Patent Application
20100047329
The present invention relates to novel vaccine carriers using liposomes having a fusogenic lipid membrane.
The immune system of animals has the function of differentiating between self and non-self and eliminating the non-self from the body. The immunological reaction in a living body that is responsible for such discrimination between self and non-self is realized by cellular immunity that utilizes MHC class I molecules or by humoral immunity that utilizes MHC class II molecules. For instance, if a living body is infected with a virus or bacterium as an infectious pathogen, it tries to eliminate such infectious pathogens by using the above-mentioned cellular immunity or humoral immunity.
Vaccination is frequently utilized as a means for preventing those infectious pathogens. In the case of humans, as well as domesticated animals and companion animals, a great variety of vaccines are utilized.
Those vaccines are roughly divided into two types, inactivated vaccines (including toxoid vaccine) and attenuated vaccines. Inactivated vaccines are such vaccines that a pathogen or toxoid is treated with formalin or other chemicals to become noninfectious and then administered in an inactivated state or they are such vaccines that only the antigen portion of the pathogen is administered; examples of this type of vaccines that can be administered to humans include triple vaccines (DPT vaccine; diphtheria pertussis tetanus vaccine), Japanese encephalitis vaccine, influenza vaccine, tetanus toxoid vaccine, etc. Attenuated vaccines, on the other hand, are those vaccines which are administered in the form of pathogens that are weakly virulent but do have infectivity, as exemplified by naturally occurring attenuated strains or artificially created strains of attenuated variants; examples of this type of vaccines that can be administered to humans include BCG vaccine, poliomyelitis vaccine, measles vaccine, rubella vaccine, mumps vaccine, varicella vaccine (chickenpox vaccine), etc.
Inactivated vaccines have the advantage of being less likely to cause pathogen-mediated infection and side effects as the result of their administration but, on the other hand, they are characterized by the ability to acquire only humoral immunity. Attenuated vaccines, on the other hand, use live pathogens, so they have the disadvantage that their virulence might for some reason be restored to develop side effects. What is more, except in the case of some attenuated vaccines (such as poliomyelitis vaccine) that are to be administered orally, vaccines are generally administered by intramuscular injection, so they have additional problems in that the antibody titers of IgG antibodies may increase but those of antibodies in other classes (such as IgA and IgM) will not and that they cannot fully induce the cellular immune response which is important for protection against infection.
Infection with infectious pathogens starts with those pathogens invading the body from mucosal surfaces as in the nasal, tracheal, intestinal and ocular mucosa, so if cellular immunity can be induced by vaccination, the invasion of pathogens into the body can be halted at the border. Although the mucosal membranes in the living body cover the surfaces of tract lumens such as oral cavity, nasal cavity, digestive tract and reproductive organs, as well as the mucosal surfaces of the eyes, what is constantly functioning on those surfaces is mucosal immunity that mainly involves secretory IgA and mucosa-associated lymph tissues against pathogenic microorganisms (e.g. viruses and bacteria), dietary antigens, and non-self foreign substance to which the mucosal membranes are constantly exposed. The mucosal immunity halts the invasion of those non-self foreign substances into the body by exhibiting diverse actions such as suppression of incorporation of protein antigens from the mucosal surfaces, inhibition of the adsorption of bacteria or viruses on the mucosal epithelia, and neutralization of viruses with which epithelial cells have been infected.
However, as mentioned above, many cases of the conventional vaccination have had the problem of failing to increase the antibody titers of antibodies in non-IgG classes (such as IgA and IgM) or to fully induce the cellular immune response, so no immune response that involves the production of antigen-specific antibodies (secretory IgA) can be effectively induced on mucosal surfaces which are sites of infection with infectious pathogens. To solve these problems, studies have been made on the assumption that antigen-specific immune response could be induced both systemically and on the mucosal surfaces at various parts of the body by administering antigens via mucosal surfaces as in oral immunization or nasal immunization.
With a view to imparting such mucosal immunity, attempts have been made to incorporate antigens in liposomes and administering them to mucosal membranes as vaccines. It was shown, for example, that by incorporating a Salmonella enterica serovar Enteritidis antigen as an immunogen in liposomes and dropping it onto the eyes of chickens, systemic immune response could be induced to thereby inhibit the invasion of Salmonella enterica serovar Enteritidis through the intestinal lumen (Non-Patent Document 1 and Non-Patent Document 2).
However, in the field of vaccine production, it is desired to develop vaccine carriers that can achieve even more efficient increases in the antibody titers of various classes of antibodies (in humoral immune response) and in cellular immune response.
Non-Patent Document 1: Fukutome, K. et al., Development and Comparative Immunology, 25, 2001, 475-484;.
Non-Patent Document 2: Li, W. et al, Development and Comparative Immunology, 28, 2004, 29-38.
An object of the present invention is to prepare novel vaccine carriers that can be used to produce vaccines that are capable of efficient induction of humoral and cellular immune responses. Another object of the present invention is to provide vaccines that are capable of efficient induction of humoral and cellular immune responses.
