Immunoactive composition and pertussis vaccine containing same

Abstract

The invention belongs to the technical field of biology, and relates to an immunocompetence composition and a pertussis vaccine containing the immunocompetence composition. The immunocompetence composition comprises pertussis Bordetella outer membrane vesicles and attenuated pertussis toxins in a weight ratio of (5-50): (5-25). Effective protective immunity can be triggered in the process of preventing pertussis infection, pertussis bacteria in the lung are effectively eradicated, and bacterial colonization in the trachea and the nasal cavity is remarkably reduced.

Description

Technical Field

[0001] This application belongs to the field of biotechnology and relates to an immunocompetent composition and a pertussis vaccine containing the same. Background Art

[0002] Pertussis is a highly contagious respiratory disease caused by the Gram-negative bacterium Bordetella pertussis (B.Pertussis, Bp) and can pose a life-threatening risk in infancy and childhood. It usually occurs in periodic epidemics, with major outbreaks every few years followed by a decline, which may be related to the duration of immune protection and immune memory after infection. Pertussis remains a major global public health problem, and vaccination is the main measure for preventing infection and controlling the spread of this disease.

[0003] There are two types of vaccines recommended for preventing pertussis, whole cell pertussis (wP) vaccine and acellular pertussis (aP) vaccine. The wP vaccine consists of a suspension of heat-killed bacteria and was introduced in the 1940s. The aP vaccine consists of purified pertussis antigens and is a new generation of acellular pertussis vaccine developed in the 1980s and 1990s. However, due to safety concerns about the whole cell pertussis component, wP was subsequently replaced by aP in children. Although in the Expanded Programme on Immunization, the pertussis vaccine has achieved a high vaccination coverage worldwide, pertussis has not been eradicated in any country. In contrast, the incidence of this disease has recently increased, especially in countries that have switched from the wP vaccine to the aP vaccine. Epidemiological and experimental evidence indicates that current aP vaccines help reduce pertussis-related morbidity and mortality, but they do not provide long-lasting immunity or sufficient protection against the infection and transmission of epidemic strains. Given the high rate of pertussis infection and its impact on public health, effective control of the disease may require not only stopping its occurrence but also preventing infection and transmission.

[0004] Studies have shown that natural infection and wP vaccine can trigger effective Th1 and Th17 responses, and there are resident CD4 T memory cells in the lungs. These CD4 T cells are present in respiratory tissues and express CD44 + CD62L low CD69 + / CD103 +Phenotypes and provide long-term protective immunity against Bordetella pertussis bacteria. On the other hand, aP vaccines induce strong antibody and Th2 polarized immune responses, with weaker Th1 and Th17 responses. Therefore, the ability of aP vaccines to provide long-term protection and clear bacteria is significantly inferior to that of wP vaccines. Therefore, there is an urgent need to develop and study new pertussis vaccine compounds that can induce a broader and more persistent protective immune response, especially at the level of respiratory mucosa, which may help improve pertussis control, although developing new and promising pertussis candidate vaccines and obtaining approval is a challenge.

[0005] However, a promising alternative strategy is outer membrane vesicles (OMVs), which are outer membrane vesicles naturally shed by bacteria. OMVs have the optimal size for uptake by immune cells, and their surfaces are exposed to a range of antigens and Toll-like receptor (TLR) activators, making them an attractive vaccine platform. In fact, vaccines based on outer membrane vesicles have been successfully developed against Neisseria meningitidis serogroup B. A pertussis vaccine based on Bordetella pertussis outer membrane vesicles (OMVsBp) contains a similar antigen composition and morphological structure to the whole-cell pertussis vaccine and induces a similar immune response. Studies have shown that OMVsBp induce effective mucosal and Th17-type responses, as well as high levels of systemic immune responses, via the nasal route, thereby preventing Bp colonization in the respiratory tract (including the nasal cavity) and potentially preventing transmission.

[0006] On the other hand, multi-component vaccines maximize the potential to kill pathogens by exerting synergistic effects between antigens. In pertussis, the antigens pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (Prn, a Bordetella pertussis adhesin) are components of aP vaccines, and BrkA, Vag8, Prn, and fimbriae 2&3 have also been described as potential candidate antigens. Among them, PT is an important protective vaccine antigen and is the main component of all current aP vaccines. It has been reported that PT-specific antibodies, whether actively induced or passively administered, can protect mice from lethal infection. The reason can be explained that neutralization and opsonophagocytosis mediated by anti-PT antibodies play a crucial role in preventing pertussis. In addition, PT is also a mitogen with excellent adjuvant properties. There is evidence that the problem faced by all acellular pertussis vaccines is a relatively rapid decline in immunity, especially to pertussis vaccines. Therefore, improving the persistent immune response against PT may provide better immune protection.

[0007] Lipopolysaccharide (LPS) plays a dual role in Gram-negative bacteria, serving as both a key virulence factor and a protective antigen. However, due to the lack of an O-specific polysaccharide domain, the LPS of Bp is called lipooligosaccharide (LOS), which is different from that of most Enterobacteria such as Escherichia coli and Salmonella. The LOS of Bp consists of lipid A and a branched dodecasaccharide core composed of unusual sugars with free amino and carboxyl groups, but without an O-specific polysaccharide, which has also been reported as a potential vaccine component. A study by Kubler-Kielb et al. showed that conjugates of Bp LPS-derived core oligosaccharide (OS) with proteins bound to amino-oxidized bovine serum albumin through their terminal Kdo residues and could induce high levels of LOS-specific IgG in mice. The antisera induced by these conjugates had bactericidal effects against Bp, and their titers were correlated with the antibody levels measured by ELISA. Although Bp LOS is a poor immunogen due to its low molecular weight (∼2,500 Kd), serum LOS antibodies can still be found in children recovering from pertussis. However, as a promising vaccine candidate, the levels and functions of the immune responses induced by outer membrane vesicles and their LOS have not been elucidated. In addition, based on existing research evidence, it was found that OMVs and PT induce protective immune responses through different mechanisms. However, it is not yet clear whether combining these two components will help address the current challenges faced by pertussis vaccines.

[0008] Although both OMVsBp and PT are major antigens of pertussis vaccines, and OMVsBp has been reported as a vaccine and can provide partial protection against pertussis disease, the protective efficacy it induces is still insufficient. PT, as a key antigen component of acellular pertussis vaccines (aP), is the main component for aP vaccines to exert their effects. However, it has poor antibody persistence, cannot directly kill bacteria, cannot prevent the clearance of infected bacteria, and has limitations in the protective efficiency produced. Summary of the Invention

[0009] Based on this, one or more embodiments of the present application provide an immunologically active composition and a pertussis vaccine containing the same.

[0010] In the first aspect of the embodiments of the present application, an immunologically active composition is provided, which comprises Bordetella pertussis outer membrane vesicles and attenuated pertussis toxin in a weight ratio of (5 - 50) : (5 - 25).

[0011] In some embodiments of the present application, the weight ratio of the Bordetella pertussis outer membrane vesicles to the attenuated pertussis toxin is (5 - 15) : (5 - 15).

