WO2024088138A1 - 质膜透化灭活口服疫苗 - Google Patents

质膜透化灭活口服疫苗 Download PDF

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WO2024088138A1
WO2024088138A1 PCT/CN2023/125248 CN2023125248W WO2024088138A1 WO 2024088138 A1 WO2024088138 A1 WO 2024088138A1 CN 2023125248 W CN2023125248 W CN 2023125248W WO 2024088138 A1 WO2024088138 A1 WO 2024088138A1
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lipopeptide
inactivated
vaccine
oral
pathogen
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PCT/CN2023/125248
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English (en)
French (fr)
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姜亦微
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宁波明亦生物科技有限公司
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Publication of WO2024088138A1 publication Critical patent/WO2024088138A1/zh

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  • the present invention relates generally to biotechnology and, more particularly, to inactivated oral vaccines prepared by permeabilizing plasma membranes and their use.
  • Vaccines are extremely important in human and non-human animal health, and there is a constant need for new or better vaccines.
  • vaccine development is challenging and requires meeting the often conflicting requirements of safety and efficacy.
  • the advantage of attenuated live vaccines is that they can stimulate both humoral and cellular immunity through a near-natural infection process; however, attenuated live vaccines may cause disease in immunosuppressed individuals.
  • inactivated vaccines avoid the risk of pathogen infection and disease, there are other defects.
  • conventional inactivated vaccines can only stimulate humoral immunity, and basically cannot stimulate cellular immunity, so the protection is weak and the duration is short, and the dose and number of vaccinations need to be increased.
  • the injection delivery method commonly used for inactivated vaccines may bring new safety and effectiveness issues. Since inactivated vaccines cannot break through the host's biological barriers through natural infection and induce an immune response, most inactivated vaccines can only be delivered by injection, but the pain and panic caused by the injection can lead to a stress response, which causes immunosuppression and then weakens the immune response, ultimately leading to poor vaccination results.
  • the injection of veterinary inactivated vaccines usually requires the capture and restraint of animals. These operations will further enhance the stress response and affect the effectiveness of vaccination.
  • Oral administration is the safest and most convenient way to deliver vaccines. More importantly, oral vaccines can stimulate cellular and humoral immunity at both the mucosal and body levels, thereby building a deeper anti-infection defense line and intercepting microbial pathogens outside the body with effective mucosal immunity.
  • inactivated oral vaccines face the challenge of antigens being completely lost due to degradation by digestive enzymes in the digestive tract. Therefore, the form or dosage form of inactivated oral vaccine antigens must have the ability to be indigestible and efficiently absorbed. How to protect the antigens and enable them to efficiently cross the digestive tract barrier and be transported to immune cells is the key to oral inactivated vaccines.
  • M cells are highly differentiated cells in the epidermis covering the organized lymphoid follicles of the small and large intestines. M cells sample a wide range of molecules, perform protein recognition, and transport antigens by transporting bacteria, viruses, parasites, and non-infectious particles to the subordinate lymphoid tissues, the Peyer's patches. Given their central role in initiating mucosal immune responses, M cells have become the delivery target for artificial vaccine vectors that allow antigens to cross the cell barrier of the digestive tract.
  • the antigen preparation process of inactivated influenza vaccine is (i) inoculating the selected strain into chicken eggs for amplification, (ii) purifying the virus, (iii) double inactivation with formaldehyde and ⁇ -propiolactone, (iv) removing the inactivating agent, and (v) separating high-efficiency antigens with detergents.
  • the conventional method of purifying antigens is incompatible with the technical requirements of inactivated oral vaccines. After purification, the "naked" antigens will be easily degraded and lost by digestive enzymes in the digestive tract. If they are coated for protection, it involves complex processes and high costs.
  • the present invention provides an oral vaccine with simple process and low cost, as well as a preparation method and use thereof.
  • the oral vaccine of the present invention inactivates pathogens or attenuated vaccines thereof by permeabilizing the plasma membrane with a permeabilizing agent, and can be prepared without conventional steps such as separating antigens (such as viruses) and/or artificially coating antigens.
  • the present invention is based on the unexpected discovery of the inventors: contrary to the teaching in the prior art that BT lipopeptide and vaccine should be delivered separately (Chinese patent application number CN201810354453.2), when BT lipopeptide is orally administered together with a live attenuated vaccine, BT lipopeptide does not cause immune failure of the live attenuated vaccine.
  • the inventors also unexpectedly found that when the pathogen is inactivated by permeabilization with a permeabilizing agent containing only BT lipopeptide as a surfactant component, oral inoculation of BT lipopeptide inactivated vaccine has a much higher protective power than oral inoculation of high temperature inactivated vaccine.
  • the present invention provides an inactivated oral vaccine comprising: A live pathogen or a live vaccine thereof, wherein the inactivated pathogen or the live vaccine thereof comprises a permeabilized plasma membrane and its integral membrane proteins, wherein the pathogen is a pathogen comprising a plasma membrane, and the permeabilizing agent comprises a lipopeptide as a surfactant.
  • the pathogen is selected from the group consisting of enveloped viruses, bacteria, fungi, internal parasites, and cancer cells.
  • the plasma membrane and its integral membrane protein are the natural plasma membrane and its integral membrane protein of the pathogen or its live vaccine.
  • the permeabilizing agent only permeabilizes the phospholipid bimolecular membrane. In one embodiment, the permeabilizing agent only permeabilizes the phospholipid bimolecular membrane without changing the bilayer membrane structure of the phospholipid molecules. In one embodiment, the permeabilizing agent does not separate the integral membrane protein from the phospholipid, and maintains the natural conformation of the integral membrane protein.
  • the vaccine further comprises the leaked contents of the pathogen or a live vaccine thereof after permeabilization.
  • the vaccine is inactivated by a step including permeabilization of plasma membrane of the pathogen or a live vaccine thereof.
  • the step of plasma membrane permeabilization results in a live pathogen count of ⁇ 10 1 /ml and/or a dead pathogen count of >10 9 /ml.
  • the inactivated oral vaccine is an oral solution, powder, tablet, capsule, drench or oral bolus.
  • the inactivated oral vaccine is a vaccine administered by direct oral administration, water load, food load, drench, or oral bolus.
  • the permeabilizing agent does not comprise a surfactant other than the lipopeptide.
  • the lipopeptide is the only surfactant.
  • the lipopeptide comprises a natural lipopeptide and/or an artificially synthesized lipopeptide.
  • the natural lipopeptide is selected from the group consisting of surfactin, fengycin, iturin, daptomycin, polymyxin, mucin, and a lipopeptide produced from Brevibacillus texasporus.
  • the lipopeptide produced from Bacillus texasporus comprises a lipopeptide having a sequence shown in any one of SEQ ID NO: 1-9 or any combination thereof.
  • the pathogen is selected from the group consisting of Coronaviridae, Influenza virus, Paramyxoviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Baculovirus, Retroviridae, African swine fever virus, Acinetobacter, Bacillus, Borrelia, Campylobacter, Clostridium, Arc Bacteria, Escherichia coli, Enterococcus, Neisseria, Haemophilus, Mannheimia, Mycobacterium, Mycoplasma, Pasteurella, Pseudomonas, Salmonella, Staphylococcus, Streptococcus, cocci, nematodes, tapeworms and cancer cells.
  • the pathogen is selected from the group consisting of Newcastle Disease Virus and Vibrio parahaemolyticus.
  • the inactivated oral vaccine comprises a pharmaceutically acceptable carrier, or comprises an ingredient that modulates immunity or promotes health or growth, such as an adjuvant, or a lipopeptide produced from Bacillus texasporus.
  • the lipopeptide produced from Bacillus texasporus comprises a lipopeptide having a sequence shown in any one of SEQ ID NO: 1-9 or any combination thereof.
  • a method for preparing the inactivated oral vaccine described herein comprising: permeabilizing the plasma membrane of a pathogen or a live vaccine thereof, wherein the permeabilizing agent comprises a lipopeptide as a surfactant.
  • the method does not include the step of isolating the antigen and/or the step of artificially coating the antigen.
  • the method does not include the step of lipid or bile encapsulation or coating the antigen.
  • the method when inactivating pathogens, does not include other inactivation methods other than permeabilization inactivation, such as high temperature inactivation methods.
  • the permeabilizing agent does not comprise a surfactant other than the lipopeptide.
  • the pathogen or a live vaccine thereof is permeabilized to a plasma membrane such that the number of live pathogens is ⁇ 10 1 /ml and/or the number of dead pathogens is >10 9 /ml.
  • the lipopeptide comprises a natural lipopeptide and/or an artificially synthesized lipopeptide.
  • the natural lipopeptide is selected from the group consisting of surfactin, fengycin, iturin, daptomycin, polymyxin, mucin, and a lipopeptide produced from Brevibacillus texasporus.
  • the lipopeptide produced from Bacillus texasporus comprises a lipopeptide having a sequence shown in any one of SEQ ID NO: 1-9 or any combination thereof.
  • the present invention provides an oral composition comprising the inactivated oral vaccine described herein.
  • the oral composition comprises food, drinking water, or a beverage.
  • the food is selected from the group consisting of a milk replacer, a grower feed, a finisher feed, a pre-brooding feed, and a brooding feed.
  • the composition comprises one or more of cereals, soy flour, isolated soy protein, soy oil, soy fat, skim milk, fish meal, meat meal, bone meal, blood meal, plasma protein, whey, rice bran, wheat bran, sweeteners, minerals, vitamins and salts.
  • the cereal is selected from the group consisting of barley, soy, wheat, triticale, rye, corn and rice.
  • the oral composition comprises an ingredient that modulates immunity or promotes health or growth, preferably a BT lipopeptide, which is mixed with or separated from an inactivated oral vaccine.
  • a BT lipopeptide comprises a BT lipopeptide produced from Bacillus texasporus, preferably a lipopeptide having a sequence shown in any one of SEQ ID NO: 1-9 or any combination thereof.
  • the oral composition may comprise a BT lipopeptide, which is mixed with or separated from an inactivated oral vaccine.
  • the concentration of the BT lipopeptide may be above 100 ppm, for example 150 ppm, 200 ppm, 250 ppm, 300 ppm, 330 ppm, 400 ppm, 450 ppm, 500 ppm, 550 ppm, 600 ppm, 650 ppm, 700 ppm, 750 ppm, 800 ppm, 850 ppm, 900 ppm or 950 ppm.
  • the present invention provides use of a permeabilizing agent for preparing an inactivated oral vaccine, wherein the use is achieved by perforating the plasma membrane of the pathogen to cause leakage of the contents, thereby inactivating the pathogen; wherein the permeabilizing agent comprises a lipopeptide as a surfactant.
  • the inactivated oral vaccine is an inactivated oral vaccine described herein.
  • the lipopeptide comprises a natural lipopeptide and/or an artificially synthesized lipopeptide.
  • the natural lipopeptide is selected from the group consisting of surfactin, fengycin, iturin, daptomycin, polymyxin, mucin, and a lipopeptide produced from Brevibacillus texasporus.
  • the lipopeptide produced from Bacillus texasporus comprises a lipopeptide having a sequence shown in any one of SEQ ID NO: 1-9 or any combination thereof.
  • the present invention provides use of the inactivated oral vaccine described herein in the preparation of a medicament for treating and/or preventing an infection or disease caused by a pathogen in a subject.
  • the present invention provides a method for treating and/or preventing an infection or disease caused by a pathogen in a subject, comprising administering to the subject the inactivated oral vaccine described herein.
  • an inactivated oral vaccine as described herein for use in treating and/or preventing an infection or disease caused by a pathogen in a subject.
  • the infection or disease is selected from: influenza, viral hepatitis, African swine fever, porcine reproductive and respiratory syndrome, colibacillosis, salmonellosis, necrotic enteritis, vibriosis, bovine shipping fever pneumonia, gonorrhea, urinary tract infection, coccidiosis, nematodiasis, tapeworm disease and cancer.
