WO2011005183A1 - New composition - Google Patents
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- WO2011005183A1 WO2011005183A1 PCT/SE2010/050798 SE2010050798W WO2011005183A1 WO 2011005183 A1 WO2011005183 A1 WO 2011005183A1 SE 2010050798 W SE2010050798 W SE 2010050798W WO 2011005183 A1 WO2011005183 A1 WO 2011005183A1
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- vaccine
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- 229940115920 streptococcus dysgalactiae Drugs 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- AWDRATDZQPNJFN-VAYUFCLWSA-N taurodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 AWDRATDZQPNJFN-VAYUFCLWSA-N 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 125000000341 threoninyl group Chemical class [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 230000029069 type 2 immune response Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 241000724775 unclassified viruses Species 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 239000012646 vaccine adjuvant Substances 0.000 description 1
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Classifications
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- C12N2760/20111—Lyssavirus, e.g. rabies virus
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Definitions
- the present invention relates to a composition comprising at least one ISCOM complex and at least one internal antigen which is not a surface antigen and not in the form of a part of a whole micro-organism. It further regards to the composition for use as an immune stimulating medicine or vaccine, especially for use in eliciting T ceil respond including CTL respond. It a!so regards a composition for use as an immune stimulating medicine or vaccine for low responders.
- the invention also relates to a composition comprising at least one ISCOM complex for use as an immune stimulating or immune modulating medicine or vaccine for the stimulation of dendritic cells in elderly.
- the invention relates to a process for preparing the composition and a kit.
- the invention encompasses ISCOM/ISCOM-Matrix formufations that are used to enhance and broaden the immune responses to enhance the leveJ and/or quality of immune responses to accessible vaccine antigens or by revealing antigens hidden in the whole microorganisms/viruses and to broaden the immune responses to non-surface antigens revealed by disintegration of intact micro-organisms and to evade immune suppression exerted by an intact microorganism incfuding viruses. It also includes stimulation of specific antibodies in various subclasses, cell mediated immune responses including Th land Th2 and cytotoxic T cell responses i.e. balanced immune responses to achieve immune protection. It also includes fast immune response for required situations.
- the invention also includes the use of ISCOM/I SCOM- Matrix adjuvant system to turning non-responding individuals to immune responders.
- Adjuvants are used to enhance vaccines either for prophylaxes or therapy.
- present vaccines need improved effect to be efficient in various vaccine fields.
- vaccine that may need improved effects are Rabies, RSV and influenza vaccines. These need improvement by reducing the number of non- and low-responders.
- Influenza virus has a strong tendency to evade the immune response evoked by vaccines of today by escape mutants.
- Rabies virus is another virus, which should function given after infection.
- RSV respiratory syncytial virus
- Other target vaccines are in the Herpes virus family.
- rabies virus infections A special use for a vaccine is in connection with rabies virus infections.
- the rabies vaccine is mostly applied after a suspected infection with the rabies virus i.e. posts infection.
- the vaccine has to induce a fast and potent immune response to precede the disease, in the case of rabies, to prevent death.
- An efficient adjuvant which concerns most kinds of vaccines, shall not only induce high levels of immune responses, but also high quality responses including antibody and ceil mediated immune responses i.e. right type of immunity to achieve immune protection.
- the specific immunity means immune response(s) directed to a specific component or components of the agent against which the vaccine is intended.
- the vaccine must, therefore, contain and expose that component(s), i.e. the vaccine antigen(s), which might be a protein a part of protein or a carbohydrate generally linked to a protein and then named glycoprotein.
- the antigens selected for the conventional vaccines and used in conventional vaccines for induction of immune protection are generally surface proteins, which are exposed on the surface of the pathogen being target for the vaccine. With regard to protective antibodies i.e.
- CMI virus neutralizing
- CTL cytotoxic T-cells
- CMI might be even more important than antibodies for protection against intracellular parasites.
- CMI might cover cross protection to other variants/isolates than the antibody arm of the immune system covers.
- EP 0 109 942 B1 discloses ISCOM complexes produced by solubilizing microorganisms creating a mixture of solubilizing agent and eel! or microorganism fragments. Charged monomeric antigenic proteins with hydrophobic regions are complex bound to the solubilizing agent. By separating the charged monomelic antigenic proteins from the sofubNtzing agent in the presence of, or by directly transferring them to, one or more glycosides with hydrophobic and hydrophi ⁇ c regions presented in a concentration of at least the critical micelle concentration an
- immunogenic complex is produced.
- the rest of the fragments are removed before the complex according is produced, while it is being produced, or afterwards.
- These ISCOM complexes will mainly comprise surface antigens or membrane antigens that are hydrophobic and no internal antigens.
- the present invention reveals internal antigens besides externally exposed antigens of disease provoking microorganisms (pathogen) and make them immunogenic by use of ISCOM Matrix formulations.
- the pathogen might be a whoie (complete microorganism including viruses) or disintegrated microorganism.
- the whole microorganism may not expose the internal antigens to the immune system that evokes immune responses unless the adjuvant according to the invention is present.
- To broaden the immune response to include internal antigens to participate in the immune protection may also contribute to increase fast immune protection particularly after post exposure use and also to long lasting immunity.
- the present invention relates to a composition
- a composition comprising at least one ISCOM complex and at least one internal antigen which is not a surface antigen and not in the form of a part of a whole micro-organism. It further regards to the composition for use as an immune stimulating medicine or vaccine, especially for use in eliciting T cell respond including CTL respond.
- Vaccines are mostly based on whole microorganisms or subunits that promote immune responses including both antibody and T cell responses against surface structures.
- the vaccine antigens are subunits i.e. most often the surface proteins, but also
- the internal proteins are revealed by disintegrating the agent e.g. the microorganism to expose other proteins/antigens that are immunogenic and of immune protective value.
- the disintegration is e.g. done by solubilization of the agent/microorganism.
- Products from the ISCOM technology are used to enhance the immunogenicity of the accessible antigens i.e. surface antigens and the antigens revealed by the disruption of the agent against which the vaccine is prepared.
- the present invention is addressing the advantage of, besides evoking immune response to the antigens covered by conventional vaccines, also to cover internal antigens.
- These may be nucleoprotei ⁇ s or intracellular non-structurai proteins of the agents including viruses and intracellular pathogens that might be revealed e.g. in cells used as expression vectors for immune stimulation.
- a broadened effect compared to or in contrast to conventional vaccine techniques in the field is obtained by making internal antigens and intracellular antigens accessible by disrupting the pathogen including cells or by making those available for immune induction by use of the whole microorganism.
- the invention is also targeting the vaccine production process by using different methods for formulating the ISCOM components i.e. in the same sequence as disrupting the pathogen or using preformed ISCOM Matrix when preparing the final adjuvanted vaccine.
- the invention is improving vaccines by making increased number of protective vaccine antigens available i.e. broadening the immune response and by stimulation of CMI.
- the adjuvant is playing an essential role by enhancing the CMI arm but also the antibody mediated immunity of the immune system.
- ISCOM the classical ISCOM is the ISCOM-particle with antigen(s) included, physically inserted into the iSCOM structure.
- the term "ISCOM" is also used in a more general sense including both ISCOM and ISCOM Matrix type of preparations.
- ISCOM Immune response covers afso immune responses.
- Sub ⁇ nit/Component(s) are antigen, part of antigen, antigenic determinant/epitope including protein/peptide carbohydrate moieties optionally linked to i.e. glycoprotein.
- Protective immunity means alternatively immune protection alternatively immune defense or defense.
- Immune stimulation includes stimulation of immune protection.
- Variants includes subtypes, types, subzero-types and sero-types are of similar species of microorganism/virus for which a broadened immune responses are intended to involve in immune protection.
- Vaccine antigen includes whole microorganism/virus, subunit, antigen determinant, epitope etc. intended for induction of any type of immune response.
- Agents include any type of microorganism/virus products of microorganisms like toxins and allergens.
- Immune defense includes defense.
- Cross protection means immune protection to additional variants, subtypes, types, subzero- types and sero-types are of similar species of microorganism/virus that conventional vaccines do not cover by immune protection.
- Identification may be done by e.g., serological testing or nucleotide typing or any other state-of- the-art method.
- Pathogen means any type of microorganism/virus part thereof e.g. toxin or allergen.
- Parasite might include any type of microorganism/virus e.g. virus is an intracellular parasite.
- internal/intraceilular means components in virus, microorganism, bacteria! celis, eukaryote cells including mammalian cells, insect cells or yeast cells.
- Responder(s) are individual(s) responding to immunization/vaccination, while non-responder(s) are individual(s) not responding to immunization/vaccination.
- Disruption includes disintegration or any word that means breaking cell or virus membranes or taking viruses or cells apart.
- Broaden immune response means compared to conventional vaccine formulation to include additional variants, subtypes, types, subzero-types and sero-types that are of similar species of microorganism/virus for which a broadened immune response(s) are intended to involve in immune protection.
- Figure legends
- Matrix M and ISCOM formulated rabies vaccine induces high titers of antigen specific antibodies of both !gG1 and !gG2a subclasses already after primary immunization.
- Matrix M consisted of 83% Matrix A and 17% Matrix C.
- Matrix M and ISCOM formulated rabies vaccine induces higher titers of antigen specific lgG2a antibodies than the corresponding formulations without Matrix M.
- Matrix M consisted of 83% Matrix A and 17% Matrix C.
- Virus neutralizing (EL)SA) serum antibody response in mice is detected already after priming in Matrix M adjuvanted vaccine and is further enhanced after booster.
- 3A Primary response.
- 3B Secondary response after booster.
- Matrix Wl consisted of 83% Matrix A and 17% Matrix C.
- FIG. 4 Rabies virus-neutralizing antibody titers (OIE approved serum neutralization test) in Grey Fox. Eight foxes per group, age 2-4 years, were vaccinated days 0 and 28 with (Group 1) WRV + AI(OH) 3 ; (Group2) WRV+ Matrix M; ⁇ Group 3) Commercial adjuvanted Rabies vaccine (Group 4) Non-vaccinated controls. Serum samples were taken at days 0, 21 , and 42. Matrix M consisted of 83% Matrix A and 17% Matrix C. Figure 5. Protein profiles in SDS-PAGE of (A) Whole Rabies Virus (WRV) and (B) Disintegrated
- CiRV Rabies Virus
- FIG. 6 Western blot analysis of sera from mice immunized with WRV (iane 2 and 3) or DiRV (lane 4 and 5) with (lane 3 and 5) and without (iane 2 and 4) Matrix-M adjuvant. 6A, blotted against WRV of Pitman Moore strain. 6B, blotted against TS80 strain. Matrix M consisted of 83%
- Matrix M consisted of 83% Matrix A and 17% Matrix C.
