CA2236538C - Influenza vaccine compositions - Google Patents
Influenza vaccine compositions Download PDFInfo
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- CA2236538C CA2236538C CA2236538A CA2236538A CA2236538C CA 2236538 C CA2236538 C CA 2236538C CA 2236538 A CA2236538 A CA 2236538A CA 2236538 A CA2236538 A CA 2236538A CA 2236538 C CA2236538 C CA 2236538C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55583—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55588—Adjuvants of undefined constitution
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
Abstract
The invention provides a vaccine composition in the form of a kit comprising a first container containing an antigenic preparation comprising influenza antigen or antigens; and a second container containing an effective adjuvant amount of a chitosan. The antigenic preparation in the first container preferably comprises haemagglutinin and neuraminidase influenza antigens.
Description
INFLUENZA VACCINE COMPOSITIONS
The invention relates to a vaccine composition in the form of a kit comprising a first container containing an antigenic preparation comprising influenza virus antigens and a second container containing a mucosal adjuvant. The invention also relates to a method of immunising a patient against influenza by administering the said composition to the patient, and a method of enhancing the immunogenicity of an influenza viral antigen, particularly when administered intranasally, by co-administering therewith the said adjuvant.
Current influenza vaccines consist of either inactivated whole virus, disrupted virus (split vaccines) or purified preparations of the membrane glycoproteins haemagglutinin (HA) and neuraminidase (NA) sub-unit vaccines. Haemagglutinin and neuraminidase are the antigens to which protective antibody responses are directed, haemagglutinin being the major protective antigen. Estimates of the efficacy of these parenterally administered vaccines vary greatly. Such vaccines are believed to act primarily by eliciting circulating anti-haemagglutinin IgG antibodies that transudate into the lower respiratory tract.
M.L. Clements eta/, J. Clinical Microbiology 24, 157-160,1986, have previously reported that both secretory IgA and serum lgG participate in immunity to influenza virus. Moreover, in mice, a number of published studies have demonstrated the importance of respiratory IgA to protection against influenza infection. It has also been found that an advantage of stimulating a local IgA response to influenza is that it is often of a broader specificity than the serum response and thus can provide cross-protection against viruses possessing haemagglutinin molecules different from those present in the vaccine. Accordingly, influenza vaccines that elicit both local secretory and serum anti-haemagglutinin responses should provide superior immunity to current vaccines. However, parenteral vaccination (intramuscular, sub-cutaneous etc) is not effective at eliciting local antibody production, if there has been no previous mucosal exposure (e.g. infection).
In order to stimulate the mucosal immune system, the vaccine must be applied topically to a mucosal surface.
Mucosal administration of influenza vaccine would have a number of advantages over traditional parenteral immunisation regimes. Paramount amongst these are more effective stimulation of the local mucosal immune system of the respiratory tract and the likelihood that vaccine uptake rates would be increased because the fear and discomfort associated with injections would be avoided. Accordingly, a number of attempts have been made to develop mucosal influenza vaccines. A drawback however is that inactivated vaccines are often poorly immunogenic when given mucosally.
For example, Kuno-sakai et al (vaccine 12: 1303-1310, (1994) have shown that administration of inactivated vaccine to humans gave strong mucosal and serum antibody responses and was effective in preventing infection by live vaccine virus. However, in order to achieve such results, Kuno-sakai et al administered three times the commercially available dose, an approach which is not considered to be commercially viable. In order to overcome this problem, different approaches to improving the immunogenicity of flu vaccines given orally or intranasally have included the use of the B sub-unit of cholera toxin (CTB) as an adjuvant (see S. Tamura et al, vaccine, 6, 409, (1988), encapsulation of the vaccine in a variety of microspheres (see Z.
Moldoveanu et al, J.inf.Dis. 167, 85-90 (1993), and the use of live attenuated strains (see H.F.Maassab et al, vaccines, Plotkin S.A and Mortimer F.A Jr (eds) W.B. Saunders Philadelphia p435 (1993). To date however, no practical means of enhancing the immunogenicity of mucosally administered flu vaccines has been developed.
It has now been found by the Applicants that by administering influenza antigens such as the haemagglutinin and neuraminidase antigens in combination with a particular chitosan derivative, it is possible to achieve good IgG and good IgA responses. More particularly , the present Applicants have now found that if a chitosan is co-administered intranasally with influenza antigens such as the neuraminidase and haemagglutinin antigens, or if a host is predosed with a chitosan prior to administration of influenza antigens, good systemic and local immune responses are produced.
Chitosans are derivatives of chitin or poly-N-acetyl-D-glucosamine in which the greater proportion of the N-acetyl groups have been removed through hydrolysis.
Chitosans have previously been used in pharmaceutical formulations and are disclosed in EP-A-0460020 as mucosal absorption enhancers, However, EP-A-0460020 does not disclose or suggest that the chitosan could provide an adjuvant effect when administered in a vaccine composition.
Accordingly, in a first aspect, the invention provides a vaccine composition in the form of a kit comprising a first container containing an antigenic preparation comprising influenza antigen or antigens; and a second container containing an effective adjuvant amount of a chitosan.
The influenza antigens can be, for example, inactivated whole influenza virus, or purified or partially purified antigens. In an alternative embodiment, the influenza antigens can be present in a carrier molecule, for example as part of a fusion protein.
The antigenic preparation preferably contains haemagglutinin and neuraminidase influenza virus antigens. More particularly, the antigenic preparation preferably is inactivated whole influenza virus (WIV) or a composition containing purified haemagglutinin and neuraminidase influenza virus antigens.
Where a purified haemagglutinin and meuraminidase composition is employed, it is preferred that the purified haemagglutinin and neuraminidase antigens are present in the form of rosettes. The rosettes preferably are particles with a radius in the range 10 to 25 nanometres. It is preferred that the rosettes are substantially free of lipid and, moreover, it is preferred that the purified haemagglutinin and neuraminidase antigen preparation as a whole is substantially free of lipids.
