WO2006076383A2 - Composition et technique permettant d'induire une reponse de vaccin de protection - Google Patents

Composition et technique permettant d'induire une reponse de vaccin de protection Download PDF

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WO2006076383A2
WO2006076383A2 PCT/US2006/000866 US2006000866W WO2006076383A2 WO 2006076383 A2 WO2006076383 A2 WO 2006076383A2 US 2006000866 W US2006000866 W US 2006000866W WO 2006076383 A2 WO2006076383 A2 WO 2006076383A2
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cells
antigen
cxcllo
vaccine
cxcr3
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WO2006076383A3 (fr
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Mitchell Krathwohl
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Rosetta Stone Labs, Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to compositions and methods for preparing vaccines. More particularly, the invention relates to compositions and methods for increasing the immune response to vaccine antigens.
  • Vaccines are important for prevention of a variety of diseases, and can be particularly effective for prevention of viral disease. Certain immunological principles govern vaccine efficacy, but these principles are not well understood. Generally, infection with a wild-type pathogenic virus will produce long-lasting immunity that protects against illness when the host is re-exposed to the same virus. This, of course, is only of benefit if the host survives the initial infection with the pathogenic virus. In some cases, live virus such as the vaccinia virus vaccine used for smallpox can provide long-term protection but may also cause lymphadenopathy, fever, and life-threatening disease.
  • T H 1 cellular
  • T H 2 antibody
  • the T H I response predominates in mice when live virus is used to immunize, while the T H 2 response predominates when outer membrane proteins of the virus are used as vaccine (Fogg, et al, J. Virol. (2004) 78: 10230-10237).
  • CD8 CTLs generally appear about one week after acute viral infection, and their numbers rapidly increase to peak at 2-3 weeks after infection. The peak often corresponds to the period when the virus is being cleared by the host.
  • CD8 cells exert their effects through two main mechanisms: direct attack on virus-infected cells and secretion of interleukins (ILs) and cytokines such as IFN- ⁇ , TNF- ⁇ , and IL-2 that may also play a role in clearing virus-infected cells.
  • ILs interleukins
  • cytokines such as IFN- ⁇ , TNF- ⁇ , and IL-2
  • Immune induction efficiency is increased if immunogen (antigen) is presented by antigen-presenting cells (APCs) such as macrophages and dendritic cells.
  • APCs antigen-presenting cells
  • Immature dendritic cells are derived from the same bone marrow precursors as macrophages.
  • DCs Dendritic cells
  • MHC major histocompatibility complex
  • a dendritic cell takes up pathogenic organisms, it becomes activated, stimulating secretion of cytokines. If the DC fails to be activated, it induces tolerance to the antigens it bears.
  • DC maturation therefore represents a key control point in the decision for immunity versus tolerance.
  • DC After encountering antigen in the context of a danger signal, DC undergo a program of maturation that enables them to efficiently induce an antigen- specific T cell immune response.
  • a large diversity of danger signals have been defined that serve to promote DC maturation, including microbial constituents, cytokines, and UV light (Bell, et ah, "Dendritic Cells," Advances in Immunology (1999) 72: 255-324).
  • DC also can amplify such danger signals by autocrine and paracrine release of cytokines such as interferon ⁇ (IFN- ⁇ ), which has been shown to act as a natural adjuvant through its effects on DC maturation (Proietti, et ah, J. Immunol.
  • IFN- ⁇ interferon ⁇
  • DC under appropriate stimulation can both secrete and respond to IFN- ⁇ , but it is not clear how IFN- ⁇ mediates DC maturation.
  • DC maturation has been shown to involve members of the MAP kinase family of signaling proteins (Ardeshna, et ah, Blood (2000) 96 (3): 1039-1046), yet EFN- ⁇ has not been shown to induce this pathway in DC.
  • interferons are known to induce expression of a number of proteins such as chemokines with downstream effector functions. Whether chemokines might work as downstream signals or amplifiers of maturation signals has remained in question.
  • Peptide vaccines provide an alternative to whole-virus vaccine and may pose less risk than do whole-virus vaccines.
  • Whole-virus vaccine may, for example, pose a risk of transmission of the vaccine strain to unvaccinated individuals. Since the beginning of the United States Armed Forces smallpox vaccination program in December 2002, for example, there have been 30 reported cases of accidental contact transmission (MMWR Morb Mortal WkIy Rep (U.S. Centers for Disease Control, 2004) 53: 103-105: Garde et ah. JAMA (2004) 291: 725-727).
  • Protein and peptide vaccines, DNA vaccines, and other vaccine formulations provide alternatives to whole microbe vaccines that are less likely to produce unwanted side-effects and are more likely to be easier to produce.
  • Peptides are poorly immunogenic in the absence of co-administered adjuvants, however.
  • compositions and methods for improving the immunogenicity and protective immune response provided by these vaccine formulations are needed.
  • the present invention provides a composition comprising a therapeutically effective amount of one or more CXCR-3 binding chemokines, such as human interferon-gamma inducible protein (EP-IO, also known as CXCLlO), that, when administered in conjunction with one or more antigens, stimulate a protective immune response to the same or an immunologically equivalent antigen.
  • CXCR-3 binding chemokines such as human interferon-gamma inducible protein (EP-IO, also known as CXCLlO)
  • the invention also provides a vaccine composition comprising a therapeutically effective amount of and at least one CXCR-3 binding chemokine, such as IP-10, and at least one immunogen to stimulate immune protection against future infection by an infectious agent.
  • the at least one immunogen is a live attenuated virus, an inactivated virus, a viral or bacterial protein, or a peptide derived from a viral or bacterial protein.
  • Proteins and peptides can also comprise modified proteins and peptides that differ from the wild-type protein or peptide by amino-acid substitution or other modification, particularly those modifications that may decrease degradation of the protein or peptide or increase its immunogenicity.
  • DNA, peptide nucleic acids, or other immunogens may also comprise compositions of the invention.
  • the invention comprises a vaccine comprising an immunogenic amount of at least one antigen chosen from among at least one bacterial antigen, at least one viral antigen, at least one fungal antigen, at least one tumor antigen, or a combination thereof, an of at least one CXCR3 -binding chemokine effective to induce maturation of lymph-node derived dendritic cells.
  • the invention also provides a method for stimulating a ThI -type immune response to an antigen administered as a vaccine.
  • a CXCR3 -binding chemokine such as, for example, CXCLlO or I-TAC, is administered to stimulate dendritic cells (DC) in the tissues to mature and promote the development of a ThI -type immune response to the vaccine antigen.
  • DC dendritic cells
  • the invention provides a method for inducing a ThI -type response to peptide and protein antigens that might most commonly induce a Th2-type immune response.
  • the invention also provides peptides comprising the amino acid sequence DSNFFTEL for use in subunit vaccines to promote a protective response to Vaccinia virus.
  • Fig. 1 is a series of photographs of microscopy illustrating that CXCLlO induces morphologic and phenotypic changes in DC characteristic of maturation. Photographs illustrate results of treatment as follows: Control - Ia; LPS - Ib; IP-IO - Ic; MIP-3-beta - Id. Treatment of monocyte derived dendritic cells (MDDC) with CXCLlO causes cells to develop extensive dendritic processes (Ic), similar to LPS-treated cells (Ib).
  • MDDC monocyte derived dendritic cells
  • Fig. 2 is a graph illustrating that lymph node-derived dendritic cells (LNDC) treated with CXCLlO for 48 hours produce IL-12 by ELIspot assay as shown by the increased number of spots formed as compared to control.
  • LNDC lymph node-derived dendritic cells
  • Fig. 3 is a graph of the proliferation index illustrating that DC cultured with CXCLlO become potent stimulators of T cells. Allogeneic T cells were cultured in various ratios with MDDC that had been grown with or without CXCLlO. After 7 days, the total number of cells was determined and expressed as a ratio of the number of input cells. The proliferation index is indicated on the Y axis and the ratio of T- cells to dendritic cells is indicated on the X axis. Each DC ratio was determined in triplicate.
  • Fig. 4 is a graph of the proliferation index following allogeneic T-cell stimulation by co-culture with CD34-derived DC, at the ratios indicated on the X axis, that had been grown with or without CXCLlO. After 7 days, the total number of cells was determined and expressed as a ratio of the number of input cells. The proliferation index is indicated on the Y axis and the ratio of T-cells to dendritic cells is indicated on the X axis. Each DC ratio was determined in triplicate.
  • Fig. 5 shows survival diagrams of treated DC. LNDC were cultured without growth factors in the presence or absence of CXCLlO. Cell viability was determined daily by trypan blue exclusion. All cultures were set up in triplicate.
  • Fig. 6 is a series of photos showing the results of mice in several treatment groups injected with vaccinia virus. Only mice in the CXCLIO+peptide group were protected from infection as shown by the lack of tail ulcers.
  • Fig. 7 is a bar graph of the quantitative results of mice injected with vaccinia virus in each of several treatment groups. Mice were injected twice with CXCLlO with or without vaccinia peptide, with peptide alone or normal saline alone. Four weeks after the first injection, all mice were injected with vaccinia virus intradermally at the base of the tail and the development of tail ulcers was monitored. Ulcer severity was scored on a 0 to 4+ scale for each group. The graph shows quantitative counts of tail ulcer formation.
  • Fig. 8 is a bar graph illustrating induction of peptide-specific cytotoxic T cells by CXCLlO administration with a vaccinia-derived peptide. Spleens were removed from all groups of mice, and splenocytes were tested for proliferation against the injected vaccinia peptide. After 5 days, the total number of cells was enumerated using a fluorescence-based method for each group. The stimulation index is indicated on the Y axis and the treatment group is indicated on the X axis.
  • Fig. 9. is a line graph illustrating the percent of specific lysis when splenocytes were stimulated with peptide for 5 days, and then incubated with target cells that had been pulsed with peptide and labeled with the fluorescent tracer BCECF. Specific lysis was calculated for each treatment group. The ratio of effector to target cells is indicated on the X axis and the percentage of specific lysis is indicated on the Y axis.
  • the present invention provides a composition comprising a therapeutically effective amount of a CXCR3 -binding chemokine and at least one immunogen for administration as a vaccine to stimulate immune protection against future infection by an infectious agent.
  • the method of the invention provides a method for increasing the immune response to one or more antigens administered as a vaccine.
  • increasing the immune response it is meant that the immune response, and especially the ThI immune response, is increased over that induced by antigen alone.
  • the CXCR3-binding chemokine is interferon-inducible protein (IP-IO), also known as CXCLlO.
  • IP-IO interferon-inducible protein
  • the invention also provides a method for stimulating a Thl-type immune response to an antigen administered as a vaccine.
  • the invention provides a method for inducing maturation of dendritic cells to promote a cellular immune response to antigen by administering a therapeutically effective amount of a CXCR3-binding chemokine such as, for example, IP-10, 1-TAC, or Mig sufficient to stimulate dendritic cell maturation in the tissues in which the antigen is delivered.
  • a CXCR3-binding chemokine such as, for example, IP-10, 1-TAC, or Mig sufficient to stimulate dendritic cell maturation in the tissues in which the antigen is delivered.
  • the invention also provides a method for inducing a Thl-type response to peptide and protein antigens that might most commonly induce a Th2-type immune response.
  • the invention also provides a method for inducing IL- 12 production by dendritic cells.
  • CXCR3-binding chemokines such as IP-10 and I-TAC, for example
  • Peptides and protein-binding domains have been shown to retain much of the functionality of a full-length protein when their sequences are isolated from that of the full-length protein itself.
  • CXCLlO also induced IL-12 production in these DC, and enabled DC to form conjugates with T cells and stimulate T cell proliferation and interferon- ⁇ production. Survival of DC was also increased by CXCLlO administration. Furthermore, IPlO-induced maturation of dendritic cells stimulated a Thl-type immune response. By administering a vaccine comprising a combination of antigen and IP-10, a CD8 T cell response and a protective response against viral challenge were induced.
  • a peptide vaccine according to the present invention was used by the inventors, for example, to protect mice from experimental challenge with Vaccinia virus infection.
  • CXCLlO as well as inducible T cell- ⁇ chemoattractant (I-TAC, CXCLl 1) and monokine-induced by ⁇ -interferon (Mig, CXCL9) are chemokines that control leukocyte migration via their binding to chemokine receptor CXCR3.
  • vaccines are prepared for injection into a human or mammalian subject.
  • Injectable vaccines can be prepared as liquid solutions or suspensions. Solid forms can be prepared that are suitable for solution in, or suspension in, liquid prior to injection. The preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with a pharmaceutically acceptable carrier that is compatible with the active ingredient. Suitable carriers include, but are not limited to, water, dextrose, glycerol, saline, ethanol, and combinations thereof.
  • the vaccine may contain additional agents such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine.
  • Vaccines may be conventionally administered parenterally using subcutaneous or intramuscular injection.
  • Other modes of administration may include oral administration, nasal administration, rectal administration, and vaginal administration, which may involve combining the immunogen with pharmaceutically acceptable carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, or other carrier.
  • Compositions for oral administration may form solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • a vaccine of the present invention can be administered by enteric-coated capsule, for example, for release of the polypeptide into the lumen of the intestine.
  • Vaccines may be delivered intravenously, although it should be taken into account that IV administration may be associated with an increased risk of side-effects.
  • Vaccines may be inhaled, and antigens may be pegylated (attached to at least one polyethylene glycol moiety) to increase the half-life of the antigen in the tissues.
  • Peptide or other immunogen may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, mandelic, oxalic, and tartaric. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, and histidine.
  • Vaccine is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, taking into account, for example, the subject's age and overall health, and the degree of protection desired. Precise amounts of active ingredient (peptide immunogen) to be administered depend on the judgment of the practitioner. Suitable dosage ranges generally require several hundred micrograms of active ingredient per vaccination.
  • regimes for initial administration and booster vaccinations which should be determined by the judgment of the practitioner.
  • Dosage of vaccine will depend on the route of administration and will vary according to the size of the host.
  • Adjuvants for use in combination with immunogen for vaccination include, but are not limited to, aluminum hydroxide or phosphate, also known as alum, commonly used as 0.05 to 0.1 percent solution; aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70° C for 30 seconds to 101 0 C for 2 minutes.
  • compositions of the present invention can comprise vials containing a combination of one or more antigens comprising, for example, whole virus, proteins, or peptides, and CXCLlO.
  • vials containing, individually, CXCLlO and one or more antigenic compositions are also provided by the invention.
  • the invention provides compositions for delivery of vaccine "cocktails" which can increase the cellular immune response to one or more infectious agents.
  • IP-10 can be administered concurrently with the target antigen or antigens. IP-10 may also be administered within an effective time prior to the administration of antigen or within an effective time following administration of antigen. IP-IO may therefore be part of a vaccine administered to a subject or may be an immunotherapeutic agent delivered prior to or after vaccination in order to stimulate the subject's immune system to generate a ThI- type response to the infectious agent to which the vaccine is directed.
  • the invention provides compositions and methods for administering vaccines for bacterial and viral infectious agents, such as, for example, human immunodeficiency virus (HIV), vaccinia virus (for smallpox vaccine), herpes simplex virus (HSV), influenza virus, Ebola virus, Dengue virus, and SARS-CoV.
  • a vaccine is prepared using a polypeptide comprising SEQ ID NO: 1 to provide a protective response to Vaccinia virus infection. It is to be understood that such a polypeptide may comprise additional amino acids at the C-terminus or N- terminus, and that functionally equivalent amino acid substitutions may be made to the amino acid sequence described by SEQ ID NO: 1.
  • polypeptides of the invention may be provided through various means and in combination with other antigenic compositions, adjuvants, and other agents.
  • Polypeptides of the invention may also be provided in the form of nucleic acids encoding the polypeptides, from which the polypeptide may be expressed. Given the polypeptide sequence, it is well within the expertise of one of skill in the art to predict nucleotide sequences that would encode such a polypeptide.
  • the invention also provides compositions and methods for administering vaccines containing tumor-associated antigens in order to generate a cellular immune response to tumor cells for the treatment of cancer.
  • the immune system recognizes tumor-specific antigen(s), usually through the help of an APC such as a dendritic cells.
  • APCs engulf tumor-specific antigens, break them into peptides and display peptide fragments on the surface of their own cells, complexed with major histocompatibility complex (MHC) proteins located on the surface of the APC.
  • MHC major histocompatibility complex
  • CD8+ cells bind to these complexes on the APC. Once activated, CD8+ cytotoxic T lymphocytes can migrate into tumor masses and cause cytolysis of tumor cells.
  • a variety of tumor-specific antigens have been identified for specific types of cancer, such as prostate cancer and melanoma.
  • previously-identified antigens can be administered in conjunction with BP-IO to promote a cell-mediated immune response to tumor cells expressing the antigen.
  • specific antigens have not been identified.
  • a CXCR3 -binding chemokine such as IP-IO can be administered in conjunction with acid-eluted peptides derived from autologous tumors.
  • a procedure for obtaining these types of peptides has been described by Zitvogel et al. (J. Exp. Med. (1996) 183: 87-97).
  • Viral, bacterial, fungal, or tumor antigens may also be provided as one or more fusion peptides containing cell-permeable peptide sequences for delivery of the peptide antigen or antigens to the interior of a dendritic cell.
  • Additional cytokines or chemokines may also be provided in conjunction with a vaccine as provided by the present invention in order to increase or modulate the immune response to the antigen or antigens of choice in the vaccine.
  • Many such immune-modulating cytokines and chemokines are known to those of skill in the art of immunology and vaccine development.
  • CXCLlO was purchased from Peprotech (Rocky Hill, NJ) and certified endotoxin-free by the manufacturer.
  • GM-CSF was a kind gift from Immunex Corporation.
  • IL-4, TNF- ⁇ were also from Peprotech.
  • PBMCs from normal human donors were purchased from AllCells.
  • PBMCs were incubated in 75 cm 2 flasks for 2 hours at 37° C to adhere monocytes. Flasks were washed extensively with PBS to remove non-adherent cells. The remaining cells were removed by scraping and routinely consisted of >70% CD 14+ cells. Resulting monocytes were cultured in 24 well plates at 5 x 10 5 cells/ml in RPMI 1640 + 10% heat-inactivated FBS (Hyclone) + antibiotics, and 100 ng/ml GM- CSF and IL-4. Cells were cultured in triplicate with or without the chemokine CXCLlO at 100ng/ml added at the beginning of culture and replaced every 3-5 days, or added only at day 7 of culture.
  • Bone marrow-derived CD34+ cells were purchased from AllCells and were >95% CD34+. Cells were cultured at 5 x 10 4 cells/ml in RPMI 1640 + 10% FBS with 100 ng/ml GM-CSF and 2.5 ng/ml TNF- ⁇ . Cells were cultured in triplicate either with or without the chemokine CXCLlO at 100 ng/ml added at day 0 and replenished every 3-5 days or else added only at day 7. Cells were harvested for further analysis on day 10-12.
  • Lymph node tissue was cut into small pieces 2 x 2 x 2 mm and cultured on Transwell insert supports in 24 wells plates, leaving the top surface exposed to air and the bottom surface resting on a membrane with 5 ⁇ m pores.
  • the bottom wells contained RPMI 1640 + 10% FBS and antibiotics.
  • Purified lymph node DC were cultured with or without CXCLlO (100 ng/ML) for 3 days, then labeled with a fluorescent tracking dye (Cell Tracker Green, CTG) according to the manufacturer's instructions and placed in bottom wells and allowed to migrate into the tissue through the pores.
  • Slices were then harvested and either stained for confocal microscopy or made into single cell suspensions and analyzed for the presence of treated DC and T cell proliferation.
  • a fluorescent monoclonal antibody to CD3 was stained with slices while in the Transwell inserts for 2 hours. Slices were then placed in agar and analyzed by Confocal microscopy for the presence of CTG+ cells adjacent to CD3+ cells.
  • IL- 12 Elispot was performed using purified LNDC added to the wells of an ELIspot plate (BD) and allowed to adhere to the PVDF membrane. CXCLlO (100 ng/ml) was added to some cultures of cells, and cells were incubated for 48 hours to allow IL-12 production. Plates were then harvested, and IL-12 was detected by a sandwich assay as per the manufacturer's instructions. Spots were counted visually. Each well was tested in triplicate, and the entire assay was performed at least twice with identical results.
  • CXCLlO 100 ng/ml
  • Allogeneic T cells were purified from peripheral blood mononuclear cells by column purification for T cell proliferation assay. MDDC or CD34-derived DC were washed and mixed with T cells in the indicated ratios. Triplicate determinations were made for each ratio. Cells were cultured for 7 days, and the total number of cells was then counted using a hemocytometer. The entire experiment was performed at least twice with identical results.
  • Monocytes were cultured in RPMI1640 + 10% FBS and antibiotics with GM-CSF and IL-4 for 7 days for the intracellular signaling assay. Either normal saline or CXCLlO (100 ng/ml) was added to the cultures for 10 minutes, then cultures were placed on ice. Surface markers (CDlIc and HLA-DR) were stained using monoclonal antibodies, then cells were fixed and permeabilized with Cytofix/Cytoperm. Fluorescent monoclonal antibodies to p-ERK, p-JNK or p-p38 were then added and cells were stained for 1 hour. Cells were then washed and analyzed by flow cytometry.
  • lymph node derived cells were washed and grown in RPMI 1640 + 10% FBS but without addition of GM-CSF. CXCLlO was added to some cultures of LNDC set up in triplicate at 100 ng/ml. Cells viability was determined by trypan blue exclusion daily.
  • CXCLlO was added to some cultures on the day of culture initiation and replaced every 3-5 days, and the resulting cells were analyzed after 7-9 days.
  • CXCLl 0-treated cells appeared similar to cells matured with E. coli LPS.
  • DC markers typical of mature cells including HLA-DR, CD83, CD40, CD80, and CD86.
  • CXCLlO resultsed in the down-regulation of the monocyte marker CD 14.
  • the inventors further tested the ability of CXCLlO to affect monocyte maturation when added only after 7 days of culture with GM-CSF and IL-4.
  • CXCLlO increased expression of maturation markers HLA-DR, CD83, CD86 and CD80 but not CD40.
  • CXCLlO appears to promote the full maturation of MDDC.
  • DC derived from CD34+ hematopoietic progenitors appear to resemble Langerhans cells as well as cells resembling interstitial DC, and may be better at stimulating CD8 T cells than MDDC.
  • CD34+ hematopoietic progenitor cells were cultured with GM-CSF and TNF- ⁇ , either with or without CXCLlO, for 7 days.
  • CXCLlO resulted in increased expression of maturation markers on these CD34-derived DC.
  • Intracellular flow cytometry for IL- 12 also confirmed that progenitors treated with CXCLlO were fully mature DC.
  • CXCLlO also induces maturation of CD34-derived DC.
  • CD4 T cells were analyzed for intracellular expression of IFN- ⁇ and CXCLlO plus tetanus toxoid-treated DC induced a population of CD4 T cells that expressed significant levels of IFN- ⁇ , while control DC and DC treated with CXCLlO alone did not. CXCLIO-treated DC therefore appear to be potent stimulators of T cells.
  • CXCLlO significantly promoted phosphorylation of JNK and p38 while also promoting de-phosphorylation of ERK. Since de-phosphorylation of ERK suggests that CXCLlO may also promote DC survival and LPS has been shown to increase the survival of DC in cultures grown without GM-CSF or IL-4, purified LNDC were cultured with or without CXCLlO in media without the growth factor GM-CSF, and cellular viability tested periodically for 6 days to test whether CXCLlO treatment would increase survival of growth factor-derived DC. CXCLlO significantly increased survival of DC. CXCLlO therefore induces signaling in DC through pathways known to be involved in maturation.
  • Example 2 Mouse [057] Murine CXCLl 0 was obtained from Peprotech (Rocky Hill, NJ) and certified by the manufacturer to contain less than 0.1 ng of endotoxin per ⁇ g protein. Ovalbumin (OVA, grade VII) was obtained from Sigma-Aldrich (St. Louis, MO), and was confirmed to contain less than 0.06 EU/mg protein using the Pyrogen Plus LAL kit (Cambrex, Walkersville, MD). To select a peptide epitope, the H3L gene sequence of Vaccinia virus encoding the VP35 envelope protein was entered into the SYFPEITHI database.
  • Ovalbumin Ovalbumin
  • the octameric peptide DSNFFTEL (SEQ ID NO: l) was chosen as potentially binding to mouse H-2Kb. This peptide was synthesized by Sigma Genosys (The Woodlands, TX) at 95% purity, soluble in water, and having a concentration of 0.06 EU/mg protein. Fluorescent monoclonal antibodies to CD3, CD8 and IFN- ⁇ were obtained from Pharmingen (San Diego, CA), as was the unlabeled CD32 monoclonal antibody (Fc Block).
  • mice For studies using OVA, groups of 10 6-7-week-old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously with 50 ⁇ l of either normal saline, 30 ⁇ g/kg CXCLlO in normal saline, or 30 ⁇ g/kg CXCLlO + 0.5 mg OVA. All studies were approved by the local animal use committee. Mice were examined daily for side effects including behavioral abnormalities, local inflammation, and anorexia. Blood was sampled from the tail vein for initial tetramer analysis and processed as described herein. Two weeks later, subcutaneous injections were repeated in all animals at the same dosages.
  • mice were sacrificed and blood and spleens were tested for the presence of tetramer-positive T cells as below. The entire experiment was performed twice with similar results.
  • vaccinia infection studies an adaptation of previously published methods (Owen, D., et ah, Nature (1975) 254: 598-9; Tscharke, D and Smith, G, J. Gen. Virol. (1999) 80: 2751-5) to determine immunity to vaccinia was used.
  • mice Groups of 10 6-7-week-old C57BL/6 mice were injected subcutaneously with 50 ⁇ l of normal saline 30 ⁇ g/kg CXCLlO, 0.3 mg peptide, or 30 ⁇ g/kg CXCLlO + 0.3 mg peptide on day 0.
  • the OVA iTAG H-2 Kb SIINFEKL-PE conjugated tetramer was obtained from Immunomics (San Diego, CA) and used according to the protocol of the manufacturer. Whole blood was diluted 1:1 with EDTA/PBS and stained with an experimentally determined optimum concentration of the tetramer, and anti-CD3, CD8 and CD32 (to prevent non-specific binding) antibodies for 1 h at 4°C in the dark. Blood cells were then lysed with 10 mg/ml saponin (Sigma, St.
  • a fluorometric assay was used to determine antigen-specific cell lysis.
  • Target cells were prepared from the spleens of normal mice by making single cell suspensions of spleens, washing cells, and placing them in RPMI 1640 without serum.
  • the fluorochrome BCECF (Molecular Probes, Eugene, OR) was added to a concentration of 10 ⁇ M.
  • the vaccinia-derived peptide was also added at a concentration of 10 ⁇ g/ml.
  • Cells were incubated for 30 min at 37°C in 5% CO 2 . Cells were washed and placed in RPMI 1640 with 10% FBS and 1001.U./ml penicillin and 100 ⁇ g/ml streptomycin.
  • Effector T cells were expanded from splenocytes prepared from spleens from each mouse group. Splenocytes were cultured with 10 ⁇ g/ml of peptide in RPMI 1640 + 10% FBS for 5 days. Cells were then washed, placed in RPMI 1640 + 10% FBS with 100 I.U./ml penicillin and 100 ⁇ g/ml streptomycin, and incubated with labeled target cells in ratios of 100: 1, 50: 1 and 25: 1 in 96 well U-bottom plates. Each ratio was set up in triplicate. Wells with target cells alone served as standards for maximum retention of fluorochrome, and wells with target cells lysed with 1% Triton X-100 served as standards for maximum release. The experiment was performed twice with identical results.
  • mice were injected subcutaneously with either: 10, 20, or 30 ⁇ g/kg CXCLlO and whole OVA CXCLlO alone, or normal saline. Dosage range was selected by comparison with dosages of other chemokines that demonstrated biological effects.
  • mice Two weeks after the first injections, all groups of mice received booster injections using the same concentrations of CXCLlO and/or OVA. Blood samples were drawn from mice at 2 and 4 weeks after the first injections and analyzed for the presence of OVA-specific T cells using tetramers recognizing the OVA- derived peptide SIINFEKL. After the first injections, CXCLlO promoted the development of OVA-specific T cells in 4/10, 5/10 and 5/10 mice injected with OVA and either 10, 20, or 30 ⁇ g/kg CXCLlO respectively.
  • Injected antigen has been shown to be taken up and processed by Langerhans cells which mature and present antigen in the draining lymph nodes. IfCXCLlO were inducing the maturation of Langerhans cells in the skin, increased numbers of cells would migrate to draining lymph nodes to present antigen. Draining lymph nodes were therefore analyzed for the presence of CDl lc+ CD8 ⁇ int MHCII high CD40 hlgh mature antigen presenting Langerhans cells. Injection of CXCL 10+ OVA resulted in a large increase in the percentage of mature Langerhans cells in the draining lymph node. CXCLlO alone also increased the percentage of mature Langerhans cells, but to a lesser degree. In contrast, control mice showed very few mature Langerhans cells. Most Langerhans cells in control animals appeared to be in an immature state. These results demonstrate that CXCLlO acts in vivo to stimulate an antigen-specific CD8 T-cell response.
  • mice were then injected subcutaneously with this peptide (SEQ ID NO: 1) and CXCLlO, CXCLlO alone, peptide alone, or normal saline. Injections were repeated 2 weeks later. No side effects were observed in any of the mouse groups.
  • mice were challenged with vaccinia virus using intradermal injections at the base of the tail. The quantity of virus injected was found to be sufficient to cause an ulcer in unvaccinated animals in preliminary experiments.
  • CXCLlO effectively promotes the development of an immune response to a pathogen- derived peptide antigen that is sufficient to prevent infection.
  • Virus-specific cytotoxic T lymphocytes were then expanded for 5 days, and cytotoxic capability was assessed by adding target splenocytes pulsed with vaccinia peptide and measuring specific lysis.
  • CXCLlO therefore demonstrated the ability, when administered in conjunction with antigen, to prevent experimental infection with vaccinia virus by inducing a T-cell-mediated immune response.

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Abstract

La présente invention concerne une technique permettant d'induire une réponse immune de protection. Cette technique utilise une composition comprenant une chimiokine se liant à CXCR3 telle que CXCL10 (IP-10) administrée avec une protéine, un peptide, un polynucléotide ou un autre antigène cible.
PCT/US2006/000866 2005-01-11 2006-01-11 Composition et technique permettant d'induire une reponse de vaccin de protection WO2006076383A2 (fr)

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US6355252B1 (en) * 1997-02-21 2002-03-12 Isis Innovation Ltd. Soluble vaccinia virus protein that binds chemokines

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6355252B1 (en) * 1997-02-21 2002-03-12 Isis Innovation Ltd. Soluble vaccinia virus protein that binds chemokines

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Title
HAMILTON N.H.R. ET AL.: 'A Recombinant Vaccinia Virus Encoding the Interferon-Inducibl T-Cell Alpha Chemoattractant is Attenuated In Vivo' SCANDINAVIAN JOURNAL OF IMMUNOLOGY vol. 59, 2004, pages 246 - 254, XP003013137 *
TRIFILO M.J. ET AL.: 'CXC Chemokine Ligand 10 Controls Viral Infection in the Central Nervous System: Evidence for a Role in Innate Immune Response through Recruitment and Activation of Natural Killer Cells' JOURNAL OF VIROLOGY vol. 78, no. 2, 2004, pages 585 - 594, XP003013138 *

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