- Figure 9 Virus neutralizing (ELlSA) antibody response in mice is detected already after priming in Matrix M adjuvanted vaccine and is further enhanced after booster, 9A. Primary response. 9B.
- ELSA Virus neutralizing
- Matrix M adjuvanted DiRV, WRV or Matrix M adjuvanted WRV Spleen cells collected 14 days after the second immunization were re-stimulated with purified rabies virus N-protein or WRV for
- IL-2 was measured in spleen cetl supernatants using CBA (cytometric bead array).
- mice were immunized twice s.c. with DiRV or Matrix M adjuvanted DiRV, WRV or Matrix M adjuvanted WRV. Spieen cells collected 14 days after the second immunization were re-stimulated with purified rabies virus N-protein or WRV for 72 hours. IFN- ⁇ was measured in spleen cell supernatants using CBA (cytometric bead array).
- mice DiRV with or without Matrix M adjuvant.
- Balb/c mice were immunized twice s.c. with DiRV or Matrix M adjuvanted DiRV, WRV or Matrix M adjuvanted WRV.
- Spleen ceils collected 14 days after the second immunization were re-stimulated with purified rabies virus N-protein or WRV for
- IL-4 was measured in spleen cell supernatants using CBA (cytometric bead array).
- Figure 13 IL-5 response after re-stimulation of spleen cell from mice vaccinated with WRV or DiRV with or without Matrix M adjuvant. Balb/c mice were immunized twice s.c. with DiRV or Matrix M adjuvanted DiRV, WRV or Matrix M adjuvanted WRV. Spleen cells collected 14 days after the second immunization were re-stimulated with purified rabies virus N-protein or WRV for 72 hours. IL-5 was measured in spleen cell supernatants using CBA (cytometric bead array). Figure 14.
- Antibody responses in Balb/c mice to D-FIu antigens with or without Matrix M adjuvant following one (A, C) or two (B, D) s.c. immunizations four weeks apart.
- a and B Primatry and secondary IgG 1 response.
- C and D Primary and secondary lgG2a response.
- the antibody responses are measured against H1 N1 component (A/New Ca!edonia/20/99) in the vaccines.
- Matrix M consisted of 90% Matrix A and 10 % Matrix C.
- FIG. 15 Adjuvant effect of Matrix M on immunization with RSV, enhancement of VN antibody levels in serum.
- Cotton Rats were vaccinated with; 1 ⁇ g (filled circles) or 5 ⁇ g (filled squares) of DiRSV adjuvanted with Matrix M.
- the Matrix M consisted of 83% Matrix A and 17 % Matrix C. Two control groups were included; triangles top up were infected with live virus at day 0 and triangles top down were untreated controls until challenge at day 46.
- Matrix-M adjuvanted DiRSV induces immune protection in cotton rat human RSV model by reduction of virus replication in upper respiratory tract and iungs. Grey filled squares represent the response in Nasal washes whereas the dotted squares represent the response in Lung lavage.
- FIG. 17 Proportion (%) of CD 83 cells following stimulation with Matrix M adjuvant in an ex vivo human DC mode).
- the cells were stimulated with 200, 100 and 10 mg of Matrix A; 10, 1 and 0,1 mg of Matrix C and with 100, 10 and 1 mg of Matrix M.
- the matrix M consisted of 87% Matrix A and 17 % Matrix C.
- FIG. 1 Proportion (%) of CD 86 cells following stimulation following stimulation with Matrix adjuvant in an ex vivo human DC model.
- the cells were stimulated with 200, 100 and 10 mg of Matrix A; 10, 1 and 0, 1 mg of Matrix C and with 100, 10 and 1 mg of Matrix M.
- the matrix M consisted of 87% Matrix A and 17 % Matrix C.
- iDCs Immature DCs obtained after culture of monocytes for 5 days with GM-CSF and !L-4
- Figure 21 An ISCOM adjuvanted Neospora vaccine formulation induced potent antibody response in calves. All animals were challenged by infection with live Neospora at week 11.
- Group B - Calves were immunized s.c. with 500 mg disintegrated Neospora formulated with
- Group C- Calves were immunized s.c. with 500 mg disintegrated Neospora at days 042.
- FIG. 22 An ISCOM Matrix adjuvanted Neospora vaccine formulation induced potent IFN- ⁇ response in calves that was not down regulated by a subsequent infection
- Group B - Calves were immunized s.c. with 500 mg disintegrated Neospora formulated with
- Group C- Calves were immunized s.c. with 500 mg disintegrated Neospora at days 0 42.
- FIG. 23 Kinetics of mean IgG levels in serum (A) and milk (B) of Heifers immunized with S. A.
- Bacterin (whole killed bacteria) adjuvanted with Matrix Q or Al(OH) 3 . Serum samples and milk sera were diluted 1/5000 and 1/500 respectively in PBS for ELISA.
- Figure 24 Kinetics of mean IgG serum titers of experimental groups vaccinated with two different formulations, S.A. Bacterin and S.A. Lysate. A group given placebo (vaccine diluent) was included. The sera were analyzed against Bacterin ( Figure 24 A) or Bacterial Lysate ( Figure 24 B).
- the present invention reveals internal antigens besides externally exposed antigens of disease provoking microorganisms (pathogen) and make them immunogenic by use of ISCOM Matrix formuiations.
- the pathogen might be a whole (complete microorganism including viruses) or disintegrated microorganism.
- the examples show that disintegration reveals hidden antigens.
- an ISCOM formulation e.g. ISCOM or iSCOM matrix the "hidden" antigens are recognized because small amounts of antigens are enough for induction of immune response.
- a whole microorganism may not expose the internal antigens to the immune system that evokes immune responses unless the adjuvant according to the invention is present.
- Example 7b it is obvious that disintegration reveals internal antigens of Staphylcoccus aureus but also that the iSCOM formulation improves the immune response if the whole bacterin is used instead of a disintegrated bacteria.
- the invention relates to a composition
- a composition comprising at least one ISCOM complex and at least one internal antigen which is not a surface antigen.
- the internal antigen is not in the form of a part of a whole micro- organism.
- the internal antigen is in form of a whole microorganism. Therefore, the invention also relates to whole pathogens where the ISCOM complex increase the immunogenicity of whole pathogens/microorganisms considerably over present available vaccines including capacity to enhance internal antigens to evoke immune responses.
- the internal antigen may include (be one or more) nucleoproteins, polymerase or, it may be a member of the group of components obtained after disintegrating a whole micro-organism.
- composition according to the invention may comprise a whole or disintegrated whole micro-organism and at least one ISCOM complex.
- Such disintegrated slurry of whole microorganism will contain and expose internal antigenic components which may be a protein, a part of protein or a glycoprotein comprising a carbohydrate linked to a protein.
- the internal antigen may be a purified internal antigen, which is not a surface epitope or surface antigen and which is not a membrane protein with surface epitope(s).
- the internal antigen may be an antigen which is not reachable from the surface of the micro-organism. It could be an internally faced antigenic part of a membrane protein.
- Disintegration may be performed with enzymes, detergents, solubiljzing agents or by physical force, e.g. by pressure or mechanically e.g. with beads e.g. heavy meta! beads or small glass beads, high pressure or ultrasonic methods. Examples of useful solubitizing agents for disintegration are mentioned below.
- Disintegration is conventionally used in the formulation of influenza virus to eliminate side effects, such as headache, muscle pains and nausea partly due to high levels of !FN-y.
- safety for another effect i.e. disintegration of a pathogen is the most secure way to prohibit proliferation by killing the infectious particles.
- there are methods to confirm that no complete particles are present in a suspension of microorganism/viruses e.g. by microscopy en EM ⁇ electron microscopy).
- the invention also regards a composition comprising a slurry of one or more disintegrated microorganisms and at least one ISCOM complex.
- Solubi ⁇ sing agents may be used that are compatible with pharmaceutical use or use in vaccines and which need not be deleted after integration.
- the invention also regards a composition, wherein the least one internal antigen is a member of the group of components obtained after disintegrating a micro-organism with a solubilising agent
- a composition comprising at least one solubilising agent, at least one disintegrated type of micro-organism and at least one ISCOM complex.
- the invention may further comprise other added antigens e.g. rDNA or synthetically produced antigens or any of the above mentioned antigens which may be added to the compositions.
- the solubi ⁇ sing agent in the composition may have been diluted 2-100 times after the disintegration to make up a suitable vaccine composition.
- the ISCOM and the iSCOM matrix are adjuvant components in the composition.
- the ISCOM complex is an ISCOM comprising at least one saponin, at least one lipid and at least one type of antigen substance.
- the lipid is at least a sterol such as cholesterol and optionally also phosphatidyl choline.
- This complexes may also contain one or more other immunomodulatory (adjuvant-active) substances, and may be produced as described in EP 0 109 942 B1 , EP 0 242 380 B1 and EP 0 180 564 Bl Moreover, a transport and/or passenger antigen may be used, as described in EP 9600647-3 (PCT/SE97/00289).
- the immunogenic complex constitutes an ISCOM-matrix complex and said ISCOM-matrix complex is used together with one or more antigens intended to elicit specific immune response to included antigen(s) and/or said ISCOM-matrix complex and antigens are separate entities (units) intended to be administered in mixture or separately.
- !SCOM matrix comprises at least one glycoside and at least one lipid.
- the lipid is at least a sterol such as cholesterol and optionally also phosphatidyl choline.
- the ISCOM complexes may also contain one or more other immunomodulatory (adjuvant-active) substances, not necessarily a saponin, and may be produced as described in EP 0436 620 B1.
- the ISCOM formulation or the components thereof t. e. the saponin and the lipid e.g. the phospholipid and the cholesterol may be added either during the disintegration process or after completed disintegration.
- ISCOM Matrix formulation might be supplemented as a complete adjuvant formulation i.e. ISCOM Matrix.
- the adjuvant components are preferentially quillaja saponins, crude preparations or purified Fractions not excluding other saponins like ginseng saponins or fractions thereof e.g. other adjuvant molecules like LPS/lipid A.
