of Actinobacillus pleuropneumoniae and Klebsiella pneumoniae as described above, and MONTANIDE™ Gel 01 adjuvant.

[0011] Furthermore, the volume ratio of the MONTANIDE™ Gel 01 adjuvant to the protein and inactivated bacteria mixture is 1:9.

[0012] An application of the porcine contagious pleuropneumonia - Klebsiella pneumoniae disease bivalent vaccine as described above in the preparation of a drug for preventing and treating porcine contagious pleuropneumonia and Klebsiella pneumoniae disease.

[0013] An application of inactivated bacteria JLKP3 and JLKP9 in the preparation of a Klebsiella pneumoniae disease vaccine.

[0014] A Klebsiella pneumoniae disease vaccine, comprising inactivated bacteria JLKP3 and JLKP9.

[0015] An application of the Klebsiella pneumoniae disease vaccine as described above in the preparation of a drug for preventing and treating Klebsiella pneumoniae disease.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] The present invention has developed a combined vaccine (APP-KP combined vaccine) that can simultaneously prevent and control porcine contagious pleuropneumonia and Klebsiella pneumoniae in pigs. This vaccine has shown significant effects in reducing the body temperature of piglets, decreasing the frequency of asthma, alleviating pathological damage to the lungs, and effectively clearing Actinobacillus pleuropneumoniae (APP) and Klebsiella pneumoniae (KP) in the body. By combining the TbpB-C and AdhE protein antigens of Actinobacillus pleuropneumoniae and the inactivated bacteria antigens JLKP3 and JLKP9 of Klebsiella pneumoniae, the present invention provides strong support for the healthy development of the pig farming industry. Description of the Drawings

[0018] Figure 1 For the expression of candidate proteins, where M: Protein Marker; 1: Before induction; 2 - 3: Supernatant and precipitate of the recombinant plasmid expression after IPTG induction; 4: Purified recombinant protein.

[0019] Figure 2 For the screening of candidate antigens of Klebsiella pneumoniae; where A is the survival rate results of mice in each immunized group after challenge; B is the clinical symptom scores of mice in each group after challenge; C is the antibody titers in the sera of each group 28 days after immunization; D is the bacterial loads in the lungs, livers, and spleens of mice in each group 72 hours after infection.

[0020] Figure 3 For the evaluation of the immunization effect of the APP-KP combined vaccine in mice; where A is the survival rate of mice infected with APP1 after immunization with the combined vaccine; B is the survival rate of mice infected with APP3 after immunization with the combined vaccine; C is the survival rate of mice infected with JLKP3 after immunization with the combined vaccine; D is the survival rate of mice infected with JLKP9 after immunization with the combined vaccine; E is the antibody titer of TbpB-C in the sera of each group of mice; F is the antibody titer of AdhE in the sera of each group of mice; G is the antibody titer of JLKP3 in the sera of each group of mice; H is the antibody titer of JLKP9 in the sera of each group of mice.

[0021] Figure 4 For the evaluation of the immunization effect of the combined vaccine against porcine contagious pleuropneumonia-Klebsiella pneumoniae in piglets; where A is the monitoring of antibody titers in piglets after immunization; B is the monitoring of body temperature in piglets after infection; C is the macroscopic pathological changes and pathological section observations of piglet lungs; D is the detection of bacterial loads in each tissue. Detailed Embodiments

[0022] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are hereby described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0023] The following describes the specific implementation of the present invention in detail in combination with specific embodiments.

[0024] Example 1: Expression and purification of proteins;

[0025] 1.1 Construction of recombinant expression plasmids;

[0026] Using the genomic DNA of APP of serotype 1 as a template, the target fragment was amplified by PCR using the primers described in Table 1.

[0027] Table 1 Primer sequences for PCR of target genes

[0028]

[0029] After PCR amplification of the target gene, it was subjected to agarose gel electrophoresis, purified by a gel extraction kit, and the purified target gene and pET-32a plasmid were double-digested with BamHI and XhoI respectively. The digested target gene was ligated to the vector, and after mixing evenly, it was placed in a 16 °C low-temperature water bath for overnight ligation. Finally, the ligated recombinant plasmid was transformed into Escherichia coli DH5α competent cells by a kit, and the recombinant expression plasmids peT32a-TbpB-C and peT32a-AdhE were finally constructed. These recombinant expression plasmids were identified by enzyme digestion and sequencing, and all were consistent with the expectations, with no mutations in the sequences and successfully inserted into the pET32a vector.

[0030] Among them, the amino acid sequence of the TbpB-C protein is shown in SEQ ID NO.1, and the nucleotide sequence encoding the TbpB-C protein is shown in SEQ ID NO.2; the amino acid sequence of the AdhE protein is shown in SEQ ID NO.3, and the nucleotide sequence encoding the AdhE protein is shown in SEQ ID NO.4.

