WO2004083374A2 - Procede - Google Patents

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Publication number
WO2004083374A2
WO2004083374A2 PCT/GB2004/001266 GB2004001266W WO2004083374A2 WO 2004083374 A2 WO2004083374 A2 WO 2004083374A2 GB 2004001266 W GB2004001266 W GB 2004001266W WO 2004083374 A2 WO2004083374 A2 WO 2004083374A2
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Prior art keywords
cell
natural antibody
organism
test
molecule
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PCT/GB2004/001266
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English (en)
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WO2004083374A3 (fr
Inventor
Paul Kaye
Deborah Smith
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Imperial College Innovations Limited
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Publication of WO2004083374A2 publication Critical patent/WO2004083374A2/fr
Publication of WO2004083374A3 publication Critical patent/WO2004083374A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • A61K39/002—Protozoa antigens
    • A61K39/008—Leishmania antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39575—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from other living beings excluding bacteria and viruses, e.g. protozoa, fungi, plants

Definitions

  • the present invention relates to antigens useful as vaccines, and methods for selecting suitable antigens.
  • CD8 T cells in host protection against intracellular pathogens is well recognized , and CD8 + T cell priming is critical for successful vaccination against leishmaniasis 5,6. Whilst genome sequencing projects now provide a plethora of potential vaccine candidates 7, few criteria exist for selecting amongst them and antigen choice remains largely empirical. Furthermore, successful vaccination requires antigen targeting into the MHC class I processing pathway 8-12 and a cytokine milieu that favors CD8 + T cell priming and differentiation 4,13,14. Recent interest has focused on how antibody and complement regulate CD8 + T cell-mediated immunity.
  • Natural antibodies represent germ-line encoded IgM, IgG and IgA antibodies found in normal individuals and animals 18,19. They are present in unimmunised (naive) individuals and have low affinity compared to hypermutated antibodies generated during normal immune responses and have broad reactivity to a variety of self-antigens 19. Most natural antibodies are produced by self-renewing B-l B cells, which preferentially localize to the peritoneum is. Natural antibodies serve as innate microbial recognition receptors, recognizing various bacterial cell wall components 20. Natural anti-phosphorylcholine antibodies can protect against pneumococcal disease 20, and natural antibody - dependent complement activation plays a role in LPS clearance and protection from endotoxemia 21. However, a specific role for natural antibodies in the regulation of acquired T cell-mediated immunity during infection or vaccination has not been described.
  • the invention makes use of these features of recognition of pathogen- derived molecules by natural antibodies and uses natural antibodies to (for example) screen whole pathogen (or other vaccination target, for example cancer cell or viral-infected cell) proteomes (including bacteria, protozoa and helminths), by immunoblotting of total protein populations separated in two dimensions or displayed on protein chips. Molecules that are positive in these screens may be identified by cross-reference to proteome and genome databases. The methods of the invention are considered to constitute a considerable short cut for the identification of potentially immunogenic vaccine candidates. Vaccines harnessing the endogenous adjuvant activity of natural antibodies and free of exogenous adjuvants or delivery systems may also avoid concerns and regulatory constraints such as those associated with viral or DNA based delivery.
  • Antibodies binding to the identified antigen may also be useful a diagnostic reagents.
  • IL-4 is always detrimental to protection against intracellular pathogens.
  • Natural antibodies from rodents or man may be used to directly identify immunogenic candidate antigens (for example in relation to TB, malaria or leishmaniasis), with additional savings in costs, time and animal usage. Consistent with the findings noted above that reactivity to self antigens is both invariant across individuals and as a function of age, selection of pathogen-derived antigens recognized by natural antibodies is considered to select for antigens recognized across the majority of the human population and in both children and adults. Further, as many self antigens are recognized by both the mouse and human natural antibody repertoires either human or murine serum-derived natural antibodies may be used for selection of pathogen proteins: the minor variations between mouse and human repertoires are considered unlikely to cause a problem. Self- antigens used for isolation of these natural antibodies may be chosen on the basis of their recognition in both species. This may further ensure that vaccine candidates identified using human antibodies can be reliably screened in rodent models of protection.