- the sapoin may be chosen from Quillaja Saponaria Molina fraction A, fraction B, fraction C of Quillaja Saponaria Molina, a raw fraction of Quillaja Saponaria Molina such as spicoside, fraction Q 1 VAC, QA 1-23.
- Fractions A 1 B and C of Quillaja Saponaria Molina each represent groups or families of chemically closely related molecules with definable properties. The chromatographic conditions under which they are obtained are such that the batch-to- batch reproducibility in terms of elution profile and biological activity is highly consistent.
- one saponin fraction from Quillaja Saponaria Molina is used throughout this specification and in the claims as a generic description of a semi-purified or defined saponin fraction of Quillaja Sapona ⁇ a or a substantially pure fraction, it is important that the fraction does not contain as much of any other fraction to negatively affect the good results that are obtained when the mixtures of ISCOM or ISCOM matrix comprising essentially one fraction is used.
- the saponin preparation may, if desired, include minor amounts for example up to 40% by weight, such as up to 30 % by weight, up to 25 % by weight, up to 20 % by weight, up to 15 % by weight, up to 10 % by weight, up to 7 % by weight, up to 5 % by weight, up to 2 % by weight, up to 1 % by weight, up to 0,5 % by weight up to 0,1 % by weight of other compounds such as other saponins or other adjuvant materials.
- up to 40% by weight such as up to 30 % by weight, up to 25 % by weight, up to 20 % by weight, up to 15 % by weight, up to 10 % by weight, up to 7 % by weight, up to 5 % by weight, up to 2 % by weight, up to 1 % by weight, up to 0,5 % by weight up to 0,1 % by weight of other compounds such as other saponins or other adjuvant materials.
- the saponin fractions A, B and C according to the present invention are as described in WO 96/11711 , the B3, B4 and B4b fractions as described in EP 0 436 620; the fractions QA1-23 are as described in EP 0 3632 279 B2.
- the fractions QA-1 -2-3-4-5-6-7-8-9-10-11-12-13-14-15-16- 17-18-19-20-21 , 22 and 23 of EP 0 3632 279 B2, especially QA-7, 17-18 and 21 , may be used. They are obtained as described in EP 0 3632 279 B2, especially on page 6 and in Example 1 on page 8 and 9.
- a raw fraction of Quillaja Saponaria Molina is any saponin fraction thereof substantially freed from other non saponin components. Also partly purified saponin fraction, obtained by selection or removal of defined materials may be used. Reversed phase fractions of the Quil A may also be used. Such raw fraction wherein the saponins are not separated from each other may be produced by modern separating techniques, e.g., chromatography or extraction procedures.
- Examples of raw saponin fractions from Quillaja Sapona ⁇ a Molina are fraction Q and Q-VAC and Spicoside and any fraction comprising fractions A, B and C substantially freed from other non saponin material; any fraction comprising QS 1 , 2, 3-and up to QS23 (also named QA 1-23) substantially freed from other non saponin material Q-VAC is commercially available ⁇ Nor-Feed, AS Denmark), as is Quillaja Saponaria Molina spicoside.
- Examples of raw fractions of Quillaja Sapona ⁇ a Molina are described in WO 9003182 and K Dalsgaard: Saponin Adjuvants III, Archiv fur die Automate Virusforschung 44, 243-254 (1974).
- Fractions A, B and C described in WO 96/11711 are prepared from the lipophilic fraction obtained on chromatographic separation of the crude aqueous Quillaja Sapona ⁇ a Molina extract and elution with approximately 70% acetonitrile in water to recover the lipophilic fraction. This lipophilic fraction is then separated by semi preparative HPLC with elution using a gradient of from 25% to 60% acetonitrile in acidic water. The fraction referred to herein as "Fraction A" or
- Fraction A is, or corresponds to, the fraction, which is eluted at approximately 39% acetonitrile.
- Fraction B is, or corresponds to, the fraction, which is eluted at approximately 47% acetonitrile.
- Fraction C is, or corresponds to, the fraction, which is eluted at approximately 47% acetonitrile.
- C" is, or corresponds to, the fraction, which is eluated at approximately 49% acetonitrile. According to one embodiment a raw fraction of saponins is used.
- a raw fraction of saponins may be used together with any other purified saponin fraction, e.g. the different saponin fractions mentioned above.
- an immunogenic complex for use according to the invention, comprising from 5-99% by weight of one fraction, e.g. fraction A of Quillaja Saponaria Molina and the rest up to 100% of weight of another fraction e.g. a raw saponin fraction or fraction C of Quillaja Saponaria Molina counted on the weight of fraction A and fraction C.
- one fraction e.g. fraction A of Quillaja Saponaria Molina
- another fraction e.g. a raw saponin fraction or fraction C of Quillaja Saponaria Molina counted on the weight of fraction A and fraction C.
- an immunogenic complex for use according to the invention, comprising from 40% to 99% by weight of one fraction, e.g. fraction A of Quillaja Saponaria Molina and from 1% to 60% by weight of another fraction, e.g. a raw saponin fraction or fraction C of Quillaja Saponaria Molina counted on the weight of fraction A and fraction C.
- one fraction e.g. fraction A of Quillaja Saponaria Molina
- another fraction e.g. a raw saponin fraction or fraction C of Quillaja Saponaria Molina counted on the weight of fraction A and fraction C.
- an immunogenic complex for use according to the invention, comprising from 70% to 95% by weight of one fraction e.g. fraction A of Quillaja Saponaria Molina and from 30% to 15% by weight of another fraction, e.g. a raw saponin fraction or fraction C of Quillaja Saponaria Molina counted on the weight of fraction A and fraction C.
- one fraction e.g. fraction A of Quillaja Saponaria Molina
- another fraction e.g. a raw saponin fraction or fraction C of Quillaja Saponaria Molina counted on the weight of fraction A and fraction C.
- an immunogenic complex for use according to the invention, wherein the saponin fraction from Quillaja Saponaria Molina is chosen from any one of QA 1-22.
- the composition for use according to the invention comprises at least two different immunogenic complexes chosen from ISCOM complexes and/or ISCOM-matrix complexes, each individuaJ complex comprising one saponin fraction from Quillaja Saponaria Molina, wherein the saponin fraction in one complex is different from the saponin fraction in the other complex.
- one type of substantially pure saponin fraction or a raw saponin fraction may be integrated into one ISCOM or ISCOM matrix complex or particle and another type of substantially pure saponin fraction or a raw saponin fraction may be integrated into another ISCOM or ISCOM matrix complex or particle.
- a composition or vaccine may comprise at least two types complexes or particles each type having one type of saponins integrated into physically different particles.
- Mixtures of iSCOM and /or matrix may be used in which one saponin fraction Quillaja Saponaria Molina and another saponin fraction Quillaja Saponaria Molina are separately incorporated into different ISCOM complexes or matrix. Any combinations of weight % of the different ISCOM complexes based on their content of one fraction, e.g. fraction A and another fraction, e.g. any raw saponin fraction or fraction C of Quillaja Saponaria Molina respectively may be used.
- the mixtures may comprise from, 0,1 to 99,9 by weight, 5 to 95% by weight, 10 to 90% by weight 15 to 85% by weight, 20 to 80% by weight, 25 to 75% by weight, 30 to 70% by weight, 35 to 65% by weight, 40 to 60% by weight, 45 to 55% by weight, 40 to 60%, by weight, 50 to 50% by weight, 55 to 45% by weight, 60 to 40% by weight, 65 to 35% by weight, 70 to 30% by weight, 75 to 25% by weight, 80 to 20% by weight, 85 to 15% by weight, 90 to 10% by weight, 95 to 05% by weight, 50 to 99% by weight , 60 to 90% by weight , 70 to 90% by weight, 70-99 by weight, 75 to 85% by weight%, of ISCOM complexes comprising one saponin fraction, e.g.
- fraction A of Quillaja Saponaria Molina and the rest up to 100 % in each case of interval of ISCOM complexes comprising another saponin fraction, e.g. any raw fraction, e.g. fraction C of Quillaja Saponaria Molina, counted on the content of the sum fractions A and C of Quillaja Saponaria Molina in the ISCOM complexes.
- another saponin fraction e.g. any raw fraction, e.g. fraction C of Quillaja Saponaria Molina, counted on the content of the sum fractions A and C of Quillaja Saponaria Molina in the ISCOM complexes.
- the above figures relate to combinations of any saponin fraction of Quillaja Saponaria Molina integrated into the same of different ISCOM or ISCOM matric complex or particle e. g. fraction A in combination with any of fractions C, B and a raw fraction e.g. fraction Q.
- the composition for use according to the invention comprises fraction A in combination with at least one of fractions C and Q, in the same or different ISCOM complexes and/or ISCOM-matrix complexes.
- a combination of fraction A and fraction C is used in the same or in different particles.
- Such combinations may consist of 30-70%, 80-99%, 80-95%, 80-92%, 83- 99%, 83-95%, 83-92%, of fraction A and the rest up to 100% of fraction C based on the weight of the saponin fractions in the same or in different particles.
- these saponin compositions are called Matrix M.
- compositions for use according to the invention further comprising at least one other adjuvant.
- This further andjuvant may be a saponin fraction from Quillaja Sapona ⁇ a Molina, which may not bound to or integrated into the immunogenic complex.
- Such other adjuvants and saponines or glucosides that are not integrated into the ISCOM or ISCOM matrix may be mixed into the composition.
- examples of other adjuvants that can be incorporated in the ISCOM and ISCOM matrix are any adjuvant, natural or synthetic, with desired imunomodulatory effect, e.g.
- MDP muramyl dipeptide
- DDA poly anions such as Dextran sulphate, lipopolysaccarides such as saponins (other than Quil A),
- the internal antigen may be chosen from internal components in micro-organisms such as virus, bacteria, parasites, yeast cells, eukaryotic cells, including mammalian cells, insect cells.
- bacteria examples include Escherichia, Staphylococci, e.g. Staphylococcus aureus and coagulase negative Staphylococcus, Streptococci e.g. Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus agalactiae and Streptococcus uber is Haemaophiius, e.g. H.
- influenzae Bordetella, e.g. B. pertussis, Vibrio, e.g. V. cholerae, Salmonella, e.g. S. typhi, S. paratyphi, preferably adherence factor in CoIi, e.g. pili K 88 and porin protein in e.g. Salmonella or outer membrane proteins from B. pertussis and Neisseria meningitidis.
- CoIi e.g. pili K 88
- porin protein e.g. Salmonella or outer membrane proteins from B. pertussis and Neisseria meningitidis.
- Orthomyxoviridae such as influenza A 1 B 1 C, RSV
- Paramyxoviridae especially measles virus, mumps virus, parainfluenza 1 ,2,3 and 4.viruses, canine distemper virus and rinderpest virus
- Rhabdoviridae especially rabies virus
- Retroviridae especially feline leukemia virus and bovine leukemia virus
- Herpesviridae especially Pseudorabies, Rabies, e.g.
- Bat Lyssa viruses such as the Duvenhagen strain, Rabies internal N - and P- proteins Coronaviridae, Togaviridae, such as EEE.WEE.VEE (eastern, western and Venezuela equine encephalitis), yellow fever virus, especially bovine virus diarrhea virus, and European swine fever virus Arenaviridae, Poxviridae, Bunyaviridae, Iridioviridae, especialiy African swine fever virus and among unclassified viruses, and Marburg/ Ebola virus.
- non-enveloped viruses with non-hydrophobic proteins are Picornaviridae, e.g.
- mycoplasma examples include M. pnemoniae, mycoides, bovis, suis, orale, salvarium, homtnis and fermentans.
- Protoza such as Toxoplasma, e.g. Toxoplasma gondii, Plasmodium, e.g. Plasmodium vivax, malariae, faiciparium, Generaleria parvum ovale and Filaroidae, preferably Parafilaria and Onchocerca, Entamoeba histolytica, anaplasma of various types, Schistosoma such as Schistosoma haematobium, mansoni, japonicum, and Trypanosoma, e.g. Trypanosoma gambiense, brusei or congolesi and Neospora caninum.
- Toxoplasma e.g. Toxoplasma gondii
- Plasmodium e.g. Plasmodium vivax
- malariae faiciparium
- Filaroidae preferably Parafilaria and Onchocerca
- Entamoeba histolytica anaplasma of various types
- the internal antigens derive from RSV virus, Rabies virus, influenza virus, Neospora or Staphylococcus aureus.
- the antigens are in form of disintegrated whole cells e.g. from RSV virus, Rabies virus, influenza virus, Neospora or Staphylococcus aureus, which may be present in the medium used for disintegration, which medium may be diluted as mentioned herein.
- the antigens are harbored in the whole microorganism.
- the composition may further also comprise whole micro-organisms which may be live and attenuated. These are not disintegrated.
- the antigens are in form of whole cells e.g. from RSV virus, Rabies virus, influenza virus, Neospora or Staphylococcus aureus.
- the internal antigen may be integrated into an ISCOM complex, mixed with an ISCOM matrix complex or mixed with an ISCOM complex or coupled on to an ISCOM complex or ISCOM matrix complex.
- the ISCOM complex may comprise another antigen, which could be but need not be an internal antigen and which could be a surface antigen.
- the invention further relates to the use of internal Rabies virus antigens, such as the N- and P proteins, disintegrated Rabies virus cells with or without solubilisation agent, which may be diluted and to the use of whole Rabies virus which may be attenuated.
- internal Rabies virus antigens such as the N- and P proteins
- disintegrated Rabies virus cells with or without solubilisation agent, which may be diluted
- solubilisation agent which may be diluted
- whole Rabies virus which may be attenuated.
- These may be mixed with ISCOM or iSCOM matrix as mentioned above or integrated into ISCOM complex.
- cells used for antigen production as for instance the insect cell e.g. the Spodeptera frug ⁇ perda (Sf9) expresses the vaccine antigen e.g. the nucleoprotein (NP) the non-structural (NS) protein of rabies virus.
- the vaccine antigen e.g. the nucleoprotein (NP) the non-structural (NS) protein of rabies virus.
- composition according to the invention may further also comprise non- internal antigens. These may be membrane proteins and determinants exposed on the surface of microorganisms.
- the composition may also comprise one or more additives such as pharmaceutically acceptable excipients, carriers and/or diluents.
- Suitable pharmaceuticaily acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
- the use of such media and agents for pharmaceutically active substances is weli known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- the invention also relates to a composition comprising at least one ISCOM complex and at least one internal antigen which is not a surface antigen for use as an immune stimulating medicine or vaccine.
- the invention relates to the use of the compositions described herein for the preparation of an immune stimulating medicine or vaccine.
- the compositions may be used in eliciting T cell respond including CTL respond.
- the compositions are useful as an immune stimulating medicine or vaccine for low responders. Low responders may be sick people, elderly people or juveniles.
- composition according to the invention stimulates the two arms of the immune system.
- the B-ce!l derived immune response triggering immune globulins such as igG are stimulated.
- T ceil derived immune response is triggered by e.g. IL-2 production as are the TH1 type of ceils with e.g. IFN- ⁇ production as well as the TH1 type of ceils by e.g. IL-4 and IL-5 production.
- the invention also relates to a process for preparing a composition comprising at least one ISCOM complex and at least one interna!
- antigen which is not a surface antigen and not in the form of a part of a whole micro-organism, characterized in that a saponin, cholesterol and a lipid are mixed with a lysed or disintegrated cell suspension of cells and solubilising agent without removal of any cell components, where after the soiubilising agent is removed or diluted.
- compositions may be used for administration to any type of mammal e.g. human or animal species.
- anima! species to which the formulations according to the invention may be administrated are companion animals such as cats, dogs, horses, birds such as parrots, economical important species such as cattle, e.g. bovine species, swines, sheep, goats.
- compositions may be used for prophylactic treatment as immunomodulating or - stimulating agents or vaccines.
- ISCOM matrix may be used as immunomoduiating agent e.g. for elderly.
- iSCOM matrix together with antigens or ISCOMs may be used as vaccine or as immunomodulating or - stimulating agents e.g. for elderly.
- compositions may be used for post infection treatment as vaccine.
- ISCOM matrix together with antigens or ISCOMs may be used as vaccine against rabies or influenza post infection.
- the pharmaceutical composition could be adapted to oral, parenteral, or topical use and could be administered to the patient as tablets, capsules, solutions, suspensions or the like.
- parenteral administration refers to the administration not through the alimentary canal but rather by injection through some other route, as subcutaneous, intramuscular,
- compositions could contain at least 0.1 % by weight of an active compound according to the.
- the amount of the active ingredient that is contained in such compositions is so high that a suitable dosage is obtained.
- At ieast 1 international Unit may be used for humans e.g. at least about 2 i.U. for humans and about 0,5 I. U. may be used for dogs i.e. at least 1 I. U. for dogs.
- the skilled person would know how to adopt the dose for bigger animals depending on the weight.
- Suitable components to formulate ISCOM-Matrix including quillaja saponin components, lipids, and detergent may be added to the disintegrated agent harboring harboring the antigens or added before the disintegration.
- ISCOM-Matrix or optionally iSCOMs are formed with integrated antigens when the detergent keeping quiilaja components and lipids soiubiiised is removed.
- the complex formation is completed by removal of the detergent/disintegration agent or by dilution of the mixture (agent components, detergent/disintegration agent lipid and quillaja components) so that the detergent cannot keep lipids and quillaja components soiubiiised.
- the vaccine formulation contains a complex adjuvant formulation with ISCOM Matrix, but optionally also a combination ISCOMs, ISCOM-Matrix and components including the vaccine antigens from the disrupted agent.
- the detergents or solubslizing agents may be, but not restricted to, non-ionic, ionic or Zwitterionic detergent or detergent based on gallic acid which is used in excess.
- suitable non-ionic detergents are polyglycoi esters and polyglycol ethers with aliphatic or arylaliphatic acids and alcohols. Examples of these are alkylpolyoxyethylene ethers with the general formula CnH2n
- glycosides mentioned below can also be used, especially saponin. These are, however, weak detergents and should be used together with other detergents.
- suitable ionic detergents are cholic acid detergents such as e.g. desoxycholate and choiate. Even conjugated detergents such as e.g. taurodesoxycholate, glycodesoxycholate and glycocholate can be used.
- Possible Zwitter-ionic detergents are lysolecitin and synthetic lysophospholipids. Even mixtures of the above-mentioned detergents can be used.
- Soiubi ⁇ zing can also be performed with alcohofs, organic solvents or small amphipathic molecules such as heptane-1 , 2, 3-triol, hexane-1 , 2, 3-triol, acetic acid, mixtures thereof or with detergents.
- the invention further relates to a composition comprising at least one iSCOM complex for use as an immune stimulating or immune modulating medicine or vaccine for the stimulation of in immunologically low responders such as non healthy individuals, genetically defect individuals, juveniles, infants or elderly.
- the invention especially regards the stimulation of dendritic cells of such individuas.
- ISCOM matrix without or with antigens (e.g. mixed therewith) and iSCOM complexes with antigens may be used for prophylactic treatment, vaccination or post infection treatment of elderly.
- the dendritic cells may be chosen from CD 80, CD 83, CD 86 and chimocine CCR 7.
- the elderly may be chosen from the species mentioned above e.g. a human being.
- the invention also relates to a kit comprising at least two compartments, wherein one compartment comprises an ISCOM complex comprising at least one internal antigen, which is not a surface antigen and not in the form of a part of a whole micro-organism and the other compartment comprises a prescription for use or wherein the first compartment comprises an ISCOM matrix complex and the other compartment comprises at least one internal antigen, which is not a surface antigen and not in the form of a part of a whole micro-organism .
- the at least one internal antigen in the kit may be a member of the group of components obtained after disintegrating a micro-organism.
- saponin fractions relate both to a composition comprising at least one ISCOM complex and at least one internal antigen, which is not a surface antigen, (claim 1), a method for preparing a composition comprising at least one ISCOM complex and at least one internal antigen (claim 21) a kit (claim 22) and to a composition comprising at least one ISCOM complex e.g. ISCOM matrix for use as an immune stimulating or immune modulating medicine or vaccine for the stimulation of dendritic cells in elderly (claim 24).
- a composition comprising at least one ISCOM complex and at least one internal antigen which is not a surface antigen
- ICR mice supplied by CDC were used for the vaccination and challenge experiments with rabies virus nucleoprotein (Example 1A), Balb/c mice were used for all other mouse studies.
- Cotton rats (50-100 gram) kindly provided by Dr. Pedro A. Piedra, (Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA) were used for the vaccination and challenge experiments with respiratory syncytial virus (Example 2).