  • the present invention provides use of the inactivated oral vaccine described herein in the preparation of a feed for improving weight gain and/or food conversion in a subject.
  • the present invention provides a method for improving weight gain and/or food conversion in a subject, comprising administering to the subject an inactivated oral vaccine described herein.
  • the subject is poultry, livestock, aquatic animals or companion animals.
  • the feed is applied by in ovo feeding, spraying to poultry in droplet form or immersion of aquatic animals.
  • the beneficial effects of the present invention include: (1) The inactivated oral vaccine described herein is orally delivered to the digestive tract, and the digestive enzymes in the digestive tract are used to naturally degrade the leaked contents and remove potential intramembrane antigen molecules, thereby selectively retaining the perforated plasma membrane and its integral membrane proteins as antigen molecules, which are ultimately absorbed to induce mucosal and systemic immune responses, completely eliminating the antigen purification step and cost in the inactivated vaccine production process; (2) The inactivated oral vaccine described herein does not require additional protective treatment of the antigen, such as treatment to protect the antigen from degradation in the digestive tract (such as lipid coating, etc.).
  • animal means any vertebrate or invertebrate, including but not limited to humans, horses, goats, sheep, cows, pigs, chickens, turkeys, hens, geese, ducks, dogs, cats, parrots, fish, crabs, shrimp (e.g., Penaeus vannamei), freshwater turtles, etc.
  • Attenuation refers to a reduction in the ability of a virus, bacterium, fungus or parasite to cause disease in animals.
  • live vaccine refers to a live microbial preparation made by artificially attenuating a pathogen or selecting avirulent or microvirulent strain of a pathogen from nature, sometimes called a live attenuated vaccine.
  • activation refers to making a virus, bacterium, fungus, parasite or cancer cell irreversibly lose the ability to replicate or proliferate.
  • Inactivating agent refers to a substance or energy that makes a virus, bacterium, fungus, parasite or cancer cell irreversibly lose the ability to replicate or proliferate.
  • plasma membrane permeabilization and “plasma membrane perforation” are synonymous, both referring to damage to the integrity of the plasma membrane resulting in leakage of contents, but without changing the overall structure of the phospholipid bilayer of the membrane, or the conformation of integral membrane proteins or their binding to the phospholipid bilayer.
  • plasma membrane dissolution refers to the removal of phosphorus from the plasma membrane.
  • the lipid bilayer is converted into phospholipid micelles, while integral membrane proteins are separated from phospholipids and denatured.
  • surfactant refers to an amphiphilic substance that can reduce the surface tension of the interface between liquids. Different types of surfactants have different types of destructive effects on the plasma membrane structure. Detergent surfactants dissolve the plasma membrane, while lipopeptide surfactants permeabilize but do not dissolve the plasma membrane.
  • Lipopeptide surfactants can include natural and synthetic lipopeptide surfactants. Natural lipopeptides are secondary metabolites produced by microorganisms, generally have a long lipid chain and 7-25 amino acid residues, and are shown to have antibacterial, antiviral, antifungal, antitumor and immunomodulatory functions. Natural lipopeptide surfactants include but are not limited to surfactins, fengycins and iturins, daptomycin, polymyxins, viscosin and BT lipopeptides.
  • BT lipopeptide may refer to a single lipopeptide or a mixture of multiple lipopeptides produced from Brevibacillus laterosporus texasporus, in particular a single lipopeptide or a mixture of multiple lipopeptides comprising SEQ ID NO: 1-9 sequences disclosed in CN201380056514.2 and CN201810354453.2.
  • Brevibacillus texasporus e.g., ATCC PTA-5854
  • NRPS nonribosomal peptide synthetase
  • BT peptides or "BT lipopeptides,” according to their newly resolved N-terminal structures, and both terms are interchangeable in this disclosure), of which BT1583 is the most abundant variant (WO/2005/074626).
  • the cationic peptides (as mixtures or individual peptides isolated from Brevibacillus texasporus) exhibit broad-spectrum antibacterial activity in vitro (BT Function #1).
  • the high 16S rDNA sequence identity (98.5%) between PTA-5854 and Brevibacillus laterosporus type strains classifies Brevibacillus laterosporus as a subspecies of Brevibacillus laterosporus, wherein Brevibacillus laterosporus subsp. texasporus is defined as a Brevibacillus laterosporus strain that produces non-ribosomal peptides from BT NRPS (or BT peptides).
  • Genome sequencing of at least two Brevibacillus laterosporus strains has validated this taxonomy.
  • the BT peptide or BT lipopeptide can be a mixture of natural BT NRP variants (including SEQ ID NO: 1-9) isolated from Brevibacillus texasporus using the separation and detection methods disclosed in WO/2005/074626 and (Wu, X., Ballard, J. and Jiang, Y.W. (2005). Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus. Appl Environ Microbiol 71, 8519-8530), but the separation and detection methods do not contain a reversed-phase HPLC step for resolving BT NRP variants.
  • a person skilled in the art can easily prepare BT peptides or BT lipopeptides using the methods described in these references.
  • the BT peptides or BT lipopeptides are characterized and the amino acid sequences are provided in Table 1 below. Table 1 describes the structures of 9 BT NRP variants and their typical relative abundance in such mixtures.
  • Table 1 Summary of BT NRP variants (Hmp stands for 2-hydroxy-3-methylpentanoic acid, Dhb stands for 2,3-dehydro-2-aminobutyric acid, which is synonymous with dehydrothreonine (Dht), Vol stands for valinol, the subscripts D and E represent the D-chirality and (E)-configuration in the amino acid, respectively, and % indicates the relative abundance of the variant in the BT NRP variants as a mixture isolated from Bacillus texasporus cells when determined by mass spectrometry).
  • Hmp 2-hydroxy-3-methylpentanoic acid
  • Dhb stands for 2,3-dehydro-2-aminobutyric acid, which is synonymous with dehydrothreonine (Dht)
  • Vol stands for valinol
  • the subscripts D and E represent the D-chirality and (E)-configuration in the amino acid, respectively
  • % indicates the relative abundance of the variant in the BT NRP variants as
  • feed or “food” are synonymous and refer to any substance or mixture of substances prepared, sold or provided to animals (such as livestock or domesticated animals) or humans for consumption in order to provide at least part of the nutritional requirements of animals or humans and/or for the purpose of preventing or treating nutritional disorders in animals, and containing amino acids, antioxidants, carbohydrates, flavoring agents, enzymes, fats, minerals, non-protein nitrogen products, proteins, vitamins and/or binders, and may contain granulating agents, colorants, foaming agents and/or fragrances.
  • water is a feed or food ingredient
  • water-containing beverages are feed or food.
  • the present invention includes an inactivated oral vaccine.
  • the main component of the inactivated oral vaccine can be a plasma membrane-permeabilized or perforated enveloped virus, bacteria, fungi, internal parasites or cancer cells.
  • the main component of the inactivated oral vaccine can be a phospholipid bilayer membrane separated from a plasma membrane-permeabilized or perforated enveloped virus, bacteria, fungi, internal parasites or cancer cells or an isolate containing a phospholipid bilayer membrane.
  • the antigen of the inactivated oral vaccine can include a perforated phospholipid bilayer membrane and its integral membrane protein derived from an enveloped virus, bacteria, fungi, internal parasites or cancer cells.
  • the main component of the inactivated oral vaccine of the present invention can be an enveloped virus, bacteria, fungus or endoparasite with a permeabilized or perforated plasma membrane, or an attenuated live vaccine of an enveloped virus, bacteria, fungus or endoparasite.
  • the main component of the inactivated oral vaccine can be a phospholipid bilayer membrane separated from an enveloped virus, bacteria, fungus or endoparasite with a perforated plasma membrane (or a live vaccine of an enveloped virus, bacteria, fungus or endoparasite) or an isolate containing a phospholipid bilayer membrane.
  • the antigen of the inactivated oral vaccine can contain a perforated phospholipid bilayer membrane and its integral membrane protein derived from an attenuated enveloped virus, bacteria, fungus or endoparasite (or a live vaccine of an enveloped virus, bacteria, fungus or endoparasite).
  • the present invention includes a method for preparing an inactivated oral vaccine, comprising mixing a pathogen or a live vaccine thereof with a permeabilizing agent, so that the plasma membrane is perforated to cause the contents to leak out and be inactivated.
  • the pathogen may be an enveloped pathogen, including enveloped viruses, bacteria, fungi, internal parasites or cancer cells.
  • the permeabilizing agent used for inactivation is a lipopeptide surfactant.
  • Lipopeptide surfactants include natural and synthetic lipopeptide surfactants (lipopeptide surfactant).
  • the permeabilizing agent used for inactivation is a BT lipopeptide.
  • the BT lipopeptide may include one or more sequences selected from SEQ ID NO: 1-9, and BT lipopeptides isolated from Bacillus texas.
  • the BT lipopeptide is a purified water-soluble BT lipopeptide.
  • the BT lipopeptide may be a mixture isolated from Brevibacillus texasporus.
  • BT lipopeptide can be used as an inactivating agent and/or adjuvant.
  • the concentration of BT lipopeptide is sufficient to make the number of live pathogens of the pathogen or its live vaccine ⁇ 10 1 /ml and/or the number of dead pathogens>10 9 /ml.
  • the final concentration of BT lipopeptide in the inactivated oral vaccine can be 100ppm or more, such as 100ppm, 500ppm, 1000ppm, 1500ppm, 2000ppm, 2500ppm, 3000ppm, 3500ppm, 4000ppm, 4500ppm, 5000ppm or 5500ppm.
  • the method for inactivating oral vaccines described herein may not include the step of separating the antigen and/or the step of artificially coating the antigen.
  • the method does not include the step of lipid or bile encapsulation or coating the antigen.
  • the method when the pathogen is inactivated, does not include other inactivation methods other than permeabilization inactivation, such as high temperature inactivation.
  • the permeabilizing agent does not include a surfactant other than lipopeptides.
  • the plasma membrane of the pathogen or its live vaccine is permeabilized so that the number of live pathogens is ⁇ 10 1 /ml and/or the number of dead pathogens is >10 9 /ml.
  • the lipopeptide comprises a natural lipopeptide and/or an artificially synthesized lipopeptide.
  • the natural lipopeptide is selected from: surfactin, fengycin, iturin, daptomycin, polymyxin, mucin and a lipopeptide produced from Bacillus texasporus.
  • the lipopeptide produced from Bacillus texasporus comprises a lipopeptide having a sequence shown in any one of SEQ ID NO: 1-9 or any combination thereof.
  • the lipopeptide produced from Brevibacillus texasporus is a mixture of lipopeptides having sequences shown in SEQ ID NOs: 1-9.
  • the present invention includes dosage forms of plasma membrane permeabilized inactivated oral vaccines, including but not limited to oral liquid, powder, tablet, capsule, irrigation liquid and oral push injection liquid.
  • the present invention also includes the delivery mode of inactivated oral vaccine.
  • the delivery mode of inactivated oral vaccine includes but is not limited to autonomous direct oral administration, water load, food load, gavage and oral push injection.
  • the delivery mode of inactivated oral vaccine also includes but is not limited to the following special mode, inactivating vaccine liquid is injected into the amniotic sac of bird egg embryo (feeding in egg), and inactivating vaccine is sprayed on birds (especially young chicks) in the form of thick droplets for pecking, and aquatic animals (especially their seedlings) are immersed in inactivated vaccine liquid.
  • the present invention also includes food/feed or drinking water/beverages to which oral inactivated vaccines are added.
  • the plasma membrane permeabilized inactivated vaccine is incorporated into drinking water or animal feed, such as a cereal-based feed, for example, a food/feed containing at least one cereal selected from the group consisting of barley, soy, wheat, triticale, rye, corn, rice, and combinations thereof.
  • the vaccine may be administered to the patient.
  • the present invention is added to a wide variety of feeds.