The present inventors revealed that the above-stated problems of the present invention could be solved by using liposomes containing a fusogenic lipid (succinylated poly(glycidol)), and this finding has led to the accomplishment of the invention. Stated specifically, the present invention can solve the aforementioned problems by providing vaccine carriers comprising liposomes containing succinylated poly(glycidol).
By using the above-described vaccine carriers that comprise liposomes containing succinylated poly(glycidol), efficient vaccines can be obtained that achieve significant increases in antibody titers as compared with the case of using vaccine carriers that comprise the conventional liposomes. In addition, the vaccines prepared by using the above-described vaccine carriers are capable of efficient induction of not only humoral immunity but also cellular immunity.
As described above, the present invention is characterized by providing vaccine carriers comprising liposomes that contain succinylated poly(glycidol) (SucPG). The advantage of using the vaccine carriers comprising such liposomes is that irrespective of whether the immunogen contained in the liposomes is an inactivated vaccine or an attenuated vaccine and whatever is the route of administration, not only the antibody titers of IgG antibodies but those of other classes of antibodies can also be elevated and what is more, cellular immunity as well as humoral immunity can also be induced.
The cellular immunity that is induced from the use of the vaccine carrier of the present invention which comprises liposomes containing succinylated poly(glycidol) (SucPG) is believed to have been accomplished by internalizing the immunogen within antigen-presenting cells. To be more specific, using the vaccine carrier of the present invention, one can incorporate the immunogen into the lumens of liposomes and can hence internalize the immunogen within the antigen-presenting cells. As a result, so it was assumed, the immunogen incorporated into the antigen-presenting cell combined as a self-component with an MHC class I molecule and presented itself as an antigen on the antigen-presenting cell, eventually inducing cellular immunity.
The succinylated poly(glycidol) (SucPG) as used herein is an amphiphilic compound characterized by having an alkyl group. Having an alkyl group, SucPG can be anchored to a liposome membrane. The alkyl group in SucPG preferably contains 6 to 24 carbon atoms and, more preferably, contains alkyl groups having 6 to 18 carbon atoms. The most preferred alkyl group is an n-decyl group having 10 carbon atoms. SucPG has the skeleton of the main chain being similar to those of amphiphilic polyethylene glycol and side chains with a carboxyl group, so it is characterized in that it stabilizes the liposome membrane in a neutral environment but that in an acidic environment, the carboxyl group on side chains is protonated to induce membrane fusion. By incorporating the SucPG into the liposome membrane, the resulting liposome (SucPG-liposome) comes to develop fusogenicity in an acidic environment. In other words, when the SucPG-liposome is incorporated into the antigen-presenting cell by endocytosis, the pH in the lysosome drops. Then the SucPG-liposome exhibits its fusogenicity and fuses with the lysosome membrane to cause the encapsulated antigenic substance to be released into the cytoplasm (internalization of the antigen).
The SucPG that is to be used in the present invention can be prepared by reacting the synthetic polymer poly(glycidol) with succinic anhydride in N,N-dimethylformamide at 80° C. for 6 hours.
What is characteristic of the present invention is that succinylated poly(glycidol) is added to the lipid that composes the liposome used in the vaccine carrier. To be more specific, the vaccine carrier of the present invention contains succinylated poly(glycidol) in an amount of 10 to 40 wt %, preferably 20 to 35 wt %, most preferably 30 wt %, of the lipid that composes the liposome.
The vaccine carrier of the present invention can be used for transmucosal administration of the immunogen contained within the liposome. The term “mucosa” or “mucosal membrane” as used herein collectively refers to sites that cover the inner surfaces of the lumens of hollow viscera such as the digestive organs, respiratory organs, and genitourinary organs and their free surfaces are always wet with secretions from mucosal glands and goblet cells. Mucosal membranes to which the vaccine carrier of the present invention can be applied include membranes of the oral cavity, throat, nasal cavity, aural cavity, conjunctival sac, vagina, and anus. By applying the immunogen-containing liposome to these mucosal surfaces, the immunogen can be incorporated into the body via the mucosal surfaces.
The vaccine carrier of the present invention can also be used for delivery by non-transmucosal administration of the immunogen contained within the liposome of the vaccine carrier. In the present invention, routes other than the transmucosal route may include intraperitoneal administration of the immunogen contained within the liposome of the vaccine carrier. For instance, when the immunogen is administered intraperitoneally, it can be incorporated into the body from the surfaces of organs in the abdominal cavity, as exemplified by the gastrointestinal tract, genital organs, liver, and pancreas. By thus administering the immunogen into the body, the immunogen can be incorporated into antigen-presenting cells ubiquitously present in the body.
Lipids that compose the liposome in the present invention include, for example, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidic acids or long-chain alkyl phosphates or phosphatidylglycerols, and cholesterols (Chol). When preparing liposomes in the present invention, the lipids listed above may be used either independently or in combination of any two or more of those lipids.