[0012] In some embodiments of the present application, the attenuated pertussis toxin is a genetically mutated attenuated pertussis toxin.

[0013] In some embodiments of the present application, relative to the pertussis toxin of the strain numbered CMCC58003, the S1 subunit of the attenuated pertussis toxin has the following mutations: R9K and Q129G.

[0014] In the second aspect of the embodiments of the present application, there is provided the use of the immunocompetent composition in the preparation of a pertussis vaccine.

[0015] In some embodiments of the present application, the dosage form of the pertussis vaccine includes an injection.

[0016] In the third aspect of the embodiments of the present application, there is provided a pertussis vaccine, which includes the immunocompetent composition and an immunoadjuvant.

[0017] In some embodiments of the present application, the pertussis vaccine includes 10 - 100 μg / mL of the outer membrane vesicles of Bordetella pertussis, 10 - 50 μg / mL of the attenuated pertussis toxin, and the immunoadjuvant.

[0018] In some embodiments of the present application, the immunoadjuvant includes an aluminum hydroxide adjuvant.

[0019] In some embodiments of the present application, the concentration of the aluminum hydroxide adjuvant in the pertussis vaccine is 100 - 1000 μg / mL.

[0020] Details of one or more embodiments of the present application are set forth in the following description, and other features, objects, and advantages of the present application will become apparent from the specification and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] To more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0022] Figure 1 Shows the morphological characteristics, particle size distribution, and protein composition of OMVsBp;

[0023] Figure 2 Shows a schematic diagram of the immunization schedule;

[0024] Figure 3 Shows the immunization effects of OMVsBp, OMVsBp combined with PTgd or aP mixed vaccine on intraperitoneal immunized mice;

[0025] Figure 4Changes in the levels of anti-PT(a), anti-Prn(b), anti-FHA(c), anti-Vag8(d), and anti-LOS(e) in the lung tissues of mice immunized intraperitoneally with OMVsBp or OMVsBp mixed with PTgd vaccine;

[0026] Figure 5 Results of the study on the levels of specific antibodies in the trachea of mice induced by intraperitoneal immunization;

[0027] Figure 6 Results of the study on the levels of specific antibodies in the nasal cavity of mice induced by intraperitoneal immunization;

[0028] Figure 7 Results of the detection of antigen-specific functional antibody levels induced by intraperitoneal immunization of mice;

[0029] Figure 8 Results of the detection of cytokines produced by splenocytes of mice immunized intraperitoneally with the vaccine after restimulation with antigen or peptide;

[0030] Figure 9 Local proliferation of CD4+ TRM cells in the lungs of mice after administration of different formulated vaccines via different immunization routes;

[0031] Figure 10 Results of the study on the protective effect of nasal challenge in mice against pertussis. Detailed implementation manners

[0032] The present application will be further described in detail below in conjunction with the accompanying drawings, implementation manners, and examples. It should be understood that these implementation manners and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing the implementation manners and examples and are not intended to limit this application.

[0034] Term

[0035] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0036] As used herein, the terms "and / or", "or / and", and "and / or" include any one of two or more related listed items, and also include any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0037] In this application, terms such as "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, mean greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0038] As used herein, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more than two of the listed items.

[0039] In this article, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0040] In this article, "preferred", "better", "more preferred", "it is advisable" are only used to describe the implementation modes or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0041] In this application, "further", "even further", "especially", etc. are used for descriptive purposes and indicate differences in content, but should not be construed as a limitation on the protection scope of this application.

[0042] In this application, "optionally", "optional", and "option" mean having or not having, that is, either one of the two alternative options of "having" or "not having". If the term "optional" appears in multiple places in a technical solution, without special instructions, and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0043] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0044] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0045] In this application, regarding a numerical interval (i.e., a numerical range), without special instructions, the optional numerical values are considered continuous within the above numerical interval, and include the two numerical endpoints of this numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Without special instructions, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group composed of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0046] The temperature parameter in this application, without special limitations, allows both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0047] In this application, %(w / w) and wt% both represent weight percentages, %(v / v) represents volume percentage, and %(w / v) represents mass-volume percentage.

[0048] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When this application involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When this application involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated into this application as references, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description of this application.

[0049] wP: whole cell pertussis vaccine; aP: acellular pertussis vaccine; LOS: lipooligosaccharide of pertussis; T RM : tissue-resident memory cell; OMVsBp: outer membrane vesicles of Bordetella pertussis; i.p.: intraperitoneal injection; PT: pertussis toxin; PTgd: genetically detoxified pertussis toxin; SBA: serum bactericidal assay; OPA: opsonophagocytic assay.

[0050] Although the currently used acellular pertussis (aP) vaccines have shown considerable efficacy in preventing diseases, they cannot prevent the infection and transmission of Bordetella pertussis bacteria. To address the global resurgence of pertussis, next-generation vaccines that can provide broad and long-lasting immunity are needed. In the embodiments of this application, the outer membrane vesicles of Bordetella pertussis (OMVsBp) combined with the genetic detoxification of pertussis toxin (PTgd) can induce a protective immune response. Compared with the OMVsBp and aP vaccines alone, co-immunization with OMVsBp and PTgd leads to a more Th17-type immune response and generates more resident memory CD4 T cells in the lung region. When conducting respiratory infection experiments, the vaccine formulation simultaneously inoculated with OMVsBp and PTgd showed a stronger induction of protective immunity. This combined method effectively eradicated Bordetella pertussis bacteria in the lungs of mice and significantly reduced bacterial colonization in the trachea and nasal cavity. This combination formula has not been reported currently and is an innovative vaccine composition for preventing pertussis disease.

[0051] In a first aspect, an embodiment of the present application provides an immunocompetent composition, which comprises Bordetella pertussis outer membrane vesicles and attenuated pertussis toxin in a weight ratio of (5 - 50) : (5 - 25). The weight ratio of the Bordetella pertussis outer membrane vesicles to the attenuated pertussis toxin is, for example, 5:5, 5:10, 5:15, 5:20, 5:25, 10:5, 10:15, 10:25, 20:5, 20:10, 20:15, 20:25, 35:5, 35:10, 35:15, 35:20, 35:25, 50:5, 50:10, 50:15, 50:20.

[0052] In some examples thereof, the weight ratio of the Bordetella pertussis outer membrane vesicles to the attenuated pertussis toxin is (5 - 15) : (5 - 15).

[0053] The present application does not particularly limit the attenuation method of the attenuated pertussis toxin, which may be chemically detoxified / attenuated PT or PT detoxified / attenuated by gene mutation. The present application does not particularly limit the strain source of the detoxified / attenuated pertussis toxin, including but not limited to the strain numbered CMCC58003. The present application does not particularly limit the mutation corresponding to the detoxification / attenuation by gene mutation. Based on PTs with different amino acid sequences, corresponding mutation schemes can be selected. For example, relative to the pertussis toxin of the strain numbered CMCC58003, the S1 subunit of the attenuated pertussis toxin has the following mutations: R9K and Q129G.

[0054] The present application does not limit the preparation method of OMVsBp. OMVsBp can be OMVsBp prepared from natural strains or OMVsBp with attenuated toxicity prepared from genetically modified strains.

[0055] The combination of OMVsBp and PT overcomes the deficiency of a single component in inducing a protective immune response. This combination co-immunization exerts a synergistic effect and can more effectively clear the bacteria colonized in the lungs, trachea, and nose than any single combination. In the present application, the combined use of OMVsBp and PT elicits an effective protective immunity in the prevention of pertussis infection. This combined method effectively eradicates the pertussis bacteria in the lungs of mice and significantly reduces the bacterial colonization in the trachea and nasal cavity, and is an effective novel pertussis vaccine.

[0056] In a second aspect, the present application provides the use of the immunocompetent composition in the preparation of a pertussis vaccine.

[0057] The present application does not particularly limit the dosage form of the pertussis vaccine, including but not limited to injection.

[0058] In a third aspect, a pertussis vaccine provided in an embodiment of the present application includes the immunologically active composition and an immunoadjuvant.

[0059] In some examples, the pertussis vaccine includes 10 - 100 μg / mL (such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 μg / mL) of the outer membrane vesicles of Bordetella pertussis, 10 - 50 μg / mL (such as 10, 15, 20, 25, 30, 35, 40, 45, 50 μg / mL) of the attenuated pertussis toxin, and the immunoadjuvant.

[0060] In some examples, the immunoadjuvant includes an aluminum hydroxide adjuvant.

[0061] In some examples, the concentration of the aluminum hydroxide adjuvant in the pertussis vaccine is 100 - 1000 μg / mL (such as 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μg / mL).

[0062] The definition of each component in the pertussis vaccine of the present application is based on the human dosage. 0.5 mL of the pertussis vaccine is one dose. When used in other animals, such as immunizing mice, the dosage can be reduced as appropriate, such as 1 / 10 or 1 / 5 of the human dosage.

[0063] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following examples, the guidance given in the present application is preferably referred to, and it can also be in accordance with the experimental manuals or conventional conditions in the art, or in accordance with the conditions recommended by the manufacturer, or refer to the experimental methods known in the art.

[0064] In the following specific examples, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0065] 1. Method

[0066] 1.1 Preparation of aP vaccine

[0067] The Bordetella pertussis CS strain (CMCC58003) is derived from the National Medical Culture Collection Center. An aP vaccine is prepared using the CS strain. Among them, the preparation of the attenuated pertussis toxin in the aP vaccine mainly involves genetically modifying the sequence encoding the S1 subunit of pertussis toxin (PT) in the CS strain. Through genetic modification, the amino acid arginine (R)-9 is replaced with lysine (K), and the amino acid glutamate (Q)-129 is replaced with glycine (G), thereby preparing an attenuated pertussis toxin (PT-9K / 129G) with an S1 subunit mutant. The mutant strain producing this attenuated pertussis toxin is denoted as the Bordetella pertussis JWCS-PTgd-001 strain. Gene sequencing confirmed the existence of this strain. The sequencing results showed that the genomic difference of the Bordetella pertussis JWCS-PTgd-001 strain compared to the CS strain is only the introduction of the above S1 subunit mutation. The identification result of the gene-attenuated S1 subunit sequence has been uploaded to GenBank (accession number: OR881390.1).

[0068] The steps for genetically modifying the CS strain include:

[0069] Genetic mutation of the CS strain is carried out through bacterial chromosomal homologous recombination technology. In this implementation case, a two-step modification strategy is adopted: in the first step, the ptx gene promoter and ptxA sequence are completely replaced with a resistance gene; in the second step, the ptxp3 promoter sequence and the E9K / R129G double mutant S1 subunit gene sequence (ptxp3-ptg) are used to replace the resistance gene.

[0070] In the first implementation method, the homologous recombination vector pMVBP-Chl is independently designed. The CS strain (MVCS) preserved by the company is subjected to second-generation sequencing to obtain the whole-genome sequence. The 25-15000bp sequence upstream of ptxp is selected as the upstream homologous region, the complete chloramphenicol gene (Chl) sequence is used as the targeted modification region, and the 25-15000bp sequence downstream of ptxpA is used as the downstream homologous region. It is introduced into the MVCS bacteria by electroporation, and positive clones expressing chloramphenicol are screened out and named JWCS-Chl. The No. 001 bacteria are selected for the second implementation.

[0071] In the second implementation method, the homologous recombination vector pMVBP-Ptg is independently designed. The upstream and downstream homologous regions are the same as those in the first implementation method. The ptxp3-ptgd is used as the targeted modification region and is introduced into the MVCS-Chl-001 bacteria by electroporation. Positive clones with the ptxp3-ptgd genotype are screened out and named JWCS-PTgd. The No. 01 bacteria are selected for subsequent verification.

[0072] Specific experimental steps:

[0073] (1) Preparation of Competent Cells

[0074] 1. Take 100 μL of glycerol stock and streak it on a plate, incubate at 35 °C for 4 days.

[0075] 2. Use an inoculation loop to pick a single colony and transfer it to 3 test tubes containing 5 mL of MSS liquid medium, incubate at 35 °C and 220 rpm for 2 days until the bacterial solution becomes turbid.

[0076] 3. Transfer it to 150 mL of MSS liquid medium, incubate at 35 °C and 220 rpm for about 16 h.

[0077] 4. Place the shaking flask on ice for pre-cooling, centrifuge to harvest the bacterial cells.

[0078] 5. Discard the supernatant, add ultrapure water equal to the volume of the original culture medium to the shaking flask, gently pipette with a 10 mL pipette tip to resuspend the bacterial cells, and centrifuge at 4 °C to precipitate the bacterial cells.

[0079] 6. Repeat step 5 once.

[0080] 7. Discard the supernatant, add glycerol water (concentration range: 1% - 25%) with a volume of 1 / 10 of the original culture medium, resuspend and centrifuge at 4 °C.

[0081] 8. Discard the supernatant, resuspend the bacterial cells with glycerol water to obtain competent cells, aliquot 200 μL per tube, and store at -80 °C in the refrigerator.

[0082] (2) Electroporation

[0083] 1. Take 200 μL of competent cells and place them on ice to thaw.

[0084] 2. Take the working plasmid (dosage range: 0.1 - 5 μg) and add it to the competent cells, flick gently several times, and place the mixture on ice for 5 - 30 min.

[0085] 3. Add the above mixture to a 0.1 cm electroporation cuvette (the electroporation cuvette has been pre-cooled at -20 °C).

[0086] 4. Use a Bio-Rad electroporator, set the electric shock voltage (range: 1.5 kV - 3.5 kV), capacitance 25 μF, resistance 200 Ω, give 1 - 2 electric shocks, immediately transfer to 1 mL of room temperature MSS medium after the electric shock, and incubate at 36 °C and 220 rpm for 4 - 8 h.

[0087] 5. Transfer the bacterial solution to a 1.5 mL sterile EP tube, centrifuge at 5000 rpm for 5 min.