- Neospora caninum Aberdeen Angus calves fifteen five months, from a beef herd located at INTA-Balcarce, Argentina kindly provided by Dr. D.P. Moorei , (Instituto Nacional de Tecnologia Agropecuaria (INTA), Argentina) were used for the vaccination experiments with Neospora caninum
- tachyzoites (Example 6). Sero-epidemiological data from year 2000 showed a low endemic prevalence of neosporosis in the experimental herd i.e. ⁇ 1%.
- the calves were allocated in dog- proof pens, calves were provided with water ad-libidum, standard hay and commercial cattle concentrate.
- Example 7 Twefve primigravid Holstein dairy heifers in the last trimester of gestation belonging to the dairy herds of INTA Rafaela Experiment Station were used in the experiment (Example 7). The animals were injected subcutaneously in the supramammary lymph node area at approximately 40 d and 14 d before expected calving date. Only animals free from S. aureus IMI and with normal udder development at 40 d before expected calving date were included in the trial.
- Viruses Rabies viruses Pitman Moore (PM) 1 ERA, TS80 and Pasteur RtV strains, propagated in VERO, cells were obtained inactivated without adjuvant. Commercial Rabies virus vaccines were obtained from the pharmacy.
- Respiratory syncytial virus Tracy strain was kindly provided by Dr. Pedro A. Piedra (Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA). The Long strain of human RS virus (ATCC VR-26) was kindly supplied by Dr Claes Orveil (Huddinge University Hospital, Swiss). RSV was propagated in HEP-2 or MA 104 cells (ECACC number
- Neospora canin ⁇ m tachyzoites of the NC-1 strain was kindly provided by D. P. Moore, In ⁇ tituto Nacional de Tecnologia Agropecuaria (INTA), Argentina.
- N. canin ⁇ m tachyzoites were propagated in VERO cells monolayer and harvested when 80% of the cells were infected. The tachozoites were released from the cells by sequential passage of the cell monolayer through 21 , 23, 25 and 27 gauge needies and subsequently washed in sterile PBS, counted with a haemocytometer and finally used either to formulate the iive inoculums or to obtain the disintegrated antigen extract.
- Preparation of experimental vaccine antigens Preparation of experimental vaccine antigens
- Spodeptera frugiperda (Sf9) cells were grown in monolayer (Reid-Sanden FL, Sumner JW,
- Rabies virus (ERA-CB20M or TS80 strain) propagated in VERO cells was purified and concentrated by conventional sucrose density ultracentrifugation. The purified concentrated virus was re-suspended in PBS. The virus antigen concentration was measured by amino-acid analysis (Aminosyraanalys laboratories Uppsala, Sweden). The concentrated rabies virus preparations were killed with 0.2% betapropioiactone (BPL) and further disintegrated by desoxychoiate treatment. The pH in the PBS was adjusted to 7.7 and sodium desoxycholate was added to a final concentration of 1.25% and Tween 80 was added to a final concentration of 0.02%. The virus was incubated for 3 hours at 20°C. The various virus preparations and vaccines were quantified in international units IU using a bioassay (EVL, Utrecht, The
- Rabies nucleo proteins were prepared from the disintegrated preparation of TS80. Lyophilized TS80 was dissolved in 1 ml ddVV, final concentration 2.76 mg/ml (18 IU/mi, 6.5 IU/mg) according to amino acid analysis. The virus was layered over 10% sucrose for density centrifugation in a Centrikon T-1075 uitracentrifuge, rotor 55:5, at 30 000 rpm for 2 h at 6 0 C. The supernatant, the sucrose gradient and the pellet were separated. The pellet was dissolved in 200 ml PBS. The protein concentration was estimated using the Bradford assay and SDS-PAGE analysis of purified protein fractions revealed the presence of proteins in the pellet fraction.
- Concentrated virus was loaded on a sucrose discontinuous gradient (10 to 40% w/v) and centrifugated at 18000 rpm in a SW55Ti rotor (Beckman) for 3 hours. Six fractions and the pellet were analyzed by Western Blot with anti-F antibody (Synagys) and culturing of virus verified the virus containing fractions. Fraction 6 (from top to bottom) and the pellet containing virus and F protein were selected for the next steps. The partially purified virus was disintegrated with 2% of b-octyl gtucoside (OG) at 37°C under soft rotation for 1 hour (in the presence of 2 ⁇ g/mL of aprotinin).
- OG b-octyl gtucoside
- Disintegrated virus was loaded on a sucrose discontinuous gradient (10 to 30% w/v) and centrifugated at 42000 rpm in a SW55Ti rotor (Beckman) for 1 hour. Five fractions were analyzed for virus and the presence of F protein by Western Blot. The 2nd fraction (from top to bottom) was selected showing high signals for F protein. SDS-PAGE stained with s ⁇ ver nitrate and Western blot showed that fractions had a complex pattern of proteins including most virus proteins it was named disintegrated virus (DiRSV). It also contained cellular proteins.
- DIRSV disintegrated virus
- influenza virus (H3N2) was obtained as a commercial non-adjuvanted vaccine.
- Live Tachyzoites (2x10 9 ) were partially purified by ge! filtration on a sephadex chromatography column (Amersham Biosciences, Uppsala, Sweden). The collected fractions were sedimented by centrifugation at 150Og. The live parasites obtained were used for immunization of animals in Group A and for challenge infection. The live parasites were further processed into a
- the parasites were suspended in 1 m! of 10 mM Tris-hydrochloride containing 2 mM of phenylmethytsulfonylfluoride (Sigma Chemical Co., St. Louis, MO, USA) and disrupted by ultrasonic treatment (Sonifier 450, Branson Ultrasonic Co., USA) in an ice-bath, and centrifuged at 10,00Og for 20 min at 4 0 C.
- the protein content of the recovered pellet was determined by the Micro BCA protein assay method (Pierce, Rockford, USA), and the supernatant aliquoted and cryo- preserved at -8O 0 C until use as disintegrated experimental vaccines and as antigen for stimulation of whole blood celts in iFN- ⁇ assay.
- Experimental vaccine formulations
- ISCOMs were prepared according to published standard technology (e.g. EP 0 109 942 B1 , EP 0 242 380 B1 and EP 0 180 564 B1). Briefly, 1 mg of recombinant Rabies N-protein, DiRV or DiRSV were mixed with 1 mg of each cholesterol and Phosphatidyl choline prepared in 20% MEGA-10 and appropriate amount of respective saponin preparation (Fraction A, Fraction C or semipurified "Quil A), The detergent was removed by dialysis. Preparations were filtered through 0,2 mm filter, and the antigen and saponin content was analysed.
- the iive tachyzoites were suspended in PBS and adjusted to 1x10 8 per calf dose in 3 ml PBS and packed in 5 ml steriie syringes.
- the live parasites were either used for immunization (Group
- Disintegrated tachyzoites were mixed to contain 500 ⁇ g
- Neospora antigen supplemented with 750 ⁇ g Matrix Q and in a final dose volume of 2 mi (Group
- the experimental vaccine consisted of a Staphylococcus aureus capsular polysaccharide type 5 strain (Reynolds). The organism was kept in frozen stocks at -8O 0 C and activated in brain heart infusion by overnight incubation at 35 0 C. One hundred ⁇ l of this culture were seeded on Tryptic soy agar added with 2% NaCI and incubated overnight at 37 0 C. The culture was washed with PBS (pH 7.4), resuspended to achieve a final concentration of 1 x 10 9 colony forming units (cfu)/ml and inactivated with 0.5% formalin for 24 hs at 37 0 C.
- PBS pH 7.4
- Sterility of this formulation was evaluated by plating 100 ⁇ l on blood agar plates by duplicate.
- the vaccine was formulated using an alum-based adjuvant system (vaccine 1) and a saponin-based Matrix Q adjuvant.
- a placebo consisting of sterile saline solution was used as control.
- Vaccine 1 Staphylococcus aureus capsular polysaccharide type 5 strain (Reynolds). The organism was kept in frozen stocks at -80 0 C and activated in brain heart infusion by overnight
- Vaccine 2 consisted of the same S. aureus capsular polysaccharide type 5 strain grown overnight under the same conditions. Following formalin inactivation, cells were washed twice with PBS, cell density was adjusted to 1 x 10 9 cfu/mL and 1 mL of culture was resuspended in 8
- Matrix M 1 (WO 2004004762), wherein compositions comprising ISCOM matrix based on fraction A and C of Quil A in physically different ISCOM matrix complex particles are described and Matrix-Q raw fraction of Quil A (WO 9003184), were supplied by Isconova AB. 0 Analyses of antibody responses
- Antibodies to rabies virus was determined by indirect ELiSA using antigens of either N-BV or whole rabiesvirus (Smith JS, Sumner JW and Ruomillat LF. Enzymen immuno assay for rabies antibody in hybridoma cultuture fluids and its application to differentiation of street and laboratory strains of rabies virus J Clin 1984; 19 267-272.)
- mice used in the experiment 1 were female 5-weeks-old females (CDC-ICR). Al! mice were tested for rabies virus neutralizing (VN) antibodies prior to use; none had rabies virus antibodies or VN antibodies at the time of inocculation. Blood was collected from the mice during the experimental immunized at two weeks interval to determine VN antibodies and anti-N-protein by ELISA.
- VN rabies virus neutralizing
- Indirect ELISA detecting IgGI and lgG2a antibodies was run according to standard protocols using HRP-labelied anti mouse IgGI and lgG2a antibody conjugates. The antigen was coated onto Nunc ELISA plates using 50 mM carbonate pH 9,6. The enzyme reaction was visualized using TMB. Rabies Virus-Neutralization antibodies
- VN-antibodies in sera from Grey Foxes were analyzed according to OiE.
- VN-ELISA an alternative in vitro test based on blocking of virus neutralizing monoclonal antibodies was used ("VN-ELISA").
- the test was run by EVL, The Netherlands according to Rooijakkers, E., Groen, J., Uittenbogarrd, J., van Herwijnen, J. & Osterhaus, A. (1996).
- HRP Horseradish peroxidase-conjugated
- the buffer was eliminated and replaced with 200 ⁇ l/well of blocking buffer (0.06 M carbonate/bicarbonate with 4% of skimmed milk (Nestle ® , Argentina) and incubated at 28 0 C for 45 min.
- the wells were washed four times with 0.01 M PBS-0.05% Tween-20 (PBS-T) plus 4% miik.