Phophatidylcholines that are used as lipids for composing the liposome in the present invention include dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleyl phosphatidylcholine (DOPC), yolk lecithin (egg PC), etc.
Phophatidylethanolamines that are used as lipids for composing the liposome in the present invention include dioleyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine, dipalmityol phosphatidylethanolamine, distearoyl phosphatidylethanolamine (DSPE), etc.
Phophatidylserines that are used as lipids for composing the liposome in the present invention include dioleyl phosphatidylserine (DOPS), dipalmitoyl phosphatidylserine (DPPS), etc.
Phophatidic acids or long-chain alkyl phosphates that are used as lipids for composing the liposome in the present invention include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dicetyl phosophate, etc.
Phophatidylglycerols that are used as lipids for composing the liposome in the present invention include dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, etc.
Among the compounds listed above, those which are particularly preferred for use are DOPE, DPPC, DSPC, DPPS, DSPE, Chol, etc.
When the above-listed lipids are to be used in admixture, the proportions at which the respective lipids are incorporated can be determined as appropriate by the desired size of liposomes, the desired fluidity, etc. In the present invention, liposomes are preferably prepared by mixing DOPE and DPPC at 1:1.
Liposomes are classified as MLV (multilamellar vesicles), DRV (dehydration-rehydration vesicles), LUV (large unilamellar vesicles) or SUV (small unilamellar vesicles), etc. depending on their structures or the method of their preparation. The liposome of the present invention which contains succinylated poly(glycidol) (SucPG) is also available in various types of liposomes including MLV, DRV, LUV and SUV that are composed of multiple layers.
To prepare the SucPG-containing liposomes, any conventionally known methods of liposome production may be employed. A variety of methods for liposome production have heretofore been known in the technical field of interest.
To give examples of some common methods for liposome production, the following may be mentioned: (1) a lipid is dissolved in a suitable organic solvent (such as, chloroform, ether, etc.) and the solvent is distilled off under vacuum to form a thin lipid film, which is then hydrated (or swollen) in water by mechanical agitating means; (2) a lipid is dissolved in an organic solvent (such as ether or ethanol) and the resulting solution is injected into high-temperature warmed water by suitable means (such as a syringe or a nozzle) under pressure at a constant rate, in the process of which the organic solvent is distilled off or diluted, whereby the lipid forms a double layer to prepare liposomes; (3) a lipid is mixed with a surfactant (such as cholic acid or deoxycholic acid) to form micelles in an aqueous solution and the resulting micelle solution is deprived of the surfactant (such as cholic acid or deoxycholic acid) by a suitable operation such as dialysis or gel filtration so as to prepare liposomes; (4) an organic solvent having a lipid dissolved therein is added to an aqueous phase, which is sonicated to form a W/O emulsion which is then deprived of the organic solvent to form a gel which is allowed to undergo phase inversion by mechanical agitation so as to prepare liposomes; (5) a thin film of lipid is mixed with an aqueous solvent so that it is hydrated or swollen and the container is mechanically vibrated to separate the thin lipid film from its inner surfaces and, thereafter, the separated thin lipid film is sonicated or passed through an orifice of a given size by means of a French press, a pressurized filtering apparatus, or an extruder so as to prepare liposomes; and (6) liposomes are freeze-dried and then rehydrated with an aqueous solvent to thereby prepare liposomes.
With a view to supplementing the immuno-augmenting activity, the vaccine carrier of the present invention may further contain an adjuvant. Adjuvants that can be contained in the vaccine carrier of the present invention include monophosphoryl lipid A, cytokine, lectin, etc.
The immunogen that can be contained in the vaccine carrier of the present invention may include (but not limited to) any of the immunogens with which humans or animals (mammals, fishes, etc.) are desirably vaccinated. Examples of the immunogens include immunogens derived from bacteria, immunogens derived from viruses, and immunogens derived from protozoa.
In the case, for example, that the case of applying the vaccine carrier of the present invention is applied to immunogens in humans, the following may be contained in the vaccine carrier: a virus-derived immunogen selected from among an influenza virus antigen, a SARS virus antigen, an AIDS virus antigen and the like; or a bacterium-derived immunogen selected from among pathogenic Escherichia coli O-157 antigen, Salmonella antigen, Staphylococcus aureus antigen, Aeromonas antigen, tubercule bacillus antigen and the like; or a protozoan-derived immunogen selected from among trypasonoma antigen, coccidium antigen, malaria antigen, theileria antigen and the like.