An example of a haemagglutinin/neuraminidase preparation suitable for use in the compositions of the present invention is the "Fluvirin" product manufactured and sold by Evans Medical Limited of Speke, Merseyside, United Kingdom, and see also S.
Renfrey and A. Watts, Vaccine, 1994, Volume 12, Number 8, pp 747-752.
Whole influenza virus vaccines can be prepared from killed influenza virus strains in known fashion. As will be appreciated, such inactivated whole virus preparations are a precursor to the purified preparations described above.
The vaccine compositions of the invention are preferably adapted for delivery to a mucosal surface, and most preferably are adapted for oral or intranasal delivery.
Preferably the chitosan used in the compositions of the invention is water-soluble.
Preferably the chitosan is present in the second container in a solution which has a pH in the range of 5.5 to 6.5.
The chitosan may advantageously be a deacetylated chitin which is at least 75% and preferably is at least 80% deacetylated. More preferably the chitosan is at least 85% de-acetylated, and particularly preferably is 88% to 90% de-acetylated.
A particular de-acetylated chitosan is the "Sea Cure G210" chitosan (Poly D-glucosamine hydroglutamate) available from Pronova Biopolymer of Drammen, Norway. This chitosan which is in the form of its glutamate derivative, has > 80% deacetylation.
In a further aspect, the invention provides a method of immunising a host against infection with influenza, which method comprises administering to the host:
(i) from a first container an antigenic preparation comprising an influenza virus antigen or antigens as hereinbefore defined and (ii) from a second container an effective adjuvant amount of a chitosan as hereinbefore defined.
The contents of the first and second containers can be administered at substantially the same time, ie simultaneously or within a few minutes of each other, or they may be administered at spaced intervals. For example, the host may be pre-dosed with the chitosan and the antigenic preparation administered subsequently.
The first and second containers can be entirely separate or can be constituted by separate chambers of the same applicator device. Where the containers are separate, they could be provided in the form of a kit comprising first and second aerosol dispensers, or first and second syringes, by way of example. Where the first and second containers form part of the same applicator, they could for example, be defined by two barrels of a multibarrel syringe. Such applicators containing an influenza antigenic preparation in one chamber and a chitosan composition in another chamber form a further aspect of the invention.
In a further aspect, the invention provides a method of inducing a protective mucosal immune response and a systemic immune response by administering to a patient (i) from a first container an antigenic preparation comprising an influenza virus antigen or antigens as hereinbefore defined and (ii) from a second container an effective adjuvant amount of a chitosan as hereinbefore defined.
In a still further aspect, the invention provides a method of inducing a protective IgA mucosal immune response and an IgG systemic immune response by administering to a patient (i) from a first container an antigenic preparation comprising an influenza virus antigen or antigens, and (ii) from a second container an effective adjuvant amount of a chitosan, wherein the antigenic preparation and chitosan are as hereinbefore defined.
In another further aspect, the invention provides a method of enhancing the immune response of influenza virus antigens by co-administering therewith from a separate container a chitosan as hereinbefore defined.
The compositions of the invention can be presented for administration by a number of routes but preferably they are presented for administration mucosally, and in particular intranasally. Intranasal compositions can be formulated for example as liquids or dry powders, for administration as aerosols or drops.
Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents and the like.
The present invention also contemplates the provision of means for dispensing intranasal formulations of influenza virus antigens and chitosan.
A dispensing device may, for example, take the form of an aerosol delivery system, and may be arranged to dispense only a single dose, or a multiplicity of doses. Thus the first and second containers may be adapted for use in an aerosol dispensing device.
The antigenic preparation will be administered to the patient in an amount effective to stimulate a protective immune response in the patient.
For example, the antigenic preparation may be administered to humans in one or more doses, each dose containing 1-250 microgrammes and more preferably 1-50 microgrammes of protein prepared from each virus strain.
For example, where haemagglutinin and neuraminidase preparations are prepared from three virus strains, e.g. 2 x Influenza A and 1 x Influenza B, a total dose of viral protein administered could be in the range 3-150 microgrammes.
The invention will now be illustrated, but not limited, by reference to the drawings and the following examples.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates the serum IgG anti-haemagglutinin response in mice immunised with inactivated whole influenza A/Texas (WIV). Each bar represents the log geometric mean titre of five mice. The error bars represent 1 standard error of the geometric mean.
Figure 2 illustrates the nasal IgA anti-influenza A/Texas/WIV response in mice immunised with WIV. As with Figure 1, each bar represents the geometric mean titre of four mice, and the error bars represent 1 standard error of the geometric mean.
Figures 3a and 3b illustrate the determination of nasal and pulmonary WIV specific antibody secreting cells (ASC) in mice immunised with WIV, using ELISPOT.
PREPARATION OF CHITOSAN FORMULATION
A solution of 1% chitosan glutamate, a medium viscosity de-acetylated chitin having greater than 80% deacetylation, was prepared by dissolving the chitosan glutamate in 0.5% W/V sodium chloride by stirring at room temperature for 48 to 60 hours. The pH of the resulting solution was increased to 5.8-6.0 using OA M Na2HPO4. The grade of chitosan glutamate used was a medium viscosity grade sold under the trade name "Sea Cure + G210" (Product Code 27150210), by Pronova Biopolymer of Drammen, Norway.
PREPARATION OF INFLUENZA ANTIGENIC FORMULATIONS
Purified Surface Antigen (PSA) 2A. Influenza purified surface antigen (PSA) containing Influenza A/Texas protein, available from Evans Medical Limited, Speke, Merseyside, United Kingdom, was made up in phosphate buffered saline (PBS) to give a protein concentration of approximately 1 mg/ml. The PSA
consists almost entirely of the spike protein haemagglutinin (HA), although it does contain some neuraminidase.
WHOLE INFLUENZA VACCINE (WIV) 2B. Whole influenza vaccine (WIV) of the A/Texas strain was made up to 1 mg/ml protein concentration in PBS.