  • the inactivated vaccine of the present invention may be mixed with drinking water or feed for livestock selected from the group consisting of milk replacer, grower feed, finisher feed, pre-starter feed, starter feed, water, and combinations thereof.
  • Examples of food/feed ingredients also include cereals, soy flour, isolated soy protein, isolated soy oil, isolated soy fat, skim milk, fish meal, meat meal, bone meal, blood meal, plasma protein, whey, rice bran, wheat bran, and may also include sweeteners, minerals, vitamins, and salts.
  • the present invention also includes a post-inoculation purification method (post-inoculation purification) for enriching high-efficiency plasma membrane surface antigens, including oral delivery of the inactivated oral vaccine described herein.
  • post-inoculation purification for enriching high-efficiency plasma membrane surface antigens, including oral delivery of the inactivated oral vaccine described herein.
  • the inactivated oral vaccine described herein is orally delivered to the digestive tract, and the digestive enzymes in the digestive tract naturally degrade the leaked contents and remove potential intramembrane antigen molecules, thereby selectively retaining the perforated plasma membrane and its integral membrane proteins as antigen molecules, which are ultimately absorbed and induce mucosal and systemic immune responses, completely eliminating the antigen purification steps and costs in the inactivated vaccine production process.
  • an inactivated vaccine whose main component is an enveloped virus, bacteria, fungus, internal parasite or cancer cell with a permeabilized or perforated plasma membrane is orally delivered, and the digestive enzymes in the digestive tract degrade the leaked contents and remove potential intramembrane antigen molecules, thereby selectively retaining the perforated plasma membrane and its integral membrane proteins as the antigen molecules that are actually delivered in the end.
  • the invention also includes methods of improving weight gain and feed conversion in growing livestock or companion animals by orally providing a plasma membrane permeabilizing inactivated vaccine in an effective amount sufficient to prevent and/or treat microbial infection in the animal.
  • the present invention also includes a method for preventing and/or treating microbial infection in an animal by orally providing a plasma membrane permeabilizing inactivated vaccine in an effective amount sufficient to prevent and/or treat microbial infection in the animal.
  • the present invention also relates to compositions and/or methods for preventing and/or treating diseases in animals, comprising orally administering to animals a plasma membrane permeabilized inactivated vaccine.
  • treatment may involve reducing pathogens in the animal.
  • pathogens that may be reduced may include, but are not limited to, Coronaviridae, Influenza viruses, Paramyxoviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Baculoviruidae, Retroviridae, African swine fever virus, Acinetobacter, Bacilli, Snails, Borrelia, Campylobacter, Clostridia, Vibrio, E.
  • Exemplary diseases that can be prevented and/or treated by the present invention include, but are not limited to, Newcastle Disease, influenza, viral hepatitis, African swine fever, porcine reproductive & respiratory syndrome, colibacillosis, salmonellosis, necrotic enteritis, vibrosis, bovine shipping fever pneumonia, gonorrhea, urinary track infection, coccidiosis, nematodiasis, cestodiasis and cancer.
  • the method of the present invention may also include oral administration of an isolated BT peptide.
  • the BT peptide may be administered on the same day as the inactivated oral vaccine of the present invention, and/or administered 1 day or several days after the inactivated oral vaccine of the present invention is administered.
  • the BT peptide may be administered for several consecutive days, for example, for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive days.
  • BT peptide in order to save the dosage of BT peptide, can be administered at intervals (pulse administration), for example, at intervals of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days.
  • BT peptide can be administered once or more times, such as 2 times, 3 times, 4 times, or 5 times.
  • the method of the present invention may also include combining the BT peptide with the inactivated oral vaccine of the present invention into a single formulation and then orally administering the formulation.
  • the BT lipopeptide in the examples herein was prepared using the methods disclosed in WO/2005/074626 and (Wu, X., Ballard, J. and Jiang, Y.W. (2005). Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus. Appl Environ Microbiol 71, 8519-8530) and the texasporus Brevibacillus strain PTA-12309 (deposited in the American Type Culture Collection) published in CN201280020898.8. Specifically, the texasporus Brevibacillus strain PTA-12309 cells were cultured in 1 liter of LB at 37°C in a shaker for 3 days.
  • the culture was spun in a centrifuge at 3,000 rpm for 15 minutes. The supernatant was collected, 500 grams of ammonium sulfate was added and dissolved. The sample was centrifuged at 3,000 ⁇ g for 15 minutes in a centrifuge. The precipitate was dissolved in 200 ml of distilled water. The solution was then boiled for 15 minutes and then cooled on ice. The sample was filtered with a 0.2 ⁇ m filter (Nalgene). The filtrate was mixed with 0.2 liters of chloroform at room temperature with a stirring bar for 20 minutes. The mixture was separated into two phases by centrifugation at 3,000 rpm in a centrifuge for 15 minutes.
  • BT peptides or BT lipopeptides can be easily prepared by those skilled in the art using the methods described in these references. BT peptides or BT lipopeptides are characterized, and the amino acid sequences are provided in Table 1.
  • the first embodiment of the present invention is a "mistake” in breeding production. It is an unexpected success that the oral inoculation of BT lipopeptide and the weak live vaccine of enveloped virus "wrongly" mixed is still effective for immunization.
  • BT lipopeptide is delivered to animals vaccinated by injection or spray through drinking water as a separate adjuvant, that is, the BT lipopeptide adjuvant is isolated from the vaccine in time and space.
  • the delivery method of Newcastle disease live vaccine was previously used in the hatchery after hatching.
  • the delivery method was changed to 2-day-old commercial chicken farms through drinking water. Because 100ppm of BT lipopeptide drinking water was required according to the original process on the same day, the Newcastle disease weak live vaccine and BT lipopeptide were mixed together in the drinking water for animals to drink. The survival, health and growth of this batch of chickens were normal, and the Newcastle disease antibody titer at 25 days of age was also completely normal.
  • Treatment group 1 was a negative control group, which was not vaccinated and was gavaged with normal saline at 2 days of age, 1 ml/.
  • Treatment group 2 was a positive control group, which was gavaged with Newcastle disease attenuated live vaccine (La Sota strain, Xinpik, Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.) at 2 days of age, 1.5 portions/.
  • Treatment groups 3-5 were gavaged with inactivated Newcastle disease vaccine preparations, treatment group 3 used lipopeptide single inactivated vaccine (lipopeptide + La Sota strain), and treatment groups 4 and 5 used detergent + lipopeptide double inactivated vaccine (detergent + lipopeptide + La Sota strain).
  • the inoculum of treatment group 3 was prepared by mixing BT lipopeptide with the above-mentioned weak live vaccine to form an aqueous solution containing 1000ppm BT lipopeptide and 1.5 doses of vaccine (inactivated)/ml, and the solution was administered orally at 1ml/each on the 2nd day of age.
  • the inoculum of treatment group 4 was prepared by mixing Triton X-100 detergent, BT lipopeptide with the above-mentioned weak live vaccine to form an aqueous solution containing 1000ppm BT lipopeptide, 2% Triton X-100 and 1.5 doses of vaccine (inactivated)/ml, and the solution was administered orally at 1ml/each on the 2nd day of age;
  • the inoculum of treatment group 5 was prepared by mixing Triton X-100 detergent, BT lipopeptide with the above-mentioned weak live vaccine to form an aqueous solution containing 1000ppm BT lipopeptide, 2% Triton X-100 and 4.5 doses of vaccine (inactivated)/ml, and the solution was administered orally at 1ml/each on the 2nd day of age.
  • the BT lipopeptide in the inoculum administered orally in treatment groups 3-5 is not only an inactivator for making inactivated vaccines, but also a vaccine adjuvant.
  • the determination of Newcastle disease antibody titer was completed according to the method described in the Chinese national standard GB/T16550-2020, and the determination of Newcastle disease vaccine virus content followed the "Virus median lethal dose and infectious dose (LD 50 , ELD 50 , ID 50 , EID 50 , TCID 50 ) determination method" of the "Chinese Veterinary Pharmacopoeia (2010 Edition, Part III)".
  • the Newcastle disease antibody titer data at 25 days of age showed that the antibody levels of the oral inoculation of BT lipopeptide single inactivated vaccine (treatment group 3) and oral inoculation of attenuated live vaccine (treatment group 2) were similar; while the antibody levels of the oral inoculation of double inactivated vaccine (treatment groups 4 and 5) and no vaccination (treatment group 1) were the same, both were zero.
  • the classic highly pathogenic Texas GB strain is a standard attack strain for testing the protective power of Newcastle disease vaccine designated and provided by USDA (United States Department of Agriculture), and is stored internally by the inventor. All treatment groups were challenged with Texas GB strain at 28 days of age, and each chicken was injected with 10 5 EID 50 of Texas GB virus into the breast muscle, and then observed for one week, and the number of survivors in each treatment group was counted, and the survival rate of each group was calculated.
  • inactivating agent inactivation method
  • the form of the antigen are crucial for inactivating oral vaccines.
  • surfactant dissolves the phospholipid bilayer membrane, that is, forms phospholipid micelles, causing the integral membrane proteins to detach from the phospholipids and denature; while lipopeptides only permeabilize the phospholipid bilayer membrane, do not significantly change the phospholipid bilayer membrane structure, do not cause the integral membrane proteins to detach from the phospholipids, and maintain the natural conformation of the integral membrane proteins.
  • BT lipopeptide produced by Bacillus texasporus is also a non-ribosomal peptide. Oral BT lipopeptide is completely undigested. Without wishing to be bound by theory, this may mean that BT lipopeptide is not only not degraded by proteases, peptidases or lipases in the digestive tract, but also that BT lipopeptide inhibits proteases, peptidases and lipases from degrading the perforated plasma membrane and integral membrane proteins embedded therein.
  • natural lipopeptide surfactants such as BT lipopeptide may have an anti-digestive effect on the bilayer membrane structure of phospholipid molecules and integral membrane proteins bound thereto, which is conducive to the absorption and delivery of permeabilized plasma membranes and their integral membrane proteins by M cells; while the protein molecules of the contents leaked by plasma membrane permeabilization are completely "naked” and are naturally degraded by proteases in the digestive tract.
  • Such differentiated digestion and degradation may actually play a role in the "post-inoculational purification" of pathogen plasma membrane surface antigens, which is conducive to the establishment of effective mucosal and systemic immunity by plasma membrane permeabilization inactivated oral vaccines.
  • Example 2 A conclusion or speculation from the analysis of the results of Example 2 is that any pathogen with a plasma membrane can be permeabilized by lipopeptide surfactants to prepare inactivated oral vaccines. To test this speculation, the following test of oral BT lipopeptide inactivated Vibrio vaccine was conducted.
  • Vibrio disease is the number one disease in shrimp farming. Although the research on oral inactivated vaccines for Vibrio parahaemolyticus has been going on for more than 30 years, there is still no vaccine of practical value for shrimp farming.
  • Existing oral "vaccines" for Vibrio such as high-temperature inactivated vaccines for Vibrio, usually need to be added to the feed for long-term continuous feeding. No oral vaccine that meets the conventional definition of vaccines and stimulates protective power with a single dose has been disclosed or reported. Unlike vertebrates, shrimp do not have a classical specific immune system that produces fixed immunoglobulin specificity through DNA-level gene recombination.
  • a plasma membrane permeabilized inactivated vaccine of pathogenic Vibrio parahaemolyticus was prepared using BT lipopeptide, and the traditional high-temperature inactivated vaccine of Vibrio parahaemolyticus was used as a control to compare the protection of single-dose inoculation of white shrimp 10 days after infection.
  • the strain was isolated from shrimps that died of Vibrio disease in Ningbo, Zhejiang. The strain carries the pirAB toxin gene, and its pathogenicity has been verified by laboratory challenge tests (pathogenic Vibrio parahaemolyticus can be used as long as it meets the pathogenicity requirements).