  • the screening method may be primarily suited to any situation where there is proteomic / genomic data available to identify the proteins of interest.
  • the natural antibody-binding antigen is considered to act almost as if the antigen had been given as a DNA vaccine, so any situation where DNA vaccination may be appropriate could be replaced or adapted by using antigens selected in this way.
  • Antigens selected using methods of tlie invention may also be administered via a DNA vaccine. Additional immune enhancement above that provided by natural antibodies alone may be achieved by incorporating immunomodulators e.g. cytokines into a DNA vaccine. IL-4 may also be co-administered (or encoded by a DNA vaccine).
  • tumours are intracellular infections (viral bacteria and protozoal), although the principles may also apply to extracellular helminth worms, though it is unclear whether CD8 T cells are needed here for vaccine induced protection.
  • Tumours are also candidates, as the method of the invention may be used to identify potential tumour specific antigens for vaccination. Currently, most studies use crude lysates of tumours for vaccination or cell-based therapies.
  • a first aspect of the invention provides a method for screening for a candidate for an antigen for use in a vaccine against a target cell or organism comprising the steps of
  • test molecule or sample binds to the natural antibody (3) selecting a test molecule or sample which binds to the natural antibody as a candidate for an antigen for use in a vaccine.
  • the test molecule or sample may be or comprise a polypeptide.
  • the test molecule may be a polypeptide (including a fragment of a naturally occurring polypeptide) or, for example, a glycoprotein or a lipoprotein.
  • the test sample may comprise one or more polypeptides and/or glycoproteins and/or lipoproteins.
  • the test molecule or test sample may be derived directly from the target cell or organism.
  • the test molecule or components of the test sample may be recombinant; this may be useful, for example, when seeking to confirm the identity of a molecule identified as binding to a natural antibody using the method of the invention with a test molecule or test sample derived directly from the target cell or organism.
  • derived directly from the target cell or organism is included derivation from a culture of the target cell type or cells from the target organism, as is well known to those skilled in the art.
  • the test molecule or sample may be a single test molecule or sample, for example to confirm the identity of a natural antibody-binding molecule or sample (which may be, for example, a cbromatographic or other fraction which may contain more than one molecular species).
  • the test molecule or sample may form part of a test population of molecules or samples derivable from the target cell or organism.
  • the method may be performed on multiple test molecules or samples from the target cell or organism. This may be useful as an initial screen in search of antigens which bind to a natural antibody.
  • this may be sufficient to permit identification of a single molecular species which binds to the natural antibody, but in other cases (for example when the initial screen is performed on complex (multi-molecule) samples rather than on separated test molecules) it may be necessary to perform further screens, for example on less complex fractions of the sample or samples identified as positive (antibody binding) in the initial screen.
  • the test population may be in the form of an array of molecules or samples derivable from the target cell or organism.
  • the test population may be an array representing the proteome of the target cell or organism.
  • Such protein microarrays or chips consist of large number of proteins (which generally are recombinant) attached in an ordered array to a solid support, which then can be screened for enzymatic activities, physical properties or interactions with other proteins or small molecules (MacBeath G (2002) Protein microarrays and proteomics. Nat Genet 32 Suppl:526-32). Improvement in the generation of protein microarrays requires generic approaches for protein production and derivatisation., and is the focus of intensive current research, which includes a dedicated call under the European Commission 6 th Framework Programme for Research, Technological Development and Demonstration.
  • the test population may be in the form of a chromatographic or electrophoretic separation of molecules derivable from the target cell or organism.
  • the test population may be in the form of a separation by SDS-polyacrylamide gel electrophoresis, or by two- dimensional gel electrophoresis, of molecules derivable from the target cell or organism.