Consider then the case of applying the vaccine carrier of the present invention to immunogens in animals; in this case, any one of the antigens derived from pathogens of important infectious diseases in domestic animals may be contained and examples of the antigens include:
for chickens, bacterium-derived immunogens such as Salmonella enterica serovar Enteritidis antigen, Haemophilus paragallinarum antigen; virus-derived immunogens such as chick influenza virus antigen, Newcastle disease virus antigen, infectious bronchitis virus antigen; protozoan-derived immunogens such as leucocytozoon antigen, eimeria antigen;
for pigs, a virus-derived immunogen such as infectious gastroenteritis virus antigen; a bacterium-derived immunogen such as Bordetella bronchiseptica antigen; and protozoan-derived immunogens such as toxoplasma antigen, eimeria antigen;
for cows, a virus-derived immunogen such as bovine viral diarrhea/mucosal disease virus antigen; bacterium-derived immunogens such as Staphylococcus aureus antigen and Mycobacterium avium var paratuberculosis antigen; and protozoan-derived immunogens such as theileria antigen, babesia antigen and the like;
for horses, virus-derived immunogens such as equine rhinopneumonitis virus antigen and equine influenza virus antigen; and protozoan-derived immunogens such as trypanosoma antigen, babesia antigen; and for fishes, bacterium-derived immunogens such as vibrio antigen, aeromonus antigen; virus-derived immunogens such as infectious pancreatic necrosis virus antigen, iridovirus antigen; and protozoan-derived immunogens such as ichthyobodo protozoan antigen, hexamita protozoan antigen.
To a lipid composition consisting of dipalmitoyl phosphatidylcholine (DPPC) (Sigma) and dioleyl phosphatidylethanolamine (DOPE) (Sigma) at a molar ratio of 1:1 (each being 10 μmoles), SucPG was added at a lipid weight ratio of 10%, 20% or 30% to prepare three kinds of SucPG-containing liposomes with different SucPG concentrations. The SucPG to be used in Example 1 was of a type having a C10 n-decyl group as an alkyl group and it was synthesized by a documented method (Kono K. et al., J. Controlled Release, 68, 225-235 (2000); Kono K. et al., Biochim. Biophys. Acta, 1325, 143-154 (1997); or Kono K. et al., Biochim. Biophys. Acta, 1193, 1-9 (1994)). Specifically, poly(epichlorohydrin) was subjected to reaction in dimethylformamide in the presence of potassium acetate at 175° C. for 6 hours to prepare poly-glycidyl acetate, which in turn was subjected to reaction in methyl carbitol in the presence of potassium acetate at 150° C. for 1 hour to synthesize poly(glycidol). The thus synthesized polymer poly(glycidol) was reacted with succinic anhydride in N,N-dimethylformamide at 80° C. for 6 hours to prepare SucPG.
To prepare liposomes, the procedure described in Non-Patent Document 1 may be adopted. Specifically, 2 μmoles of DPPC, 2 μmoles of DOPE, and SucPG were dissolved in an organic solvent and mixed in a conical flask. The lipids were dried on a rotary evaporator and placed for 30 minutes under vacuum in a desiccator. As a model antigen, 4 mg/mL of ovalbumin (OVA) was added and the mixture was incubated at 35-40° C. for 3 minutes, followed by vigorous vortexing to disperse the lipid film. In this way, the model antigen OVA was encapsulated in the liposomes. Any OVA that was not encapsulated in the liposomes was removed by repeated centrifuging at 14000 g for 20 minutes at 4° C. so as to purify the liposomes having the model antigen OVA encapsulated therein. Prepared in this way were multilamellar vesicles (OVA-SucPG-liposomes) (MLV).
The purpose of this Example was to study the immune response from transmucosal administration of a vaccine in the SucPG-containing liposomes prepared in Example 1, as compared with a vaccine in the SucPG-free liposomes.
The vaccine in the SucPG-containing liposomes was prepared as described in Example 1. On the other hand, the vaccine in the SucPG-free liposomes was a vaccine in multilamellar vesicles (OVA-liposomes) (MLV) that were prepared by encapsulating OVA in a lipid composition consisting of DPPC and DOPE at a molar ratio of 1:1.
The thus prepared two types of vaccines, one in the OVA-SucPG-liposomes and the other in the OVA-liposomes, as well as a vaccine solely composed of OVA were administered transnasally to BALB/c mice twice at a 7-day interval, each time to give 100 μg per mouse of OVA.
Seven days after the final administration of the immunogen, 0.1 ml of blood was taken from the orbital venous plexus and the serum collected from the blood was used to study the production of anti-OVA antibodies (IgM, IgG, and IgE) by the ELISA procedure. Also, seven days after the final administration, the intestinal fluid was collected and studied for the production of anti-OVA antibodies (IgA and IgG) by the ELISA procedure. In addition, the immune serum obtained was used to make an analysis for anti-OVA-IgG subclasses by the ELISA procedure.
The results are shown in
From the results shown in
In addition, the results shown in
Further in addition,
Accordingly, the following general observations were obtained from the transmucosal administration of the vaccine in the SucPG-containing liposomes: it was capable of efficient antigen introduction into antigen-presenting cells, eventually inducing high antibody production in the blood as well as high antibody production in the intestinal tract; and it was potentially capable of inducing not only humoral immunity but also cellular immune response.
Since it was shown in Example 2 that immunization with the vaccine in the SucPG-containing liposomes by transmucosal administration of the antigen had the potential to induce cellular immune response, Example 3 was conducted to study an ability to exert the cellular immune response for the case of using the vaccine in the SucPG-containing liposomes.