[0088] 6. After discarding 850 μL of the supernatant, gently pipette the remaining liquid to resuspend, streak on a sheep blood-containing Jiang's plate with kana antibiotic, and incubate in a 35 °C incubator for 5 - 7 days.

[0089] (3) Positive clone screening

[0090] 1. Pick 4 - 6 monoclonal colonies from the above electrotransformation plate and transfer them to 3 mL of MSS liquid medium. Incubate at 35 °C and 220 rpm until the bacterial solution becomes turbid;

[0091] 2. Aspirate the bacterial solution for serial dilution. Take the bacterial solutions of two dilution factors, 10 -8 and 10 -9 and spread them evenly on a sheep blood - containing Bordet - Gengou plate. Incubate in an incubator at 35 °C for 5 - 7 days;

[0092] 3. Take several sterile 96 - well plates and add 10 μL of sterile water to the bottom of each well;

[0093] 4. After picking monoclonal colonies on the blood plate with a sterile plastic rod, place them in a sterile 96 - well PCR plate, one clone per well, and disperse the bacteria in the sterile water;

[0094] 5. Use a multi - channel pipette to aspirate the above liquid and spot it successively at the same positions on the antibiotic - free and antibiotic - containing sheep blood - containing Bordet - Gengou plates. Incubate in an incubator at 35 °C for 1 - 3 days;

[0095] 6. According to the growth of clones on the two types of plates, pick the corresponding positive clones, transfer them to 3 mL of MSS liquid medium, and incubate at 35 °C and 220 rpm until the bacterial solution becomes turbid for subsequent verification.

[0096] (4) Verification results

[0097] Through two - step screening, the Bordetella pertussis strain MVCS - PTgd expressing the gene of detoxified pertussis toxin was successfully screened and named MVCS - PTgd - 01.

[0098] This strain grows on a Bordet - Gengou agar plate (BD, 248220) containing 20% defatted sheep blood. Collect the colonies and inoculate them into 50 mL of liquid Modified Stainer - Scholte medium (MSS). Sub - culture the culture twice in the same medium (each culture for 48 h). Inoculate the sub - cultured culture into 200 L of MSS medium and culture it in a 500 L fermenter for 36 - 48 h. The culture conditions include: temperature controlled at 36 ± 1 °C, ventilation rate controlled at 0.1 - 0.4 vvm, and rotation speed controlled at 50 - 200 rpm. When the culture reaches OD600nm ≥ 12, centrifuge using a continuous - flow centrifuge to harvest the bacteria and the supernatant.

[0099] Purify the concentrated supernatant using cation - exchange chromatography and type - II hydroxyapatite chromatography columns to obtain PTgd (attenuated pertussis toxin) and FHA (filamentous hemagglutinin) respectively.

[0100] The Prn protein was released from the bacteria by heat treatment (60 ± 2 °C), clarified by continuous flow centrifugation and depth filtration, and purified Prn (Bordetella pertussis adhesin) was obtained by anion exchange chromatography and ammonium sulfate precipitation.

[0101] The above antigens were treated with 0.035 - 0.05% formaldehyde (w / v) to obtain PTgd-S, FHA-S, and Prn-S. After stabilization, the pertussis antigens (30 μg / mL PTgd; 16 μg / mL FHA; 16 μg / mL Prn) were adsorbed by aluminum hydroxide adjuvant (prepared in the laboratory, 0.45 mg / mL Al 3+ ) to obtain the aP vaccine (see Table 1).

[0102] 1.2 Preparation of OMVsBp

[0103] The preparation method of OMVsBp is as follows: On a bordt - gengou (BG) agar plate, the working seed of Bordetella pertussis strain (JWCS - PTgd - 001) was cultured at 34 - 36 °C for 72 h. Then the bacterial lawn was collected and scaled up from 50 mL to 500 mL in SS liquid medium. The culture was inoculated into a 50 L MSS medium (34 - 36 °C, pH 7.2) fermenter and fermented until the bacteria grew to the stationary phase. Subsequently, it was centrifuged at 20,000 g for 30 min, and the supernatant was collected. Then the supernatant was ultrafiltered using a filtration membrane with a molecular weight of 300 kDa (PALL, 2.5 m 2 surface area). The precipitate obtained by ultrafiltration was resuspended in a 20 mM trihydrochloric acid (pH 8.5) solution containing 1 wt% sodium deoxycholate, then ultracentrifuged at 100,000 g at 4 °C for 2 h. The centrifuged supernatant was discarded, and the precipitate was resuspended in a 3 wt% sucrose solution. Finally, it was homogenized at a pressure of 600 bar.

[0104] The structure of OMVsBp was characterized by electron microscopy, and the types and abundances of proteins in Bordetella pertussis outer membrane vesicles were analyzed by quantitative proteomics methods.

[0105] 1.3 Immunization

[0106] NIH female mice aged 8 - 10 weeks from SPF (Beijing) Biotechnology Co., Ltd. (production license number: SCXK(Beijing)2019 - 0010) were selected, with 6 mice in each cage, and maintained in a pathogen - free environment. The mice were randomly divided into 4 groups (18 mice in each group) for intraperitoneal immunization. Group 1 was given OMVsBp (2 μg / dose) twice on day 0 and day 28. Group 2 was given OMVsBp supplemented with PTgd (2 μg / dose; 2 μg / dose, named OMVsBp + PTgd group) twice on day 0 and day 28. Group 3 (positive control group) was given an aP vaccine twice on day 0 and day 28. Group 4 (negative control group) was given normal saline twice on day 0 and day 28.

[0107] On the 56th day, for some mice (6 mice) in each group, 0.5 mL of tribromoethanol was injected intraperitoneally. After the mice were anesthetized, serum was collected and stored at - 20 °C for antigen - specific antibody analysis. The remaining mice (12 mice) in each group were stimulated with aerosol, and the mice were euthanized with carbon dioxide. After euthanasia, the lungs and spleens of the mice were taken for cytokine analysis. The lungs, nasal cavities, and tracheas of each group were rinsed, and the tissue lavage fluid was collected and stored to detect the levels of antigen - specific IgG and IgA antibodies.

[0108] The formulations of each dose are shown in Table 1. The research design is as Figure 2 shown.

[0109] 1.4 Serological detection

[0110] 1.4.1 ELISA for detecting antigen - specific antibody titers

[0111] The indirect ELISA method was used to detect the presence of IgG and IgA antibodies against each component of the candidate vaccine in mouse serum or tissue lavage fluid. In this laboratory, coated antigen purified components (PT, FHA, Prn, Vag8, BrkA, LOS) and positive control sera for BrkA, Vag8, and LOS were prepared.

[0112] Briefly, microtiter plates (Nunc MaxiSorpTM, 44240421) were coated with specific antigens at a concentration of 100 ng / well (4 °C, overnight). After washing, the uncoated sites were blocked with a blocking buffer of PBST containing 1.0% BSA. The serum to be tested or standard serum was diluted 2 - fold on the plate. After incubation at room temperature for 1 h, the plates were washed. Goat anti - mouse IgG - HRP (IgA - HRP) was added to each well and incubated for 1 h. After the final wash, ABTS substrate was added and reacted in the dark for 30 minutes, and then the stop solution (1 wt% SDS) was added.