Preparation of a Chitosan/Influenza Admixture A 1 :1 mixture of the chitosan glutamate solution and the PSA or WIV
solution was prepared to give an intranasal vaccine composition containing 0.5% chitosan glutamate and 0.5 mg/ml influenza antigen.
Control solutions containing the same concentrations of WIV or PSA
but not chitosan glutamate, were also prepared.
Mice Immunisation Studies The compositions prepared as described in the Examples above were administered to groups of five adult (6-8 weeks) female BALB/c mice as follows:
Group Formulation Dose/mice (total protein) A WIV + chitosan 1Ogg (separate administration) B WIV + chitosan 10/ug (admixture C PSA + chitosan 1Opg (separate administration) D PSA + chitosan 1O,ug (admixture) E WIV (A/Texas) 10lug F PSA (A/Texas) 1O/ag SUBSTITUTE SHEET (RULE 26) Immunisations were given intranasally in 20,ul volumes (10,ul per nostril) to the mice in groups B, D, E, F. The group B and D mice received the preparations described in Example 3 above, whilst the group E and F control mice received the formulations of Examples 2B and 2A respectively and no chitosan. The mice in groups A and C first received 5/11 per nostril of the chitosan composition of Example 1 and then, following a recovery time of 5 minutes, were given 5/11 per nostril of the influenza formulations of Examples 2B and 2A respectively. The immunisation and sampling regime used is shown in Table 1.
Immunisation and sampling regime Immunisation Day Sample Day At sampling points 1 and 2, all mice from each group were sample bled. Sampling at sample point 3 was performed by cardiac puncture, following which the heads of the mice were removed and their nasal passages lavaged with 1 ml PBS + 1 % bovine serum albumin.
Antibody and ELISPOT Assays The immune responses stimulated by the immunisation regimes described above were analysed in conventional fashion using the ELISPOT
and ELISA techniques. Thus, an Enzyme Linked Immunosorbant Assay (ELISA) was used to determine the serum IgG anti-A/Texas response, and the results are shown in Figure 1 and Table 2 below. The ELISA technique was also used to determine the nasal IgA anti-A/Texas response and the results of this assay are shown in Figure 2 and Table 3 below. In all assays whole influenza vaccine (WIV) was used as antigen. ELISA endpoint titres were calculated as sample dilutions yielding a result of 0.3 OD units for IgG
and 0.1 OD units for IgA assay after background control (without sample) subtraction.
RESULTS
Serum anti-WIV serum response (Table 2 and Figure 1) Group Post-Dose 1 Post-Dose 2 Post-Dose 3 Seroconversion= GMT' Seroconversion GMT Seroconversion GMT
A. WIV+chitosan 5/5 3005 5/5 29708 5/5 44338 (separate) B. WIV+chitosan 5/5 2038 5/5 41426 5/5 63157 (admixture) C. PSA+chitosan 4/5 271 5/5 9314 5/5 30599 (separate) D. PSA+chitosan 5/5 1017 5/5 69331 5/5 144224 (admixture) E. WIV (A/Texas) 5/5 499 5/5 3800 5/5 6044 F. PSA (A/Texas) 0/5 49 2/5 89 4/4 428 * No. positive/No. tested + Geometric Mean Titre Nasal IqA anti-WIV response Nasal IQA anti-A/Texas response EU/ug total IqA (Individual responses -geometric mean + sem) Group Individual IgA Geometric Mean Responses (EU/pg) (EU/pg) A. WIV+chitosan 342 351 405 240 246 310 t 65 (separate) B. WIV+chitosan 263 100 328 447 250 249 t 126 (admixture) C. PSA+chitosan 74 265 142 14 100 83 t 52 (separate) D. PSA+chitosan 283 199 289 193 490 274 t 92 (admixture) E. WIV (A/Texas) 52 89 156 67 254 104 t 61 F. PSA (A/Texas) 7 9 5 4 6 t 2 Antibody Secreting Cells (ASC) Specific for WIV in the Nasal and Pulmonary Mucosa Lymphocytes were isolated from the nasal mucosa and lung parenchyma of groups of five mice at the third sampling point. Lymphocytes from individual mice were pooled and assayed for cells secreting IgA, IgG
and IgM anti-flu antibodies using ELISPOT. The results are shown in Figures 3a and 3b. In Figures 3a and 3b, the antibody secreting cells detected by the ELISPOT assay are expressed as the number of positive cells/107 total cells (lymphocytes). Figure 3a illustrates the effect of chitosan on the nasal and pulmonary immunogenicity of the PSA preparation of Example 2A
whereas Figure 3b illustrates the effect of chitosan on the nasal and pulmonary immunogenicity of the WIV preparation of Example 2B. The results shown in Figures 3a and 3b illustrate that chitosan enhances the IgA
ASC response to both PSA and WIV, the greater enhancement being observed with PSA. The strongest ASC responses were generated in the nasal mucosa with IgA ASC being dominant. Pulmonary responses were predominantly IgG secreting cells.
In general, greater ASC responses were induced by both PSA and WIV
when they were formulated with chitosan, than when the chitosan was administered first. In the case of the IgA ASC response, the formulation containing both PSA and chitosan produced a greater response than was obtained when chitosan was administered first. However, there was no significant difference between the IgA ASC responses induced by the formulation containing both WIV and chitosan, and WIV preceded by the chitosan.
The results set out above illustrate that good immune responses can be obtained by administering influenza antigens and chitosans separately via the mucosal route. This offers an advantage in that it enables the potential problems associated with administration by injection to be avoided.
The aforementioned examples are merely exemplary of the present invention and are not intended in any way to limit the scope of the invention which is defined solely by the Claims appended hereto.
The invention relates to a vaccine composition in the form of a kit comprising a first container containing an antigenic preparation comprising influenza virus antigens and a second container containing a mucosal adjuvant. The invention also relates to a method of immunising a patient against influenza by administering the said composition to the patient, and a method of enhancing the immunogenicity of an influenza viral antigen, particularly when administered intranasally, by co-administering therewith the said adjuvant.