- Negative and strong positive controls (C++) sera, and serum samples were diluted 1/100 in PBS/0.75 M EDTA/EGTA (pH 6.3) plus 4% skimmed milk.
- Conjugate controls serum free
- IgG 1 ZIgG 2 rate wells were filled with 100 ⁇ l of 1/100 dilution of anti-bovine IgGi or IgG 2 mAbs (SerotecTM, Oxford, UK) in PBS-T and incubated during 30 min. Each serum was simultaneously evaluated with both mAbs in the same plate. After four washings 100 ⁇ l of anti- mouse IgG mAb conjugated to peroxidase (Jackson ® ), diluted 1/1000 was added and incubated on a shaker for 30 min. After four washings, 100 ⁇ l of 3% H 2 O 2 /0.04 M ABTS were added.
- mice Female mice (BALB/c) 10-12 weeks old, were immunized twice according to Table 3.
- mice were sacrificed and spleen removed and a single ceil suspension prepared.
- the splenocytes (5x10 s cells/well in 200 ⁇ l) were plated in 96-weli plates and were stimulated for 72 hours with either WCRV (2.5 ⁇ g/ml), or purified rabies N-protein (0.1 ⁇ g/ml), or Con A as positive control (2.5 ⁇ g/ml) or sterile RPMI- medium as negative contro!,.
- Supernatants were collected and stored in -70 0 C until analysis with cytometric bead array (CBA, BD Bioscience), in order to determine cytokine concentrations.
- Cytokines analyzed were, for T cells in general; interleukin (IL)-2 and for Thi cells; interferon (IFN)-gamma and for Th2 cells; IL-4 and IL-5. Data collection and analysis were performed on a FACSCanto flow cytometer.
- IL interleukin
- IFN interferon
- Splenocytes (2.5x10 5 cells/well in 100 ml) were stimulated with either whole inactivated rabies virus (Abs, 2.5 mg/ml, 0.5 mg/ml and 0.1 mg/ml), or rabies N protein (2.5 mg/ml, 0.5 mg/ml and 0.1 mg/ml), or rabies G protein (2.5 rrig/m!, 0.5 mg/ml and 0.1 mg/ml), or Concanavalin A (Con A) as positive control (2.5 mg/mi) or sterile RPMI culture medium as negative control, for 42 hours.
- Cellular proliferation i.e.
- DNA synthesis was measured using a BrdU-ELISA assay (coiorimetric) according to the manufacturer ' s protocol (Roche Diagnostics GmbH, Germany). Absorbance was measured on a spectrophotometer at the test wavelength 370 nm and the reference wavelength 492 nm.
- Splenocytes (5x10 s cells/well in 200 ml) were plated in 96-well plates and were stimulated with either whole inactivated rabies virus (2.5 mg/ml, 0.5 mg/ml and 0.1 mg/ml), or rabies N protein (2.5 mg/ml, 0.5 mg/ml and 0.1 mg/mi), or rabies G protein (2.5 mg/ml, 0.5 mg/ml and 0.1 mg/ml), or Con A as positive control (2.5 mg/mi) or sterile RPMI-medium as negative control, for 72 hours.
- rabies virus 2.5 mg/ml, 0.5 mg/ml and 0.1 mg/ml
- rabies N protein 2.5 mg/ml, 0.5 mg/ml and 0.1 mg/mi
- rabies G protein 2.5 mg/ml, 0.5 mg/ml and 0.1 mg/ml
- Con A as positive control (2.5 mg/mi) or sterile RPMI-medium as negative control, for
- cytometric bead array CBA
- Cytokines analyzed were, for T cells in general; interleukin (IL)-2 and for Th1 cells; interferon (IFN)-g and forTh2 cells; IL-4 and IL-5. Data collection and analysis were performed on a FACSCanto flow cytometer.
- Immune stimulation was performed as mentioned Serrano-Martinez et al., (2007). Briefly, 0.9 ml of heparinised whole blood was dispensed into each of two wells of 24-well tissue cuiture plates (Ceilstar Greiner, USA) and cultured with 0.1 m! of PBS (unstimulated control), concanava ⁇ n A (Con-A, Sigma, St. Louis, USA) at 10 ⁇ g/mi to ensure cellular ability to respond to stimulation and secrete IFN- ⁇ , and with disintegrated antigen from the N. caninum NC-1 strain (1 ⁇ g/ml). Heparinised whole blood samples were incubated in a 5% CO 2 atmosphere for 16h at 37 0 C. Plasma was harvested from each well and frozen at -20 0 C until testing. To assess IFN- ⁇ production, plasma samples were tested using a commercial ELISA kit (Bovigam iFN- ⁇ kit, CSL, Australia), according to the manufacturer's recommendations.
- Rats were immunized twice at 3 week intervals (on days 0 and 21) with the DiRSV, prepared as described above, in 1 ⁇ g or 5 ⁇ g doses adjuvanted with Matrix-M (24 ⁇ g/dose) in a total volume of 200 ⁇ L (Table 5).
- the vaccine or controls (placebo or infectious virus) were injected intramuscularly (i.m.) in volumes of 100 ⁇ Ls in each leg of the rat (see table 1 below). Animals in all groups except in group 5 were challenged infected on day 46 under lightly anaesthetize (Isoflurane) with a dose if 10 5 PFU RSV strain Tracy in 100 ⁇ L.
- Lunglavage iung & URT
- Virus isoiation & PFU
- Virus neutralization was performed according to standard and published procedures (for references see, e.g., Hu et al., Clin Exp Immunol 113 p.235, 1998).
- Group A were inoculated intravenously day 0 with 10 8 live tachzoites while animals in Groups B through E received 2 subcutaneous inoculations laterally on the neck on day 0 and the second dose was given on the other side 4 weeks later. All calves were challenged with 1x10 8 tachyzoites of NC-1 strain by intravenous inoculation at week 11. Fifteen five months old Aberdeen Angus calves, were randomly distributed into 5 experimental groups with three animais per group were observed daily throughout the experimental period.
- iDCs Immature dendritic cells
- Immature DCs were further cultured for 24h in the presence of either; medium, LPS (1 ⁇ g/mL), Matrix-A at 200 ⁇ g/mL, 100 ⁇ g/mL, 10 ⁇ g/mL, Matrix-C at 10 ⁇ g/mL, 1 ⁇ g/mL, 0,1 PgZmL 1 Matrix M at 100 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL. Thereafter, the DCs were analyzed for the following surface proteins: CD 11 c, CD14, CD83, CD86 and HLADR using monoclonal antibodies for identification and for quantification by flow cytometry (FACscan Beckton Dickinson, San Jose California, US).
- Example 1 Qualitative improvement of traditional Rabies vaccines by Matrix M adjuvant formulation and virus particle disintegration
- Rabies infection is a zoonotic fatal infection of warm-blooded animals.
- the only modus operandi for protection available for animals and man is vaccination; prophylactic to prevent disease or after expected virus exposure as post-exposure treatment together with hyper-immune serum. Post exposure treatment of animals after suspected rabies virus exposure is not allowed or practised.
- Rabies vaccines used for man and animals are similar, differing in that adjuvants i.e. Alum adjuvants (AI(OH) 3 or AIPO 3 ) are used in most animal rabies vaccines while no adjuvants are used in man.
- the present vaccines are conventional, they induce predominantly a TH2 type of response and have not faced development for the last 50 years.
- Example 1 A-D explore and demonstrate the beneficial effects of including Matrix M as adjuvant in rabies virus vaccines to induce broader protective immune responses (1a) including also internal antigens e.g., the rabies N-protein; (1b) improving magnitude and quality of antibody responses in mice (1), in Grey Fox (2), as demonstrated by Western biot analysis (3) and by improved performance of two commercially available WRV vaccines (4).
- Example 1A ISCOM formulation triggers internal rabies N-protein to induce Protective immunity
- This example was designed to explore whether an interna! virus protein adjuvanted with a potent adjuvant such as Matrix M can induce immune protection.
- a recombinant Rabies virus nucleoprotein (N-protein) produced in insect ceils transformed by Bacculovirus (see M & M) was used excluding the presence of other rabies virus components.
- the rabies N-protein formulated as ISCOMs (see M&M section) vaccine was administered (SC, IM and IP) to mice in 1 and 5 ⁇ g doses and was compared to a 25 ⁇ g dose (SC, IM) of the non-adjuvanted N-protein vaccine (see tables 1.1 and 1.2 for experimental setup and results).
- the experimental vaccines were administered days 0 and 7 for a primary immunization, being the standard for testing rabies vaccines according to the NIH test.
- the 25 ⁇ g dose of the non-adjuvanted N-protein vaccine was selected since preliminary experiments indicated that such a dose was required to detect immune protection according to the NIH test.
- the N-protein ISCOMs were also immunogenicity tested in Balb/c mice (see Table 1.3). Blood samples for sera were taken at days 14, 29, 45, 58 and 72. The sera were tested in ELiSA against the recombinant Rabies N-protein and Rabies virus. Table 1.1 Experimental setup and Protection to challenge 1 infection. Mice immunized 2 with 25 ⁇ g of non-adj ⁇ vanted Rabies N-protein.
- This example shows that protective immunity can be induced with Rabies virus N-protein provided that a potent adjuvant is used.
- additional protective mechanism(s) besides virus- neutralizing antibodies to the G-protein
- the protective immunity evoked by the N-protein ISCOM is not due to protective antibody responses since the antibody titers to N-protein and WRV were iow at the time for challenge.
- the protective immunity must be dependent on cell-mediated immunity, which most likely includes Th1 and CTL responses. The fact that protection was induced rapidly is particularly important for a post-exposure vaccine effect.
- This example demonstrates that a fast protective immune response can be induced by the internal rabies virus N-protein after ISCOM formulation.
- the optimal immunization protocol for the ISCOM adjuvanted experimental vaccine was not applied i.e. a first dose day 0 and a boost week 4 to 6.
- the possibility to use the N-protein is likeiy to broaden the protective immunity e.g. to also include protection against ( B)bat Lyssa viruses e.g. the Duvenhagen strain.
- the results are novel in view of; (I) no rabies G-protein was present, (II) the dose (1-5 ⁇ g) was low (compared to 25 ⁇ g of non-adjuvanted antigen), (III) the time iapse of one week after completed priming is short. Moreover, a long-lasting immune response was induced.