  • Vaccine preparation The Vibrio parahaemolyticus liquid with a concentration of 1x10 9 cfu/ml was subjected to a high temperature treatment at 100°C for 15 minutes, and the number of live bacteria after heating was confirmed to be ⁇ 10 1 cfu/ml. A high temperature inactivated vaccine containing 10 9 dead bacteria/ml was prepared, and the vaccine was packaged into 50 ml/bottle and stored frozen at -20°C. The Vibrio parahaemolyticus liquid with a concentration of 3x10 9 cfu/ml was mixed with a 1500ppm BT lipopeptide aqueous solution at a volume ratio of 1:2.
  • the number of live bacteria after mixing was confirmed to be ⁇ 10 1 cfu/ml.
  • the BT lipopeptide inactivated vaccine containing 10 9 dead bacteria/ml and 1000ppm BT lipopeptide was prepared, and the vaccine was packaged into 50 ml/bottle and stored frozen at -20°C.
  • Feed loading Take the two frozen vaccines mentioned above, thaw them in a water bath at room temperature, and spray them on the surface of No. 05 shrimp compound feed (Wuxi Tongwei Biotechnology Co., Ltd.) at a weight ratio of 1:10. Stir for 5 minutes and then dry them to prepare a high-temperature inactivated vaccine-loaded feed containing 10 8 dead bacteria/ml and a BT lipopeptide-inactivated vaccine-loaded feed containing 10 8 dead bacteria/ml and 100 ppm BT lipopeptide. Take a 1500 ppm BT lipopeptide aqueous solution and spray it on the surface of the above-dried high-temperature inactivated vaccine-loaded feed at a weight ratio of 1:15. Stir for 5 minutes and then dry them to prepare a BT adjuvant high-temperature inactivated vaccine-loaded feed containing 100 ppm BT lipopeptide and 10 8 dead bacteria/ml.
  • Table 3 Comparative test data on the protective efficacy of a single oral dose of BT lipopeptide inactivated vaccine and high temperature inactivated vaccine
  • the relative survival rate of treatment group 5 which was orally vaccinated with a single dose of BT lipopeptide inactivated vaccine, exceeded 40%, while the relative survival rate of treatment group 3, which was orally vaccinated with a single dose of high-temperature inactivated vaccine, was zero, and the relative survival rate of treatment group 4, which was orally vaccinated with a single dose of high-temperature inactivated vaccine with 100 ppm BT lipopeptide as an adjuvant, was still zero.
  • Example 3 Although the inactivated oral vaccine of Vibrio BT lipopeptide disclosed in Example 3 has made a great step forward in the practicality of shrimp Vibrio vaccine, its protective power is still insufficient.
  • BT lipopeptide itself has the function of regulating nonspecific immunity and promoting animal health and growth, the following combination comparison test (Table 4) was conducted to test the protective power of the combination scheme of "single dose vaccination of BT lipopeptide inactivated Vibrio vaccine + continuous feeding of BT peptide".
  • the vaccine preparation and feed loading, oral inoculation and injection challenge methods in this example are the same as those in Example 3.
  • the feed containing 200 ppm BT lipopeptide is prepared by spraying a 1500 ppm BT lipopeptide aqueous solution on the feed at a ratio of 1:7.5, stirring and mixing, and then drying.
  • the body length gain and weight gain of the survivors of each treatment were measured on the 1st and 21st days, and the number of survivors was measured on the 21st day to calculate the relative survival rate.
  • the relative survival rate of treatment group 4 which was only immunized (with BT lipopeptide inactivated vaccine) but without adding BT lipopeptide in the feed, was 38%, which was similar to the result in Example 3, that is, it only had partial protection; the relative survival rate of treatment group 3, which only added BT lipopeptide in the feed but not immunized, was 40%, which also had only partial protection; however, the relative survival rate of treatment group 5, which was both immunized and added BT lipopeptide in the feed, was 99%, showing almost complete protection against the virus attack.
  • oral immunization with the inactivated BT lipopeptide vaccine has a growth-promoting function.
  • the survivors of the immunized treatment groups 4 and 5 had significantly better increases in body length and weight than those of the non-immunized treatment groups 2 and 3.
  • the field experiment was conducted in a small shed earth pond shrimp farm in Ningbo.
  • the 37 sheds of the same batch and the same conditions were divided into two treatment groups.
  • the control group (20 sheds) was neither immunized (with BT lipopeptide inactivated vaccine) nor given BT peptide, but only given No. 05 shrimp compound feed.
  • the experimental group (17 sheds) was fed on the first day of the experiment. Feed the feed loaded with BT lipopeptide inactivated vaccine (10 8 dead Vibrio/g) + 200ppm of BT peptide; to save the use of BT peptide, feed loaded with 200ppm of BT peptide was only fed on the 8th day, that is, BT peptide was no longer used continuously.
  • Shrimp (10 per shed) were taken on the 1st, 8th and 15th days, body length was measured, and the average value of the treatment group was calculated. No clinical vibriosis occurred during the trial.
  • Table 6 Field trial data of shrimps using the combination of “BT lipopeptide inactivated vaccine + alternating pulse BT peptide”
  • Example 5 still involves two preparations, which may cause confusion and operational errors in production applications. Therefore, it is necessary to further simplify and try to combine the two preparations into a single preparation.