Current influenza vaccines consist of either inactivated whole virus, disrupted virus (split vaccines) or purified preparations of the membrane glycoproteins haemagglutinin (HA) and neuraminidase (NA) sub-unit vaccines. Haemagglutinin and neuraminidase are the antigens to which protective antibody responses are directed, haemagglutinin being the major protective antigen. Estimates of the efficacy of these parenterally administered vaccines vary greatly. Such vaccines are believed to act primarily by eliciting circulating anti-haemagglutinin IgG antibodies that transudate into the lower respiratory tract.
M.L. Clements eta/, J. Clinical Microbiology 24, 157-160,1986, have previously reported that both secretory IgA and serum lgG participate in immunity to influenza virus. Moreover, in mice, a number of published studies have demonstrated the importance of respiratory IgA to protection against influenza infection. It has also been found that an advantage of stimulating a local IgA response to influenza is that it is often of a broader specificity than the serum response and thus can provide cross-protection against viruses possessing haemagglutinin molecules different from those present in the vaccine. Accordingly, influenza vaccines that elicit both local secretory and serum anti-haemagglutinin responses should provide superior immunity to current vaccines. However, parenteral vaccination (intramuscular, sub-cutaneous etc) is not effective at eliciting local antibody production, if there has been no previous mucosal exposure (e.g. infection).
In order to stimulate the mucosal immune system, the vaccine must be applied topically to a mucosal surface.
Mucosal administration of influenza vaccine would have a number of advantages over traditional parenteral immunisation regimes. Paramount amongst these are more effective stimulation of the local mucosal immune system of the respiratory tract and the likelihood that vaccine uptake rates would be increased because the fear and discomfort associated with injections would be avoided. Accordingly, a number of attempts have been made to develop mucosal influenza vaccines. A drawback however is that inactivated vaccines are often poorly immunogenic when given mucosally.
For example, Kuno-sakai et al (vaccine 12: 1303-1310, (1994) have shown that administration of inactivated vaccine to humans gave strong mucosal and serum antibody responses and was effective in preventing infection by live vaccine virus. However, in order to achieve such results, Kuno-sakai et al administered three times the commercially available dose, an approach which is not considered to be commercially viable. In order to overcome this problem, different approaches to improving the immunogenicity of flu vaccines given orally or intranasally have included the use of the B sub-unit of cholera toxin (CTB) as an adjuvant (see S. Tamura et al, vaccine, 6, 409, (1988), encapsulation of the vaccine in a variety of microspheres (see Z.
Moldoveanu et al, J.inf.Dis. 167, 85-90 (1993), and the use of live attenuated strains (see H.F.Maassab et al, vaccines, Plotkin S.A and Mortimer F.A Jr (eds) W.B. Saunders Philadelphia p435 (1993). To date however, no practical means of enhancing the immunogenicity of mucosally administered flu vaccines has been developed.
It has now been found by the Applicants that by administering influenza antigens such as the haemagglutinin and neuraminidase antigens in combination with a particular chitosan derivative, it is possible to achieve good IgG and good IgA responses. More particularly , the present Applicants have now found that if a chitosan is co-administered intranasally with influenza antigens such as the neuraminidase and haemagglutinin antigens, or if a host is predosed with a chitosan prior to administration of influenza antigens, good systemic and local immune responses are produced.
Chitosans are derivatives of chitin or poly-N-acetyl-D-glucosamine in which the greater proportion of the N-acetyl groups have been removed through hydrolysis.
Chitosans have previously been used in pharmaceutical formulations and are disclosed in EP-A-0460020 as mucosal absorption enhancers, However, EP-A-0460020 does not disclose or suggest that the chitosan could provide an adjuvant effect when administered in a vaccine composition.
Accordingly, in a first aspect, the invention provides a vaccine composition in the form of a kit comprising a first container containing an antigenic preparation comprising influenza antigen or antigens; and a second container containing an effective adjuvant amount of a chitosan.
The influenza antigens can be, for example, inactivated whole influenza virus, or purified or partially purified antigens. In an alternative embodiment, the influenza antigens can be present in a carrier molecule, for example as part of a fusion protein.
The antigenic preparation preferably contains haemagglutinin and neuraminidase influenza virus antigens. More particularly, the antigenic preparation preferably is inactivated whole influenza virus (WIV) or a composition containing purified haemagglutinin and neuraminidase influenza virus antigens.
Where a purified haemagglutinin and meuraminidase composition is employed, it is preferred that the purified haemagglutinin and neuraminidase antigens are present in the form of rosettes. The rosettes preferably are particles with a radius in the range 10 to 25 nanometres. It is preferred that the rosettes are substantially free of lipid and, moreover, it is preferred that the purified haemagglutinin and neuraminidase antigen preparation as a whole is substantially free of lipids.
An example of a haemagglutinin/neuraminidase preparation suitable for use in the compositions of the present invention is the "Fluvirin" product manufactured and sold by Evans Medical Limited of Speke, Merseyside, United Kingdom, and see also S.
Renfrey and A. Watts, Vaccine, 1994, Volume 12, Number 8, pp 747-752.
Whole influenza virus vaccines can be prepared from killed influenza virus strains in known fashion. As will be appreciated, such inactivated whole virus preparations are a precursor to the purified preparations described above.
The vaccine compositions of the invention are preferably adapted for delivery to a mucosal surface, and most preferably are adapted for oral or intranasal delivery.
Preferably the chitosan used in the compositions of the invention is water-soluble.
Preferably the chitosan is present in the second container in a solution which has a pH in the range of 5.5 to 6.5.
The chitosan may advantageously be a deacetylated chitin which is at least 75% and preferably is at least 80% deacetylated. More preferably the chitosan is at least 85% de-acetylated, and particularly preferably is 88% to 90% de-acetylated.