- Example Ib Matrix M improves rabies virus vaccine formulations measured by magnitude and quality of antibody responses
- mice 1 st immunization in mice ( Figure 1A and B).
- the antibody responses (IgGI and lgG2a) to Rabies virus antigens were measured by ELlSA (see M&M).
- the highest antibody level after one immunization was induced by a DiRV formulated as ISCOM 2 vaccine inducing approximately 50 fold higher IgGI titres than the non-adjuvanted WRV corresponding to a conventional rabies virus vaccine.
- the two Matrix M adjuvanted preparations induced about 5-fold higher antibody titers than the WRV.
- the animals were immunized at weeks O and 4, serum samples were taken at weeks 3 and 6.
- Serum from mice immunized with DiRV without Matrix M formulation (lanes 2 in Fig 6 A and B) detected more protein bands than the sera from mice immunized with WRV without Matrix M formulation (lanes 4 in Fig 6A and B).
- the disintegrated virus exposed more proteins that stimulated specific antibody formation e.g. N and P proteins.
- no N or P, being internal proteins were detected from mice immunized with non-adjuvanted whole virions.
- the rabies virus in order to broaden the immune response to internal antigens, the rabies virus can be disintegrated.
- a potent adjuvant like Matrix-M is used to stimulate the immune system, resulting in induction of immune responses to the revealed antigens.
- Matrix M enhances immunogenicity of small and low amount) of rabies virus antigens.
- To stimulate induction of immune responses also to minor components of a vaccine(s) is an important property of an adjuvant; to induce immune protection as well as for antigen saving in vaccines.
- Matrix M strongly potentiates the antibody response in mice to both rabies vaccines. ( Figures 7- 9). In particular the lgG2a and the VN responses, (measured by blocking ELISA) were enhanced. The antibody responses developed also faster after addition of Matrix M. Compared to the corresponding preparations without Matrix M both Matrix M adjuvanted vaccines induced high [gG2a and VN titers already after the 1 sl administration after the 2 nd immunization. Thus, a dos-sparing potential was demonstrated.
- Matrix M adjuvanted rabies vaccines induced high levels of antibody already after one (primary) immunization, including functional protective Virus Neutralizing (VN) antibodies.
- VN functional protective Virus Neutralizing
- a fast functional protective immune response is more important for a rabies virus than for other vaccines in view of the fact that it is used post-exposure to inhibit a suspected virus infection.
- the combined immune protection exerted by two arms of the immune system has added immune protective effect.
- the inclusion of immune response to the N protein of rabies virus broaden the immunity to rapidiy induce a protective immune response.
- Example 1c Matrix IVI adjuvanted rabies virus formulations induce T-helper 1 (T H 1) and TH2 responses in mouse
- T cell responses are analyzed in mice after immunization with whole rabies virus (WRV) or disintegrated rabies virus (DiRV) with and without addition of Matrix M.
- IL- 2 production is indicative for strong T cell responses
- IFN- ⁇ production is produced by TH1 type T cells
- iL-4 and IL-5 are produced by TH2 type T cells.
- a combination of IL-2 and IFN-y production are essential Th1 components for combating virus infections.
- mice were vaccinated twice with different formulations as indicated in Table 3. Two weeks after the second immunization, spleen ceils were re-stimulated with N-protein or WRV. The supernatants of the stimulated spleen cells were screened for production of IL-1, IFN- ⁇ , IL-4 and IL-5.
- Disintegrated rabies virus of TS80 strain as described in experiment 1 b.
- the experimental vaccines were diluted in PBS and were given as 200 ⁇ l injections s.c. in the neck. Results
- T cell responses to experimental Rabies virus vaccine formulations were measured as profile of the cytokine production after re-stimulation in vitro of spleen cells. 1L-2 production after re-stimulation with rabies virus N-protein.
- Figure 10 it is clearly demonstrated that mice immunized with Matrix M adjuvanted N-DiRV or WRV responded with enhanced production of IL-2 detected after re-stimulation of spleen cells in vitro with rabies N- protein, demonstrating that a strong T cell response to the Rabies N-protein was induced by both DiRV and WRV formuiated with Matrix.
- IL-2 production after re-stimulation with whole rabies virus Similar results, measured as IL-2 production, was obtained after re-stimulation of the spleen ceils with WRV ( Figure 10).
- the IFN- ⁇ production after re-stimulatio ⁇ with rabies virus N-protein Similarly to the IL-2 production, it is clearly demonstrated that Matrix M adjuvant enhanced DiRV and WRV to induce the production of IFN- ⁇ ( Figure 11.) detected after re-stimulation of the spleen cells with rabies N- protein. Production of Th2 cytokines, IL-4 and 1L-5, after re-stimuiation with rabies virus N-protein or whole rabies virus (WRV).
- Matrix M did not enhance the IL-4 or IL-5 production after re- stimulation with Rabies virus N-protein or WRV ( Figures 12 and 13). Contrary, the mice vaccinated with non-adjuvanted DiRV or WRV rather produced somewhat higher levels of IL-4 and IL-5 after re-stimulation with N-protein or WRV than mice vaccinated with the Matrix M adjuvanted formulations.
- Matrix M enhances the cell mediated TH1 immune responses to both WRV and DiRV virus formulations, which is reflected by the Th1/Th2 ratio.
- the cellular TH2 type response was not enhanced by the Matrix M adjuvant even though a potentiation of serum IgGI responses was noted (see Example 1b (1).
- Both WRV and DiRV virus formulations evoked high levels of antibody and cell mediated immune responses well above the levels of those induced by rabies vaccines available today and above expectations. Both arms of the immune response are essential components to optimize immune protection both for prophylaxis and for post exposure immune treatment of rabies virus infection.
- This experiment was designed to explore the enhancing effect of Matrix M on a commercial disintegrated non-adjuvanted influenza vaccine (D-FLU) in a mouse model. Considerations were taken to level of immune response, quality and antigen sparing. Moreover, the duration of the immune response is an additional important factor.
- mice 18 g female Baib/c mice were immunized as indicated in Table 4. The mice were immunized s.c. at weeks 0 and 4. Blood samples for testing were taken at weeks 3 and 6. The antigen specific antibody responses in IgG 1 and igG2a subclasses at weeks 3 and 6 are shown in figure 14 (A-D).
- Matrix M formulation consists of a mixture of 90% ug Matrix A and 10% Matrix C.
- mice immunized with the 30 fold reduced human vaccine dose (1.5 ⁇ g) formulated with the Matrix M responded with clear cut IgGI and lgG2a antibody responses.
- Mice immunized with the commercial D-FLU vaccine formulation alone did not respond with lgG2a antibody and hardty with detectable levels of IgGl Mice immunized with a 300 fold reduced human dose (0.15 ⁇ g) alone or formulated with the Matrix M required two
- the Matrix M formulated D-FLU vaccine induced high antibody levels, almost as high levels (93 and 91% for IgGI and lgG2a respectively), as the ten-fold higher dose, while the commercial vaccine alone only stimulated lower levels of igG1 antibody, 73 and 65% compared to the Matrix adjuvanted groups and no igG2a antibodies. No side effects were recorded in the immunized mice.
- the conventional vaccine induced iow levels of antibody, even with ten-foid higher doses than the Matrix M adjuvanted D-FLU. Above all, the commercial vaccine induced low quality immune responses. The low level is particularly obvious at the first immunization when the commercial vaccine formulation did not induce detectable antibody responses. An early response with good quality immune response in individuals, that are not earlier vaccinated or infected with influenza virus is important, since particularly young children being in that situation are at risk group to develop very serious illness to a natural infection with influenza virus.
- the low dose required of the Matrix M adjuvanted experimental vaccine to develop high levels and high quality antibodies is important not only for the fast response but above all for antigen sparing, decreasing production costs and increasing production capacity.
- the low quality of the response to the commercial vaccine is indicated by the lack of lgG2a.
- the Matrix M formulated D-FLU induced an unforeseen increase of quality to the commercial vaccine.
- the insufficient quality of the present FLU vaccines is well documented with regard to protection, duration of immune response requiring yearly revaccinations and quality of immune response.
- the shortcomings of poor immunogentcity of present commercially available influenza vaccines are particularly prominent in elderly.
- the experiments demonstrate that a Matrix M adjuvanted vaccine would fill unmet needs with regards to: (I) quality, level and duration of antibody response as weli as an early effect in immunologically na ⁇ ve indviduals.
- Example 3 Disintegrated experimental RSV vaccine adjuvanted with Matrix M induces potent immune protection in cotton rat
- Respiratory syncytial virus causes disease in man and particularly in infants and elderly, the disease can be serious. So far no therapy or vaccine is available.
- Cotton rats were immunized twice at days 0 and 21 with 1 ⁇ g or 5 ⁇ g doses DiRSV adjuvanted with Matrix M (24 ⁇ g/dose) in a total volume of 200 ⁇ L.
- the vaccine or controls (placebo or infectious virus) were injected intramuscularly (i.m.) in volumes of 100 ⁇ l in each leg of the rat (see Table 5). Animals in all groups except in group 5 were challenge infected on day 46 under light anaesthetize (Isoflurane) with a dose if 10 s PFU RSV strain Tracy in 100 ⁇ L. Table 5.
- VN antibodies Virus neutralizing (VN) antibodies was measured in serum samples from all animals in each group. After one immunization, the infected animals in Group 4 responded with the highest levels of VN antibodies in serum, a level that hardly changed over time not even after challenge infection day 49. The animals immunized with the 1 and 5 ⁇ g doses of DiRSV adjuvanted with Matrix M responded with VN titers at day 21 , the higher dose induced higher antibody levels. After the second immunization at day 21 , the levels of VN antibodies increased and remained at this level during and after challenge infection at day 45. No anamnestic response was detected in these Groups after challenge infection (Figure 15).
- Immune protection was measured by virus isolation in the upper respiratory tract and lungs, expressed as PFU in nasal wash and lung lavage (Figure 16).
- the higher dose of the DiRSV Matrix M formulation induced a protective immune response in the lung that was of the same magnitude as that induced by infection i.e. 260-fold reduction of virus excretion following challenge infection compared to virus excretion in non-immunized animals.
- the reduction of virus replication in the URT was about 50-fold (compared to non-immunized animals).