  • the single preparation tested contained BT lipopeptide inactivated vaccine (10 9 dead Vibrio bacteria/g) + BT peptide (2000 ppm). Feed loaded with BT lipopeptide inactivated vaccine (10 8 dead Vibrio bacteria/g) + 200 ppm of BT peptide was fed at P35, P45, P55 and P65, and the feeding amount each time was 1/4 of the daily amount (as the experimental group).
  • the control group was fed with feed and freeze-dried powder of Bdellovibrio ("Hunting Bdellovibrio Hero", Guangzhou Shunzhou Biotechnology Co., Ltd.) or functional feed with Chinese herbal formula ("Xia Youkang”, Ningbo Sansheng Biotechnology Co., Ltd.).
  • the feeding method of freeze-dried powder of Bdellovibrio and functional feed with Chinese herbal formula was carried out according to the product instructions.
  • the yield per unit area of the experimental group, the yield per unit area of the control group and the number of small sheds without disease throughout the whole process were calculated.
  • Table 7 summarizes the field comparison tests of different breeding production modes, and the results show that the use of the above single preparations can also effectively improve production indicators.
  • the size of the control group is at least twice that of the experimental group.
  • Table 7 Summary of the results of the field trial of shrimp with a single formulation of "BT lipopeptide inactivated vaccine + BT peptide"

Abstract

本发明提供了质膜透化灭活口服疫苗及其制备方法，所述灭活口服疫苗包含被透化剂透化而灭活的病原体，质膜及其整合膜蛋白，其中病原体选自下组：包膜病毒、细菌、真菌、体内寄生虫和癌细胞，并且透化剂包含脂肽作为表面活性剂。

Description

质膜透化灭活口服疫苗
本申请要求申请日：2022年10月25日，申请号：202211310154.1，发明名称为“质膜透化灭活口服疫苗”的中国发明专利申请的优先权权益。
技术领域
本发明一般涉及生物技术，更具体地，涉及通过透化质膜制备的灭活口服疫苗及其使用。
背景技术
疫苗在人类和非人类动物健康中极为重要，对新的或更好的疫苗有持续的需求。然而，疫苗研发富有挑战性，需要同时满足安全性和有效性这两个经常对立的要求。减毒(attenuated)活疫苗的优势在于能够通过近乎自然的感染过程同时激发体液和细胞免疫，然而，减毒活疫苗可能使处于免疫抑制状态的个体致病。
在另一方面，虽然常规灭活疫苗避免了病原体感染致病的风险，但是存在其他缺陷。首先，即便包含了佐剂，常规灭活疫苗仅能激发体液免疫，基本无法激发细胞免疫，因而保护力弱且持续时间短，需要增加接种的剂量和次数。其次，对灭活疫苗常用的注射递送方式又可能带来新的安全性和有效性问题。由于灭活疫苗无法通过自然感染突破宿主的生物屏障而引发免疫应答，绝大多数灭活疫苗只能被注射递送，但注射引起的疼痛和惊恐会导致应激反应，而引起免疫抑制进而减弱免疫应答，最终导致疫苗接种效果不佳。另外，兽用灭活疫苗的注射通常还需要捕捉和束缚动物，这些操作会进一步强化应激反应，影响疫苗接种效果。
口服是疫苗递送最安全和便捷的方式，更重要的是，口服疫苗可以同时刺激粘膜和机体两个层面的细胞和体液免疫，因而构建更具纵深的抗感染防线，以有效的粘膜免疫将微生物致病原拦截于机体外。然而，灭活口服疫苗面临抗原被消化道中的消化酶降解而彻底丧失的挑战，因此，灭活口服疫苗抗原的形态或剂型必须具备不被消化且被高效吸收的能力。如何保护抗原并使之高效穿越消化道屏障被转运至免疫细胞，是口服灭活疫苗的关键。
肠道对抗原的自然吸收为口服疫苗抗原递送提供了技术灵感。M细胞 (Microfold or M cells)是覆盖小肠和大肠组织淋巴滤泡(organized lymphoid follicles)的表皮中高度分化的细胞。M细胞通过将细菌、病毒、寄生虫以及非传染性颗粒物转运至下属淋巴组织潘式斑(the Peyer’s patches)，对众多分子进行采样、蛋白识别以及抗原转运。基于其在启动粘膜免疫应答中发挥的核心作用，M细胞成为使抗原穿越消化道细胞屏障的人工疫苗载体的递送目标。利用M细胞偏好摄取含有脂质双层膜结构颗粒物的特性，采样脂质材料的人工包被(encapsulation)技术被开发出来用于保护和递送口服疫苗抗原，然而制备以M细胞为直接递送目标的人工载体疫苗，例如脂质体和胆汁体(liposome and bilosome)疫苗，涉及复杂的工艺和高昂的成本，使它们的实用范围限于“高端疫苗”。
为避免或减少无效和低效抗原竞争导致的免疫资源浪费，制备常规灭活疫苗时需要分离纯化高效能抗原。例如，流感灭活疫苗的抗原制备流程是(i)将选定的毒株接种到鸡胚蛋中扩增，(ii)纯化病毒，(iii)用甲醛和β-丙内酯双重灭活，(iv)清除灭活剂，(v)用去污剂分离高效能抗原。然而，纯化抗原的常规与灭活口服疫苗的技术要求不相容，纯化后“裸露的”抗原会在消化道中被消化酶轻易降解而丧失，若进行包被保护，则又涉及复杂的工艺和高昂的成本。
因此，本领域中需要工艺简单且成本低廉的口服疫苗。
发明内容
针对现有技术的缺陷，本发明提供了一种工艺简单且成本低廉的口服疫苗以及其制备方法和用途。本发明的口服疫苗通过透化剂透化质膜而灭活病原体或其减毒疫苗，无需分离抗原(例如病毒)和/或人工包被抗原等常规步骤就可制备。
本发明基于发明人的出乎意料的发现：与现有技术中应当分开递送BT脂肽与疫苗的教导(中国专利申请号CN201810354453.2)相背离，当将BT脂肽与减毒活疫苗一起口服施用时，BT脂肽未导致减毒活疫苗的免疫失败。发明人还出乎意料地发现，当用仅包含BT脂肽作为表面活性剂成分的透化剂透化而灭活病原体时，BT脂肽灭活疫苗口服接种有远高于高温灭活疫苗口服接种的保护力。
在一方面，本发明提供了灭活口服疫苗，其包含质膜被透化剂透化而灭 活的病原体或其活疫苗，其中灭活的病原体或其活疫苗包含经透化的质膜及其整合膜蛋白，其中病原体是包含质膜的病原体，并且透化剂包含脂肽作为表面活性剂。
在一个实施方案中，病原体选自下组：包膜病毒、细菌、真菌、体内寄生虫和癌细胞。
在一个实施方案中，质膜及其整合膜蛋白为病原体或其活疫苗的天然质膜及其整合膜蛋白。在一个实施方案中，透化剂仅透化磷脂双分子膜。在一个实施方案中，透化剂仅透化磷脂双分子膜而不改变磷脂分子双层膜结构，在一个实施方案中，透化剂不使整合膜蛋白脱离磷脂，保持整合膜蛋白自然构象。
在一个实施方案中，疫苗还包含病原体或其活疫苗在透化后的外泄内容物。
在一个实施方案中，疫苗通过包括对病原体或其活疫苗进行质膜透化(permeabilization of plasma membrane)的步骤而灭活。
在一个实施方案中，质膜透化的步骤使得活病原体数<101/ml和/或死病原体数>109/ml。
在一个实施方案中，灭活口服疫苗是口服液、粉剂、片剂、胶囊、灌服液或口腔推注液。
在一个实施方案中，灭活口服疫苗是通过直接口服、饮水负载、食物负荷、灌服或口腔推注施用的疫苗。
在一个实施方案中，透化剂不包含除脂肽外的表面活性剂。
在一个实施方案中，脂肽是唯一的表面活性剂。
在一个实施方案中，脂肽包含天然脂肽和/或人工合成脂肽。
在一个实施方案中，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的脂肽。
在一个实施方案中，产自德克萨斯侧孢短芽孢杆菌的脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
在一个实施方案中，病原体选自下组：冠状病毒科、流感病毒、副粘病毒科、疱疹病毒科、痘病毒科、肝病毒科、杆状病毒属、反转录病毒科、非洲猪瘟病毒、不动杆菌属、杆菌属、疏螺旋体属、弯曲杆菌属、梭菌属、弧 菌属、大肠杆菌、肠球菌属、奈瑟菌属、嗜血菌属、曼氏杆菌属、分枝杆菌属、支原体属、巴斯德氏菌属、假单胞菌属、沙门氏菌属、葡萄球菌属、链球菌属、球虫、线虫、绦虫和癌细胞。
在一个实施方案中，病原体选自下组：新城疫病毒和副溶血弧菌。
在一个实施方案中，灭活口服疫苗包含药学可接受的载体，或者包含调节免疫或促进健康或生长的成分，例如佐剂，或者产自德克萨斯侧孢短芽孢杆菌的脂肽。在一个实施方案中，产自德克萨斯侧孢短芽孢杆菌的脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
在另一个方面，提供了制备本文所述的灭活口服疫苗的方法，其包括：对病原体或其活疫苗进行质膜透化，其中透化剂包含脂肽作为表面活性剂。
在一个实施方案中，方法不包括分离抗原的步骤和/或人工包被抗原的步骤。例如，方法不包括将抗原进行脂质或胆汁包裹或包被的步骤。
在一个实施方案中，当对病原体进行灭活时，方法不包括透化灭活外的其他灭活方式，例如高温灭活方式。
在一个实施方案中，透化剂不包含除脂肽外的表面活性剂。
在一个实施方案中，对病原体或其活疫苗进行质膜透化，使得活病原体数<101/ml和/或死病原体数>109/ml。
在一个实施方案中，脂肽包含天然脂肽和/或人工合成脂肽。
在一个实施方案中，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的脂肽。
在一个实施方案中，产自德克萨斯侧孢短芽孢杆菌的脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
在又一个方面，本发明提供了口服组合物，其包含本文所述的灭活口服疫苗。
在一个实施方案中，口服组合物包含食物、饮用水或饮料。
在一个实施方案中，食物选自下组：代乳品、生长饲料、肥育饲料、育雏前饲料和育雏饲料。
在一个实施方案中，组合物包含谷类、大豆粉、分离的大豆蛋白、大豆油、大豆脂肪、脱脂乳、鱼粉、肉粉、骨粉、血粉、血浆蛋白质、乳清、稻糠、麦麸、甜味剂、矿物、维生素和盐中的一种或多种。
在一个实施方案中，谷类选自下组：大麦、大豆、小麦、小黑麦、黑麦、玉米和稻米。
在一个实施方案中，口服组合物包含调节免疫或促进健康或生长的成分，优选BT脂肽，所述BT脂肽与灭活口服疫苗是混为一体的或分离的。例如，BT脂肽包含产自德克萨斯侧孢短芽孢杆菌的BT脂肽，优选地，具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。口服组合物可以包含BT脂肽，所述BT脂肽与灭活口服疫苗是混为一体的或分离的。BT脂肽的浓度可以为100ppm以上，例如150ppm、200ppm、250ppm、300ppm、330ppm、400ppm、450ppm、500ppm、550ppm、600ppm、650ppm、700ppm、750ppm、800ppm、850ppm、900ppm或950ppm。
在又一个方面，本发明提供了透化剂用于制备灭活口服疫苗的用途，该用途通过将病原体的质膜穿孔引起内容物外泄而灭活病原体实现；其中透化剂包含脂肽作为表面活性剂。
在一个实施方案中，灭活口服疫苗是本文所述的灭活口服疫苗。
在一个实施方案中，脂肽包含天然脂肽和/或人工合成脂肽。
在一个实施方案中，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的脂肽。
在一个实施方案中，产自德克萨斯侧孢短芽孢杆菌的脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
在又一个方面，本发明提供了本文所述的灭活口服疫苗在制备药物中的用途，所述药物用于治疗和/或预防受试者中由病原体引起的感染或疾病。
在又一个方面，本发明提供了治疗和/或预防受试者中由病原体引起的感染或疾病的方法，其包括对受试者施用本文所述的灭活口服疫苗。
在又一个方面，提供了本文所述的灭活口服疫苗，用于治疗和/或预防受试者中由病原体引起的感染或疾病。
在上述方面的一个实施方案中，感染或疾病选自：流感、病毒性肝炎、非洲猪瘟、猪生殖与呼吸综合征、大肠杆菌病、沙门氏菌病、坏死性肠炎、弧菌病、牛航运热肺炎、淋病、尿路感染、球虫病、线虫病、绦虫病和癌症。
在又一个方面，本发明提供了本文所述的灭活口服疫苗在制备饲料中的用途，所述饲料用于改善受试者中的体重增加和/或食物转化。
在又一个方面，本发明提供了用于改善受试者中的体重增加和/或食物转化的方法，其包括对受试者施用本文所述的灭活口服疫苗。
在上述方面的一个实施方案中，受试者是禽类、家畜、水生动物或伴侣动物。在上述方面的一个实施方案中，饲料通过蛋内饲喂、以液滴形式喷洒于禽类或沉浸水生动物施加。
本发明的有益效果包括：(1)本文所述的灭活口服疫苗口服递送至消化道，利用消化道中的消化酶自然降解外泄的内容物而清除潜在的膜内抗原分子，从而选择性地保留穿孔质膜及其整合膜蛋白作为抗原分子，最终被吸收而引发粘膜和全身免疫应答，完全省去灭活疫苗制作过程中的抗原纯化步骤及成本；(2)本文所述的灭活口服疫苗无需对抗原进行额外的保护处理，例如保护抗原免于消化道降解的处理(如脂质包被等)。
具体实施方式
定义
为了促进本发明的理解，下文定义了许多术语。本文定义的术语具有与本发明相关领域普通技术人员通常理解的含义。
如本文中使用的，“动物”意指任何脊椎动物或无脊椎动物，包括但不限于人、马、山羊、绵羊、牛、猪、鸡、火鸡、母鸡、鹅、鸭、犬、猫、鹦鹉、鱼、蟹、虾(例如南美白对虾)、淡水龟等。
如本文中使用的，“减毒”是指降低病毒、细菌、真菌或寄生虫对动物的致病能力。
如本文中使用的，“活疫苗”指用人工的方法使病原体减毒或从自然界筛选某病原体的无毒株或微毒株所制成的活微生物制剂，有时称减毒活疫苗。
如本文中使用的，“灭活”是指使病毒、细菌、真菌、寄生虫或癌细胞不可逆地失去复制或增殖能力。“灭活剂”是指使病毒、细菌、真菌、寄生虫或癌细胞不可逆地失去复制或增殖能力的物质或能量。
如本文中使用的，“质膜透化”和“质膜被穿孔”同义，都是指使质膜完整性受损而导致内容物外泄，但不改变膜的磷脂分子双层总体结构，也不改变整合膜蛋白的构象或其与磷脂分子双层的结合。
如本文中使用的，“质膜溶解”(plasma membrane dissolution)是指将磷 脂分子双层转化为磷脂胶束(phospholipid miscelle)，同时使整合膜蛋白脱离磷脂和变性。
如本文中使用的，“表面活性剂”(surfactant)是指能降低液体与液体间等界面表面张力的两亲性物质。不同类型的表面活性剂对质膜结构有不同类型的破坏作用，去污剂(detergent)表面活性剂溶解质膜，而脂肽表面活性剂透化但不溶解质膜。
脂肽表面活性剂可以包括天然和人工合成的脂肽表面活性剂。天然脂肽是由微生物产生的次级代谢产物，一般有一个长脂链和7-25个氨基酸残基，被显示具有抗细菌、抗病毒、抗真菌、抗肿瘤和免疫调剂功能。天然脂肽表面活性剂包括但不限于表面活性素(Surfactins)、芬枯草菌素(Fengycins)和伊枯草菌素(Iturins)、达托霉素(Daptomycin)、多粘菌素(Polymyxins),粘液菌素(Viscosin)和BT脂肽。
如本文中使用的，“BT脂肽”可以是指产自德克萨斯侧孢短芽孢杆菌(Brevibacillus laterosporus texasporus)的单个脂肽或多个脂肽的混合物，特别是包含CN201380056514.2和CN201810354453.2所披露的SEQ ID NO：1-9序列的单个脂肽或多个脂肽的混合物。德克萨斯侧孢短芽孢杆菌(Brevibacillus texasporus)(例如ATCC PTA-5854)是先前鉴定的土壤细菌，其表达非核糖体肽合成酶(NRPS，由在GenBank登录号AY953371下的操纵子编码)，以产生13个氨基酸残基的相关阳离子NRP变体的家族/混合物(“BT肽”或“BT脂肽”，根据其新近分辨的N末端结构，并且两个术语在本公开内容中是可互换的)，其中BT1583是最丰富的变体(WO/2005/074626)。阳离子肽(作为从德克萨斯侧孢短芽孢杆菌中分离的混合物或个别肽)在体外展示广谱抗菌活性(BT功能#1)。在PTA-5854和侧孢短芽孢杆菌(Brevibacillus laterosporus)类型菌株之间的高度16S rDNA序列同一性(98.5％)，将德克萨斯侧孢短芽孢杆菌分类为侧孢短芽孢杆菌的亚种，其中侧孢短芽孢杆菌德克萨斯亚种(Brevibacillus laterosporus subsp.texasporus)定义为产生来自BT NRPS(或BT肽)的非核糖体肽的侧孢短芽孢杆菌菌株。至少两个侧孢短芽孢杆菌菌株(LMG 15441和GI-9)的基因组测序已验证该分类学。两个基因组(分别在GenBank登录号AFRV00000000和EMBL登录号CAGD01000001至CAGD01000061下公开)均含有完整的BT NRPS操纵子，与AY953371具有99％DNA序列同一性，即使未知这些侧孢短芽孢杆菌菌株是BT肽的生产者。 “德克萨斯侧孢短芽孢杆菌”、“侧孢短芽孢杆菌德克萨斯亚种”和“德克萨斯侧孢短芽孢杆菌(B.texasporus)”因此是同义的。
BT肽或BT脂肽可以是使用WO/2005/074626和(Wu,X.,Ballard,J.和Jiang,Y.W.(2005).Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus.Appl Environ Microbiol 71,8519-8530)中公开的分离和检测方法，从德克萨斯侧孢短芽孢杆菌中分离的天然BT NRP变体(包括SEQ ID NO:1-9)的混合物，但所述分离和检测方法不含分辨BT NRP变体的反相HPLC步骤。本领域技术人员可使用这些参考文献中所述的方法，容易地制备BT肽或BT脂肽。表征BT肽或BT脂肽，并且氨基酸序列在下表1中提供。表1描述了9种BT NRP变体的结构及其在此类混合物中的通常相对丰度。
表1：BT NRP变体的概括(Hmp代表2-羟基-3-甲基戊酸，Dhb代表2,3-脱氢-2-氨基丁酸，其与脱氢苏氨酸(Dht)同义，Vol代表缬氨醇，下标D和E分别表示氨基酸中的D型手性和(E)-构型，并且％指示当用质谱法测定时，在作为从德克萨斯侧孢短芽孢杆菌细胞中分离的混合物的BT NRP变体中的变体相对丰度)。
如本文中使用的，“饲料”或“食物”同义，是指为了对动物或人提供营养需求的至少一部分，和/或用于预防或治疗动物中的营养性病症的目的，而制备、销售或提供给动物(如家畜或驯化动物)或人消耗的，含有氨基酸、抗氧化剂、碳水化合物、调味剂、酶、脂肪、矿物、非蛋白质氮产物、蛋白质、维生素和/或粘合剂中的任何物质或物质混合物，并且可以含有造粒剂、着色剂、起泡剂和/或芳香剂。作为至关重要的营养物，水是饲料或食物成分， 而且含水饮料是饲料或食物。
本发明的灭活口服疫苗
本发明包括灭活口服疫苗。灭活口服疫苗的主要成分可以为质膜被透化或穿孔了的(plasma membrane-permeabilized or perforated)包膜病毒、细菌、真菌、体内寄生虫或癌细胞。灭活口服疫苗的主要成分可以为分离自质膜被穿孔了的包膜病毒、细菌、真菌、体内寄生虫或癌细胞的磷脂分子双层膜或含有磷脂分子双层膜的分离物。灭活口服疫苗的抗原可以包含源自包膜病毒、细菌、真菌、体内寄生虫或癌细胞的、被穿孔的磷脂双分子层膜及其整合膜蛋白。
本发明的灭活口服疫苗的主要成分可以为质膜被透化或穿孔了的包膜病毒、细菌、真菌或体内寄生虫，或者包膜病毒、细菌、真菌或体内寄生虫的减毒(attenuated)活疫苗。灭活口服疫苗的主要成分可以为分离自被质膜穿孔了的包膜病毒、细菌、真菌或体内寄生虫(或者包膜病毒、细菌、真菌或体内寄生虫的活疫苗)的磷脂分子双层膜或含有磷脂分子双层膜的分离物。灭活口服疫苗的抗原可以包含源自减毒包膜病毒、细菌、真菌或体内寄生虫(或者包膜病毒、细菌、真菌或体内寄生虫的活疫苗)的、被穿孔的磷脂双分子层膜及其整合膜蛋白。
制备本发明的灭活口服疫苗的方法
本发明包括制备灭活口服疫苗的方法，包括将病原体或其活疫苗与透化剂混合，使质膜被穿孔引起内容物外泄而灭活。病原体可以是有包膜的病原体，包括包膜病毒、细菌、真菌、体内寄生虫或癌细胞。在一个例示性实施方案中，用于灭活的透化剂是脂肽表面活性剂。脂肽表面活性剂包括天然和人工合成的脂肽表面活性剂(lipopeptide surfactant)。天然脂肽表面活性剂的例子包括：表面活性素(Surfactins)、芬枯草菌素(Fengycins)、伊枯草菌素(Iturins)、达托霉素(Daptomycin)、多粘菌素(Polymyxins)、粘液菌素(Viscosin)和BT脂肽。在一个进一步的例示性实施方案中，用于灭活的透化剂是BT脂肽。BT脂肽可以包括一种或多种选自SEQ ID NO:1-9的序列、来自德克萨斯侧孢短芽孢杆菌的分离的BT脂肽。BT脂肽是纯化的水溶性的BT脂肽。BT脂肽可以是从德克萨斯侧孢短芽孢杆菌中分离的混合物。在本 文中，BT脂肽可以用作灭活剂和/或佐剂。在本文的灭活口服疫苗中，BT脂肽的浓度足以使病原体或其活疫苗的活病原体数<101/ml和/或死病原体数>109/ml。例如，BT脂肽在灭活口服疫苗中的终浓度可以为100ppm以上，例如100ppm、500ppm、1000ppm、1500ppm、2000ppm、2500ppm、3000ppm、3500ppm、4000ppm、4500ppm、5000ppm或5500ppm。
本文所述的灭活口服疫苗的方法可以不包括分离抗原的步骤和/或人工包被抗原的步骤。例如，方法不包括将抗原进行脂质或胆汁包裹或包被的步骤。在一个实施方案中，当对病原体进行灭活时，方法不包括透化灭活外的其他灭活方式，例如高温灭活方式。在一个实施方案中，透化剂不包含除脂肽外的表面活性剂。在一个实施方案中，对病原体或其活疫苗进行质膜透化，使得活病原体数<101/ml和/或死病原体数>109/ml。在一个实施方案中，脂肽包含天然脂肽和/或人工合成脂肽。在一个实施方案中，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的脂肽。在一个实施方案中，产自德克萨斯侧孢短芽孢杆菌的脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。在一个实施方案中，产自德克萨斯侧孢短芽孢杆菌的脂肽是以SEQ ID NO:1-9所示的序列的脂肽的混合物。
剂型和施用
本发明包括质膜透化灭活口服疫苗的剂型，包括但不限于口服液、粉剂、药片、胶囊、灌服液和口腔推注液。
本发明还包括灭活口服疫苗的递送方式。灭活口服疫苗的递送方式包括但不限于自主直接口服、饮水负载、食物负荷、灌服和口腔推注。灭活口服疫苗的递送方式还包括但不限于以下特殊方式，将灭活疫苗液体注入鸟类蛋胚的羊膜囊中(蛋内饲喂)，以及将灭活疫苗以粗液滴形式喷洒在鸟类(尤其是幼雏)身上供啄食，将水生动物(尤其是其幼苗)沉浸在灭活疫苗液体中。
本发明还包括添加了口服灭活疫苗的食物/饲料或饮水/饮料。在一个例示性实施方案中，质膜透化灭活疫苗被纳入到饮用水或动物饲料中，如基于谷类的饲料，例如含有至少一种选自下组的谷类的食物/饲料：大麦、大豆、小麦、小黑麦(triticale)、黑麦(rye)、玉米、稻米及其组合。实际上，可以将 本发明添加到极其多种饲料中。可以将本发明的灭活疫苗与选自下组的家畜用饮用水或饲料混合：代乳品(milk replacer)、生长饲料(grower feed)、肥育饲料(finisher feed)、育雏前饲料(pre-starter feed)、育雏饲料(starter feed)、水及其组合。食物/饲料成分的例子还包括谷类、大豆粉、分离的大豆蛋白、分离的大豆油、分离的大豆脂肪、脱脂乳、鱼粉、肉粉、骨粉、血粉、血浆蛋白质、乳清、稻糠、麦麸，并且还可以包含甜味剂、矿物、维生素和盐。
本发明的灭活口服疫苗的应用