[0113] The IgG levels of PT, FHA, Prn, BrKA, Vag8, and LOS antibodies were detected using different plates (Corning, 42592). The secondary antibody was goat anti-mouse IgG-AP. PNPP substrate and 3M sodium hydroxide stop solution were used. The optical density was read at 405 nm using an MDSPECTRAMAX M4 / EL X 800.

[0114] For the detection of lavage fluid IgA, serum titers were used in the case of standard sera. ELISA units (IU / EU) were determined by the SoftMax Pro analysis software according to the four-parameter logistic reference curve of the standard sera. If the IgG IU was below the LLQ, half of the LLQ value was assigned for the calculation of geometric mean IU / EU.

[0115] 1.4.2 Serum bactericidal assay (SBA)

[0116] The target strain Tohama I (BAA-589) was obtained from ATCC, and fresh bacterial suspensions were prepared to reduce experimental variation and stored at -70 °C. The bacterial suspension in the freezer vial was diluted to approximately 2.7×10 9 CFU / mL, 0.65 mL per vial, and 10 μL of this SBA bacterial suspension was added to each reaction system in the serum bactericidal assay. Briefly, the sera to be tested in each group were heat-inactivated (56 °C for 30 minutes) before testing. The final volume of the reaction solution for the serum bactericidal assay was 40 μL, containing 10 μL of the above SBA bacterial suspension, 20 μL of the three-fold diluted serum to be tested, and 10 μL of human complement (Pel-freez). In addition, control group A (inactive complement) and control group B (active complement) were also set up to calculate the non-specific killing rate of the active complement without antibodies. After incubation (37 °C, 120 min) of each group's reaction solution, 10 μL was pipetted onto a BG blood agar plate, and parallel controls were set for each sample. Incubate at 37 °C, 5.0% CO2 for 3 - 5 days.

[0117] After incubation, the colonies were counted, and the SBA titer was calculated as the reciprocal of the serum dilution at which more than 50% bacterial killing was achieved compared to the control group. Based on the initial serum dilution in the well, the lowest SBA titer was 1:4. Therefore, the LLOD (lower limit of detection) was defined as 1:4. If the test serum did not achieve a 50% reduction in CFU in the SBA assay, the titer was designated as 1:2.

[0118] 1.4.3 Antigen absorption assay

[0119] The antigen absorption assay verified the contribution of LOS to the pertussis omv functional antibodies. 100 μg / mL of pertussis diphtheria LOS was mixed with each immune serum in equal volume and incubated at 37 °C for 30 min. After detecting anti-los antibodies by indirect ELISA, SBA and OPA analyses were performed using this solution.

[0120] 1.4.4 Opsonophagocytosis (OPA) assay

[0121] The OPA target stain Tohama I (BAA - 589) was obtained as previously described. HL - 60 cells were obtained from ATCC (CCL240) and maintained, passaged, and differentiated into granulocyte - like cells for 5 days at 37 °C and 5% CO2 in 100 mM dimethylformamide (DMF). Cell viability was detected by trypan blue exclusion cell counting (>90%).

[0122] Briefly, heat - inactivated test sera were serially diluted (3 - fold) in 96 - well round - bottom microtiter plates (10 μL / well) with HBSS (Hanks balanced salt solution containing 0.1% gelatin, containing Ca 2+ , Mg 2+ ) and incubated with bacterial cells (1000 - 2000 CFU / well) at 37 °C for 30 minutes on an orbital shaker (MIULAB, MIX - 1500); after the opsonization step, human complement (Pel - frzeez, 34010, [final concentration, 12.5%]) was mixed with differentiated HL60 cells at a volume ratio of 4:1. Then, the cell - complement mixture was added to the bacterium - serum mixture (final volume, 80 μL / well). Control groups A (inactive complement) and B (active complement) contained all reagents except serum, and the non - specific killing rate of active complement was calculated. Incubate at 750 rpm for 50 minutes at 37 °C (phagocytosis step). After incubation, 10 μL was taken from each well and spread on B - G blood agar plates and incubated at 35 °C for 3 - 4 days. Colonies were counted after incubation, and the OPA titer was calculated as the reciprocal of the serum dilution that achieved >50% bacterial killing compared to the CFU of the control group. The lowest OPA titer was 1:8, based on the dilution in the starting well of the serum. Therefore, the LLOD (lower limit of detection) was defined as 1:8. When the test serum did not achieve a 50% reduction in CFU in OPA, its titer was set at 1:4.

[0123] 1.5 Flow cytometry

[0124] 1.5.1 Intravascular staining for differentiating circulating cells and lung - retained cells

[0125] To differentiate blood - borne circulating cells and cells within the lung, mice were injected with anti - mouse PE - CD45 antibody (eBioscience) 10 minutes before euthanasia and lung resection. Circulating cells were labeled with the antibody (CD45+), which cannot penetrate tissues to stain tissue - resident memory T cells, resulting in them remaining unstained (CD45 -). This method allows the evaluation of CD4 tissue - resident memory (TRM) cells in the lung based on the presence of CD69 and / or CD103.

[0126] 1.5.2 Preparation of splenic T lymphocytes and B lymphocytes

[0127] The spleen was aseptically removed, and the single-cell suspension was gently homogenized through a 40-μm filter (BD Falcon, BD Biosciences) into incomplete medium (Gibco) (supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin antibiotic, and 2.92 mg / mL l-glutamine). The spleen and lung tissue homogenate samples were lysed for red blood cells with red blood cell lysis buffer. The cells obtained through this procedure were >; the viability was determined to be 99% by trypan blue dye exclusion. Isolation and FACS analysis of lung and nasal tissue cells The lung and nasal tissue were minced and digested with collagenase D (1 mg / mL; Sigma-Aldrich) and DNase I (20 U / mL; Sigma-Aldrich). Next, the lung and nose were passed through a 40-μm cell strainer to obtain a single-cell suspension, followed by red blood cell lysis. The cells were incubated with CD16 / CD32 Fcγriii (1:100) to block IgG Fc receptors. The cells were incubated with LIVE / DEAD Aqua (Invitrogen), and then surface stained with fluorescent dye-conjugated anti-mouse antibodies against various markers. To detect cytokines, the cells were stimulated with PMA (50 ng / mL) and ionomycin (500 ng / mL) at 37 °C for 4 h in the presence of brefeldin A (5 μg / mL). The surface antibodies used were: CD45R-PE, CD3-BV421, CD44-BV605 (BioLegend), CD62L-PE-CF594 (BD), CD103-BV786, CD4-APC-eF780, and CD69-FITC (eBioscience). Fluorescent negative or non-specific isotype antibodies were used as controls. Flow cytometry analysis was performed using Diva software (BD Biosciences) to acquire data. The results were analyzed using FlowJo software (TreeStar).