The vaccine in the OVA-SucPG-liposomes prepared in Example 1 was administered transnasally to BALB/c mice twice at a 7-day interval, each time to give 100 μg per mouse of OVA. Seven days after the final administration, the mice were sacrificed and the spleen was collected and subjected to density-gradient centrifugation to purify the spleen lymphocytes.
From the purified spleen lymphocytes, total RNA was extracted using TRIzol™ (Invitrogen) and IFN-γ which was an index of cellular immunity and IL-4 were checked for mRNA expression by the RT-PCR technique.
First of all, cDNA was synthesized from the total RNA. The total RNA (1 μg to 5 μg) was mixed with an oligo(dT)12-18 primer (500 ng) and dNTP Mix (10 nmol) to make a total volume of 12 μL; the mixture was subjected to reaction at 65° C. for 5 minutes and quenched on ice. Subsequently, 4 μL of 5× First-Strand Buffer, 2 μL of 0.1 M DTT and 1 μL of RNaseOUT™ Recombinant Ribonuclease Inhibitor (40 units/μL) (Invitrogen) were added and, following a 2-minute reaction at 42° C., SuperScript™ II reverse transcriptase (Invitrogen) was added in an amount of 200 units. Following an additional 50-minute reaction at 42° C., the reverse transcriptase was inactivated by performing a reaction at 70° C. for 15 minutes.
One microliter of the resulting cDNA, 2.5 pmol of each of the primers identified below, 37.5 nmol of MgCl2, 10 nmol of dNTP Mix, and 1 unit of Taq DNA Polymerase (Invitrogen) were added to a PCR buffer to make a total volume of 25 μL and PCR reaction was performed in TaKaRa PCR Thermal Cycler MP TP3000 (TaKaRa). The PCR reaction consisted of a 5-minute reaction at 94° C., followed by 35 cycles, in which each consisting of a 94° C.×45-sec reaction, a 60° C.×45-sec reaction and a 72° C.×2-min reaction, and the final reaction at 72° C. for 7 minutes. Following the PCR, the sample was electrophoresed on a 2% agarose gel and made visible by staining with ethidium bromide.
In Example 3, the following primers were used to amplify the mouse IFN-γ gene:
forward primer: 5′-tgcatcttggcttgcagctcttcctcatggc-3′ (SEQ ID NO: 1) and
reverse primer: 5′-tggacctgtgggttgttgacctcaaacttggc-3′ (SEQ ID NO: 2);
and the following primers were used to amplify the mouse IL-4 gene:
forward primer: 5′-ccagctagttgtcatcctgctcttctttctcg-3′ (SEQ ID NO: 3) and
reverse primer: 5′-cagtgatgtggacttggactcattcatggtgc-3′ (SEQ ID NO: 4), each of these primers being available from CLONTEC.
As primers for amplifying a control mouse G3PDH gene, the following were used:
forward primer: 5′-accacagtccatgccatcac-3′ (SEQ ID NO: 5) and
reverse primer: 5′-tccaccaccctgttgctgta-3′ (SEQ ID NO: 6).
The results of measurement for the mRNA expression of IFN-γ and IL4 are shown in
In Example 3, the purified spleen lymphocytes were also cultured in an OVA-supplemented medium for 5 days and the amounts of IFN-γ and IL-4 that were released into the supernatant of the culture were measured as indices of cellular immunity and humoral immunity, respectively. To quantify the IFN-γ and IL-4 in the supernatant of the culture, the relevant quantitation kits (Endogen) were used.
The results of quantitation of the amounts of IFN-γ and IL-4 released into the supernatant of the culture of spleen lymphocytes are shown in
From the results described in Examples 2 and 3, it has been shown that the vaccine in the SucPG-containing liposomes of the present invention, when it is used in transnasal immunization, can induce not only humoral immunity but also cellular immunity and this indicates that the SucPG-containing liposomes of the present invention are useful as an antigen carrier for transmucosal vaccines.
The purpose of this Example was to study the immune response from non-transmucosal administration of a vaccine in the SucPG-containing liposomes prepared in Example 1, as compared with a vaccine in the SucPG-free liposomes.
The vaccine in the SucPG-containing liposomes was prepared as described in Example 1. On the other hand, the vaccine in the SucPG-free liposomes was a vaccine in multilamellar vesicles (OVA-liposomes) (MLV) that were prepared by encapsulating OVA in a lipid composition consisting of DPPC and DOPE at a molar ratio of 1:1.
The thus prepared two types of vaccines, one in the OVA-SucPG-liposomes and the other in the OVA-liposomes, as well as a vaccine solely composed of OVA were administered intraperitoneally to BALB/c mice twice at a 7-day interval, each time to give 100 μg per mouse of OVA.
Seven days after the final administration of the immunogen, 0.1 ml of blood was taken from the orbital venous plexus and the serum collected from the blood was used to study the production of anti-OVA antibodies (IgM, IgG, and IgE) by the ELISA procedure. In addition, the immune serum obtained was used to make an analysis for anti-OVA-IgG subclasses by the ELISA procedure.