- the severe symptoms following RSV infection of man is due to the viral replication in the lungs. Discussion and conclusions
- RSV infection causes respiratory tract infections at all ages but in infants and elderly the infections often become severe and cause morbidity and occasional mortality. There is no therapy directly targeting the virus infection. The prevention has to rely on vaccination as we understand today. However today, there is no vaccine for man against RSV infection. In the veterinary field there are vaccines but their efficacy is low and not very much used. The development of an efficient RSV vaccine is cumbersome and more than 50 years of research has not yet been accomplished with a protective vaccine. It is believed that a balanced Th1/Th2 response with IFN-y producing T cells is an important feature for the virus clearance.
- DiRSV antigen Since only 5% of the DiRSV antigen constitute the protective F-protein, it is likely that other viral antigens revealed by disintegration contribute to the immune protection. Matrix M adjuvanted DiRSV vaccine is likely to fill the unmet need of a potent RSV vaccine.
- Matrix M is analyzed on human immature dendritic cells (iDCS) being the main actors in the initiation of the immune response in man.
- IDCs Immature Dendritic cells
- CD83 expression the capacity of Matrix M to initiate iDCs activation measured as CD83 expression and the differentiation measured by the expression of the communication molecule CD 86 i.e. surface molecules that communicate with the lymphocytes to enter the specific immune response.
- Matrix M and its constituents Matrix A and Matrix C were evaluated by measuring the production of CD86 after cuituring iDCs with Matrix M.
- Figure 18 shows that Matrix M and its constituents Matrix A and Matrix C induce expression of CD86 i.e. differentiate iDCs facilitating communication with lymphocytes.
- Matrix M activates, differentiate human DCs measured by the surface molecules CD 83 and 86, which are essential for induction of acquired i.e. specific immune responses.
- CD 86 was selected as a read-out for this example since DCs of elderly humans are hampered in expressing this molecule resulting in immune compromised individuals prone to develop severe disease following exposure and infection with e.g. RSV or influenza virus.
- Matrix M is a novel way, facilitating vaccine development for elderly and optionaliy also for other immune deficiencies in man or animals.
- Example 5 Matrix formulations activates and differentiate dendritic cells (PCs) derived from human blood monocytes from elderly
- White blood cells were collected from ten anonymous volunteers 60 years of age or older.
- Monocytes were obtained and cultures in the presence of GWIC-SF and IL-4 to receive immature DC (iDC) as described in Materials and Methods.
- the iDCs were stimulated either with; medium, LPS, Matrix M, DiRSV adjuvanted with Matrix M at two doses or an ISCOM formulation with integrated envelope proteins of RSV (described in Hu et al., Clin Exp Immunol., 113, p 325, 1998). Thereafter, the DCs were analyzed for the following surface proteins: CD11c, CD14, CD80, CD83, CD86 and (Class It antigen (major human histocompatibility complex, MHC class II) using monoclonal antibodies for identification and for quantification by flow cytometry.
- iDC immature DC
- the expression of CD14 was down-regulated following culture of the monocytes, thus indicating that iDC was received.
- the iDCs derived from elderly volunteers were cultured for 24h with DiRSV Matrix M vaccine or controls and then analyzed for surface receptors. After 24h stimulation with DiRSV Matrix M vaccine the expression of activation markers on the DC were increased to the same extent as the positive control (LPS stimulation) demonstrating that the experimental vaccine activated and differentiated the iDC into activated DC.
- Figures 19 and 20 show that stimulation of iDCs with ISCOM or Matrix M adjuvanted DIRSV increased the expression of the activation marker CD83, co-stimulatory molecules CD86 and CD80 and
- HLADR expression This differentiation is essential for the function of DCs to initiate the immune response to the RSV antigens. Discussion and Conclusion
- Example 6 Disintegrated Neospora antigens formulated with Matrix Q adjuvant is a potential vaccine candidate
- Neospora caninum (Nc) infection causes abortion and economic losses in cattle worldwide. Although there is no treatment or proven vaccine to prevent infections or disease in cattle (Dubey et al., 2007), it has been shown that cattle experimentally inoculated with live tachyzoites prior to mating developed protective immunity against vertical transmission (Innes et al., 2001). Moreover, cows with latent ⁇ /e ⁇ spora-infection develop protective immunity against foetopathy caused by experimental inoculation (Williams et al., 2003) or a natural second exposure to the parasite (McAllister et al., 2000). Protective mechanisms are associated with induction of type 1 immune response including IFN- ⁇ production (Innes et al., 2002).
- an effective vaccine should be based on an immune response that includes IFN- ⁇ production.
- This example shows that the concept of using disintegrated Nc as vaccine antigen together with Matrix Q adjuvant induce high quality immune response encompassing both antibody and cell mediated immunity, particularly IFN- ⁇ production that is expected to contribute to immune protection.
- We compared some immune parameters induced in calves inoculated with live tachyzoites (proposed as a vaccine candidate) and calves inoculated with disintegrated Nc antigens adjuvanted with Matrix Q. It is shown that the experimental Matrix Q vaccine formulation gives superior immune response to the live vaccine.
- IgGI /lgG2 ratio N. caninum specific IgG-, and IgG 2 antibody responses presented as IgGI /lgG2 ratio are shown in Table 7. Clear differences in the distribution of the antibody response into subclasses between Groups A and B were recorded. Calves in Group A receiving live tachyzoits responded with a dominant lgG2 profile, i.e. with a ratio ⁇ 1. In contrast, a ratio >1 was recorded in calves from Group B receiving disintegrated tachyzoit adjuvanted with Matrix Q. After the challenge infection (week 12) similar lgG1/lgG2 ratios were maintained in both groups.
- inoculation of live tachyzoites before mating prevents not only the abortion, but also vertical transmission (Innes et al., 2001; Williams et al., 2007).
- inoculation of live tachyzoites is not a choice for a commercial vaccine.
- Spread of new infections and reversion to pahogenicity are only two of many disadvantage of using live vaccines.
- the development of inactivated vaccines to control bovine neosporosis is still needed, commercial vaccines available today are based on inactivated whole tachyzoites and has only efficacy around 50%.
- the availability of inactivated immunogens that generates protective immune responses equivalent to the immunity induced by live tachyzoites is of major significance.
- the iFN- ⁇ response increase to a maximum level in ail groups except in the two groups receiving antigen without adjuvant.
- live tachyzoites and non-adjuvanted disintegrated antigens apparently, skew or down-regulate the IFN- ⁇ response facilitating the infection, which is overcome by Matrix adjuvant.
- Matrix adjuvant The importance of the IFN- ⁇ response is the notion that animals immunized with non-adjuvanted tacyzoite formulation after challenge infection developed an anamnestic response implicating infection by the parasite.
- this study shows the potential of using disintegrated antigen of Neospora caninum together with a Matrix adjuvant in a safe and inactivated vaccine, to induce a broad immune response equivalent to the immunity induced by inoculation of live tachyzoites.
- the Matrix adjuvanted vaccine is inducing a better quality vaccine promoting IFN- ⁇ response in contrast to a "live vaccine" formulation that down regulates the IFN- ⁇ response occurring at a subsequent infection.
- Example 7a Immune responses to vaccination against a Staphylococcus aureus CP5 Bacterin in heifers
- the objective of this example was to compare the humoral immune response in serum and m ⁇ k to a staphylococcal capsular polysaccharide type 5 bacterin formulated with two different adjuvants; Matrix Q or AI(OH) 3 .
- a placebo consisting of sterile saline solution was used as control.
- Tabie Type of sample and sampling frequency
- the Matrix Q adjuvant increased the antibody response to S. Aureus CP5 bacteri ⁇ in both serum and miik.
- the response in serum was substantially higher and of longer duration.
- AI(OH) 3 did not at al promote an antibody response, in contrast, Matrix Q adjuvanted Bacteiin stimulated to high levels of antibodies in milk.
- the bacterin without any adjuvant did not induce any detectable antibodies at all.
- Example 7b Humoral immune responses to vaccination against Staphylococcus aureus CP5 Bacterin and CP5 Lysate in heifers
- the objective of this example was to evaluate the humoral response generated by (1) a Staphylococcus aureus CP5 whole cell vaccine and (2) a S. aureus CP5 lysate vaccine, both formulated with Matrix Q adjuvant and (3) a placebo consisting of sterile saline solution plus Matrix-Q adjuvant was used as control.
- the vaccines are formulated as described in M&M. Twelve primigravid Holstein dairy heifers in the last trimester of gestation were used in the experiment. The animals were randomly allocated in 3 groups. Each group received one of the different formulations injected subcutaneously in the supramammary lymph node area at approximately 40 and 14 days before expected calving. Samples were taken as described in Table 9. The sera were tested for antibody response against whole bacteria and a bacteria lysate (disintegrated bacteria) in ELISA. The results are shown in Figures 24 A and B.
Abstract
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Cited By (2)
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WO2013051994A1 (en) | 2011-10-03 | 2013-04-11 | Moreinx Ab | Nanoparticles, process for preparation and use thereof as carrier for amphipatic of hydrophobic molecules in fields of medicine including cancer treatment and food related compounds |
WO2014163558A1 (en) | 2013-04-01 | 2014-10-09 | Moreinx Ab | Nanoparticles, composed of sterol and saponin from quillaja saponaria molina process for preparation and use thereof as carrier for amphipatic of hydrphobic molecules in fields of medicine including cancer treatment and food related compounds |
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US10751410B2 (en) | 2013-09-19 | 2020-08-25 | Novavax, Inc. | Immunogenic middle east respiratory syndrome coronavirus (MERS-CoV) compositions and methods |
WO2017041100A2 (en) | 2015-09-03 | 2017-03-09 | Novavax, Inc. | Vaccine compositions having improved stability and immunogenicity |
SG11202009206QA (en) | 2018-03-19 | 2020-10-29 | Novavax Inc | Multivalent influenza nanoparticle vaccines |
US10953089B1 (en) | 2020-01-27 | 2021-03-23 | Novavax, Inc. | Coronavirus vaccine formulations |
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EP4176871A1 (en) | 2011-10-03 | 2023-05-10 | Canqura Oncology Ab | Nanoparticles, process for preparation and use thereof as carrier for amphipatic of hydrphobic molecules in fields of medicine including cancer treatment and food related compounds |
WO2014163558A1 (en) | 2013-04-01 | 2014-10-09 | Moreinx Ab | Nanoparticles, composed of sterol and saponin from quillaja saponaria molina process for preparation and use thereof as carrier for amphipatic of hydrphobic molecules in fields of medicine including cancer treatment and food related compounds |
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