A particular de-acetylated chitosan is the "Sea Cure G210" chitosan (Poly D-glucosamine hydroglutamate) available from Pronova Biopolymer of Drammen, Norway. This chitosan which is in the form of its glutamate derivative, has > 80% deacetylation.
In a further aspect, the invention provides a method of immunising a host against infection with influenza, which method comprises administering to the host:
(i) from a first container an antigenic preparation comprising an influenza virus antigen or antigens as hereinbefore defined and (ii) from a second container an effective adjuvant amount of a chitosan as hereinbefore defined.
The contents of the first and second containers can be administered at substantially the same time, ie simultaneously or within a few minutes of each other, or they may be administered at spaced intervals. For example, the host may be pre-dosed with the chitosan and the antigenic preparation administered subsequently.
The first and second containers can be entirely separate or can be constituted by separate chambers of the same applicator device. Where the containers are separate, they could be provided in the form of a kit comprising first and second aerosol dispensers, or first and second syringes, by way of example. Where the first and second containers form part of the same applicator, they could for example, be defined by two barrels of a multibarrel syringe. Such applicators containing an influenza antigenic preparation in one chamber and a chitosan composition in another chamber form a further aspect of the invention.
In a further aspect, the invention provides a method of inducing a protective mucosal immune response and a systemic immune response by administering to a patient (i) from a first container an antigenic preparation comprising an influenza virus antigen or antigens as hereinbefore defined and (ii) from a second container an effective adjuvant amount of a chitosan as hereinbefore defined.
In a still further aspect, the invention provides a method of inducing a protective IgA mucosal immune response and an IgG systemic immune response by administering to a patient (i) from a first container an antigenic preparation comprising an influenza virus antigen or antigens, and (ii) from a second container an effective adjuvant amount of a chitosan, wherein the antigenic preparation and chitosan are as hereinbefore defined.
In another further aspect, the invention provides a method of enhancing the immune response of influenza virus antigens by co-administering therewith from a separate container a chitosan as hereinbefore defined.
The compositions of the invention can be presented for administration by a number of routes but preferably they are presented for administration mucosally, and in particular intranasally. Intranasal compositions can be formulated for example as liquids or dry powders, for administration as aerosols or drops.
Compositions for administration as nasal drops may contain one or more excipients of the type usually included in such compositions, for example preservatives, viscosity adjusting agents, tonicity adjusting agents, buffering agents and the like.
The present invention also contemplates the provision of means for dispensing intranasal formulations of influenza virus antigens and chitosan.
A dispensing device may, for example, take the form of an aerosol delivery system, and may be arranged to dispense only a single dose, or a multiplicity of doses. Thus the first and second containers may be adapted for use in an aerosol dispensing device.
The antigenic preparation will be administered to the patient in an amount effective to stimulate a protective immune response in the patient.
For example, the antigenic preparation may be administered to humans in one or more doses, each dose containing 1-250 microgrammes and more preferably 1-50 microgrammes of protein prepared from each virus strain.
For example, where haemagglutinin and neuraminidase preparations are prepared from three virus strains, e.g. 2 x Influenza A and 1 x Influenza B, a total dose of viral protein administered could be in the range 3-150 microgrammes.
The invention will now be illustrated, but not limited, by reference to the drawings and the following examples.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates the serum IgG anti-haemagglutinin response in mice immunised with inactivated whole influenza A/Texas (WIV). Each bar represents the log geometric mean titre of five mice. The error bars represent 1 standard error of the geometric mean.
Figure 2 illustrates the nasal IgA anti-influenza A/Texas/WIV response in mice immunised with WIV. As with Figure 1, each bar represents the geometric mean titre of four mice, and the error bars represent 1 standard error of the geometric mean.
Figures 3a and 3b illustrate the determination of nasal and pulmonary WIV specific antibody secreting cells (ASC) in mice immunised with WIV, using ELISPOT.
PREPARATION OF CHITOSAN FORMULATION
A solution of 1% chitosan glutamate, a medium viscosity de-acetylated chitin having greater than 80% deacetylation, was prepared by dissolving the chitosan glutamate in 0.5% W/V sodium chloride by stirring at room temperature for 48 to 60 hours. The pH of the resulting solution was increased to 5.8-6.0 using OA M Na2HPO4. The grade of chitosan glutamate used was a medium viscosity grade sold under the trade name "Sea Cure + G210" (Product Code 27150210), by Pronova Biopolymer of Drammen, Norway.
PREPARATION OF INFLUENZA ANTIGENIC FORMULATIONS
Purified Surface Antigen (PSA) 2A. Influenza purified surface antigen (PSA) containing Influenza A/Texas protein, available from Evans Medical Limited, Speke, Merseyside, United Kingdom, was made up in phosphate buffered saline (PBS) to give a protein concentration of approximately 1 mg/ml. The PSA
consists almost entirely of the spike protein haemagglutinin (HA), although it does contain some neuraminidase.
WHOLE INFLUENZA VACCINE (WIV) 2B. Whole influenza vaccine (WIV) of the A/Texas strain was made up to 1 mg/ml protein concentration in PBS.
Preparation of a Chitosan/Influenza Admixture A 1 :1 mixture of the chitosan glutamate solution and the PSA or WIV
solution was prepared to give an intranasal vaccine composition containing 0.5% chitosan glutamate and 0.5 mg/ml influenza antigen.
Control solutions containing the same concentrations of WIV or PSA
but not chitosan glutamate, were also prepared.
Mice Immunisation Studies The compositions prepared as described in the Examples above were administered to groups of five adult (6-8 weeks) female BALB/c mice as follows:
Group Formulation Dose/mice (total protein) A WIV + chitosan 1Ogg (separate administration) B WIV + chitosan 10/ug (admixture C PSA + chitosan 1Opg (separate administration) D PSA + chitosan 1O,ug (admixture) E WIV (A/Texas) 10lug F PSA (A/Texas) 1O/ag SUBSTITUTE SHEET (RULE 26) Immunisations were given intranasally in 20,ul volumes (10,ul per nostril) to the mice in groups B, D, E, F. The group B and D mice received the preparations described in Example 3 above, whilst the group E and F control mice received the formulations of Examples 2B and 2A respectively and no chitosan. The mice in groups A and C first received 5/11 per nostril of the chitosan composition of Example 1 and then, following a recovery time of 5 minutes, were given 5/11 per nostril of the influenza formulations of Examples 2B and 2A respectively. The immunisation and sampling regime used is shown in Table 1.