[0128] 1.6 Cytokine-specific ELISPOT assay

[0129] Single-cell suspensions were obtained from the spleens of untreated and immunized mice by passing them through a 40-μm cell strainer. For the corresponding different vaccine groups, the stimulant consisted of one or more mixed peptides of OMV, PTgd, FHA, or Prn (5 μg / mL). The relevant stimulant was incubated with 1×10 6 splenocytes in a 24-well plate at 37 °C for 72 h. The enzyme-linked immunospot assay (ELISPOT) was used to count the proportion of antigen-specific antibody-secreting cells. The number of spots was counted using a microscope.

[0130] 1.7 Bacterial challenge test

[0131] Before infecting the mice, a strain of Bordetella pertussis working bacteria (ATCC, 18323) was taken out from the liquid nitrogen tank. Before infection, the bacterial suspension was diluted to 5×10 10 CFU / mL (aerosol infection route) with 1% casein solution. Through the improvement of the method described by Sato et al., mouse respiratory tract infection was caused by aerosol attack. On the 56th day after immunization, each group of mice was infected with 1.5×1011 CFU through the nebulizer of an inhalation exposure system (glass-col, 099CA-4212, USA). The aerosol challenge program was set as: preheating for 10 min - atomizing for 30 min - descending for 30 min, and ultraviolet sterilization for 30 min. At two time points, 24 hours and 6 days (n = 6) after bacterial exposure, the mice were anesthetized and sacrificed for dissection. The lungs and tracheas of each mouse were homogenized in 1 mL of 1% casein solution respectively, and separate nasal lavage fluids were taken for bacterial isolation and culture.

[0132] 1.8 CFU counting

[0133] 10 μL aliquots of serial dilutions (3-fold) of the homogenates of the 3 kinds of tissues were replicated onto BG agar plates and the colonies were counted after incubation at 35 °C for 4 days. The results were reported as the average CFU number per individual mouse (lung, trachea, and nasal lavage).

[0134] 1.9 Statistical analysis

[0135] For the analysis of the CFU count in the animal lungs, the Shapiro-Wilk test (http: / / www.cistatcalc.blogspot.com.ar / 2013 / 10 / shapiro-wilk-test-calculator.html) was used to evaluate the normality of the data. After confirming that the CFU data was normally distributed, one-way analysis of variance (ANOVA) and Bonferroni's multiple comparison test (GraphPad Prism) were used for statistical analysis. A difference was considered significant when p < 0.05.

[0136] 2. Results and analysis

[0137] 2.1 Structural characterization of OMVsBp

[0138] The results are shown in Figure 1 . Figure 1Figure a shows the electron microscopy examination of OMV. Membrane vesicle structures with a diameter of approximately 80 - 120 nm were observed under the electron microscope, and the rod length = 100 nm. Figure b shows the particle size of OMV measured by a laser diffraction particle size analyzer. The OMVs of three batches (corresponding to 3 curves) did not aggregate, showing a similar uniform size distribution, with an average size between 40 and 400 nm. Figure c shows the protein composition of OMV. The proteins present in OMV were determined by quantitative LC-MS analysis. The abundance of each protein is expressed as a percentage of the total detected protein content.

[0139] Examine the morphological characteristics of purified OMVsBp by electron microscopy ( Figure 1 Figure a). Measure the particle diameter of OMVsBp using a laser particle size analyzer (Malvern Zetasizer Nano ZS90) ( Figure 1 Figure b), and obtain the protein composition of OMVsBp by proteomic analysis ( Figure 1 Figure c). Under the electron microscope with negative staining, intact nanoparticles with a diameter of 100 nm were observed, which is consistent with the results reported in other studies, indicating that naturally formed Bordetella pertussis outer membrane vesicles present as spherical particles with a diameter of 50 - 200 nm. The DLS data confirmed that the particle diameter of OMVsBp is approximately 134.8 nm (pdi = 0.181) ( Figure 1 Figure b).

[0140] To further characterize OMVsBp, LC-MS analysis was used to characterize the total protein composition. A total of 313 proteins were identified and quantified in OMVsBp. Obviously, these numbers do not include all the proteins present in the omv, because Bordetella pertussis has more than 3500 genes encoding 289 proteins. However, this LC-MS method identified and quantified the most abundant proteins in OMVsBp, revealing significant differences in protein abundance. Among the proteins identified in OMVsBp, the top 15 proteins with the highest abundance accounted for 85.24% of the total protein content of OMVsBp. These proteins include various toxins and virulence factors related to Bordetella pertussis pathogenicity, such as FHA, Vag8, BrkA, Prn, cya, TCF, etc. The four subunits of pertussis toxin (PT) are ranked as: ptxA (NO.33), ptxD (NO.68), ptxB (NO.76), ptxC (NO.85) (data not shown), indicating that although PT can be detected in the outer membrane vesicles, its content is extremely low, usually below 0.5%. Among the vaccine-related antigens, FHA and the autotransporter Vag8 have the highest content in OMVsBp, accounting for 34.83% and 12.72% respectively. Another autotransporter BrkA ranks 7th in abundance in the omv (2.61%), while the abundance of another aP vaccine component Prn is relatively low (0.75%).

[0141] Among them, the conditions of the LC-MS method include: After the sample is separated in a size exclusion chromatography column at a flow rate of 0.5 ml / min with 0.85 wt% sodium chloride as the mobile phase in a Thermo U3000 chromatograph, each component enters the mass spectrometry detector in sequence for detection.

[0142] 2.2 Composition and grouping of the vaccines used in the study

[0143] Table 1

[0144]

[0145] 2.3 Schematic diagram of the immunization schedule (see Figure 2 )

[0146] Taking NIH female mice at 8 - 10 weeks old as the research objects, they were randomly divided into 4 inoculation groups, with 6 mice in each group for intraperitoneal immunization. The mice were inoculated with OMVsBp or OMVsBp + PTgd vaccine on day 0 and day 28 respectively. The mice inoculated with the aP vaccine prepared in item 1.1 were used as the positive control, and the mice injected with normal saline were used as the negative control. After 56 days of immunization, antigen-specific antibody analysis of serum, lung, nose, and trachea tissues was performed (n = 6). And the lungs and spleens of the mice were taken for cytokine analysis. 12 mice in each group were used for aerosol challenge, the colonies were numbered from the lung, nose, and tracheal lavage fluids respectively, and the protective effect of the pertussis vaccine was evaluated at 24 h and 144 h respectively.

[0147] After the antigens purified in this study were fractionally purified, they were identified by SDS-PAGE and specific serum. These proteins were adsorbed with aluminum hydroxide and then used to immunize NIH mice. The composition of each vaccine preparation is shown in Table 1. To evaluate the effects of OMVsBp and PTgd on inducing protective immune responses, the mice were intraperitoneally immunized according to Figure 2 the immunization schedule shown, and the corresponding time points were strictly observed.