The results are shown in
From the results shown in
In addition, the results shown in
Accordingly, the following general observations were obtained from the non-transmucosal administration of the vaccine in the SucPG-containing liposomes: it was capable of efficient antigen introduction into antigen-presenting cells, eventually inducing high antibody production; and it was potentially capable of inducing not only humoral immunity but also cellular immune response.
Since it was shown in Example 4 that immunization with an antigen using the SucPG-containing liposomes had the potential to induce cellular immune response, Example 5 was conducted to study an ability to exert the cellular immune response for the case of using the SucPG-containing liposomes.
The OVA-SucPG-liposomes prepared in Example 1 were administered intraperitoneally to BALB/c mice twice at a 7-day interval, each time to give 100 μg per mouse of OVA. Seven days after the final administration, the mice were sacrificed and the spleen was collected and subjected to density-gradient centrifugation to purify the spleen lymphocytes. Using the thus purified spleen lymphocytes, IFN-γ and IL-4 were measured for mRNA expression by the RT-PCR technique and the amounts of IFN-γ and IL-4 released into the culture supernatant were measured by the ELISA procedure as described in Example 3.
The results of measurement for the mRNA expression of IFN-γ and IL4 are shown in
In addition, the results of quantitation of the amounts of IFN-γ and IL-4 released into the supernatant of the culture of spleen lymphocytes are shown in
From the results described in Examples 4 and 5, it has been shown that the vaccine in the SucPG-containing liposomes of the present invention, even when it is used in non-transnasal immunization, can induce not only humoral immunity but also cellular immunity as in the case of transnasal immunization and this indicates that the SucPG-containing liposomes of the present invention are also useful as an antigen carrier for non-transmucosal vaccines.
The purpose of this Example was to study the immune response from ophthalmic (transmucosal) administration to chickens of a vaccine in SucPG-containing liposomes containing Salmonella enteritidis antigen as an immunogen.
The Salmonella enteritidis antigen, or the immunogen to be used in this Example, was prepared in the following manner. First, Salmonella enteritidis (strain 1227) was inoculated in a heart infusion medium (Nissui Pharmaceutical Co., Ltd.) and following cultivation at 37° C. for 14 hours, 7×1014 CFU of the bacterium Salmonella enteritidis was harvested. The harvested bacterium Salmonella enteritidis was inactivated by denaturation with an excess amount of formalin; following the removal of formalin, sonication was conducted to prepare an antigenic fluid. A vaccine in SucPG-containing liposomes containing the thus prepared Salmonella enteritidis antigen (vaccine in Salmonella enteritidis antigen-SucPG-liposomes) was prepared by a method that was basically the same as the procedure described in Example 1.
The thus prepared vaccine in Salmonella enteritidis antigen-SucPG-liposomes was administered once to 5 chickens (white leghorn) 3 weeks old after birth by dropping onto the eyes to give 100 μg per chick of Salmonella enteritidis antigen.
At days 14 and 35 after the administration of the immunogen (designated as “2 wks (5-wk old)” and “5 wks (8-wk old)”, respectively), 2.0 mL of blood was collected from the wing vein (also called as basilic vein) and using the serum collected from the blood sample, the production of anti-Salmonella enteritidis antigen antibody (IgG or IgA) was studied by the ELISA procedure. As a control, there was used serum that had been obtained from chick individuals that were yet to be immunized with the Salmonella enteritidis antigen (designated as pre (3-wk old)).
The results are shown in
From the results shown in
From these results, it has been shown that immunizing chickens by ophthalmic (transmucosal) administration of the vaccine in Salmonella enteritidis antigen-SucPG-liposomes can induce high antibody production in the blood.
The purpose of this Example was to study the immune response from transnasal (transmucosal) administration to mice of a vaccine in SucPG-containing liposomes containing Trypanosoma brucei antigen as an immunogen.
To obtain the T. brucei antigen, the immunogen to be used in this Example, protozoa T. brucei were collected. The method of collection was in accordance with a published method (Lanham, S. M., Nature, 218, 1273-1274 (1968)). Specifically, protozoa T. brucei (1×105 parasites) were inoculated in the abdominal cavities of Wistar rats (Japan SLC, Inc.) and 4 days later, whole blood was collected from their hearts. The collected blood was treated with heparin (10 units/mL) for inhibition of protection against coagulation, and the buffy coat was collected by centrifuging (1300 g×10 min). The protozoa were purified and harvested from the collected buffy coat by means of a DE52 cellulose (Whatman) column. The harvested protozoa T. brucei were ground by sonication to obtain the T. brucei antigen. A vaccine in SucPG-containing liposomes containing the thus prepared T. brucei antigen (vaccine in T. brucei antigen-SucPG-liposomes) was prepared by a method that was basically the same as the procedure described in Example 1.