Immunisation and sampling regime Immunisation Day Sample Day At sampling points 1 and 2, all mice from each group were sample bled. Sampling at sample point 3 was performed by cardiac puncture, following which the heads of the mice were removed and their nasal passages lavaged with 1 ml PBS + 1 % bovine serum albumin.
Antibody and ELISPOT Assays The immune responses stimulated by the immunisation regimes described above were analysed in conventional fashion using the ELISPOT
and ELISA techniques. Thus, an Enzyme Linked Immunosorbant Assay (ELISA) was used to determine the serum IgG anti-A/Texas response, and the results are shown in Figure 1 and Table 2 below. The ELISA technique was also used to determine the nasal IgA anti-A/Texas response and the results of this assay are shown in Figure 2 and Table 3 below. In all assays whole influenza vaccine (WIV) was used as antigen. ELISA endpoint titres were calculated as sample dilutions yielding a result of 0.3 OD units for IgG
and 0.1 OD units for IgA assay after background control (without sample) subtraction.
RESULTS
Serum anti-WIV serum response (Table 2 and Figure 1) Group Post-Dose 1 Post-Dose 2 Post-Dose 3 Seroconversion= GMT' Seroconversion GMT Seroconversion GMT
A. WIV+chitosan 5/5 3005 5/5 29708 5/5 44338 (separate) B. WIV+chitosan 5/5 2038 5/5 41426 5/5 63157 (admixture) C. PSA+chitosan 4/5 271 5/5 9314 5/5 30599 (separate) D. PSA+chitosan 5/5 1017 5/5 69331 5/5 144224 (admixture) E. WIV (A/Texas) 5/5 499 5/5 3800 5/5 6044 F. PSA (A/Texas) 0/5 49 2/5 89 4/4 428 * No. positive/No. tested + Geometric Mean Titre Nasal IqA anti-WIV response Nasal IQA anti-A/Texas response EU/ug total IqA (Individual responses -geometric mean + sem) Group Individual IgA Geometric Mean Responses (EU/pg) (EU/pg) A. WIV+chitosan 342 351 405 240 246 310 t 65 (separate) B. WIV+chitosan 263 100 328 447 250 249 t 126 (admixture) C. PSA+chitosan 74 265 142 14 100 83 t 52 (separate) D. PSA+chitosan 283 199 289 193 490 274 t 92 (admixture) E. WIV (A/Texas) 52 89 156 67 254 104 t 61 F. PSA (A/Texas) 7 9 5 4 6 t 2 Antibody Secreting Cells (ASC) Specific for WIV in the Nasal and Pulmonary Mucosa Lymphocytes were isolated from the nasal mucosa and lung parenchyma of groups of five mice at the third sampling point. Lymphocytes from individual mice were pooled and assayed for cells secreting IgA, IgG
and IgM anti-flu antibodies using ELISPOT. The results are shown in Figures 3a and 3b. In Figures 3a and 3b, the antibody secreting cells detected by the ELISPOT assay are expressed as the number of positive cells/107 total cells (lymphocytes). Figure 3a illustrates the effect of chitosan on the nasal and pulmonary immunogenicity of the PSA preparation of Example 2A
whereas Figure 3b illustrates the effect of chitosan on the nasal and pulmonary immunogenicity of the WIV preparation of Example 2B. The results shown in Figures 3a and 3b illustrate that chitosan enhances the IgA
ASC response to both PSA and WIV, the greater enhancement being observed with PSA. The strongest ASC responses were generated in the nasal mucosa with IgA ASC being dominant. Pulmonary responses were predominantly IgG secreting cells.
In general, greater ASC responses were induced by both PSA and WIV
when they were formulated with chitosan, than when the chitosan was administered first. In the case of the IgA ASC response, the formulation containing both PSA and chitosan produced a greater response than was obtained when chitosan was administered first. However, there was no significant difference between the IgA ASC responses induced by the formulation containing both WIV and chitosan, and WIV preceded by the chitosan.
The results set out above illustrate that good immune responses can be obtained by administering influenza antigens and chitosans separately via the mucosal route. This offers an advantage in that it enables the potential problems associated with administration by injection to be avoided.
The aforementioned examples are merely exemplary of the present invention and are not intended in any way to limit the scope of the invention which is defined solely by the Claims appended hereto.
Claims (19)
1. A kit for providing an influenza vaccine, said kit comprising:
a first container containing an antigenic preparation comprising influenza antigen or antigens; and a second container containing an effective adjuvant amount of a chitosan, wherein the chitosan is a deacetylated chitin which is at least 80%
deacetylated.
a first container containing an antigenic preparation comprising influenza antigen or antigens; and a second container containing an effective adjuvant amount of a chitosan, wherein the chitosan is a deacetylated chitin which is at least 80%
deacetylated.
2. A kit according to claim 1, wherein the antigenic preparation in the first container comprises haemagglutinin and neuraminidase influenza antigens.
3. A kit according to claim 2, wherein the antigenic preparation in the first container is a whole influenza virus preparation.
4. A kit according to claim 2, wherein the antigenic preparation in the first container comprises purified haemagglutinin and neuraminidase influenza antigens.
5. A kit according to claim 4, wherein the purified haemagglutinin and neuraminidase influenza antigens are present in the form of rosettes having a radius in the range 10 to 25 nanometres.
6. A kit according to any one of claims 1 to 5, wherein the antigenic preparation and the chitosan are adapted for mucosal administration.
7. A kit according to any one of claims 1 to 6, wherein the chitosan is at least 85% deacetylated.
8. A kit according to claim 7, wherein the chitosan is 88% to 90%
deacetylated.
deacetylated.