[0148] 2.4 Specific antibody levels induced by intraperitoneal immunization of mice

[0149] Immunization effects of OMVsBp, OMVsBp mixed with PTgd or aP vaccine by intraperitoneal injection in mice: The levels of specific IgG antibodies against PT(a), Prn(b), FHA(c), BrkA(d), Vag8(e), and LOS(f) in serum were determined by quantitative indirect ELISA. 6 mice in each group were given the drug 2 times (on day 0 and day 28), 2 μg each time. Normal saline and aP vaccine were used as the negative control and positive control respectively. The results were expressed as the geometric mean ± SD of each group of mice on day 56.

[0150] Intraperitoneal immunization of mice with OMVsBP or OMVsBP supplemented with PTgd can effectively induce specific circulating antibodies against vaccine antigens, especially against PT( Figure 3 panel a in Figure 3 panel c in Figure 3 panel d in Figure 3 panel e in Figure 3 panel f in Figure 2 ).( Figure 3 panel b in Figure 3 panel a in Figure 3 panel b - e in Figure 3 panel a in Figure 3 panel a in Figure 3 panel f in

[0151] 2.5 OMV - derived proteins, especially LOS, induce high levels of specific antibodies in the lungs via the intraperitoneal route

[0152] Changes in the levels of specific antibodies in the lungs of mice induced by intraperitoneal immunization as shown: Changes in the levels of anti - PT (a), anti - Prn (b), anti - FHA (c), anti - Vag8 (d), and anti - LOS (e) in the lung tissues of mice intraperitoneally immunized with OMVsBP or OMVsBP mixed with PTgd vaccines. Eighteen mice in each group were administered the drugs 2 times (on day 0 and day 28), 2 μg each time. Normal saline and aP vaccine were used as negative control and positive control respectively. The antigen - specific IgG antibody titers in the lung lavage fluid were measured by quantitative indirect ELISA. The results are expressed as the geometric mean ± SD of each group of mice on day 56.

[0153] After intraperitoneal immunization of each vaccine group, bronchoalveolar lavage fluid (BALF) was collected, and the levels of key antigen-specific IgG antibodies were measured by indirect ELISA. The levels of anti-PT, FHA, Prn, Vag8, and LOS antibodies in BALF increased or decreased following the trend of serum antibody levels. The combination group of OMVsBp and PT induced anti-PT antibody levels similar to those of the aP vaccine ( Figure 4 a). Due to the low Prn content in OMVsBp, the induced anti-Prn antibody levels were significantly lower than those in the aP vaccine group ( Figure 4 b). In contrast, FHA was highly expressed in OMVsBp, and thus high levels of anti-FHA antibodies could be detected in all components containing OMVsBp ( Figure 4 c). In addition, in mice immunized with OMV preparations (i.p.), specific antibodies against Vag8 ( Figure 4 panel d in Figure 4 and LOS (

[0154] 2.6 The levels of OMV-derived protein-specific antibodies induced by the intraperitoneal route via the trachea depend on their abundance in OMVs

[0155] Study on the levels of specific antibodies in the trachea of mice induced by intraperitoneal immunization: Observe the IgG levels of anti-Pt (a), anti-Prn (b), anti-Fha (c), and anti-Vag8 (d) in the tracheal tissues of mice injected intraperitoneally with OMVsBp or a mixed vaccine of OMVsBp and PTgd. Six mice in each group were administered the drug twice (on day 0 and day 28), 2 μg each time. Normal saline and acP vaccine were used as negative and positive controls respectively. The antigen-specific antibody titers in tracheal grinding fluid were measured by quantitative indirect ELISA. The results were expressed as the geometric mean ± SD of each group of mice on day 56.

[0156] To compare the antibody levels induced by vaccination via different immunization routes, considering the common infection routes of respiratory bacteria, the effectiveness of intranasal immunization of mice was also evaluated. After intraperitoneal immunization, only a weak antibody response of anti-Pt IgG was detected in the bronchoalveolar lavage fluid of mice in the OMVsBp group ( Figure 5 panel a in Figure 5 and specific Prn IgG was only detected in the aP group ( Figure 5 panel b in Figure 5In the figure d). Compared with the OMVsBp and aP groups, the OMVsBp group containing PTgd induced higher levels of anti-FHA antibodies. There was no significant difference in the Vag8 antibody levels between the OMVsBp group and the OMVsBp + PTgd group, and relatively high antibody levels were maintained in the bronchoalveolar lavage fluid.

[0157] 2.7 Only antibodies against the highly abundant proteins of OMVs induced via the intraperitoneal route were weak, but could be detected in the nasal tissue.

[0158] Study on the induction of specific antibody levels in the nasal cavity of mice by intraperitoneal immunization: IgG levels of anti-pt(a), anti-fha(b), and anti-vag8(c) in the nasal tissue of mice immunized intraperitoneally with OMVsBp or OMVsBp mixed with PTgd vaccine. Six mice were in each group and were administered twice (on day 0 and day 28), with 2 μg each time. Normal saline and aP vaccine were used as negative and positive controls respectively. The antigen-specific antibody titers in the nasal lavage fluid were measured by quantitative indirect ELISA. The results were expressed as the geometric mean ± SD of each group of mice on day 56.

[0159] In addition to detecting antigen-specific antibodies in serum and BALF, IgG antibodies in the nasal lavage fluid were also detected to observe the circulating ability of total IgG antibodies in the initial segment of the respiratory tract. After intraperitoneal immunization, only the highly abundant FHA (34.83%) and Vag8 (12.72%) in OMVs had weakly detectable specific antibodies in the nasal lavage fluid ( Figure 6 in figure b and Figure 6 in figure c), while BrkA and Prn induced lower antibody levels due to their low expression in OMVs and could not migrate to the nasal cavity (data not shown). The PT in OMVsBp promoted the migration of FHA antibodies to the nasal cavity, which was not observed in the aP vaccine ( Figure 6 in figure a). This also indicates that although the three components of the pertussis vaccine induced high levels of circulating antibodies, the antibody levels in local tissues, especially in the nasal cavity and trachea, were low, making it difficult for the pertussis vaccine to prevent pertussis invasion.

[0160] 2.8 Vaccines containing OMVs induced superior functional activity in mice via the intraperitoneal route.

[0161] The antigen-specific functional antibody levels induced by intraperitoneal immunization in mice were confirmed: serum bactericidal activity (SBA) titers (a) and opsonophagocytosis activity (OPA) titers (b) against Bordetella pertussis Tohama I (BAA-589) in mice immunized intraperitoneally with OMVsBp or a mixed vaccine of OMVsBp and PTgd. The SBA or OPA titers in sera of different immunized groups were detected using a homologous vaccine strain and rabbit (SBA) or human (OPA) complement. Six mice were in each group, and they were administered twice (on day 0 and day 28), with 2 μg each time. Normal saline and the aP vaccine served as negative and positive controls respectively. The results were expressed as the geometric mean ± SD of each group of mice on day 56.