The thus prepared vaccine in T. brucei antigen-SucPG-liposomes was administered transnasally to five BALB/c mice (Japan SLC, Inc.) 6 weeks old after birth to give 100 μg per mouse of T. brucei antigen, and 2 weeks later the same amount of T. brucei antigen was additionally boosted transnasally to effect immunization.
Fourteen days after the initial administration of the immunogen (Day 14) and seven days after the final administration of the immunogen (Day 21), 0.1 ml of blood was taken from the orbital venous plexus and the serum collected from the blood was used to study the production of anti-T. brucei antigen antibodies (IgG and IgM) by the ELISA procedure. As a control, there was used serum that had been obtained from mouse individuals that were yet to be immunized with the T. brucei antigen (Day 0).
The results are shown in
From the results shown in
From these results, it has been shown that immunizing mice by transnasal (transmucosal) administration of the vaccine in T. brucei antigen-SucPG-liposomes can induce high antibody production in the blood.
The purpose of this Example was to study the immune response from transnasal (transmucosal) administration to cows of a vaccine in SucPG-containing liposomes containing Staphylococcus aureus antigen as an immunogen.
To prepare the S. aureus antigen, the immunogen to be used in this Example, S. aureus (strain Cowan I) was first inoculated in an LB medium (Nissui Pharmaceutical Co., Ltd.) and following cultivation at 37° C. for 14 hours, the bacterium S. aureus was harvested. The harvested bacterium S. aureus was inactivated by denaturation with an excess amount of formalin; following the removal of formalin, sonication was conducted to prepare an antigenic fluid. A vaccine in SucPG-containing liposomes containing the thus prepared S. aureus antigen (vaccine in S. aureus antigen-SucPG-liposomes) was prepared by a method that was basically the same as the procedure described in Example 1.
The thus prepared vaccine in S. aureus antigen-SucPG-liposomes was administered transnasally to three Holstein milking cows to give 5 mg per cow of S. aureus antigen, and 14 days later the same amount of S. aureus antigen was additionally boosted transnasally to effect immunization.
Fourteen days after the initial administration of the immunogen (Day 14) and 7 and 14 days after the final administration of the immunogen (Day 21 and Day 28), 5 ml of blood was taken from the cervical vein and the serum collected from the blood was used to study the production of anti-S. aureus antigen antibodies (IgA and IgG) by the ELISA procedure. As a control, there was used serum that had been obtained from milking cow individuals that were yet to be immunized with the S. aureus antigen (Day 0).
The results are shown in
From the results shown in
Fourteen days after the initial administration of the immunogen (Day 14) and 7 and 14 days after the final administration of the immunogen (Day 21 and Day 28), milk was collected from the cows and studied for the production of anti-S. aureus antigen antibodies (IgG and IgA) by the ELISA procedure. As a control, there was used milk that had been obtained from milking cow individuals that were yet to be immunized with the S. aureus antigen (Day 0).
The results are shown in
From the results shown in
From these results, it has been shown that immunizing milking cows by transnasal (transmucosal) administration of the vaccine in S. aureus antigen-SucPG-liposomes can induce markedly high antibody production in both blood and milk.
The purpose of this Example was to study the immune response from oral (transmucosal) administration to carp of a vaccine in SucPG-containing liposomes containing Aeromonas salmonicida antigen as an immunogen.
The A. salmonicida antigen, or the immunogen to be used in this Example, was prepared in the following manner. First, A. salmonicida (strain T1031) was inoculated in a heart infusion medium and following cultivation at 20° C. for 24 hours, the bacterium A. salmonicida was harvested and the harvested bacterium A. salmonicida was inactivated by denaturation with an excess amount of formalin; following the removal of formalin, sonication was conducted to prepare an antigenic fluid. A part of the A. salmonicida antigen was immediately utilized as an immunogen whereas another part was utilized to prepare a vaccine in SucPG-containing liposomes containing the A. salmonicida antigen. The vaccine in SucPG-containing liposomes containing the inactivated A. salmonicida antigen (vaccine in A. salmonicida antigen-SucPG-liposomes) was prepared by a method that was basically the same as the procedure described in Example 1.
The thus prepared vaccine in A. salmonicida antigen-SucPG-liposomes was orally administered three times at 2-wk intervals to six carp (distributed from Aquatic Life Conservation Research Center, Research Institute of Environment, Agriculture and Fisheries, Osaka Prefectural Government) for immunization to give 200 μg per carp of the A. salmonicida antigen. As a control, only the A. salmonicida antigen was orally administered three times at 2-wk intervals to four carp for immunization to give 200 μg per carp of the A. salmonicida antigen.
At the initial administration of the immunogen (Day 0), at its second administration (Day 14), at its third administration (Day 28) and 14 days after its final administration (Day 42), 1 ml of blood was taken from the caudal peduncle of each carp and the serum collected from the blood sample was used to study the production of anti-A. salmonicida antigen antibody by the ELISA procedure. As a control, there was used serum that had been obtained from carp individuals immunized with the A. salmonicida antigen only.