9. A kit according to any one of claims 1 to 8, wherein the chitosan is water-soluble.
10. A kit according to any one of claims 1 to 9, wherein the chitosan is present in the second container in a solution which has a pH in the range of 5.5 to 6.5.
11. A kit according to claim 10, wherein the pH of the chitosan solution is approximately 5.8 to 6.
12. A kit according to any one of claims 1 to 1 1, wherein the first and second containers are constituted by separate chambers in the same dispensing device.
13. A kit according to claim 12, wherein the first and second containers are constituted by two barrels of a multi-barrel syringe.
14. A kit according to any one of claims 1 to 13, wherein the antigenic preparation and the chitosan are adapted for intranasal administration.
15. A kit according to any one of claims 1 to 1 3, wherein the antigenic preparation and the chitosan are adapted for oral administration.
16. A kit according to any one of claims 1 to 14, wherein the first and second containers are adapted to cooperate with a dispenser for administering the contents thereof intranasally.
17. A pharmaceutical product for providing an intranasal influenza vaccine, said product comprising a dispensing device adapted to deliver a composition intranasally, in combination with a kit as defined in any one of claims 1 to 14.
18. A pharmaceutical product according to claim 17, wherein the dispensing device is an aerosol delivery system.
19. The use of a chitosan for the manufacture of an adjuvant composition in one container for enhancing the immune response of influenza virus antigens formulated for co-administration therewith from a separate container, the chitosan being a deacetylated chitin which is at least 80%
deacetylated.
deacetylated.
Applications Claiming Priority (3)
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| GB9522351.7 | 1995-11-01 | ||
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| PCT/GB1996/002680 WO1997016208A1 (en) | 1995-11-01 | 1996-11-01 | Influenza vaccine compositions |
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| CA2236538A1 CA2236538A1 (en) | 1997-05-09 |
| CA2236538C true CA2236538C (en) | 2011-05-10 |
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| EP (1) | EP0859632B1 (en) |
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| GB9525083D0 (en) | 1995-12-07 | 1996-02-07 | Danbiosyst Uk | Vaccine compositions |
| GB9725084D0 (en) | 1997-11-28 | 1998-01-28 | Medeva Europ Ltd | Vaccine compositions |
| GB9914412D0 (en) * | 1999-06-22 | 1999-08-18 | Worrall Eric E | Method for the preservation of viruses,bacteria and biomolecules |
| US20020177614A1 (en) * | 2001-03-23 | 2002-11-28 | Merril Carl R. | Methods for treating nuerodegenerative diseases including alzheimer's |
| NZ530635A (en) * | 2001-06-15 | 2007-03-30 | Nordic Vaccine Technology As | Therapeutical vaccination comprising an immunogen and a targeting vehicle comprising a carrier and a antigen silmilar to the first |
| BR0214279A (en) * | 2001-11-19 | 2005-12-20 | Becton Dickinson Co | Pharmaceutical compositions in particulate form |
| WO2003086454A1 (en) * | 2002-04-18 | 2003-10-23 | Akzo Nobel N.V. | Antigen-loaded chitosan microparticles for oral vaccination |
| MXPA05011644A (en) * | 2003-04-30 | 2005-12-15 | Nastech Pharm Co | CLAUDINSaCOE UNDEREXPRESSION AS MARKERS OF TUMOR METASTASIS. |
| US20050123550A1 (en) * | 2003-05-12 | 2005-06-09 | Laurent Philippe E. | Molecules enhancing dermal delivery of influenza vaccines |
| US7588774B2 (en) * | 2003-05-12 | 2009-09-15 | Becton, Dickinson And Company | Molecules enhancing dermal delivery of influenza vaccines |
| GB0315632D0 (en) | 2003-07-04 | 2003-08-13 | West Pharm Serv Drug Res Ltd | Pharmaceutical formulations |
| BRPI0417225A (en) * | 2003-12-05 | 2007-03-06 | Becton Dickinson Co | methods of enhancing the intradermal compartment immune response and compounds useful in said methods |
| US20050129679A1 (en) * | 2003-12-15 | 2005-06-16 | Nastech Pharmaceutical Company Inc. | Method for opening tight junctions |
| JPWO2006019183A1 (en) * | 2004-08-18 | 2008-05-08 | 国立大学法人徳島大学 | Mucosal immunity, mucosal vaccine and immunization system using the sensitizer |
| US20060121055A1 (en) * | 2004-12-06 | 2006-06-08 | Becton, Dickinson And Company, Inc. | Compositions with enhanced immunogenicity |
| CN100425288C (en) * | 2005-01-28 | 2008-10-15 | 北京金迪克生物技术研究所 | Nasal cavity spraying inactivated influenza virus vaccine and its prepn process |
| CA2552596A1 (en) * | 2005-08-09 | 2007-02-09 | Solvay Pharmaceuticals B.V. | Methods and systems for determining mid-value titers |
| EP2021483B1 (en) * | 2006-05-05 | 2016-01-06 | Temasek Life Sciences Laboratory Limited | Biomolecule surface display and uses thereof |
| WO2008118970A2 (en) | 2007-03-27 | 2008-10-02 | Sea Lane Biotechnologies, Llc | Constructs and libraries comprising antibody surrogate light chain sequences |
| AU2009228058A1 (en) * | 2008-03-28 | 2009-10-01 | Sea Lane Biotechnologies, Llc | Neutralizing molecules to viral antigens |
| AU2010249046A1 (en) | 2009-05-13 | 2011-12-01 | Sea Lane Biotechnologies, Llc | Neutralizing molecules to influenza viruses |