[0162] The functional antibody levels induced by vaccination via different routes were evaluated using SBA and OPA. In addition, the effectiveness of antigen components was further demonstrated by serum absorption assays of LOS. The functional activities of antibodies induced by the vaccine were evaluated using SBA and OPA. Compared with aP, the vaccine containing omv showed stronger bactericidal activity and anti-phagocytic activity ( Figure 7 a and 7b). Intranasal immunization did not induce detectable functional antibodies (data not shown). In contrast, intraperitoneal immunization induced strong functional antibodies, and a similar trend was observed in SBA and OPA. Whether using SBA or OPA, immunization of mice with OMVsBp combined with PTgd could induce antibodies with higher functional activities, although there was no statistically significant difference compared with immunization with OMVs alone. Serum absorption assays of the LOS antigen showed that the functional antibody levels evaluated by SBA ( Figure 7 panel c in 7 and Figure 7 panel e in Figure 7 panel f in 7) and OPA (

[0163] 2.9 Vaccines containing OMV induced a broad cellular immune profile in the spleen after intraperitoneal immunization

[0164] Cytokines produced by splenocytes of mice immunized intraperitoneally with the vaccine after re-stimulation with antigen or peptide: The secretion of IFN-γ (a), TNF-α (b), IL-4 (c), IL-17 (d), and IL-6 (e) in splenocytes of mice after vaccination with OMVsBp or OMVsBp + PTgd (i.p.) was analyzed using ELISPOT. Six mice were in each group, and they were administered twice (on day 0 and day 28), with 2 μg each time. Normal saline and the aP vaccine served as negative and positive controls respectively. The results were expressed as the geometric mean ± SD of each group of mice on day 56.

[0165] After vaccinating mice intraperitoneally, in addition to evaluating antibody levels, cellular immune responses were also evaluated. Spleens were collected from mice at the end of the immunization period and splenocytes were stimulated with antigen (OMV) or different peptide libraries corresponding to the vaccine antigen (such as PT, FHA, Prn). Then, antigen-specific cytokines secreted by the stimulated splenocytes were detected using the ELISPOT method.

[0166] The number of cells secreting IFN-γ is shown in Figure 8 Figure a in Figure 8 Figure c in Figure 8 Figure b in Figure 8 Figure d in Figure 8 Figure e in

[0167] 2.10 Mice immunized with the OMV-containing vaccine had more accumulation of CD4 TRM cells in the lungs compared to the aP vaccine.

[0168] Local proliferation of CD4+ TRM cells in the lungs of mice after administration of different formulated vaccines via different immunization routes:

[0169] (a) The frequency of CD4+ TRM cells was analyzed by flow cytometry after nasal immunization (i.e., administration of OMVsBp or OMVsBp + PTgd). Then, lung cells were stimulated with OMVsBp.

[0170] (b) After intraperitoneal injection of OMVsBp or OMVsBp + PTgd, the frequency of CD4+ TRM cells was analyzed by flow cytometry. Lung cells were stimulated with OMVsBp. Lung cells were stimulated with PTgd peptide or OMVsBp, respectively. The results are presented as the geometric mean ± standard deviation (SD) of each group of mice on day 56.

[0171] Upon stimulation with OMVsBp, both vaccines containing OMVsBp induced detectable TRM cells in the lung tissue, and there was no statistical difference between the two groups ( Figure 9 Figure a in Figure 10 Figure b in

[0172] 2.11 Addition of PTgd to OMVsBp completely clears Bordetella pertussis colonization in the lungs and limits bacterial colonization in the nose and trachea of mice

[0173] Protective effect of intranasal challenge in mice against Bordetella pertussis: The cfu of lung, trachea, and nasal tissues were determined at 24 h and 14 h after intraperitoneal injection (i.p.) of OMVsBp or a mixture of OMVsBp and PTgd vaccine in mice (a - c). Normal saline and aP vaccine were used as negative and positive controls, respectively. Results are expressed as the geometric mean ± SD of each group of mice on day 56.

[0174] A mouse respiratory infection model was established to evaluate the effective protective effect of the candidate vaccine. Although there were significant individual differences among the mice used for bacterial colonization, bacteria could be detected in the tissues of all mice 24 h after respiratory infection, indicating successful establishment of the mouse model. However, when aerosolized respiratory infection was performed, the bacterial load in the nasal cavity and internal organs was relatively low 24 h later, which might be related to the self - cleaning function of the mouse nasal cavity and the difficulty of bacterial colonization in the trachea. However, due to the presence of a large number of bacteria in the lung tissue, the bacterial load showed an increasing trend at 144 h.

[0175] Our research results showed that OMVsBp + PTgd and aP vaccine administered by intraperitoneal immunization completely cleared the bacterial load in the lung tissue of mice after 144 h ( Figure 10 Figure a in). The bacteria in the trachea and nasal cavity, which are farther from the lung, did not completely disappear, but the number of bacteria decreased significantly ( Figure 10 Figure b and c in). In contrast, the ability of OMVBp and aP vaccine to clear tissue bacteria was lower than that of the OMVsBp + PTgd group. Overall, there was no significant difference between OMVsBp and aP vaccine. The trend of decrease in the number of bacteria was consistent with the distribution of our antigen - specific antibody levels. However, we observed that the lowest bacterial proliferation rate was in each group vaccinated with OMVsBp + PTgd via different routes and different organs. The results showed that the combination of OMVsBp and PTgd could effectively reduce the bacterial load and growth ability.

[0176] The technical features of the above - described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the various technical features in the above - described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0177] The above-described embodiments merely represent several implementation manners of the present application, facilitating a specific and detailed understanding of the technical solution of the present application. However, it should not be construed as a limitation of the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An immunologically active composition comprising Bordetella pertussis outer membrane vesicles and detoxified pertussis toxin in a weight ratio of (5 - 50):(5 - 25).

2. The immunologically active composition according to claim 1, wherein, The weight ratio of the Bordetella pertussis outer membrane vesicles to the detoxified pertussis toxin is (5 - 15):(5 - 15).

3. The immunologically active composition according to claim 1 or 2, wherein, The detoxified pertussis toxin is a genetically mutated detoxified pertussis toxin.

4. The immunologically active composition according to claim 3, wherein, Relative to the pertussis toxin of the strain numbered CMCC58003, the S1 subunit of the detoxified pertussis toxin has the following mutations: R9K and Q129G.

5. Use of the immunologically active composition according to any one of claims 1 to 4 in the preparation of a pertussis vaccine.

6. The application according to claim 5, wherein, The dosage form of the pertussis vaccine includes an injection.

7. A pertussis vaccine comprising the immunologically active composition according to any one of claims 1 to 4 and an immunoadjuvant.

8. The pertussis vaccine according to claim 7, comprising 10 - 100 μg / mL of the Bordetella pertussis outer membrane vesicles, 10 - 50 μg / mL of the detoxified pertussis toxin, and the immunoadjuvant.

9. The pertussis vaccine according to claim 7 or 8, wherein, The immunoadjuvant includes an aluminum hydroxide adjuvant.

10. The pertussis vaccine according to claim 9, wherein, The concentration of the aluminum hydroxide adjuvant in the pertussis vaccine is 100 - 1000 μg / mL.

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