WO1998040062A1 - Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination - Google Patents
Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination Download PDFInfo
- Publication number
- WO1998040062A1 WO1998040062A1 PCT/NL1998/000145 NL9800145W WO9840062A1 WO 1998040062 A1 WO1998040062 A1 WO 1998040062A1 NL 9800145 W NL9800145 W NL 9800145W WO 9840062 A1 WO9840062 A1 WO 9840062A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mucosal
- pharmaceutical composition
- liposomes
- antigen
- phagocytic cells
- Prior art date
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 53
- 239000000417 fungicide Substances 0.000 title claims description 8
- 239000002738 chelating agent Substances 0.000 title claims description 6
- 238000002255 vaccination Methods 0.000 title abstract description 32
- 230000000855 fungicidal effect Effects 0.000 title description 2
- 239000000427 antigen Substances 0.000 claims abstract description 30
- 108091007433 antigens Proteins 0.000 claims abstract description 30
- 102000036639 antigens Human genes 0.000 claims abstract description 30
- 241001465754 Metazoa Species 0.000 claims abstract description 25
- 229960005486 vaccine Drugs 0.000 claims abstract description 21
- 210000001539 phagocyte Anatomy 0.000 claims abstract description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 18
- 239000000126 substance Substances 0.000 claims description 18
- 239000008194 pharmaceutical composition Substances 0.000 claims description 11
- 239000000443 aerosol Substances 0.000 claims description 8
- 230000036039 immunity Effects 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 230000003612 virological effect Effects 0.000 claims description 6
- 241000282887 Suidae Species 0.000 claims description 3
- 244000144977 poultry Species 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 241000282472 Canis lupus familiaris Species 0.000 claims description 2
- 241000283086 Equidae Species 0.000 claims description 2
- 241000282326 Felis catus Species 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims description 2
- 230000003071 parasitic effect Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 241000711404 Avian avulavirus 1 Species 0.000 claims 1
- 239000003440 toxic substance Substances 0.000 abstract description 4
- 230000016379 mucosal immune response Effects 0.000 abstract description 3
- 231100000252 nontoxic Toxicity 0.000 abstract description 3
- 230000003000 nontoxic effect Effects 0.000 abstract description 3
- 210000001132 alveolar macrophage Anatomy 0.000 description 15
- 230000028993 immune response Effects 0.000 description 12
- 210000004072 lung Anatomy 0.000 description 12
- 210000004837 gut-associated lymphoid tissue Anatomy 0.000 description 8
- 210000002540 macrophage Anatomy 0.000 description 8
- 241000700605 Viruses Species 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 6
- 230000007123 defense Effects 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000008030 elimination Effects 0.000 description 6
- 238000003379 elimination reaction Methods 0.000 description 6
- 210000000987 immune system Anatomy 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 239000000470 constituent Substances 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002519 immonomodulatory effect Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 241000282412 Homo Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 230000001627 detrimental effect Effects 0.000 description 3
- 230000028996 humoral immune response Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 210000003097 mucus Anatomy 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 241000710777 Classical swine fever virus Species 0.000 description 2
- 241000711573 Coronaviridae Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000010359 Newcastle Disease Diseases 0.000 description 2
- 241000606856 Pasteurella multocida Species 0.000 description 2
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229940051027 pasteurella multocida Drugs 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 235000013594 poultry meat Nutrition 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 231100000167 toxic agent Toxicity 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 108010042685 trinitrophenyl keyhole limpet hemocyanin Proteins 0.000 description 2
- PFYHYHZGDNWFIF-UHFFFAOYSA-N (+)-DMDP Natural products OCC1NC(CO)C(O)C1O PFYHYHZGDNWFIF-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- PFYHYHZGDNWFIF-KVTDHHQDSA-N 2,5-bis(hydroxymethyl)-3,4-dihydroxypyrrolidine Chemical compound OC[C@H]1N[C@H](CO)[C@@H](O)[C@@H]1O PFYHYHZGDNWFIF-KVTDHHQDSA-N 0.000 description 1
- PFYHYHZGDNWFIF-OMMKOOBNSA-N 2R,5R-Dihydroxymethyl-3R,4R-dihydroxy-pyrrolidine Natural products OC[C@@H]1N[C@@H](CO)[C@H](O)[C@@H]1O PFYHYHZGDNWFIF-OMMKOOBNSA-N 0.000 description 1
- 241000606748 Actinobacillus pleuropneumoniae Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000588807 Bordetella Species 0.000 description 1
- 241000725585 Chicken anemia virus Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- WMTIOGUVKBSEOW-UHFFFAOYSA-N ClC1(Cl)OP(=O)OP(=O)O1 Chemical compound ClC1(Cl)OP(=O)OP(=O)O1 WMTIOGUVKBSEOW-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000223924 Eimeria Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000701063 Gallid alphaherpesvirus 1 Species 0.000 description 1
- 241000701047 Gallid alphaherpesvirus 2 Species 0.000 description 1
- 241000711450 Infectious bronchitis virus Species 0.000 description 1
- 241000702626 Infectious bursal disease virus Species 0.000 description 1
- 101710125507 Integrase/recombinase Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000000779 depleting effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000011221 initial treatment Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- -1 or adhesian factors Substances 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
Definitions
- the invention relates to the field of mucosal immunity and to vaccination methods to specifically generate mucosal immunity .
- the immune system in general serves to protect animals, including man, against disease caused by invading foreign substances by actively mounting a cellular and humoral immune response directed against antigens present on or in the foreign substances. In this way, invading micro-organisms, pathogens, toxins, or other substances are rendered harmless to the animal .
- Vaccination makes use of the immune system; vaccines can be used to effectively generate a specific and selective immune response that prevents disease caused by the pathogens against which the animal is vaccinated.
- vaccines need to be administered parenterally, i.e. via injection, to be effective; vaccines that comprise replicating microorganisms can also effectively be administered at mucosal surfaces, e.g. orally, nasally or possibly via aerosols.
- the immune system of the mucosae is distinct from the general immune system that is present in most other parts of the body.
- the mucosae are, with the skin, the parts of the body where the most frequent contact with foreign substances is found. Inhalation of particles in the air and ingestion of food and other substances generates a constant load of antigens on the mucosae of lung and gut. An active immune response generated against these day-to-day recurring antigens would be very detrimental to the mucosae; therefore in the mucosae a different type of immune response can be found. Instead of every time mounting an active cellular and humoral immune response, the mucosae respond in a more tolerant and even immunosuppressive way and act mainly via thorough removal of foreign substances before an active humoral or cellular response to those substances can be initiated .
- the main line of defense of the mucosae is constituted by a layer of mucus covering the underlying epithelial cells.
- enzymes such as lysozyme can be found that enzy atically degrade possible harmful substances.
- phagocytic cells such as macrophages, are present, to phagocytize particles and micro-organisms. In this way, most antigens are effectively removed from the mucosal surface before they can invade the body further and no specific immune response will be generated.
- BALT bronchus-associated lymphoid tissue
- GALT gut-associated lymphoid tissue
- Both BALT and GALT are capable of mounting humoral and cellular immune responses.
- both BALT and GALT typically react with the formation of IgA antibodies which are excreted and transported to the mucosal surface where they can constitute a specific component of the otherwise mainly non-specific immune response of the mucosae.
- IgA recognizes and binds to the specific antigens present which are then earlier recognized and removed by phagocytizing cells.
- BALT and GALT migration and exchange of IgA-B-cells and IgA-memory cells assures a distribution of specific IgA throughout the mucosae.
- an IgA immune response generated in the lung also results in the same IgA immune response in the gut and other mucosae (Sminia et al . , Adv . Exp . Med . Biol. 216:981, 1987), facilitating antigen recognition throughout all mucosal surfaces .
- macrophages that return from the mucosal surface into the interstitium where BALT and GALT are located, is again detrimental for eliciting a specific immune response .
- These macrophages suppress the activities of the antigen presenting cells and the formation of specific T- cells present in the mucosal lymphoid system.
- the present invention provides a solution for the above disadvantages while it still is possible to achieve effective mucosal vaccination without having to resort to live-vaccines.
- the present invention thus provides effective mucosal vaccines.
- the invention firstly provides liposomes that do not contain the toxic substance CI2MDP but that instead contain or comprise a non-toxic substance that can eliminate phagocytic cells, such as macrophages, present on the mucosal surfaces of an animal.
- liposomes containing chelating agents such as ethylenediaminetetraacetic acid (EDTA) when incorporated in liposomes, are functionally analogous to liposomes containing CI2MDP without having the toxic side effects .
- EDTA ethylenediaminetetraacetic acid
- fungicides, and small peptides such as secropines can replace CI2MDP.
- Intratracheal application of the liposomes according to the invention effectively results in depletion or elimination of alveolar macrophages, and other phagocytic cells present on the mucosal surfaces of the respiratory tract of an animal .
- the invention further provides a vaccination proto- col in which elimination of alveolar macrophages and the immunization with an antigen take place in one simultaneous event. It was however found (see the experimental part) that the obvious solution comprising vaccination or immunization with a mixture of liposomes and antigen did not result in effective mucosal vaccination. Surprisingly, however, applying liposomes comprising the antigen was very successful in generating an effective mucosal immune response and thus mucosal vaccination.
- the invention thus also provides liposomes that comprise antigen and that can be used to simultaneously eliminate alveolar macrophages and immunize or vaccinate the mucosae against a specific antigen of choice.
- antigen-comprising liposomes according to the invention can be successfully performed against both respiratory as well as enteric disease and against diseases wherein the pathogen has its pathway-of-entry via the mucosae such as lung or gut.
- antigen a wide variety of substances can be selected. Good examples are viral antigens, comprising viral structural or non structural proteins with antigenic properties, such as the E2 protein of classical swine fever virus, gE of pseudorabies virus, SI of Corona viruses.
- bacterial or protozoal or parasitic antigens comprising toxins, or adhesian factors, or constituents of the cell membrane tnat elicit an immune response in a host.
- Mucosal vaccination via food or drinking water or via the aerosal route is widely applicable.
- small animals such as dogs and cats and other pets
- animal friendly treatments are greatly appreciated by the client.
- large animals such as farm animals, such as cows, horses, pigs and poultry, the ease of application of mucosal vaccines according to the invention allows for fast applications on a wide scale.
- Newcastle Disease infectious bronchitis virus
- laryngotracheitis virus infectious bursal disease virus
- tenosynovitisreovirus malabsortion syndrome virus
- Marek ' s disease virus chicken anemia virus, Escherichia coli, Salmonella spp, Pasteurella multocida, or Eimeria spp.
- influenza virus In pigs, influenza virus, classical swine fever virus, pseudorabies virus, Estavirus, Corona virus, Escherichia coli, actinobacillus pleuropneumonia,
- Bordetella bronchisentia, or Pasteurella multocida are, among others, pathogens against which can be vaccinated with the liposomes provided by the invention.
- aerosol vaccination In humans, aerosol vaccination is not widely applied. However, parenteral vaccination is often uncomfortable to the patient to be treated. Especially aerosol and/or oral vaccination of infants and children will be positively received.
- Good examples are viral diseases such as poliomyelitis, rubella, influenza respiratory syncitial virus and various other viral and bacterial diseases .
- Vaccines can be prepared according to the invention by preparing a pharmaceutical composition of a liposome provided by the invention and a suitable carrier and/or stabilizer .
- the local destruction of alveolar macrophages by the liposomes creates enough local penetration of the mucosal surfaces by the antigen provided by the same liposomes that an effective immune response can follow. Therefore, immunization or vaccination of humans or animals with the liposomes according to the invention can be used to obtain an effective mucosal vaccination, without hampering the majority of the alveolar macrophages and thus safeguarding a solid protection of the alveolar mucosae and thus the lung.
- Liposome preparation specifically for elimination phagocytic cells is discussed in Cell Tissue Res (1984) 238:355-3.
- Uni-and multilamellar forms of liposomes exist and size and other characteristics of liposomes depends on the constituents (such as cholesterol, sphingomyeline or other naturally occurring or synthetic phospholipids of the liposomal membranes and the ratios in which these constituents are being used.
- a specific protocol for liposomes containing EDTA and viral antigen is provided herewith, however, this protocol can in no way be seen as limiting the invention.
- the liposomes can be stored or used immediately. Application of immunomodulating liposomes in mucosal vaccination.
- mice Five groups of 4 BALB/C mice each were treated intratracheally with 0.1 ml liposomes prepared as above with 90% phosphadityl choline and 10% cholesterol and containing, respectively, PBS (control), or 0.6 M
- CI2MDP or 0.2, 0.4, or 0.5 M EDTA (experimental groups). After two days the number of macrophages per lung were counted. In the experimental groups, the percentages of alveolar macrophages found were reduced to approximately 20, 40, 30, and 35% of the number in the controls, showing that EDTA can replace the toxic CI2MDP.
- Figure 1 Mean antibody titer directed against NCD in four groups of chickens vaccinated against NCD in preparations of liposomes containing NCD (1), and additionally, PBS (2) CI2MDP (3) or EDTA (4)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention provides a solution to achieve effective mucosal vaccination without having to resort to live-vaccines. The invention provides liposomes that contain a non-toxic substance that can eliminate phagocytic cells present on the mucosal surfaces of an animal. The invention further provides said liposomes containing antigen, whereby the antigen can provide the effective mucosal immune response needed.
Description
USE OF LIPOSOMES CONTAINING A CHELAΗNG AGENT, A FUNGICIDE OR A SECROPINE FOR IMPROVING MUCOSAL VACCINATION
The invention relates to the field of mucosal immunity and to vaccination methods to specifically generate mucosal immunity .