| US9849173B2 (en) | 2009-07-06 | 2017-12-26 | Variation Biotechnologies Inc. | Methods for preparing vesicles and formulations produced therefrom |
| WO2011005772A1 (en) | 2009-07-06 | 2011-01-13 | Variation Biotechnologies, Inc. | Methods for preparing vesicles and formulations produced therefrom |
| EP2308506A1 (en) * | 2009-10-02 | 2011-04-13 | Mucosis B.V. | Adjuvanted intranasal vaccine formulations |
| US9610248B2 (en) | 2010-07-06 | 2017-04-04 | Variation Biotechnologies, Inc. | Compositions and methods for treating influenza |
| CN103501811B (en) | 2011-01-13 | 2016-03-30 | 变异生物技术公司 | Be used for the treatment of compositions and the method for viral infection |
| MX357319B (en) | 2011-01-13 | 2018-07-04 | Variation Biotechnologies Inc | Methods for preparing vesicles and formulations produced therefrom. |
| CA2842860A1 (en) | 2011-07-28 | 2013-01-31 | Sea Lane Biotechnologies, Llc | Sur-binding proteins |
| US20140328876A1 (en) | 2011-11-18 | 2014-11-06 | Variation Biotechnologies Inc. | Synthetic derivatives of mpl and uses thereof |
| EP2793940B1 (en) | 2011-12-22 | 2018-11-14 | i2 Pharmaceuticals, Inc. | Surrogate binding proteins |
| AU2013208693B2 (en) | 2012-01-12 | 2017-12-07 | Variation Biotechnologies Inc. | Compositions and methods for treating viral infections |
| AU2013213345A1 (en) | 2012-01-27 | 2014-08-28 | Variation Biotechnologies, Inc. | Methods and compositions for therapeutic agents |
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| JPS57136528A (en) * | 1981-02-09 | 1982-08-23 | Hayashibara Biochem Lab Inc | Preparation of viral vaccine |
| CA1261264A (en) * | 1984-11-29 | 1989-09-26 | Shigeo Suzuki | Immunopotentiating agents and method |
| US5817753A (en) * | 1985-12-04 | 1998-10-06 | The Ohio State University Research Foundation | Antigenic modification of polypeptides |
| GB8712176D0 (en) * | 1987-05-22 | 1987-06-24 | Cosmas Damian Ltd | Drug delivery system |
| GB8723846D0 (en) * | 1987-10-10 | 1987-11-11 | Danbiosyst Ltd | Bioadhesive microsphere drug delivery system |
| GB8904370D0 (en) * | 1989-02-25 | 1989-04-12 | Cosmas Damian Ltd | Liquid delivery compositions |
| DK0454044T3 (en) * | 1990-04-25 | 1996-04-22 | Hoechst Ag | Pharmacological preparation containing polyelectrolyte complexes in microparticle form and at least one active substance |
| US5169840A (en) * | 1991-03-27 | 1992-12-08 | Nobipols Forskningsstiftelse | Diequatorially bound β-1, 4 polyuronates and use of same for cytokine stimulation |
| JP3109886B2 (en) * | 1991-12-13 | 2000-11-20 | デンカ生研株式会社 | Nasal vaccine |
| GB9202464D0 (en) * | 1992-02-05 | 1992-03-18 | Danbiosyst Uk | Composition for nasal administration |
| JPH06166635A (en) * | 1992-06-18 | 1994-06-14 | San Five Kk | Immunoadjuvant |
| GB9419979D0 (en) * | 1994-10-04 | 1994-11-16 | Medeva Holdings Bv | Vaccine compositions |
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- 1996-11-01 WO PCT/GB1996/002680 patent/WO1997016208A1/en not_active Application Discontinuation
- 1996-11-01 JP JP51716097A patent/JP4010462B2/en not_active Expired - Fee Related
- 1996-11-01 CN CN96198047A patent/CN1201396A/en active Pending
- 1996-11-01 DK DK96935149T patent/DK0859632T3/en active
- 1996-11-01 AT AT96935149T patent/ATE214615T1/en active
- 1996-11-01 DE DE69620001T patent/DE69620001T2/en not_active Expired - Lifetime
- 1996-11-01 ES ES96935149T patent/ES2174112T3/en not_active Expired - Lifetime
- 1996-11-01 PT PT96935149T patent/PT859632E/en unknown
- 1996-11-01 BR BR9611218A patent/BR9611218A/en not_active IP Right Cessation
- 1996-11-01 NZ NZ320488A patent/NZ320488A/en not_active IP Right Cessation
- 1996-11-01 US US09/066,340 patent/US6136606A/en not_active Expired - Lifetime
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- 1996-11-01 CA CA2236538A patent/CA2236538C/en not_active Expired - Fee Related
- 1996-11-01 EP EP96935149A patent/EP0859632B1/en not_active Expired - Lifetime
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1998
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- 1998-04-30 NO NO19981963A patent/NO323555B1/en not_active IP Right Cessation
Also Published As
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| JP4010462B2 (en) | 2007-11-21 |
| CN1201396A (en) | 1998-12-09 |
| NO323555B1 (en) | 2007-06-11 |
| DE69620001D1 (en) | 2002-04-25 |
| EP0859632B1 (en) | 2002-03-20 |
| CA2236538A1 (en) | 1997-05-09 |
| EP0859632A1 (en) | 1998-08-26 |
| JPH11515023A (en) | 1999-12-21 |
| US6136606A (en) | 2000-10-24 |
| ATE214615T1 (en) | 2002-04-15 |
| KR19990067255A (en) | 1999-08-16 |
| ES2174112T3 (en) | 2002-11-01 |
| PT859632E (en) | 2002-09-30 |
| WO1997016208A1 (en) | 1997-05-09 |
| AU7322796A (en) | 1997-05-22 |
| GB9522351D0 (en) | 1996-01-03 |
| BR9611218A (en) | 1999-05-25 |
| KR100555687B1 (en) | 2007-06-08 |
| DK0859632T3 (en) | 2002-06-03 |
| MX9803290A (en) | 1998-09-30 |
| DE69620001T2 (en) | 2002-11-21 |
| NZ320488A (en) | 2001-03-30 |
| NO981963L (en) | 1998-06-30 |
| NO981963D0 (en) | 1998-04-30 |
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