The results are shown in
From the results shown in
Based on these results, 14 days after the final administration of the vaccine in A. salmonicida antigen-SucPG-liposomes (Day 42), the intestinal fluid and bile were collected from the carp and studied for the production of anti-A. salmonicida antigen antibody by the ELISA procedure. As controls, there were used the intestinal fluid and bile that had been obtained from non-immunized carp individuals.
The results are shown in
From the results shown in
Furthermore, 14 days after the final administration of the vaccine in A. salmonicida antigen-SucPG-liposomes, six carp were immersed in a suspension of the bacterium A. salmonicida (1×106 cfu/mL) for 60 minutes so that they would be attacked by A. salmonicida; the subsequent transitional change of their survival rate was recorded. As controls, eight non-immunized carp individuals were similarly treated and the subsequent transitional change of their survival rate was recorded.
The results are shown in
From these results, it has been shown that even with the fish carp, immunization by oral (transmucosal) administration of the vaccine in A. salmonicida antigen-SucPG-liposomes can induce high antibody production in the blood.
The purpose of this Example was to study the immune response from transnasal (transmucosal) administration to mice of a vaccine in SucPG-containing liposomes containing Mycoplasma gallisepticum antigen as an immunogen.
The Mycoplasma gallisepticum antigen, the immunogen to be used in this Example, was prepared in the following manner. First, Mycoplasma gallisepticum (strain S6) was inoculated in Fray medium (Difco) supplemented with a fresh yeast extract and following cultivation at 37° C. for 48 hours or longer, 3.58×CFU of the bacterium Mycoplasma gallisepticum was harvested. The harvested bacterium Mycoplasma gallisepticum was inactivated by denaturation with an excess amount of formalin; following the removal of formalin, sonication was conducted to prepare an antigenic fluid. A vaccine in SucPG-containing liposomes containing the thus prepared Mycoplasma gallisepticum antigen (vaccine in Mycoplasma gallisepticum antigen-SucPG-liposomes) was prepared by a method that was basically the same as the procedure described in Example 1.
The thus prepared vaccine in Mycoplasma gallisepticum antigen-SucPG-liposomes was administered transnasally to five BALB/c mice (Japan SLC, Inc.) 5 weeks old after birth to give 100 μg per head of Mycoplasma gallisepticum antigen, and 2 weeks later the same amount of Mycoplasma gallisepticum antigen was additionally boosted transnasally to effect immunization.
Seven days after the final administration of the immunogen (Day 21), 0.1 ml of blood was taken from the orbital venous plexus and the serum collected from the blood was used to study the production of anti-M. gallisepticum antigen antibodies (IgG and IgA) by the ELISA procedure. As a control, there was used serum that had been obtained from mouse individuals that were yet to be immunized with the M. gallisepticum antigen (Day 0).
The results are shown in
From the results shown in
Accordingly, it has been shown that transmucosal administration of the vaccine of the M. gallisepticum antigen by means of the SucPG-containing liposomes can induce high antibody production in the blood and that it is potentially capable of inducing not only humoral immunity but also cellular immune response.
The purpose of this Example was to study the immune response from transnasal (transmucosal) administration to mice of a vaccine in SucPG-containing liposomes containing Newcastle disease virus antigen as an immunogen.
The Newcastle disease virus antigen, the immunogen to be used in this Example, was prepared from a commercial live vaccine of Newcastle disease which was sonicated to prepare an antigenic fluid. A vaccine in SucPG-containing liposomes containing the thus prepared Newcastle disease virus antigen (vaccine in Newcastle disease virus antigen-SucPG-liposomes) was prepared by a method that was basically the same as the procedure described in Example 1.
The thus prepared vaccine in Newcastle disease virus antigen-SucPG-liposomes was administered transnasally to five BALB/c mice (Japan SLC, Inc.) 5 weeks old after birth to give 100 μg per head of Newcastle disease virus antigen, and 2 weeks later the same amount of Newcastle disease virus antigen was additionally boosted transnasally to effect immunization.
Seven days after the final administration of the immunogen (Day 21), 0.1 ml of blood was taken from the orbital venous plexus and the serum collected from the blood was used to study the production of anti-Newcastle disease virus antigen antibodies (IgG and IgA) by the ELISA procedure. As a control, there was used serum that had been obtained from mouse individuals that were yet to be immunized with the Newcastle disease virus antigen (Day 0).
The results are shown in
From the results shown in
From these results, it has been shown that immunizing the mice by transnasal (transmucosal) administration of the vaccine in Newcastle disease virus antigen-SucPG-liposomes can induce high antibody production in the blood.
By using the above-described vaccine carriers that comprise liposomes containing succinylated poly(glycidol), efficient vaccines can be obtained that achieve marked increases in antibody titers as compared with the case of using vaccine carriers that comprises the conventional liposomes. In addition, the vaccines prepared by using the above-described vaccine carriers are capable of efficient induction of not only humoral immunity but also cellular immunity.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-030246 | Feb 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2007/052079 | 2/7/2007 | WO | 00 | 1/6/2009 |