The immune system in general serves to protect animals, including man, against disease caused by invading foreign substances by actively mounting a cellular and humoral immune response directed against antigens present on or in the foreign substances. In this way, invading micro-organisms, pathogens, toxins, or other substances are rendered harmless to the animal . Vaccination makes use of the immune system; vaccines can be used to effectively generate a specific and selective immune response that prevents disease caused by the pathogens against which the animal is vaccinated. In general, vaccines need to be administered parenterally, i.e. via injection, to be effective; vaccines that comprise replicating microorganisms can also effectively be administered at mucosal surfaces, e.g. orally, nasally or possibly via aerosols.
The immune system of the mucosae, as can for example be found in lung and gut but also in nose, mouth and eyes, is distinct from the general immune system that is present in most other parts of the body. The mucosae are, with the skin, the parts of the body where the most frequent contact with foreign substances is found. Inhalation of particles in the air and ingestion of food and other substances generates a constant load of antigens on the mucosae of lung and gut. An active immune response generated against these day-to-day recurring antigens would be very detrimental to the mucosae; therefore in the mucosae a different type of immune response can be found. Instead of every time mounting an active cellular and humoral immune response, the mucosae respond in a more tolerant and even immunosuppressive way and act mainly via thorough removal of foreign substances before an active
humoral or cellular response to those substances can be initiated .
The main line of defense of the mucosae is constituted by a layer of mucus covering the underlying epithelial cells. In the mucus, enzymes such as lysozyme can be found that enzy atically degrade possible harmful substances. Furthermore, phagocytic cells, such as macrophages, are present, to phagocytize particles and micro-organisms. In this way, most antigens are effectively removed from the mucosal surface before they can invade the body further and no specific immune response will be generated.
In addition, a common mucosal immune system can be found in close proximity with the mucosae. In the lung it is called the bronchus-associated lymphoid tissue (BALT), in the gut the gut-associated lymphoid tissue (GALT) . Both the BALT and GALT are capable of mounting humoral and cellular immune responses. However, contrary to the general immune system, where mainly IgG and IgM antibodies are formed, both BALT and GALT typically react with the formation of IgA antibodies which are excreted and transported to the mucosal surface where they can constitute a specific component of the otherwise mainly non-specific immune response of the mucosae. IgA recognizes and binds to the specific antigens present which are then earlier recognized and removed by phagocytizing cells. In and inbetween BALT and GALT, migration and exchange of IgA-B-cells and IgA-memory cells assures a distribution of specific IgA throughout the mucosae. In that way, an IgA immune response generated in the lung also results in the same IgA immune response in the gut and other mucosae (Sminia et al . , Adv . Exp . Med . Biol. 216:981, 1987), facilitating antigen recognition throughout all mucosal surfaces . The presence of macrophages, however, that return from the mucosal surface into the interstitium where BALT and GALT are located, is again detrimental for eliciting a specific immune response . These macrophages suppress the activities of the
antigen presenting cells and the formation of specific T- cells present in the mucosal lymphoid system.
In the light of the above, it is explicable that vaccination via the mucosal surfaces is generally not successful . Most vaccine antigens that for instance may be applied or administered via aerosol, intratracheally or orally to mucosal surfaces such as conjunctivae of the eyes, mouth and nose, and gut and lung will be removed in the layer of mucus by phagocytosis or enzymatic degradation. In addition, particle size limits and restricts in general the distribution of the antigen to the upper respiratory tract. Only live-vaccines, comprising live micro-organisms, that can replicate in or on the mucosae may generate enough antigenic load to induce the BALT or GALT to specifically mount an immune response. In addition, although most infectious diseases are caused by micro-organisms that invade the body via the mucosal surface, most of the time it is not possible or wanted to vaccinate with live-vaccines due to the possible virulence of such vaccines. Furthermore, vaccination via the parenteral route does not result in the formation of specific IgA- forming cells and thus does not result in specific mucosal immunity.
