USE OF LIPOSOMES CONTAINING A CHELATING AGENT, A FUNGICIDE OR A SECROPINE FOR IMPROVING MUCOSAL VACCINATION

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: 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.

Inventors: CLAASSEN HENRICUS JOHANNES HER, JEURISSEN SUSANNE HENRIETTE MA, KRAAL GEORG, CLAASSEN, HENRICUS, JOHANNES, HERMANUS, MARIA, JEURISSEN, SUSANNE, HENRIETTE, MARIA, KRAAL, GEORG
Applicants: CLAASSEN HENRICUS JOHANNES HER, JEURISSEN SUSANNE HENRIETTE MA, KRAAL GEORG, STICHTING INST DIERHOUDERIJ, STICHTING TECH WETENSCHAPP, TNO, VRIJE UNIVERSITEIT AMSTERDAM, CLAASSEN, HENRICUS, JOHANNES, HERMANUS, MARIA, JEURISSEN, SUSANNE, HENRIETTE, MARIA, KRAAL, GEORG, NEDERLANDSE ORGANISATIE VOOR TOEGEPASTNATUURWETENSCHAPPELIJK ONDERZOEK TNO, STICHTING INSTITUUT VOOR DIERHOUDERIJ EN DIERGEZONDHEID, STICHTING VOOR DE TECHNISCHE WETENSCHAPPEN
Application year: 1998
Main classification: A61K31
IPC codes: A61K31