[0031] 1.2 Expression and purification of proteins;

[0032] The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) competent cells. The next day, a single colony on the plate was picked and inoculated into an LB liquid medium containing Amp+ resistance. When cultured at 37 °C and 180 rpm until OD600nm = 0.6 - 0.8, 200 μl of IPTG with a final concentration of 1 mM was added for induction at 16 °C for 16 h. After centrifugation at 4 °C and 6000 rpm for 10 min, the supernatant was discarded, and the cells were collected. The cells were resuspended with PBS and sonicated, and then the supernatant and precipitate were collected respectively.

[0033] The supernatant collected after sonication was purified by nickel column affinity chromatography. After sonication of the bacteria, they were centrifuged at 8000 rpm for 10 min at 4°C, and the supernatant was collected and adjusted to pH 8.0 for purification. The specific operation steps refer to the Cytiva Ni column purification instructions. Finally, 5×SDS gel loading buffer was added to the 500 mM imidazole eluent, and after boiling for 10 min, 12% SDS-PAGE was performed to detect the protein expression in different parts. The results showed that both proteins were highly expressed in the supernatant, and the sizes of TbpB-C and AdhE were 41.76 kDa and 114.2 kDa respectively ( Figure 1 ).

[0034] Example 2: Screening of Klebsiella pneumoniae antigens;

[0035] 2.1 Inactivation of candidate antigens;

[0036] Eight strains of candidate Klebsiella pneumoniae (JLKP3, JLKP9, KPP19, KPP17, KPP15, KP69, K36, and KP26) were respectively placed in LB liquid medium and cultured in a shaker at 37°C until the logarithmic phase (OD 600 ≈ 1.8), and formaldehyde was added to a final concentration of 0.2%. After the bacterial solution was inactivated at 37°C for 48 h, 200 μL was taken and spread on an LB plate to test the inactivation effect. After centrifuging an appropriate amount of bacteria, they were resuspended for later use.

[0037] Among them, Klebsiella pneumoniae KPP19, KPP17, KPP15, KP69, K36, and KP26 were all isolated from the laboratory and were available for use.

[0038] Klebsiella pneumoniae JLKP3 was deposited in the China Center for Type Culture Collection. The address of the depository is Wuhan University, Wuhan, China, with a postal code of 430072. The deposit date was January 13, 2025, and the deposit number is CCTCC NO: M2025116, and the taxonomic name is Klebsiella Pneumoniae JLKP3.

[0039] Klebsiella pneumoniae JLKP9 was deposited in the China Center for Type Culture Collection. The address of the depository is Wuhan University, Wuhan, China, with a postal code of 430072. The deposit date was January 13, 2025, and the deposit number is CCTCC NO: M2025117, and the taxonomic name is Klebsiella Pneumoniae JLKP9.

[0040] 2.2 Evaluation of the mouse immune protection effect of Klebsiella pneumoniae antigens;

[0041] Take the inactivated bacteria prepared in 2.1 and mix them thoroughly with MONTANIDE™ Gel 01 adjuvant (v / v = 9:1) to formulate an inactivated vaccine, with each antigen dose being approximately 3×10 9 CFU. Take 50 SPF 6-week-old female ICR mice, 5 mice in each group, and immunize and challenge them according to the experimental protocol in Table 2:

[0042] Table 2 Screening test for Klebsiella pneumoniae antigens

[0043]

[0044] Perform the second immunization 14 days after the first immunization, and perform the challenge 14 days after the second immunization. Before the first immunization, the second immunization, and the challenge, collect blood from the tail veins of the mice in each group and aliquot the serum for freezing and storage for later use. Continuously observe the mice for 5 days after the challenge, and count the survival of the mice. 72 hours after infection, aseptically collect the lungs (Lung), livers (Liver), and spleens (Spleen) of 3 mice in each group, grind them into homogenates, dilute them to a uniform multiple with PBS, take 100 μL and spread it on LB plates, and count the number of plates after 24 hours. The results of the survival rates of the mice in each group after the challenge are shown ( Figure 2 in A), and all the immunized mice survived after infection, especially the mice in the JLKP3 group and the JLKP9 group had the best clinical status ( Figure 2 in B). The results of the antibody titers in the sera of each group 28 days after immunization are shown ( Figure 2 in C). The specific antibody titers in the sera of the JLKP3 group, the JLKP9 group, the KPP19 group, the KPP17 group, the KPP15 group, the KP69 group, the K36 group, and the KP26 group were significantly higher than those in the adjuvant control group and the PBS group. The results of the bacterial loads in the tissues (lungs, livers, and spleens) of the mice in each group 72 hours after infection are shown ( Figure 2 in D). The JLKP3 group and the JLKP9 group had the lowest bacterial loads in the tissues and organs of the mice and the best clinical status. Combining the survival rates, specific antibody titers, tissue bacterial loads, and bacterial load data, determine that the JLKP3 and JLKP9 inactivated bacteria are candidate antigens for the Klebsiella pneumoniae vaccine.

[0045] Example 3: Preparation of a porcine contagious pleuropneumonia - Klebsiella pneumoniae combined vaccine and evaluation of its immunization effect in mice;

[0046] 3.1 Preparation of a porcine contagious pleuropneumonia - Klebsiella pneumoniae combined vaccine;

[0047] Preparation process: Take the TbpB-C and AdhE proteins from Example 1 and the inactivated bacteria JLKP3 and JLKP9 from Example 2, mix them well, dilute appropriately according to their concentrations and mix evenly. Then mix the diluted proteins and inactivated bacteria with MONTANIDE™ Gel 01 adjuvant (protein and inactivated bacteria: MONTANIDE™ Gel 01 adjuvant = 9:1 (v / v)). After sufficient combination, the APP-KP bivalent vaccine was obtained.

[0048] The volume of each dose of the APP-KP bivalent vaccine is 200 μL, containing 66 μg of TbpB-C protein, 33 μg of AdhE protein, 3×10 9 CFU of inactivated JLKP3 bacteria, 3×10 9 CFU of inactivated JLKP9 bacteria and adjuvant.

[0049] 3.2 Evaluation of the immune effect of the porcine contagious pleuropneumonia - Klebsiella pneumoniae bivalent vaccine in mice;

[0050] Take 60 SPF-grade 6-week-old female ICR mice, 5 in each group, and perform immune challenge according to the experimental protocol in Table 3:

[0051] Table 3 Immune challenge protocol of the porcine contagious pleuropneumonia - Klebsiella pneumoniae bivalent vaccine in mice

[0052]

[0053] The second immunization was carried out 14 days after the first immunization, and the challenge was carried out 14 days after the second immunization. Before the first immunization, the second immunization and the challenge, the mice in each group were bled from the tail vein and the serum was aliquoted and stored frozen for later use. The results showed that the APP-KP bivalent vaccine could provide an 80% (4 / 5) protective effect against both APP1 and APP3 type infections ( Figure 3 in A and B); after infection with JLKP3 and JLKP9 type strains, all the mice in the PBS group and the adjuvant control group died, and the survival rates of the vaccine group were 100% (5 / 5) ( Figure 3 in C) and 80% (4 / 5) ( Figure 3 in D). The results of serum antibody titers showed that the specific antibody levels of TbpB-C, AdhE, JLKP3 and JLKP9 in the mice increased significantly after the first immunization, and were further enhanced after the second immunization, while the specific antibody levels in the PBS group and the adjuvant control group did not change significantly compared with those before immunization ( Figure 3 in E - H). The above results prove that the APP-KP bivalent vaccine can stimulate mice to produce high-level specific antibodies against the four antigens of TbpB-C, AdhE, JLKP3 and JLKP9, and at the same time protect mice from infections of multiple serotypes of APP and KP.

[0054] Example 4: Preparation of a Porcine Contagious Pleuropneumonia - Klebsiella pneumoniae Subtype III Pneumoniae Bivalent Vaccine and Evaluation of Its Immunization Efficacy in Piglets

[0055] 4.1 Preparation of a Porcine Contagious Pleuropneumonia - Klebsiella pneumoniae Subtype III Pneumoniae Bivalent Vaccine

[0056] The preparation process is the same as that in 3.1 above.

[0057] Each dose of the APP - KP bivalent vaccine has a volume of 2 ml and contains 1 mg of TbpB - C protein, 0.5 mg of AdhE protein, 1×10 10 CFU of heat - inactivated JLKP3 bacteria and 1×10 10 CFU of heat - inactivated JLKP9 bacteria.

[0058] 4.2 Evaluation of the Immunization Efficacy of the Porcine Contagious Pleuropneumonia - Klebsiella pneumoniae Subtype III Pneumoniae Bivalent Vaccine in Piglets

[0059] Twenty - two 4 - week - old SPF piglets were selected and immunized and challenged according to the experimental protocol in Table 4:

[0060] Table 4 Immunization and challenge protocol for piglets with the Porcine Contagious Pleuropneumonia - Klebsiella pneumoniae Subtype III Pneumoniae Bivalent Vaccine

[0061]

[0062] The second immunization was carried out 21 days after the first immunization, and the challenge was carried out 14 days after the second immunization. At the same time, tail vein blood was collected from the piglets in each group before the first immunization, the second immunization, and the challenge, and the serum was aliquoted and stored frozen for later use.