本发明还包括富集高效能的质膜表面抗原的接种后纯化方法(post-inoculation purification)，包括将本文所述的灭活口服疫苗口服递送。本文所述的灭活口服疫苗口服递送至消化道，利用消化道中的消化酶自然降解外泄的内容物而清除潜在的膜内抗原分子，从而选择性地保留穿孔质膜(perforated plasma membrane)及其整合膜蛋白作为抗原分子，最终被吸收而引发粘膜和全身免疫应答，完全省去灭活疫苗制作过程中的抗原纯化步骤及成本。在一个例示性实施方案中，将主要成分为质膜被透化或穿孔了的包膜病毒、细菌、真菌、体内寄生虫或癌细胞的灭活疫苗口服递送，由消化道中的消化酶降解外泄的内容物而清除潜在的膜内抗原分子，从而选择性地保留穿孔质膜及其整合膜蛋白作为最终实际被递送的抗原分子。
本发明还包括改善正在生长的家畜或伴侣动物中重量增加和饲料转化的方法，其通过以足以预防和/或治疗动物中微生物感染的有效量，口服提供质膜透化灭活疫苗来进行。
本发明还包括用于预防和/或治疗动物中微生物感染的方法，其通过以足以预防和/或治疗动物中微生物感染的有效量，口服提供质膜透化灭活疫苗来进行。
本发明还涉及预防和/或治疗动物中疾病的组合物和/或方法，其包括对动物口服施用质膜透化灭活疫苗。例如，治疗可以涉及减少动物体内的病原体。可减少的例示性病原体可以包括但不限于冠状病毒科(Coronaviridae)、流感病毒(Influenza viruses)、副粘病毒科(Paramyxoviridae)疱疹病毒科(Herpesviridae)、痘病毒科(Poxviridae)、肝病毒科(Hepadnaviridae)、杆状病毒属(Baculoviruidae)、反转录病毒科(Retroviridae)、非洲猪瘟病毒(African swine fever virus)、不动杆菌属(Acinetobacter)、杆菌(Bacilli)、疏螺 旋体属(Borrelia)、弯曲杆菌属(Campylobacter)、梭菌(Clostridia)、弧菌属(Vibrio)、大肠杆菌(E.coli)、肠球菌属(Enterococcus)、奈瑟菌属(Neisseria)、嗜血菌属(Haemophilus)、曼氏杆菌属(Mannheimia)、分枝杆菌(Mycobacteria)、支原体属(Mycoplasma)、巴斯德氏菌属(Pasteurellaceae)、假单胞菌属(Pseudomonas)、沙门氏菌属(Salmonella)、葡萄球菌属(Staphylococcus)、链球菌属(Streptococcus)、球虫(Coccidia)、线虫(Nematoda)、绦虫(Cestoda)和癌细胞(cancer cells)。
本发明可预防和/或治疗的例示性疾病包括但不限于新城疫(Newcastle Disease)、流感(influenza)、病毒性肝炎(Hepatitis)、非洲猪瘟(African swine fever)、猪生殖与呼吸综合征(porcine reproductive&respiratory syndrome)、大肠杆菌病(colibacillosis)、沙门氏菌病(salmonellosis)、坏死性肠炎(necrotic enteritis)、弧菌病(vibrosis)、牛航运热肺炎(bovine shipping fever pneumonia)、淋病(Gonorrhea)、尿路感染(urinary track infection)、球虫病(coccidiosis)、线虫病(nematodiasis)、绦虫病(cestodiasis)和癌症(cancer)。
本发明的方法还可以包括口服施用分离的BT肽。BT肽可以与本发明的灭活口服疫苗在同一日施用，和/或在本发明的灭活口服疫苗施用后的1天或数天施用。BT肽可以连续数日施用，例如连续2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、或30天施用。或者，为了节约BT肽的用量，BT肽可以间隔施用(脉冲施用)，例如间隔1天、2天、3天、4天、5天、6天、7天、8天、9天、10天、11天、12天、13天、14天、15天、16天、17天、18天、19天、20天、21天、22天、23天、24天、25天、26天、27天、28天、29天、或30天施用。在此情况下，BT肽可以施用1次或多次，如2次、3次、4次、5次。
本发明的方法还可以包括将BT肽与本发明的灭活口服疫苗合并为单一制剂后口服施用。
下面将结合实施例对本申请的实施方案进行详细描述，本领域技术人员将会理解，下列实施例仅用于说明本申请，而不应视为限定本申请的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。若无特殊标明，所列出的量均基于总重并以重量份数描述。本申请不应解释为受限 于所述的具体实施例。
实施例
本文实施例中BT脂肽是使用WO/2005/074626和(Wu,X.,Ballard,J.和Jiang,Y.W.(2005).Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus.Appl Environ Microbiol 71,8519-8530)中公开的方法和CN201280020898.8中公布的德克萨斯侧孢短芽孢杆菌菌株PTA-12309(保藏于美国典型培养物保藏中心)制备而成。具体地，在37℃在摇床中在1升LB中将德克萨斯侧孢短芽孢杆菌菌株PTA-12309细胞培养3天。培养物在离心机中以3,000rpm的转速旋转15分钟。收集上清液，加入500克的硫酸铵并溶解。将样品在离心机中以3,000×g离心15分钟。将沉淀物溶解在200ml蒸馏水中。然后将该溶液煮沸15分钟，然后在冰上冷却。用0.2μm的过滤器(Nalgene)过滤样品。滤液与0.2升氯仿在室温下用搅拌棒混合20分钟。在离心机中以3,000rpm离心15分钟，将混合物分离成两相。收集有机相并在真空蒸发器中进行干燥。本领域技术人员可使用这些参考文献中所述的方法，容易地制备BT肽或BT脂肽。表征BT肽或BT脂肽，并且氨基酸序列在表1中提供。
实施例1
本发明的首个实施例是养殖生产中的一次“失误”，是将BT脂肽与包膜病毒的弱活疫苗“错误”混合后口服接种仍有效免疫的意外成功。如CN201810354453.2披露的，BT脂肽作为分离式佐剂，被通过饮水递送给被以注射或喷雾接种了疫苗的动物，也就是说，BT脂肽佐剂与疫苗时空隔离。在本实施例的“失误”中，之前采用的孵化后在孵化场通过喷雾接种新城疫活疫苗(La Sota株，欣匹克，哈药集团生物疫苗有限公司)的递送方式，被改作2日龄在商品代鸡场通过饮水递送，因当天按原有流程需要进行100ppm的BT脂肽饮水，故新城疫弱活疫苗与BT脂肽被一同混合在饮水中供动物饮用。这批鸡的成活、健康和生长均正常，25日龄的新城疫抗体滴度也完全正常。
在回顾总结该批次生产时，BT脂肽佐剂与新城疫弱活疫苗混合的“冲突”方才被注意和意识到，因为，CN201810354453.2披露的“BT脂肽口服佐剂与 疫苗的组合物”中“BT佐剂作为口服活疫苗组成部分”的组合方式是被双重排除了的。首先，活疫苗自带多种天然佐剂，因而根本无需增加外源性佐剂。更重要的是，增加的佐剂不能负面影响或改变疫苗原有的基本性质。BT脂肽对质膜有很强的透化作用，对新城疫病毒这样的包膜病毒及其弱活疫苗有灭活作用。因此，将BT脂肽与包膜病毒弱活疫苗“错误”混合后口服接种并未导致免疫失败的结果实属意外。
实施例2
为验证和解析实施例1展示的“新城疫BT脂肽灭活口服疫苗”，进行了一个多重对比试验(表2)。100只鸡雏被分作5个不同免疫的处理组，每组20只。处理组1为负对照组，不接种疫苗，在2日龄灌服生理盐水，1ml/只。处理组2为正对照组，在2日龄灌服接种新城疫弱活疫苗(La Sota株，欣匹克，哈药集团生物疫苗有限公司)，1.5羽份/只。处理组3-5灌服接种灭活后的新城疫疫苗制剂，处理组3采用脂肽单灭活疫苗(脂肽+La Sota株)，处理组4和5采用去污剂+脂肽双重灭活疫苗(去污剂+脂肽+La Sota株)。处理组3的接种物为将BT脂肽与上述弱活疫苗混合配制成含1000ppm BT脂肽和1.5羽份疫苗(灭活)/ml的水液，按1ml/只在2日龄灌服。处理组4的接种物为将Triton X-100去污剂、BT脂肽与上述弱活疫苗混合配制成含1000ppm BT脂肽、2％Triton X-100和1.5羽份疫苗(灭活)/ml的水液，按1ml/只在2日龄灌服；处理组5的接种物为将Triton X-100去污剂、BT脂肽与上述弱活疫苗混合配制成含1000ppm BT脂肽、2％Triton X-100和4.5羽份疫苗(灭活)/ml的水液，按1ml/只在2日龄灌服。需要指出的是，处理组3-5灌服接种物中的BT脂肽，不仅是制作灭活疫苗的灭活剂，同时也是疫苗佐剂。新城疫抗体滴度的测定按中国国家标准GB/T16550-2020所述方法完成，新城疫疫苗毒含量的测定遵循《中国兽药典(2010年版三部)》“病毒半数致死量、感染量(LD50、ELD50、ID50、EID50、TCID50)测定法”。
表2：新城疫口服疫苗攻毒对比试验数据

25日龄的新城疫抗体滴度数据显示，BT脂肽单灭活疫苗口服接种(处理组3)和弱活疫苗口服接种(处理组2)的抗体水平接近；而双灭活疫苗口服接种(处理组4和5)与未接种任何疫苗(处理组1)的抗体水平相同，皆为零。
经典高致病Texas GB毒株是USDA(美国农业部)指定并提供的用于检验新城疫疫苗保护力的标准攻毒毒株，由发明人内部保存。所有处理组在28日龄接受Texas GB毒株攻毒，每只鸡被胸肌注射了105EID50的Texas GB病毒，然后观察一周，统计各处理组的存活数，计算各组的存活率。结果显示，BT脂肽单灭活疫苗口服接种(处理组3)和弱活疫苗口服接种(处理组2)的保护力接近，而双灭活疫苗口服接种(处理组4和5)与未接种任何疫苗(处理组1)的保护力相同，皆为零。
以上结果显示BT脂肽灭活疫苗口服与弱活疫苗功效相当，验证了实施例1中的意外发现和发明。更重要的是，在灭活步骤增加去污剂Triton X-100作为第二个表面活性剂灭活剂，则使BT脂肽灭活口服疫苗完全失效，即便仍含有等量的BT脂肽可以作为疫苗佐剂。
这些结果表明，灭活剂的种类(灭活方式)和/或抗原的形态对于灭活口服疫苗至关重要。具体地，虽然都是表面活性剂，去污剂和脂肽破坏磷脂双分子膜的方式和结果相当不同。Triton-X100等去污剂表面活性剂(detergent  surfactant)溶解磷脂分子双层膜，即形成磷脂胶束(phospholipid miscelle)，使整合膜蛋白脱离磷脂而变性；而脂肽则仅透化磷脂双分子膜，不重大改变磷脂分子双层膜结构，不使整合膜蛋白脱离磷脂，保持整合膜蛋白自然构象。
与其他微生物产生的天然脂肽表面活性剂一样，德克萨斯侧孢短芽孢杆菌产生BT脂肽也是非核糖体肽，口服BT脂肽完全不被消化，不希望受到理论约束，这可能意味着BT脂肽不仅不被消化道中的蛋白酶、肽酶或脂酶降解，而且BT脂肽抑制蛋白酶、肽酶和脂酶对所嵌入穿孔的质膜及整合膜蛋白的降解。也就是说，在消化道中，BT脂肽等天然脂肽表面活性剂对于与之结合的磷脂分子双层膜结构及整合膜蛋白可能有抗消化作用，有利于透化质膜及其整合膜蛋白被M细胞吸收递送；而质膜透化外泄的内容物蛋白分子则完全“裸露”，在消化道中被蛋白酶自然降解。如此差异化的消化降解可能实际上起到对病原体质膜表面抗原的“接种后纯化”(postinoculational purification)作用，有利于质膜透化灭活口服疫苗建立有效的粘膜和全身免疫。
实施例3
对实施例2结果分析的一个结论或推测是，可以用脂肽表面活性剂透化任何包有质膜的病原体来制备灭活口服疫苗，为检验这个推测，进行了以下口服BT脂肽灭活弧菌疫苗的试验。
弧菌病是虾类养殖的头号疾病，虽然副溶血弧菌灭活口服疫苗研究已超过30年，但目前虾类养殖仍无具实用价值的疫苗，现有弧菌口服“疫苗”，例如弧菌高温灭活疫苗，通常需要被添加到饲料中长期连续饲喂，还没有符合疫苗常规定义、单剂接种即刺激产生保护力的口服疫苗被披露或报道。与脊椎动物不同，虾没有通过DNA水平基因重组产生固定免疫球蛋白特异性的经典特异性免疫系统，目前只发现了通过mRNA替代剪切产生暂时性免疫球蛋白特异性的“类特异性免疫系统”，可能因此无论采用何种剂型或递送方式，虾疫苗都更难建立特异性免疫，保护力“记忆”持续时间更短。鉴于此，虾弧菌病成为进一步验证脂肽质膜透化灭活口服疫苗技术的试金石。
首先，用BT脂肽制备了致病性副溶血弧菌质膜透化灭活疫苗，以传统的副溶血弧菌高温灭活疫苗为对照，比较单剂接种南美白对虾10天后的攻毒保护力。用于疫苗制备和攻毒的致病性副溶血弧菌(宁波大学张德民、郭海朋 赠送)分离自浙江宁波地区的弧菌病死亡虾，该菌株携带pirAB毒素基因，其致病性经过实验室攻毒试验验证确立(致病性副溶血弧菌满足致病要求即可使用)。
疫苗制备：将浓度为1x109cfu/ml副溶血弧菌菌液进行15分钟的100℃高温处理，检测确认加热后活菌数<101cfu/ml，制成含109死菌/ml的高温灭活疫苗，按50ml/瓶分装后-20℃冰冻保存。将浓度为3x109cfu/ml副溶血弧菌菌液与浓度为1500ppm BT脂肽水溶液按1:2的体积比混合，检测确认混合后活菌数<101cfu/ml，制成含109死菌/ml、1000ppm BT脂肽的BT脂肽灭活疫苗，按50ml/瓶分装后-20℃冰冻保存。