It would therefore be very beneficial to be able to vaccinate specifically with the purpose of mounting an effective mucosal immune response without having to resort to a live-vaccine. In order to achieve this mucosal vaccination, one would have to evade the main line of defense of the mucosae and design a vaccination protocol by which the vaccine antigens reach BALT or GALT without having been phagocytized or enzymatically degraded. One particular suitable way of evading the main line of defense of the mucosae can be achieved by temporarily destructing (Rooijen and Nieuwmegen, Cell Tissue Research 236: 355, 1984) the macrophage population that can be found on the mucosal surface. Thepen et al . (J. Exp. Med. 170: 499-509, 1989) have shown that elimination of the
resident population of alveolar macrophages in the lungs of mice could be achieved via the aerosol application of liposomes containing the toxic dichloromethylene diphosphonate (CI2MDP or DMDP) . The alveolar macrophages were effectively eliminated in vivo by the effect of CI2MDP within 24 hours after administration of the liposomes. Subsequent immunisation with TNP-KLH via intratracheal vaccination resulted in a broad immune response, IgG as well as IgA antibodies directed against TNP-KLH were detected, indicating that the main line of defense of the mucosae had successfully been evaded and mucosal immunity had been established. Furthermore, the population of alveolar macrophages was replenished within 10-14 days after the initial treatment, thereby restoring the main line of defense of the alveolar mucosae.
The above experiment showed that mucosal vaccination protocols can be designed that result in effective mucosal vaccination, even without using live-vaccines. However, the above protocol has some serious disadvantages that make a practical application not really feasible.
First of all, using CI2MDP as toxic substance in the liposomes to eliminate the alveolar macrophages is not feasible in vaccines for human or animal use due to side effects caused by the inherent toxicicity of the substance due to the presence of chloride ions.
Secondly, the two-throng approach, as illustrated above, in which the macrophages are eliminated in a first step via intratracheal application, and the animal is subsequently immunized in a second step, again via intratracheal application is cumbersome. It would require that humans or animals need to be handled, treated and possibly caught twice to obtain one vaccination.
Thirdly, a total depletion or elimination of the alveolar macrophages is, albeit instrumental in obtaining a effective mucosal vaccination, in itself very detrimental for maintaining a solid protection of the alveolar mucosae and thus the lung. Any pathogen that
normally would be removed by the phagocytic activities of the macrophages could now cause disease in the lung because the first line of defense is seriously maimed by the lack of alveolar macrophages. Since it takes 10-14 days to replenish the population of alveolar macrophages, this would leave the treated human or animal unprotected against possible pathogens invading via the alveolar mucosa.
The present invention provides a solution for the above disadvantages while it still is possible to achieve effective mucosal vaccination without having to resort to live-vaccines. The present invention thus provides effective mucosal vaccines.
The invention firstly provides liposomes that do not contain the toxic substance CI2MDP but that instead contain or comprise a non-toxic substance that can eliminate phagocytic cells, such as macrophages, present on the mucosal surfaces of an animal. Surprisingly, it was found that liposomes containing chelating agents such as ethylenediaminetetraacetic acid (EDTA) when incorporated in liposomes, are functionally analogous to liposomes containing CI2MDP without having the toxic side effects . Furthermore fungicides, and small peptides such as secropines can replace CI2MDP. Intratracheal application of the liposomes according to the invention effectively results in depletion or elimination of alveolar macrophages, and other phagocytic cells present on the mucosal surfaces of the respiratory tract of an animal .
The invention further provides a vaccination proto- col in which elimination of alveolar macrophages and the immunization with an antigen take place in one simultaneous event. It was however found (see the experimental part) that the obvious solution comprising vaccination or immunization with a mixture of liposomes and antigen did not result in effective mucosal vaccination. Surprisingly, however, applying liposomes comprising the antigen was very successful in generating
an effective mucosal immune response and thus mucosal vaccination. The invention thus also provides liposomes that comprise antigen and that can be used to simultaneously eliminate alveolar macrophages and immunize or vaccinate the mucosae against a specific antigen of choice.
Since both lung and gut immunity is achieved via the vaccination methods provided by the invention, vaccination using antigen-comprising liposomes according to the invention can be successfully performed against both respiratory as well as enteric disease and against diseases wherein the pathogen has its pathway-of-entry via the mucosae such as lung or gut. As antigen, a wide variety of substances can be selected. Good examples are viral antigens, comprising viral structural or non structural proteins with antigenic properties, such as the E2 protein of classical swine fever virus, gE of pseudorabies virus, SI of Corona viruses. Other good examples can be found with bacterial or protozoal or parasitic antigens, comprising toxins, or adhesian factors, or constituents of the cell membrane tnat elicit an immune response in a host. Mucosal vaccination via food or drinking water or via the aerosal route is widely applicable. In small animals, such as dogs and cats and other pets, animal friendly treatments are greatly appreciated by the client. In large animals, such as farm animals, such as cows, horses, pigs and poultry, the ease of application of mucosal vaccines according to the invention allows for fast applications on a wide scale. In poultry where aerosol vaccination with modified live vaccines is widely applied, one can for example think of vaccination against Newcastle Disease, infectious bronchitis virus, laryngotracheitis virus, infectious bursal disease virus, tenosynovitisreovirus, malabsortion syndrome virus , Marek ' s disease virus , chicken anemia
virus, Escherichia coli, Salmonella spp, Pasteurella multocida, or Eimeria spp.