[0063] The morbidity rate, respiratory rate, LLS lung consolidation score, and pathological section score of the piglets in each group are shown in Table 5:

[0064] Table 5 Morbidity rate, respiratory rate, LLS lung consolidation score, and pathological section score of piglets in each group

[0065]

[0066] The results showed that after primary and booster immunizations, the titers of specific antibodies against TbpB - C, AdhE, and JLKP3 in the serum of piglets increased ( Figure 4 in A). Fourteen days after the second immunization, the piglets were infected with APP1 and JLKP3, and at the same time, a commercial APP vaccine was introduced as a control. There is no commercial vaccine for Klebsiella pneumoniae subtype III pneumonia. After infection, it was found that the piglets in the APP control group had clinical symptoms such as asthma (higher respiratory rate), coughing, and elevated body temperature ( Figure 4In Table 5), no respiratory symptoms were found in the APP commercial vaccine group and the APP-KP combined vaccine group (APP1). The mental state and activity were normal, and the appetite was normal. After sacrificing the piglets, it was found that most of the lungs in the APP control group were purplish-red, the lungs were swollen, some tissues were necrotic, there were suppurative foci in the lungs, and emphysema was accompanied. Only bleeding, surface fibrinous exudate and other phenomena were found in the APP commercial vaccine group and the APP-KP combined vaccine group (APP1), and the degree of lung consolidation was much lower than that in the APP control group. The main change in the KP control group was bleeding, with a large number of bleeding points the size of millet grains scattered and accompanied by swelling. Substantiated areas could be seen on the cut surface. The results of pathological tissue sections showed that compared with the APP control group and the KP control group, the APP-KP combined vaccine group (APP1 and JLKP3) and the APP commercial vaccine group had fewer inflammatory cell infiltrations in the lungs, better alveolar integrity, and lower bleeding degree (blood cell deposition) ( Figure 4 In C). In addition, a large number of APP and KP still existed in the tissues 14 days after infection. Compared with the APP control group and the KP control group, the APP-KP combined vaccine group reduced the colonization of APP and KP in the liver and lungs at the same time. The number of APP in the spleen also decreased significantly, and the effect of reducing KP in the blood was more obvious ( Figure 4 In D), which proved the important role of the APP-KP combined vaccine in clearing bacteria in the body.

[0067] In summary, the APP-KP combined vaccine plays an important role in reducing fever in piglets, reducing the frequency of asthma, alleviating lung pathological changes, and clearing APP and KP in the body, preventing various respiratory symptoms after APP and KP infection, and is a candidate vaccine for APP and KP.

[0068] The above is only the preferred embodiment of the present invention. It should be pointed out that for those skilled in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. An antigen composition, characterized in that Including AdhE and TbpB-C proteins of Actinobacillus pleuropneumoniae and JLKP3 and JLKP9 inactivated bacteria of Klebsiella hyopneumoniae; the amino acid sequence of TbpB-C protein is shown in SEQ ID NO.1, and the amino acid sequence of AdhE protein is shown in SEQ ID NO.3; the JLKP3 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025116; the JLKP9 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025117.

2. Use of the antigen composition according to claim 1 in the preparation of a combined vaccine for the prevention and treatment of porcine contagious pleuropneumonia and Klebsiella hyopneumoniae.

3. A combined vaccine for the prevention and treatment of porcine contagious pleuropneumonia and Klebsiella hyopneumoniae, comprising the antigen composition as claimed in claim 1 and MONTANIDE™ Gel 01 adjuvant.

4. The bivalent vaccine for preventing and treating porcine contagious pleuropneumonia and Klebsiella hyopneumoniae according to claim 3, characterized in that: The volume ratio of the MONTANIDE™ Gel 01 adjuvant to the protein and inactivated bacteria mixture is 1:

9.

5. Use of the bivalent vaccine for preventing and treating porcine contagious pleuropneumonia and Klebsiella hyopneumoniae according to claim 3 or 4 in the preparation of a medicament for preventing and treating porcine contagious pleuropneumonia and Klebsiella hyopneumoniae.

6. Use of inactivated bacteria JLKP3 and JLKP9 in the preparation of vaccines for the prevention and treatment of Klebsiella hyopneumoniae, characterized in that: The JLKP3 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M2025116; the JLKP9 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with the deposit number CCTCC NO: M 2025117.

7. A vaccine for preventing and treating Klebsiella hyopneumoniae, characterized in that: It comprises inactivated bacteria of JLKP3 and JLKP9; the JLKP3 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M 2025116; the JLKP9 strain was deposited in the China Center for Type Culture Collection on January 13, 2025, with a deposit number of CCTCC NO: M2025117.

8. Use of the vaccine for preventing and treating Klebsiella hyopneumoniae according to claim 7 in the preparation of a medicament for preventing and treating Klebsiella hyopneumoniae.

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