饲料负载：分别取上述两个冰冻疫苗，常温水浴化冻后，按1:10的重量比喷洒在05号对虾配合饲料(无锡通威生物科技有限公司)表面，搅拌5分钟后晾干，制成含108死菌/ml的高温灭活疫苗负载饲料以及含108死菌/ml、100ppm BT脂肽的BT脂肽灭活疫苗负载饲料。取1500ppm BT脂肽水溶液，按1:15的重量比喷洒在上述晾干的高温灭活疫苗负载饲料表面，搅拌5分钟后晾干，制成含100ppm BT脂肽、108死菌/ml的BT佐剂高温灭活疫苗负载饲料。
口服免疫：在试验第1天对各处理的平均体长约50mm的南美白对虾饲喂表3中列出的未负载和负载了疫苗的饲料，从第2天起饲喂各组的饲料均为未负载疫苗的饲料。对虾配合饲料的具体饲喂方式遵循供应商的产品说明书(05号对虾配合饲料；无锡通威生物科技有限公司)。
注射攻毒：在第11天用制备上述灭活疫苗的致病性副溶血弧菌菌株的菌液进行注射攻毒，目标攻毒强度为50％的48小时内死亡率。停饲两天，在第13天恢复正常饲喂。在第21天测定幸存者数量以计算存活率和相对存活率(Relative Survival Rate，RSP＝(处理组存活率-不免疫但攻毒对照组存活率)/(不免疫不攻毒对照组存活率-不免疫但攻毒对照组存活率))。
表3：单剂口服接种BT脂肽灭活疫苗与高温灭活疫苗的保护力比较试验数据

在第21天，即攻毒后的第10天，统计各处理组的死亡率，计算各组的相对存活率。被BT脂肽灭活疫苗单剂口服接种处理组5的相对存活率超过40％，而被高温灭活疫苗单剂口服接种的处理组3的相对存活率为零，被添加了100ppm BT脂肽作为佐剂的高温灭活疫苗单剂口服接种的处理组4的相对存活率仍为零。这些结果显示，BT脂肽灭活疫苗单剂接种有远高于高温灭活疫苗单剂接种的保护力，而且增加BT脂肽作为佐剂未能提升后者的保护力，再次显示灭活方式及抗原形态对于灭活口服疫苗的关键性。
实施例4
虽然实施例3披露的弧菌BT脂肽灭活口服疫苗使对虾弧菌疫苗在实用性方面前进了一大步，但是它的保护力仍显不足。鉴于BT脂肽本身就有调节非特异性免疫、促进动物健康和生长的作用，进行了以下组合比较试验(表4)，测试“BT脂肽灭活弧菌疫苗单剂接种+连续饲喂BT肽”组合方案的攻毒保护力。
本实施例中的疫苗制备和饲料负载、口服接种以及注射攻毒方法均与实施例3的相同。含200ppm BT脂肽的饲料是按1:7.5比例将1500ppm BT脂肽水溶液喷洒在饲料上搅拌混匀后，晾干而制成。在第1天和第21天测定各处理的幸存者体长增加和体重增加并且在第21天测定幸存者数量以计算相对存活率。
在攻毒后第10天的第21天，仅(用BT脂肽灭活疫苗)免疫但不在饲料中添加BT脂肽的处理组4的相对存活率为38％，结果与实施例3中的结果相近，即仅具部分保护力；仅在饲料中添加BT脂肽但不免疫的处理组3的相对存活率为40％，也仅具部分保护力；然而，既免疫也在饲料中添加BT脂肽的处理组5的相对存活率为99％，表现出了近乎完全的攻毒保护力。
表4：BT灭活疫苗与BT饲料添加剂组合比较试验数据
在同等的攻毒条件下，BT脂肽灭活疫苗口服免疫有生长促进的功能，免疫了的处理组4和5两组的幸存者体长增加和体重增加均大幅优于未免疫的处理组2和3。
在同等的免疫和攻毒条件下，连续饲喂BT脂肽有生长促进的作用，饲喂了200ppm BT脂肽饲料的处理组5的幸存者体长增加和体重增加优于未饲喂200ppm BT脂肽饲料的处理组4。
实施例5
实施例4中“BT脂肽灭活弧菌疫苗单剂接种+连续饲喂BT肽”组合方案在实验室弧菌攻毒试验中表现出显著促进对虾生长的作用。然而，组合方案的实用性在以下两个方面值得改进或探讨。首先，连续饲喂BT肽涉及使用成本问题；其次，在大田生产条件下，临床性弧菌病并不是在每次中都发生。因此，有必要试验“BT脂肽灭活弧菌疫苗单剂接种+脉冲饲喂BT肽”组合方案在没有临床性弧菌病发生时是否也有显著生长促进作用。
大田试验在宁波一家小棚土塘养虾场进行，将该场同批次、条件等同的37棚被分设为两个处理组。对照组(20棚)既不(用BT脂肽灭活疫苗)免疫也不使用BT肽，仅给予05号对虾配合饲料。实验组(17棚)在试验第1天饲 喂负载BT脂肽灭活疫苗(108死弧菌数/g)+BT肽200ppm的饲料；为节省BT肽的使用，仅在第8天饲喂负载BT肽200ppm的饲料，即不再连续使用BT肽。在第1、8和15天取虾(每棚10头)，测量体长，计算处理组平均值。试验期间没有发生临床性弧菌病。
表5：简化“BT脂肽灭活疫苗+BT肽组合方案”大田对虾生长试验数据
表5数据显示，实验组对虾体长在第8天就已明显超过对照组，实验组体长优势在随后的一周进一步扩大。这些大田试验结果表明，“BT脂肽灭活弧菌疫苗单剂接种+脉冲饲喂BT肽”组合方案显著促进对虾生长，无论是否有临床性弧菌病发生。
还在该大田中试进行另外的实验：将该场同批次、条件等同的31棚被分设为两个处理组。对照组(20棚)既不免疫也不使用BT肽，仅给予05号对虾配合饲料。实验组(11棚)在P31(仔虾第31天)、P45、P59、P73、P87和P101时对南美白对虾饲喂负载BT脂肽灭活疫苗(108死弧菌数/g)+BT肽200ppm的饲料；在P38、P52、P66、P80、P94和P108时饲喂负载BT肽200ppm的饲料，即不再连续使用BT肽。若存在弧菌病，会发生大面积虾死亡，则该小棚计为发生病害小棚。在收获时(大约P120)计算虾尾数。比较两组的成活率和产量。成功率＝处理组全程未发生病害小棚数目/处理组小棚总数，成活率＝各棚收获虾尾数/投苗尾数。
表6：“BT脂肽灭活疫苗+交替脉冲BT肽”组合方案对虾大田试验数据
表6数据显示，实验组所有指标均高于对照组，其中，产量差异极显著(P<0.01)，成活率差异显著(P<0.05)。大田中试结果表明，“BT脂肽灭活弧菌疫苗单剂接种+交替脉冲饲喂BT肽”组合方案显著提升对虾成活率和产量。
实施例6
虽然较实施例4方案有所简化，实施例5仍涉及两个制剂，可能在生产应用中因混淆而操作失误，因此有必要进一步的简化，尝试合并两个制剂为单一制剂。本实施例以表6所示的地点和虾场模式进行，其中“相对单产＝实验组单位面积产量/对照组单位面积产量。
试验的单一制剂包含BT脂肽灭活疫苗(109死弧菌数/g)+BT肽(2000ppm)，在P35、P45、P55和P65投喂负载BT脂肽灭活疫苗(108死弧菌数/g)+BT肽200ppm的饲料，每次的投喂量为当日量的1/4(作为实验组)。
对照组投喂饲料和蛭弧菌冻干粉(“猎弧英雄”，广州市顺州生物科技有限公司)或中草药配方功能性饲料(“虾佑康”，宁波三生生物科技有限公司)。蛭弧菌冻干粉和中草药配方功能性饲料投喂方式根据产品说明书进行。
在收获时(大约P120)，计算实验组的单位面积产量、对照组单位面积产量以及全程未发生病害小棚数目。
表7汇总了不同养殖生产模式的大田对比试验，结果显示使用上述单一制剂同样能有效提升生产指标。在这些大田试验中，对照组的规模至少是实验组的2倍。
表7:“BT脂肽灭活疫苗+BT肽”单一制剂方案对虾大田试验结果汇总

尽管已参考说明性实施例描述了本发明，但所属领域的技术人员将理解，在不背离本发明的精神及范围的情况下可做出各种其它改变、省略及/或添加且可用实质等效物替代所述实施例的元件。另外，可在不背离本发明的范围的情况下做出许多修改以使特定情形或材料适应本发明的教示。因此，本文并不打算将本发明限制于用于执行本发明的所揭示特定实施例，而是打算使本发明将包含归属于所附权利要求书的范围内的所有实施例。

Claims (10)

  1. 灭活口服疫苗，其包含质膜被透化剂透化而灭活的病原体或其活疫苗，其中灭活的病原体或其活疫苗包含经透化的质膜及其整合膜蛋白，其中病原体是包含质膜的病原体，并且透化剂包含脂肽作为表面活性剂；
    优选地，病原体选自下组：包膜病毒、细菌、真菌、体内寄生虫和癌细胞；
    优选地，疫苗还包含病原体或其活疫苗在透化后的外泄内容物；
    优选地，疫苗通过包括对病原体或其活疫苗进行质膜透化的步骤而灭活，使得活病原体数<101/ml和/或死病原体数>109/ml；
    优选地，灭活口服疫苗为口服液、粉剂、片剂、胶囊、灌服液或口腔推注液；
    优选地，灭活口服疫苗是通过直接口服、饮水负载、食物负荷、灌服或口腔推注施用的疫苗；
    优选地，透化剂不包含除脂肽外的表面活性剂。
  2. 根据权利要求1所述的灭活口服疫苗，其中脂肽包含天然脂肽和/或人工合成脂肽；
    优选地，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的BT脂肽；
    优选地，产自德克萨斯侧孢短芽孢杆菌的BT脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合；
    优选地，病原体选自下组：冠状病毒科、流感病毒、副粘病毒科、疱疹病毒科、痘病毒科、肝病毒科、杆状病毒属、反转录病毒科、非洲猪瘟病毒、不动杆菌属、杆菌属、疏螺旋体属、弯曲杆菌属、梭菌属、弧菌属、大肠杆菌、肠球菌属、奈瑟菌属、嗜血菌属、曼氏杆菌属、分枝杆菌属、支原体属、巴斯德氏菌属、假单胞菌属、沙门氏菌属、葡萄球菌属、链球菌属、球虫、线虫、绦虫和癌细胞；
    优选地，病原体选自下组：新城疫病毒和副溶血弧菌。
  3. 根据权利要求1或2所述的灭活口服疫苗，其包含药学可接受的载体，或者佐剂。
  4. 制备根据权利要求1-3中任一项所述的灭活口服疫苗的方法，其包括：
    对病原体或其活疫苗进行质膜透化而灭活，其中透化剂包含脂肽作为表 面活性剂；优选地，所述方法不包括分离抗原的步骤和/或人工包被抗原的步骤；
    优选地，当对病原体进行灭活时，方法不包括透化灭活外的其他灭活方式；
    优选地，透化剂不包含除脂肽外的表面活性剂；
    优选地，对病原体或其活疫苗进行质膜透化，使得活病原体数<101/ml和/或死病原体数>109/ml；
    优选地，脂肽包含天然脂肽和/或人工合成脂肽；
    优选地，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的BT脂肽；
    优选地，产自德克萨斯侧孢短芽孢杆菌的BT脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
  5. 口服组合物，其包含根据权利要求1-3中任一项所述的灭活口服疫苗；
    优选地，口服组合物包含食物、饮用水或饮料；
    优选地，食物选自下组：代乳品、生长饲料、肥育饲料、育雏前饲料和育雏饲料；
    优选地，组合物包含谷类、大豆粉、分离的大豆蛋白、大豆油、大豆脂肪、脱脂乳、鱼粉、肉粉、骨粉、血粉、血浆蛋白质、乳清、稻糠、麦麸、甜味剂、矿物、维生素和盐中的一种或多种；
    优选地，谷类选自下组：大麦、大豆、小麦、小黑麦、黑麦、玉米和稻米；
    优选地，口服组合物包含调节免疫或促进健康或生长的成分，优选BT脂肽，所述BT脂肽与灭活口服疫苗是混为一体的或分离的，例如产自德克萨斯侧孢短芽孢杆菌的BT脂肽，优选地，具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
  6. 透化剂用于制备根据权利要求1-3中任一项所述的灭活口服疫苗的用途，该用途通过将病原体或其活疫苗的质膜穿孔引起内容物外泄而灭活病原体或其活疫苗实现；其中透化剂包含脂肽作为表面活性剂；
    优选地，脂肽包含天然脂肽和/或人工合成脂肽；
    优选地，天然脂肽选自：表面活性素、芬枯草菌素、伊枯草菌素、达托霉素、多粘菌素、粘液菌素和产自德克萨斯侧孢短芽孢杆菌的脂肽；
    优选地，产自德克萨斯侧孢短芽孢杆菌的脂肽包含具有以SEQ ID NO:1-9中任一项所示的序列的脂肽或其任意组合。
  7. 根据权利要求1-3中任一项所述的灭活口服疫苗在制备药物中的用途，所述药物用于治疗和/或预防受试者中由病原体引起的感染或疾病。
  8. 根据权利要求7所述的用途，其中所述感染或疾病选自：流感、病毒性肝炎、非洲猪瘟、猪生殖与呼吸综合征、大肠杆菌病、沙门氏菌病、坏死性肠炎、弧菌病、牛航运热肺炎、淋病、尿路感染、球虫病、线虫病、绦虫病和癌症。
  9. 根据权利要求1-3中任一项所述的灭活口服疫苗或权利要求5的口服组合物在制备饲料中的用途，所述饲料用于改善受试者中的重量增加和/或食物转化。
  10. 根据权利要求8所述的用途，其中受试者是禽类、家畜、水生动物或伴侣动物；优选地，饲料是通过蛋内饲喂、以液滴形式喷洒于禽类或沉浸水生动物施加的饲料。
PCT/CN2023/125248 2022-10-25 2023-10-18 质膜透化灭活口服疫苗 WO2024088138A1 (zh)

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