In pigs, influenza virus, classical swine fever virus, pseudorabies virus, Estavirus, Corona virus, Escherichia coli, actinobacillus pleuropneumonia,
Bordetella bronchisentia, or Pasteurella multocida are, among others, pathogens against which can be vaccinated with the liposomes provided by the invention.
In humans, aerosol vaccination is not widely applied. However, parenteral vaccination is often uncomfortable to the patient to be treated. Especially aerosol and/or oral vaccination of infants and children will be positively received. Good examples are viral diseases such as poliomyelitis, rubella, influenza respiratory syncitial virus and various other viral and bacterial diseases .
Vaccines can be prepared according to the invention by preparing a pharmaceutical composition of a liposome provided by the invention and a suitable carrier and/or stabilizer .
Furthermore, it was found that, when using the liposomes provided by the invention, depletion or elimination of only a portion of the alveolar macrophages was needed to induce successful mucosal vaccination.
Possibly, the local destruction of alveolar macrophages by the liposomes creates enough local penetration of the mucosal surfaces by the antigen provided by the same liposomes that an effective immune response can follow. Therefore, immunization or vaccination of humans or animals with the liposomes according to the invention can be used to obtain an effective mucosal vaccination, without hampering the majority of the alveolar macrophages and thus safeguarding a solid protection of the alveolar mucosae and thus the lung.
Experimental part
Preparation of immunomodulating liposomes. Standard techniques to prepare liposomes are well known. Liposome preparation specifically for elimination phagocytic cells is discussed in Cell Tissue Res (1984) 238:355-3. Uni-and multilamellar forms of liposomes exist and size and other characteristics of liposomes depends on the constituents (such as cholesterol, sphingomyeline or other naturally occurring or synthetic phospholipids of the liposomal membranes and the ratios in which these constituents are being used. A specific protocol for liposomes containing EDTA and viral antigen is provided herewith, however, this protocol can in no way be seen as limiting the invention. Changing ratios of constituents or the constituents themselves, or replacing EDTA with another substance, for instance fungicides or peptides, or changing the antigen or antigen concentration or changing the methods by wich the liposomes are prepared will generate specific liposome preparations that will be suitable for specific applications.
1) 10 ml chloroform containing 86 mg egg phosphatidyl choline and 8 mg cholesterol is transferred to a 500 ml round bottom flask
2) The chloroform is removed by low vacuum rotation using a film evaporator at 37 C.
3) The lipid film is hydrated with 4 ml of 0.4 M EDTA pH 7.4 containing the wanted concentration of antigen by rotation.
4) The suspension is sonicated
5) The liposomes can be stored or used immediately.
Application of immunomodulating liposomes in mucosal vaccination.
A) The influence of intratracheal application of liposomes containing the toxic CI2MDP or the non-toxic
EDTA on the number of alveolar macrophages present after application. Five groups of 4 BALB/C mice each were treated intratracheally with 0.1 ml liposomes prepared as above with 90% phosphadityl choline and 10% cholesterol and containing, respectively, PBS (control), or 0.6 M
CI2MDP, or 0.2, 0.4, or 0.5 M EDTA (experimental groups). After two days the number of macrophages per lung were counted. In the experimental groups, the percentages of alveolar macrophages found were reduced to approximately 20, 40, 30, and 35% of the number in the controls, showing that EDTA can replace the toxic CI2MDP.
B) Vaccination of chickens against New Castle Disease with CI2MDP containing liposomes. Four groups of 10 chickens were either left inoculated (1, controls) or vaccinated intratracheally with inactivated NCD virus (2), liposomes containing inactivated NCD virus (3), or with CI2MDP liposomes containing inactivated NCD virus (4). At two weeks after treatment the animals were bled and the antibody titres against NCD were determined; at four weeks after treatment, the animals were infected with virulent NCD challenge virus . The controls did not develop antibody and died all after challenge, whereas the animals in groups 2 and 3 developed mean antibody titres of 1:5, and in each group 1 animal died after challenge. In group 4, the animals developed mean antibody titres of 1:8 and none of the animals died after challenge. In conclusion, the animals treated with CI2MDP and NCD containing liposomes were the best protected against disease.
C) In a similar experiment as B) , the immunomodulating effect of EDTA liposomes was compared
with that of CI2MDP containing liposomes. Groups of 5 chickens were vaccinated intratracheally with inactivated NDV that was either mixed with or contained in CI2MDP liposomes, EDTA liposomes, liposomes without depleting agent, or no liposomes, or were left unvaccinated. The antibody profiles were determined during 10 consecutive weeks . Mixing the antigen with the liposomes did not result in a discernible immune response, however, the two groups vaccinated against NCD with the preparations containing CI2MDP or EDTA liposomes had high levels of antibody against NCD, when compared to the other experimental groups or the control groups, showing that the beneficial immunomodulating effect of EDTA liposomes is comparable to that of CI2MDP liposomes. (see Figure 1)
Figure legend
Figure 1 Mean antibody titer directed against NCD in four groups of chickens vaccinated against NCD in preparations of liposomes containing NCD (1), and additionally, PBS (2) CI2MDP (3) or EDTA (4)
Claims
1. A method for eliminating phagocytic cells present on mucosal surfaces of an animal comprising administering to a mucosal surface a liposome containing a substance selected from the group of chelating agents, fungicidal agents and secropines .
2. A method for eliminating phagocytic cells present on mucosal surfaces of an animal comprising administering to a mucosal surface a liposome containing EDTA.
3. A pharmaceutical composition comprising liposomes comprising an antigen and comprising a substance that can eliminate phagocytic cells present on mucosal surfaces.
4. A pharmaceutical composition according to claim 3 wherein the antigen is selected from any of the group of viral, bacterial or parasitic antigens.
5. A pharmaceutical composition according to claims 3 or 4 wherein the substance that can eliminate phagocytic cells is selected from the group of chelating agents, or fungicides or secropines .
6. A pharmaceutical composition according to any of claims 3 to 5 wherein the substance that can eliminate phagocytic cells is EDTA.
7. A pharmaceutical composition according to any of claims 3 to 6 wherein the antigen is inactivated New Castle Disease virus .
8. A method to induce mucosal immunity comprising mucosal administration of a pharmaceutical composition according to any of claims 3 to 7.
9. A method to induce mucosal immunity comprising oral or aerosol application of a pharmaceutical composition according to any of claims 3 to 7.
10. A vaccine comprising a pharmaceutical composition according to any of claims 3 to 7.
11. A mucosal vaccine according to claim 10 for oral or aerosol application.
12. A vaccine according to claim 10 or 11 for application in animals such as dogs, cats, cows, horses, pigs or poultry .
13. A vaccine according to claim 10 or 11 for application in adult humans or in children or infants .
14. A liposome containing an antigen and a substance that can eliminate phagocytic cells present on mucosal surfaces .
15. Use of a substance selected from the group consisting of chelating agents, fungicidal agents and secropines, in particular EDTA, in the manufacture of a pharmaceutical composition for eliciting mucosal immunity.
16. Use of a substance selected from the group consisting of chelating agents, fungicidal agents and secropines, in particular EDTA, in the manufacture of a pharmaceutical composition for eliminating phagocytic cells present on mucosal surfaces.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU64241/98A AU6424198A (en) | 1998-03-11 | 1998-03-11 | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination |
| PCT/NL1998/000145 WO1998040062A1 (en) | 1997-03-11 | 1998-03-11 | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination |
| EP98909873A EP0969826A1 (en) | 1997-03-11 | 1998-03-11 | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination |
| CA002283863A CA2283863A1 (en) | 1997-03-11 | 1998-03-11 | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP97200729.8 | 1997-03-11 | ||
| EP97200729 | 1997-03-11 | ||
| PCT/NL1998/000145 WO1998040062A1 (en) | 1997-03-11 | 1998-03-11 | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1998040062A1 true WO1998040062A1 (en) | 1998-09-17 |
Family
ID=26146231
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/NL1998/000145 WO1998040062A1 (en) | 1997-03-11 | 1998-03-11 | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP0969826A1 (en) |
| WO (1) | WO1998040062A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106074403A (en) * | 2016-07-28 | 2016-11-09 | 浙江美保龙生物技术有限公司 | A kind of Pseudorabies virus liposome dilution freeze-dried products and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992003162A1 (en) * | 1990-08-24 | 1992-03-05 | The Wellcome Foundation Limited | Vaccines |
| WO1992020370A1 (en) * | 1991-05-13 | 1992-11-26 | Regents Of The University Of California | Liposomal polysaccharide vaccines |
| US5422120A (en) * | 1988-05-30 | 1995-06-06 | Depotech Corporation | Heterovesicular liposomes |
-
1998
- 1998-03-11 EP EP98909873A patent/EP0969826A1/en not_active Withdrawn
- 1998-03-11 WO PCT/NL1998/000145 patent/WO1998040062A1/en not_active Application Discontinuation
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5422120A (en) * | 1988-05-30 | 1995-06-06 | Depotech Corporation | Heterovesicular liposomes |
| WO1992003162A1 (en) * | 1990-08-24 | 1992-03-05 | The Wellcome Foundation Limited | Vaccines |
| WO1992020370A1 (en) * | 1991-05-13 | 1992-11-26 | Regents Of The University Of California | Liposomal polysaccharide vaccines |
Non-Patent Citations (4)
| Title |
|---|
| GOULD-FOGERITE S. ET AL: "Lipid matrix-based subunit vaccines: A structure-function approach to oral and parenteral immunization", AIDS RES. HUM. RETROVIRUSES, 1994, 10/SUPPL. 2 (S99-S103), USA, XP002037265 * |
| GREGORIADIS, G.: "Immunological adjuvants: a role for liposomes", IMMUNOLOGY TODAY, vol. 11, no. 3, 1990, pages 89 - 97, XP000102991 * |
| THEPEN, T. ET AL: "Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 170, no. 2, 1989, pages 499-509, XP002037267 * |
| VAN ROOIJEN N ET AL: "Efficacy of various water-soluble chelator molecules in the liposome-mediated macrophage "suicide" technique.", J PHARMACOL TOXICOL METHODS, DEC 1992, 28 (4) P217-21, UNITED STATES, XP002037266 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106074403A (en) * | 2016-07-28 | 2016-11-09 | 浙江美保龙生物技术有限公司 | A kind of Pseudorabies virus liposome dilution freeze-dried products and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0969826A1 (en) | 2000-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2201598C (en) | Vaccine compositions | |
| US7052701B2 (en) | Inactivated influenza virus vaccine for nasal or oral application | |
| US6136606A (en) | Influenza vaccine compositions | |
| EP2429581B1 (en) | Enhanced immune response in avian species | |
| JPH10500113A (en) | Improved modified live BRSV vaccine | |
| IE48402B1 (en) | Pasteurellosis vaccines | |
| HRP20040282A2 (en) | Interleukin-12 as a veterinary vaccine adjuvant | |
| WO1996011707A1 (en) | Renibacterium salmoninarum vaccine and method for its preparation | |
| CA1335959C (en) | Newcastle disease virus vaccine and method for the application thereof | |
| WO1998040062A1 (en) | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination | |
| CA2283863A1 (en) | Use of liposomes containing a chelating agent, a fungicide or a secropine for improving mucosal vaccination | |
| US5456914A (en) | Transthoracic intrapulmonary immunization against Actinobacillus pleuropneumoniae | |
| WO2020067302A1 (en) | Mucosal adjuvant | |
| EP0012718B1 (en) | Intra-respiratory vaccine, modified bacteria strain used in it, vaccine dose form and process for preparing it | |
| HU203674B (en) | Process for producing weakened turtle rhinotracheitis virus and vaccine containing them | |
| JP2005509598A (en) | How to produce antibodies ex vivo | |
| Degré et al. | Pathogenesis of sendai virus infection in mice. On the possible role of interferon on the development of disease | |
| WO1993024147A1 (en) | Lecithin adjuvanted modified live virus vaccines | |
| De Haan et al. | Liposomes and antiviral mucosal immunity | |
| UA125017C2 (en) | Liposomal adjuvant compositions | |
| WO2024088138A1 (en) | Plasma membrane-permeabilized inactivated oral vaccine | |
| Chinnah | Evaluation of the antiviral, adjuvant and immunomodulatory effects of a beta-(1, 4)-linked polymannose (acemannan) | |
| KR20220041142A (en) | Composition for mucosal administration to birds | |
| AU754675B2 (en) | Influenza vaccine compositions | |
| JPH0725694B2 (en) | Method of preventing cattle-type respiratory disease complications in domestic animals by interferon administration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
| AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG |
|
| DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
| ENP | Entry into the national phase |
Ref document number: 2283863 Country of ref document: CA Ref country code: CA Ref document number: 2283863 Kind code of ref document: A Format of ref document f/p: F |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 1998909873 Country of ref document: EP |
|
| WWP | Wipo information: published in national office |
Ref document number: 1998909873 Country of ref document: EP |
|
| REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 09381121 Country of ref document: US |
|
| NENP | Non-entry into the national phase |
Ref country code: JP Ref document number: 1998539473 Format of ref document f/p: F |
|
| WWW | Wipo information: withdrawn in national office |
Ref document number: 1998909873 Country of ref document: EP |