  • the test population may be in the form of a blot onto a membrane (for example a nylon membrane) of a two-dimensional gel of total protein from the target cell or organism. Suitable techniques, for example regarding 2-dimensional gel electrophoresis will be well known to those skilled in the art. See, for example demanderor N et al.
  • test molecules or test samples included in an array may be prepared using chromatographic or electrophoretic separation techniques (or other separation/fractionation techniques).
  • test samples in an array may be fractions from a chromatographic separation, for example a High Pressure Liquid Chromatography (HPLC) separation.
  • HPLC High Pressure Liquid Chromatography
  • the method may further comprise the step of identifying or further characterising the test compound or sample. For example, if the test compound is part of a two-dimensional electrophoresis blot, then exposure to the natural antibody may identify one or more regions or "spots" on the blot where the natural antibody binds.
  • the identity of the binding molecule(s) may be determined either through knowledge of molecules known to occupy the positions where binding occurs; or by other techniques for identifying molecules, for example using mass spectrometry techniques.
  • the material at the position/spot in question is analysed directly by protease digestion followed by mass spectroscopy (generally by MALDI-TOF (using a matrix-assisted laser desorption/ionization time of flight mass spectrometer) or by electrophoresis and electrospray ionization quadrupole time-of-flight tandem mass spectrometry), which gives masses of fragments which can be compared with a database containing the sequences of all proteins from the target organism/cell type in order to find a "match".
  • N-terminal sequencing may also be used in identifying polypeptides, as well known to those skilled in the art.
  • Antibodies of known specificity may also be used.
  • the sample may be complex, then it may be desirable to identify the constituent which binds to the natural antibody. This may be done by screening of fractions (for example chromatographic or electrophoretic) of the sample and identification (for example using mass spectroscopy techniques) of the binding constituent.
  • fractions for example chromatographic or electrophoretic
  • identification for example using mass spectroscopy techniques
  • the natural antibody used in the screening method of the invention may be a natural antibody from a human. However, this is not considered essential, even if the organism in which it is intended to use the vaccine is human. As noted above, the conservation of self-antigens to which natural antibodies bind is high and the repertoire to which natural antibodies from different species bind is very similar, so it is considered that natural antibodies from non-human (for example rodent, for example mouse or rat) may be as useful as human natural antibodies in the selection of candidate vaccine molecules.
  • Other sources of natural antibodies may include veterinary species (for example canine, feline, poultry (for example chicken or turkey) etc). These may be particularly useful in relation to diseases in such veterinary species. There are a number of veterinary diseases where this approach might be beneficial for veterinary vaccine development e.g canine leishmaniasis, canine and feline tumours, Salmonella in chickens.
  • the natural antibody may be in the form of na ⁇ ve serum, or alternatively may be a recombinant molecule or the product of a B cell hybridoma secreting a natural antibody. More than one natural antibody may be used: for example when na ⁇ ve serum is used, more than one type of natural antibody may be present.
  • the natural antibody may be, for example IgG or IgM or IgA.
  • the na ⁇ ve serum may be from a neonate. Serum may be pooled serum (ie derived from more than one idividual).
  • B cell hybridomas, plasmacytomas or myelomas secreting natural antibodies may be isolated using the methods given in Notkins (2000) Curr Top Microbiol Immunol 252, 241-250; Potter et al (2000) Curr Top Microbiol Immunol 252, 265-274.
  • Germline sequences encoding natural Ig molecules may be determined and expression constructs (for example a library of expression constructs expressing natural antibodies (the term including antibody-like molecules, as discussed below)) prepared using techniques well known to those skilled in the art.
  • PCR techniques may be used, for example based on techniques described in, for example, EP 0 368 684; Larrick et al (1989) Biochem Biophys Res Commun 160, 1250; Larrick et al (1989) Biotechnology 7, 934; Orlandi et al (1989) PNAS 86, 3833; LeBoef et al (1989) Gene 82, 371-377; