WO1998052962A1 - Oligonucleotide adjuvant - Google Patents
Oligonucleotide adjuvant Download PDFInfo
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- WO1998052962A1 WO1998052962A1 PCT/US1998/010459 US9810459W WO9852962A1 WO 1998052962 A1 WO1998052962 A1 WO 1998052962A1 US 9810459 W US9810459 W US 9810459W WO 9852962 A1 WO9852962 A1 WO 9852962A1
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- oligonucleotide
- adjuvant
- hbsag
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- antigen
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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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
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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
- A61K39/29—Hepatitis virus
- A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
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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
- 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
- A61K2039/55505—Inorganic adjuvants
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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
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
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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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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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
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- This invention relates to the field of adjuvants and their use in vaccination.
- Vaccines are widely used to stimulate animal to mount an immune response against the antigen(s) in the vaccine.
- the immune response includes an immune memory component that helps to protect the animal against future exposure to infectious agents that present an antigen in the vaccine.
- the immune response can also include a cell- mediated response component.
- Adjuvants are compounds useful in vaccine compositions.
- adjuvant will cause an animal to mount a more vigorous immune response. Adjuvants can also cause an animal to develop a stronger immune memory for the antigen presented by administration of a vaccine. Many compounds are useful as adjuvants in vaccine compositions. Since at least 1925 aluminum containing compounds have been used as adjuvants. Currently, the only adjuvants approved for use in human are aluminum salts, e.g., aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate. More than 100 compounds or formulations exhibiting some degree of adjuvant properties have been described (See e.g.,Vogel, F.K. and M.F. Powell (1995) A compendium of vaccine adjuvants and excipients, In: Vaccine Design, M.F. Powell and MJ. Newman, Eds., Pharm-Biotechnol. 6:141-228. All literature, patents and other publications cited herein are incorporated herein by reference in the entirety as background material).
- Bacterial DNA has been reported to have immunostimulatory properties including the ability to induce natural killer (NK) cell activity and the induction of cytokines such as interferon (IFN) ⁇ / ⁇ and IFN- ⁇ (Yamamoto, S., et al.. 1988. In vitro augmentation of natural killer cell activity of interferon ⁇ / ⁇ and - ⁇ with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn. J. Cancer Res. (Gann) 79:866-873). Interferon- ⁇ production was reported to be dependent on the induction of interleukin 12 (IL-12) and tumor necrosis factor- ⁇ (Halpern, M.D., et al., 1996.
- IL-12 interleukin 12
- tumor necrosis factor- ⁇ Halpern, M.D., et al., 1996.
- the CpG motif is reported to be most stimulatory when it is presented within a larger oligonucleotide that includes a sequence of -GACGTT- or -GACGTC- (WO 96/02555, col. 6, lines 11-12).
- a larger oligonucleotide that includes a sequence of -GACGTT- or -GACGTC- (WO 96/02555, col. 6, lines 11-12).
- the size required for the oligonucleotide to be active at least two reports state that testing indicated that oligonucleotides which include these sequences, and are less than 8 bases long, are non-stimulatory (Id. at col. 7, lines 19-20; Krieg, A.M. et al., 1995. Nature 374:546-549, at col. 2, lines 14-18).
- human B cells are reported to proliferate and produce polyclonal immunoglobulin in a T cell-independent manner upon culture with phosphorothioate oligonucleotides (Liang, H., et al., 1996. Activation of human B cells by phosphorothioate oligodeoxynucleotides. J. Clin. Invest. 98:1119-1129). It is known that cytotoxic T lymphocytes (CTLs) kill virally- or bacterially-infected cells when their T cell receptors recognize foreign peptides associated with MHC class I and/or class ⁇ molecules.
- CTLs cytotoxic T lymphocytes
- peptides can be derived from endogenously synthesized foreign proteins, regardless of the protein's location or function within the pathogen. By recognition of epitopes from conserved proteins, CTLs may provide heterologous protection.
- proteins secreted by or released from the pathogen are processed and presented by MHC class I and II molecules, thereby generating T-cell responses that may play a role in reducing or eliminating infection.
- oligonucleotides containing CpG motifs induce immune activation.
- a key element is the induction of transcription of cytokine genes.
- rapid induction of high levels of IL-6 reportedly occurs both in vitro and in vivo, and transcription of IL-6 message is reported to be elevated in liver, spleen and thymus tissues upon administration of stimulatory oligonucleotides (Yi, A.-K., et al, 1996. Rapid immune activation by CpG motifs in bacterial DNA. Systemic induction of IL-6 transcription through an antioxidant-sensitive pathway. J. Immunol. 157:5394-5402).
- oligonucleotides containing CpG motifs have been reported to upregulate expression of MHC class II antigens (Krieg, A.M., et al, 1995. Nature 374:546-549). Taken together, these effects indicate that CpG oligonucleotides can augment antigen presentation thereby providing an adjuvant effect. Moreover, these adjuvants can provide a method to elicit a CTL response to an antigen presented in a vaccine.
- the oligonucleotide adjuvant of this invention can include a CpG dimer
- the oligonucleotide does not satisfy the requirements of minimum lengths taught in the art. It is also found that a palindromic sequence is not required. Therefore, it is speculative whether the reported rationale for CpG oligonucleotide immunostimulatory activity applies to the oligonucleotides of the present invention.
- an oligonucleotide consisting of six bases of the sequence 5' GACGTT 3,' 5' GAGCTT 3,' or 5'TCCGGA 3' are useful as a vaccine adjuvant in animals. These oligonucleotide adjuvants results in the generation of both a cell mediated response (cytotoxic T lymphocyte, CTL) and an antibody response to antigens presented by a vaccine.
- CTL cytotoxic T lymphocyte
- an adjuvant is a compound that improves the immune response of an animal to a vaccine antigen through one or more of the following mechanisms: (1) providing a depot of antigen for slow release of the antigen to the immune system, (2) causing enhanced uptake of the vaccine antigen by macrophages and other antigen presenting cells, (3) stimulation or activation of macrophages or neutrophiles (4) chemotactic recruitment of effector cells to the site of antigen injection, (5) enhanced proliferation of antigen-specific T and B cells, (6) increasing the precursor frequency of antigen-specific T and B cells, (7) increasing the amount and persistence of specific antibody, (8) increasing the breadth of the immune response in terms of antibody isotypes, (9) expanding the response to include multiple rather than single epitopes on the infectious agent, and (10) enhanced activation of memory T and B cells.
- animal includes humans and non-human primates.
- Animal also includes domestic companion animals, e.g., cats and dogs, and livestock, e.g., chickens, cows, pigs, horses and sheep.
- livestock e.g., chickens, cows, pigs, horses and sheep.
- Animal also includes all developmental stages of animals that are responsive to vaccination. In particular, pediatric administration is appropriate in animals and humans as soon as they are capable of responding to the antigen in a particular vaccine formulation.
- an aspect of this invention is a hexamer oligonucleotide adjuvant of the sequence 5' GACGTT 3,' 5' GAGCTT 3,' or 5'TCCGGA 3'.
- the oligonucleotide is useful in the administration of vaccine compositions to animals.
- the oligonucleotide has non-natural internucleotide linkages.
- the non-natural linkages can be only at the terminal linkages, the terminal and penultimate linkages or all the internucleotide linkages.
- the internucleotide linkages are phosphorothioate linkages.
- the oligonucleotide can have mixed linkages.
- adjuvant is encapsulated in a slow release delivery vehicle.
- Another aspect of this invention is a vaccine formulation comprising one or more antigens and the oligonucleotide adjuvant.
- the formulation can be liquid or lyophilized in dosage form. Many dosage forms are known in the art and can be applied herein.
- the oligonucleotide is present in the composition at a dose of from about 10 to about 10,000 ⁇ g per dose, from about 50 to about 5,000 ⁇ g per dose or from about 100 to about 500 ⁇ g per dose.
- the antigen is one or more antigens of hepatitis B, hepatocellular carcinoma antigens induced by hepatitis B virus, herpes simplex virus, human papilloma virus, hepatitis C virus envelope or core proteins, rotavirus bovine and human reassortants hepatitis A virus, human immunodeficiency virus envelope, polymerase and core proteins, varicella, varicella zoster, Streptococcus pneumonia polysaccharide, E. coli, Haemophilus influenza polysaccharide, Mycobacterium tuberculosis, Staphylococcus, Plasmodium and Schistosoma.
- the antigen is hepatitis B surface antigen (HBsAg).
- HBsAg protein antigen can be provided as a recombinant protein product prepared from yeast or an inactivated hepatitis B virus.
- An aspect of this invention is a method of vaccination of an animal, including humans.
- the animal can be vaccinated prophylactically or therapeutically.
- the method of vaccination includes administering the oligonucleotide adjuvant of this invention and one or more antigens ⁇ that is, the vaccine can be designed against one disease target or a combination of disease targets.
- Antigens that can be used in either or both therapeutic or prophylactic vaccines include antigens of hepatitis B, hepatocellular carcinoma induced by hepatitis B virus, herpes simplex virus, human papilloma virus, hepatitis C virus envelope or core proteins, rotavirus bovine and human reassortants hepatitis A vims, human immunodeficiency virus envelope, polymerase and core proteins, varicella, varicella zoster, Streptococcus pneumonia polysaccharide, E. coli, Haemophilus influenza polysaccharide, Mycobacterium tuberculosis, Staphylococcus, Plasmodium and Schistosoma.
- the oligonucleotide and the antigen are administered contemporaneously. In other embodiments, they are administered simultaneously. In additional embodiments, the oligonucleotide and antigen are administered by intramuscular injection at the same site. In preferred embodiments of this aspect, the animal is a human.
- Vaccine compositions useful with the adjuvant of this invention can present antigens directly, e.g., in the form of a particular protein(s) or peptide(s).
- vaccines can present an antigen(s) as a part of a complex biological or biochemical conglomerate, e.g., as complete virus(es), virus-like particle(s), bacterial cell(s) or conjugate(s) including polysaccharide-protein conjugate molecules. These components can be combined to make a vaccine with multiple antigens.
- a vaccine composition including at least one antigen is formulated to include the oligonucleotide adjuvant of this invention.
- the antigen can be hepatitis B surface antigen as either a protein, a virus-like particle of recombinant protein produced in yeast (HBsAg), mammalian or insect cell culture or a peptide antigen derived from any of these.
- the oligonucleotide adjuvant can be administered simultaneously or contemporaneously with the administration of the vaccine antigen.
- simultaneously it is meant that the antigen and adjuvant are administered together in the same formulation.
- contemporaneously it is meant that the antigen and the adjuvant are administered closely in time, e.g., the adjuvant is administered within from about one minute to within about one day before or after the antigen is administered. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the antigen and adjuvant will be administered within about one minute to within about eight hours, and preferably within less than about one to about four hours.
- the oligonucleotide adjuvant and antigen are administered at the same site on the animal.
- the same site includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
- the oligonucleotide can have various internucleotide linkages. It is preferred that at least the terminal internucleotide linkages be non- natural linkages. However, the terminal and penultimate linkages, or all the linkages can be non-natural. It is particularly preferred that oligonucleotide include phosphorothioate internucleotide linkages and it is most preferred that all of the internucleotide linkages are phosphorothioate.
- an "S-ODN” is an oligodeoxynucleotide wherein all the internucleotide linkages are phosphothioate linkages. The natural phosphodiester linkage can also be appropriate in some cases..
- Non-natural linkages are well known in the art and include methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages. Dephospho-linkages are also known, as bridges between nucleotides, and include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges. "Plastic DNA,” having, for example, N-vinyl, methacryloxytethyl, methacrylamide or ethyleneimine internucleotide linkages. "Peptide Nucleic Acid” (PNA) is also useful and resists degradation by nucleases. Non-natural linkages can be mixed in the oligonucleotide. One need only test whether any particular linkage or combination of linkages in an oligonucleotide is appropriate for an adjuvant by using the assays described in the examples below
- the purity of the oligonucleotide is very important for obtaining adjuvant activity. Residual chemicals, solvents, etc., from the process of synthesis should be removed by methods known in the art including chromatography on SEPHADEXTM, reversed phase chromatography, high performance liquid chromatography, precipitation, dialysis, gel electrophoresis, or a combination thereof. Care should also be taken to ensure proper deprotection, which should be as complete as possible. Nevertheless, even with these precautions in mind, some syntheses will produce oligonucleotide that lack appropriate adjuvant activity. Therefore, it is most preferred that one test the oligonucleotide produced from each synthesis to confirm and assess the adjuvant activity.
- a further refinement to a formulation is to incorporate the oligonucleotide adjuvant into a delivery vehicle to provide for delayed release of the adjuvant over time.
- the oligonucleotide can be formulated separately or together with at least one vaccine antigen.
- means of slow release delivery vehicles are encapsulation of the oligonucleotide adjuvant into PLGA microspheres (Deasy, P. B. et al. 1989. Preparation and characterization of lactic/glycolic acid polymers and copolymers. J. MicroencapsuL, 6: 369-378) Hposomes, or bound (covalently or not) to carrier molecules such as proteins, polysaccharides or lipids.
- the hexamer oligodeoxynucleotide adjuvant elicits the generation of both a cell mediated (cytotoxic T lymphocyte, CTL) response and an antibody response to an antigen presented by a vaccine.
- a vaccine containing an antigen and the hexamer oligodeoxynucleotide adjuvant can be formulated and used as a prophylactic or therapeutic vaccine for a variety of disease targets including sexually transmitted diseases.
- a prophylactic vaccine is defined herein as a vaccine designed to elicit complete or partial protection from a disease caused by an infectious agent through the induction of specific immunity (e.g. antibodies specific for the infectious agent or its products, cytotoxic T lymphocytes (CTLs) specific for appropriately presented derivatives, e.g. peptides, associated with or produced by the infectious agent, or the activation of effector cells, e.g. activated macrophages, mediated by cytokine production occurring as a result of T cell recognition of peptides derived from the infectious agent).
- specific immunity e.g. antibodies specific for the infectious agent or its products, cytotoxic T lymphocytes (CTLs) specific for appropriately presented derivatives, e.g. peptides, associated with or produced by the infectious agent, or the activation of effector cells, e.g. activated macrophages, mediated by cytokine production occurring as a result of T cell recognition of peptides derived from the infectious agent.
- CTLs
- a therapeutic vaccine is defined as a vaccine designed to induce complete or partial remission of a chronic illness associated with an infectious agent.
- This vaccine activates cell-mediated immunity (e.g. specific CTLs) or the activation of effector cells (e.g. activated macrophages) mediated by cytokine production occurring as a result of T cell recognition of peptides derived from the infectious agent.
- the CTLs and activated macrophages lyse cells containing the infectious agent thereby eliminating the reservoir of infection. It is advantageous for a therapeutic vaccine to also induce specific antibodies to neutralize any infectious agents released from infected cells following lysis thereby preventing reinfection of host cells and tissues.
- Vaccine antigens include antigens derived from hepatitis B (e.g., S and preS-containing envelope proteins and core proteins); hepatocellular carcinoma antigens induced by hepatitis B virus, herpes simplex, human papilloma virus, hepatitis C virus (hepatitis C envelope or core proteins); rotavirus (including bovine and human reassortants); hepatitis A virus (e.g., VAQTA®); human immunodeficiency virus ((HTV) HIV envelope proteins, HIV core proteins, HTV polymerase proteins); varicella or varicella zoster; antigens derived from bacteria including purified proteins, non-protein antigens, or mixtures of protein and non-protein antigens, e.g., vaccines against Streptococcus pneumonia, (pneumococcal polysaccharide vaccines, pneumococcal polysaccharide-protein conjugate vaccines), E.
- Haemophilus influenza including polysaccharide-protein conjugate vaccines
- Mycobacterium tuberculosis vaccines and vaccines against bacteremia caused by Staphylococcus e.g., Staphylococcus aureus
- antigens derived from parasites that cause diseases such as malaria (Plasmodium falciparium) and schistosomiasis (Schistosoma mansoni); and antigenic material derived from human or animal tumors.
- the above vaccines can be directed against various serotypes of bacteria and viruses as appropriate.
- a vaccine including the adjuvant oligonucleotide of this invention can be formulated as appropriate for the subject animal.
- Formulations can be liquid or lyophilized. Many vaccine formulations are known in the art and can be used by substituting the adjuvant of this invention for the adjuvant previously known in the art.
- An example of a liquid formulation vaccine is one for hepatitis B.
- the formulation is an injectable solution containing from about 5 to about 40 ⁇ g, preferably from about 5 to about 20 or from about 5 to about 10 ⁇ g, of hepatitis B surface antigen (prepared, e.g., as described in U.S.Patent Nos.
- the formulation contains from about 10 to about 10,000 ⁇ g of the 5' GACGTT 3' oligonucleotide adjuvant, preferably from about 50 to about 5,000 ⁇ g and most preferably from about 100 to about 1,000 ⁇ g. Particular formulations may require particular amounts within these ranges, for example, about 200, 500, 750, 1,500, 2,500 or 3,500 ⁇ g, or other amounts not listed here, of the adjuvant oligonulceotide can be used.
- a pharmaceutically acceptable buffer e.g., phosphate
- the formulation contains from about 10 to about 10,000 ⁇ g of the 5' GACGTT 3' oligonucleotide adjuvant, preferably from about 50 to about 5,000 ⁇ g and most preferably from about 100 to about 1,000 ⁇ g. Particular formulations may require particular amounts within these ranges, for example, about 200, 500, 750, 1,500, 2,500 or 3,500 ⁇ g, or other amounts not listed here, of the adjuvant oligonulceotide can be used
- An injectable formulation can be prepared by mixing a double- strength liquid formulation of antigen with a double strength liquid (or previously frozen) solution of an oligonucleotide adjuvant such as 5' GACGTT 3'.
- a vaccine can be prepared by mixing from about 10 to about 1,000-2,000 ⁇ g of the oligonucleotide 5' GACGTT 3 ' in a pharmaceutically acceptable buffer with antigen formulated with aluminum hydroxyphosphate or another adjuvant (such as aluminum hydroxide, aluminum phosphate, calcium phosphate, saponins, non-ionic block copolymers, oil in water emulsions, or cytokines) and/or with excipients.
- aluminum hydroxyphosphate or another adjuvant such as aluminum hydroxide, aluminum phosphate, calcium phosphate, saponins, non-ionic block copolymers, oil in water emulsions, or cytokines
- a vaccine can be formulated to contain from about 5 to about 20 ⁇ g of HBsAg, from about 10 to about 1,000-2,000 ⁇ g of the oligonucleotide 5' GACGTT 3', and from about 0.1 to about 100 ⁇ g of a cytokine such as interleukin 12.
- a lyophilized formulation is a lyophilized live, attenuated virus such as VARIVAX® (a vaccine for varicella or chicken pox) with a buffered solution containing from about 10 to about 1,000- 2,000 ⁇ g of the oligonucleotide 5' GACGTT 3'.
- ODNs having phosphodiester or phosphorothioate internucleotide linkages were synthesized at Midland Certified Reagent Co., Midland TX.
- ODNs with phosphodiester backbones were synthesized using the 0-cyanoethyl phosphoroamidite chemistry (Sinha, N.D., Biernat, J, and Koster, H. 1983. Tetrahedron Letters 24:5843- 5846).
- ODNs with phosphorothioate backbones were synthesized as described in Iyer, R.P., Egan, W., Regan, J.B., Beaucage, S.L. 1990.
- ODNs were purified by trityl selective purification (TSP) on a reverse phase column or by high pressure liquid chromatography (HPLC) (ZORBAXTM Bioseries Oligo (21.2 x 250 mm) anion exchange column).
- TSP trityl selective purification
- HPLC high pressure liquid chromatography
- ODNs containing a terminal trityl group are selectively bound to the reverse phase column.
- the trityl is removed and the ODN eluted from the column in ammonium acetate, acetonitrile, and trifluoroacetate and then dried to remove the volatile solvents.
- HPLC procedure a buffer containing sodium sulfate and urea was used to purify the ODN.
- a desalting step on a SEPHADEXTM column aids in the removal of salts, e.g., salts of sulfate, urea or amonium, from the final product and is strongly recommended. It may be possible to use SEPHADEXTM G-25 for desalting, but is not preferred. The purification provided by a SEPHADEXTM G-10 desalting column is preferred.
- Anti-HBsAg EIA total antibody
- a microtiter plate modification of the AUSAB EIA kit (Abbott Labs, N. Chicago, IL) was used to quantify antibodies to hepatitis B surface antigen (HBsAg).
- Costar EIA 96-well flat bottom plates (Costar, Cambridge MA, #3591) were coated overnight at 4°C with recombinant HBsAg (prepared e.g., U.S.Patent Nos. 4,769,238 9/6/88; 4,935,235 6/19/90; and 5,196,194 3/23/93; all of which are incorporated herein by reference in their entirety) at 4 ⁇ g/ml in Tris- saline, pH 9.5.
- Microtiter plates were coated with HBsAg and blocked as described above. Five-fold serial dilutions were made (in duplicate) in 8 consecutive wells of the plate for each serum sample. The plates were then incubated overnight at 4°C. After 3 wash cycles with PBS (using a TiterTech plate washer), alkaline phosphatase-conjugated goat anti- mouse immunoglobulin reagents specific for mouse IgGl or mouse IgG2a isotypes (Southern Biotechnology Associates, Birmingham, AL) were added at a final dilution of 1:2000.
- the plates were washed 6 times using a Titertech plate washer, and then 60 ⁇ l per well of the enzyme substrate (p-nitrophenylphosphate (Sigma Chemical Co., St. Louis, MO) dissolved at 1 mg/mL in Tris saline, pH 9.5) was added. After 30 minutes at room temperature, the reaction was stopped with the addition of 60 ⁇ l/well of 3N NaOH. Optical densities were read at 405 nm using a Molecular Devices microplate reader. Data were collected using the Softmax computer program (version 2.32)
- L OD value of the first log 5 dilution giving an OD value below 0.1
- H OD value of the log 5 dilution closest to, but above the cutoff (0.1)
- x the well number that has the OD value L.
- the antibody concentration (c) in experimental samples is determined by comparing the endpoint titer in experimental wells with that of the standard curve by the following formula:
- A the antibody concentration of the standard
- s the log 5 titer of the standard
- t the log 5 titer of the unknown. For example, if the log 5 endpoint titer of the standard (100 ng/ml) is 2.6 and the value of the unknown is 3.4, the concentration of antibody in the unknown would be:
- K Mouse spleen cells were cultured for ⁇ 48 hours in round bottom 96-well culture plates (Costar #3799) at 2 x 10 5 cells per well in 200 ⁇ l "K” medium.
- ["K” medium consists of RPMI-1640 medium (Gibco-BRL, Grand Island, NY, Cat.
- Radioactive thymidine incorporation was quantified using a BetaPlate 1205 scintillation counter (Wallac, Turku, Finland). The counts per minute (cpm) in experimental and control wells were compared to determine a stimulation index (cpm experimental/cpm control).
- CTL assays Cytotoxic T Lymphocyte Assays
- the CTL assays were performed as reported in Ulmer, J.B., et al. 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259: 5102:1745-9. Briefly, BALB/c mice were injected three times with a vaccine formulation consisting of HBsAg and the 5' GACGTT 3' oligonucleotide. A single cell suspension of effector cells was then prepared and cultured in vitro with HBsAg peptide (28-39)-pulsed syngeneic stimulator cells. The cell suspension was assayed 7 days later for CTL activity against 51 Cr- labeled P815 cells.
- the syngeneic stimulator cells were prepared as a single cell suspension from the spleens of unimmunized BALB/c mice as follows. After lysis of red blood cells with ammonium chloride buffer (Gibco BRL ACK buffer), the cells were washed by centrifugation for 10 minutes at 1200 rpm (Jouan centrifuge model CR422), resuspended in DMEM culture medium (Gibco BRL #11965-092), and then irradiated using a 60 Co source to deliver 2,000 - 4,000 rads.
- ammonium chloride buffer Gibco BRL ACK buffer
- H-2 K d peptide HBsAg 28-39
- H-2 K d peptide HBsAg 28-39
- IPQSLDSWWTSL SEQ ID NO:l
- Antibody and cytotoxic T-cell responses to soluble hepatitis B virus (HBV) S antigen in mice implication for the pathogenesis of HBV-induced hepatitis. /. Virol 68:1418-1425.
- the cells were mixed approximately every 20 minutes for 1.5 - 2.5 hrs and then washed 3 times with RPMI-1640 medium.
- Effector cells were prepared as single cell suspensions from spleens of immunized mice as described and then co-cultured with an approximately equal number of peptide-pulsed stimulator cells for 7 days at 37° C (5% C0 2 ) in "K" medium.
- P815 (H-2 d ) mouse mastocytoma cells (ATCC, Rockville, MD) were radiolabeled by overnight culture with 0.5 - 1.2 mCi 51 Cr (Amersham, cat. # CJS.4) added to 75 cm 2 culture flasks (Costar #3376) containing ⁇ 5 x 10 5 cells per mL in a volume of 10 mL. The labeled cells were centrifuged at 1200 rpm for 5 minutes and the supernatant removed by aspiration.
- the cells were washed, counted, resuspended in DMEM culture medium at ⁇ 10 6 cells per mL and then pulsed with 10 ⁇ M HBsAg (28-39) peptide at 37° C for 2-3 hr with frequent mixing.
- the target cells were then washed and adjusted to 10 5 cells per mL for plating.
- effector cells from the 7 day restimulation cultures were harvested, washed, and added to triplicate wells of V bottom microtiter plates (Costar #3898) at 60 x 10 5 , 30 x 10 5 , 15 x 10 5 , and 7.5 x 10 5 cells per mL.
- the 51 Cr-labeled target cells were plated at 10 4 cells per well in 100 ⁇ l "K" medium to achieve effecto ⁇ target ratios of 60:1, 30:1, 15:1, and 7.5:1.
- Triplicate wells containing only target cells cultured in 0.2 mL of medium served as controls for spontaneous 51 Cr release whereas triplicate wells containing target cells cultured in 0.2 mL of medium containing 1.0 % Triton X-100 detergent (Sigma #T6878) served as controls for maximum 51 Cr release.
- the plates were incubated for 4 hours at 37°C in a 5% C0 2 incubator and then centrifuged at 1200 rpm for 5 minutes to pellet the remaining target cells.
- the supernatants (20 ⁇ l) were then harvested using an Impact multichannel pipetor (Matrix Technology, Lowell MA, model #6622) and then transferred to Betaplate filter mats (Wallac #1205-402).
- WO 96/02555 teaches that to be immunostimulatory, oligonucleotides require sequences containing CpG motifs and must be at least 8 nucleotides long (WO 96/02555, col. 13, lines 19-20, see also, Krieg, A.M., et al, 1995. Nature 374:546-549, col. 2, lines 14-18). Further, if the oligonucleotides were 8 nucleotides long they also require a palindromic sequence of at least 6 nucleotides (WO 96/02555, col. 13, lines 36-38).
- EP 0 468 520 teaches that to be imunostimulatory, oligonucleotides require a palindromic sequence of at least 6 nucleotides long to be satisfactory and must be at least 10 nucleotides long overall to be active (EP 0 468 520, col. 11, lines 34-37 & Table 7). Therefore, S-ODNs less than 8 nucleotides in length were tested for the ability to induce proliferation of mouse spleen cells in vitro.
- TCCGGA 3' (lot # 050796-115) had no activity in this experiment and was used as a control thereafter. Proliferation was measured by the incorporation into DNA of 3 H-thymidine as described above. The amount of proliferation induced with the 5' GACGTT 3' oligonucleotide was dose-dependent. The 100 ⁇ M dose resulting in a stimulation index (SI) of > 10-fold. The two oligonucleotide lots described in Table 1 were also used in examples 2 - 6.
- EXAMPLE 3 Adjuvant effect of the 5' GACGTT 3' oligonucleotide in elevating antibody titers in mice.
- the ODNs of this invention are adjuvants that elevate antibody titers raised against an antigen.
- the formulation used in this example is the simple addition of a source of HBsAg with an oligonucleotide hexamer consisting of the sequence 5' GACGTT 3'. All of the internucleotide linkages of the oligonucleotide used in these examples were phosphorothioate linkages (S-ODNs), except where indicated. However, alternative linkages can also be used.
- S-ODNs phosphorothioate linkages
- the efficacy of an oligonucleotide adjuvant candidate can be very simply tested following the method of this example. The results can be compared to the results presented here for the 5' GACGTT 3' S-ODN.
- vaccine formulations including an adjuvant in addition to 5'GACGTT3', e.g., an aluminum salt adjuvant, can be designed, tested for efficacy and compared to formulations containing only one of the adjuvants.
- the HBsAg antigen was the Recombivax HB® final aqueous product (FAP) — that is, Recombivax HB® without the aluminum adjuvant. This antigen was used in formulations with the 5' GACGTT 3' adjuvant.
- hepatitis B surface antigen examples include ENERGIX B® (SmithKline Beecham, Inc., King of Prussia, PA).
- ENERGIX B® SmithKline Beecham, Inc., King of Prussia, PA.
- hepatitis B surface antigen prepared from mammalian cells e.g., Chinese hamster ovary (CHO) cells, or insect cells, would also be appropriate.
- the mixture of HBsAg plus 5' GACGTT 3' was used to immunize BALB/c mice. The mice were bled at intervals to obtain serum that was tested for anti-HBsAg antibodies.
- mice were immunized with HBsAg plus the control palindromic CpG oligonucleotide 5' TCCGGA 3.'
- the dose of HBsAg was 1 ⁇ g and the dose of the oligonulceotides was approximately 0.3, 3.0 or 30 ⁇ g per injection.
- the isotype profile of an immune response is dependent on the activation of a population of helper T (TH) cells of the THl type or of the TH2 type. These two populations of TH cells exhibit different functions as a result of the production of distinct cytokines. THl cells produce IL-2 and interferon- ⁇ (IFN- ⁇ ) whereas TH2 cells produce IL-4 and IL-10 (Mosmann, T.R, et al, 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. /. Immunol 136:2348; Cher, D.J., and T.R. Mosmann. 1987. Two types of murine helper T cell clone.
- n 10 BALB/c mice per group i.m. injection day 0, 42; assay d. 84
- mice that received the ⁇ 30 ⁇ g dose of the 5' GACGTT 3' (plus HBsAg) co-injected into the same i.m. site developed a good anti-HBsAg titer that was 10-fold higher than the response to HBsAg alone.
- Injection of the oligonucleotide in the opposite leg from the antigen did not result in an adjuvant effect suggesting that the 5' GACGTT 3' does not simply increase innate immunity; rather, the oligonucleotide elicits a localized adjuvant effect at the site of injection or, possibly, in the draining lymph node.
- the control lot of S-ODN 5'TCCGGA3' did not produce a significant response.
- Anti-HBsAg GMT (mlU/mL)
- EXAMPLE 6 5' GACGTT 3' S-ODN enhances the specific cytotoxic T lymphocyte (CTL) response to an antigen.
- CTL cytotoxic T lymphocyte
- spleen cells from BALB/c mice were restimulated in vitro with HBsAg peptide (28-39) and then assayed 7 days later for CTL activity against 51 Cr-labeled P815 cells.
- the 5' GACGTT 3' oligonucleotide was a potent adjuvant for the induction of HBs- specific CTLs.
- the adjuvant effect of the 5 ' GACGTT 3 ' S-ODN may have a sequence specific component since injection of mice with HBsAg + a control hexamer consisting of the sequence 5' TCCGGA 3' did not result in significant activation of specific CTLs in this experiment.
- different lots of S-ODNs containing this sequence were found to have adjuvant activity in separate experiments (see example 10).
- HBsAg formulated with an aluminum adjuvant (Recombivax HB®) or with no adjuvant did not induce an HBs-specific CTL response.
- effector cells were restimulated (d. 181) with HBs 28-39 peptide pulsed spleen cells for 7d.
- %Specific lysis 100 x [(experimental cpm - spontaneous cpm) / (maximum release cpm - spontaneous)]
- HBV soluble hepatitis B virus
- Schirmbeck et al. are in agreement with the present results showing that immunization with a conventional HBV vaccine (formulated with aluminum adjuvant) does not induce a CTL response (Schirmbeck, R., et al, 1994.E. J. Immunol. 24:1088-1096). Furthermore, Schirmbeck et al.
- the 5' GACGTT 3' S-ODN adjuvant is effective in eliciting an immune response to an antigen, in this case HBsAg, when contemporaneously injected into the same intramuscular site of an animal.
- an antigen in this case HBsAg
- mice were injected with HBsAg without adjuvant. Contemporaneous injection resulted in a 4- to 14-fold enhancement of anti-HBsAg titers relative to the control group.
- the internucleotide linkages are phosphorothioates.
- S-ODNs phosphorothioate internucleotide linkages
- O-ODNs conventional phosphodiester internucleotide linkages
- the reason for the lot to lot variability of the ODNs in their ability to induce a lymphoproliferative response is unknown, however, it may relate to trace contaminants of urea or ammonium ions that may have been incompletely removed by size exclusion chromatography of large scale ODN preparations on SEPHADEXTM columns.
- Example 9 examines the sequence requirements for an adjuvant effect whereas Example 10 addresses the effect of the internucleotide linkage on adjuvanticity.
- mice were co-injected i.m. on days 0 and 42 with HBsAg protein mixed with the indicated concentration of phosphorothioate ODNs containing a CpG or a GpC motif.
- Control mice were injected with HBsAg protein formulated with or without an aluminum adjuvant.
- Anti-HBsAg titers were determined on sera collected 3 weeks post-dose 2.
- Table 9 Low responder C3H mouse response to HBsAg ⁇ S-ODN
- EXAMPLE 10 Effect of internucleotide linkage on adiuvant properties of ODNs
- Table 10 demonstrate that certain ODNs with conventional phosphodiester internucleotide linkages can have adjuvant effects when formulated with an HBsAg protein vaccine.
- Groups of 10 BALB/c mice were co-injected i.m. on days 0 and 21 with HBsAg protein mixed with 30 ⁇ g of the indicated ODNs.
- Control mice were injected with HBsAg protein formulated with or without an adjuvant.
- Anti-HBsAg titers were determined on sera collected 3 weeks post-dose 2. The results are presented as the GMT (in mlU/mL) with the standard error of the mean in parentheses.
- Table 10 Effect of internucleotide linkage on adjuvant properties of ODNs
- Vaccine formulations containing the 5' GACGTT 3', 5' GAGCTT 3 ' or 5 ' TCCGGA 3 ' adjuvant of this invention can be used to vaccinate humans against a variety of bacterial and viral disease agents.
- human refers to humans of any age that can mount an immune response to an antigen presented by the administration of a vaccine to the human.
- a human can be vaccinated against hepatitis B by administration of a vaccine formulation that includes HBsAg and the 5'GACGTT3' adjuvant.
- the amount of HBsAg in each dose, and the administration schedule, can vary as appropriate for the age of the human.
- a three dose schedule of from about 2.5 ⁇ g to about 5 ⁇ g of HBsAg can be administered at 0, 1-3 months afterward and 4-18 months afterward, preferably at 0, 2 and 6 months.
- the adjuvant in the formulation can be from about 10 ⁇ g to about 1,000 - 5,000 ⁇ g, and preferably from about 100 ⁇ g to about 1,000-2,000 ⁇ g per dose or from about 100 to about 500 ⁇ g per dose.
- a three dose schedule is appropriate.
- the HBsAg can be present in an amount about 5 ⁇ g to about 20 ⁇ g, preferably from about 5 ⁇ g to about 10 ⁇ g per dose, and the 5' GACGTT 3' adjuvant can be present in an amount from about 10 ⁇ g to about 2,000-10,000 ⁇ g, and preferably from about 100 ⁇ g to about 1,000-2,000 ⁇ g per dose or from about 100 to about 500 ⁇ g per dose.
- the schedule for administration can be at 0, 2-6 and 4-24 months, preferably at 0, 2-4 and 6-12 months.
- the HBsAg can be present in an amount about 5 ⁇ g to about 40 ⁇ g, preferably from about 10 ⁇ g to about 20 ⁇ g per dose, and the 5' GACGTT 3' adjuvant can be present in an amount from about 10 ⁇ g to about 2,000-10,000 ⁇ g, and preferably from about 100 ⁇ g to about 1,000-2,500 ⁇ g per dose or from about 100 to about 500 ⁇ g per dose.
- the schedule for administration can be at 0, 2-6 and 4-24 months, preferably at 0, 2-4 and 6-12 months.
- a therapeutic protocol of treatment for humans is designed for more regular, closely spaced administration of the vaccine.
- the HBsAg can be present in an amount about 5 ⁇ g to about 40 ⁇ g, preferably from about 10 ⁇ g to about 20 ⁇ g per dose
- the 5' GACGTT 3' adjuvant can be present in an amount from about 10 ⁇ g to about 2,000-10,000 ⁇ g, and preferably from about 50 ⁇ g to about 1,000-2,500 ⁇ g per dose or from about 100 to about 500 ⁇ g per dose.
- the schedule for administration can be about once every two to six weeks and preferably about once a month for up to about six months or a year.
Abstract
Description
Claims
Priority Applications (4)
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CA 2289741 CA2289741A1 (en) | 1997-05-19 | 1998-05-19 | Oligonucleotide adjuvant |
EP98923657A EP0983289A4 (en) | 1997-05-19 | 1998-05-19 | Oligonucleotide adjuvant |
AU75893/98A AU7589398A (en) | 1997-05-19 | 1998-05-19 | Oligonucleotide adjuvant |
JP55065898A JP2001526688A (en) | 1997-05-19 | 1998-05-19 | Oligonucleotide adjuvant |
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US4712197P | 1997-05-19 | 1997-05-19 | |
US60/047,121 | 1997-05-19 |
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WO1998052962A1 true WO1998052962A1 (en) | 1998-11-26 |
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PCT/US1998/010459 WO1998052962A1 (en) | 1997-05-19 | 1998-05-19 | Oligonucleotide adjuvant |
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EP (1) | EP0983289A4 (en) |
JP (1) | JP2001526688A (en) |
AU (1) | AU7589398A (en) |
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WO2011109833A2 (en) | 2010-03-05 | 2011-09-09 | President And Fellows Of Harvard College | Induced dendritic cell compositions and uses thereof |
WO2013117348A1 (en) * | 2012-02-09 | 2013-08-15 | Carsten Kirschning | Agonists and antagonists of toll-like receptor (tlr) 13 |
US9556439B2 (en) | 2012-02-09 | 2017-01-31 | Bavarian Nordic A/S | Agonists and antagonists of toll-like receptor (TLR) 13 |
Also Published As
Publication number | Publication date |
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EP0983289A4 (en) | 2001-04-25 |
AU7589398A (en) | 1998-12-11 |
EP0983289A1 (en) | 2000-03-08 |
CA2289741A1 (en) | 1998-11-26 |
JP2001526688A (en) | 2001-12-18 |
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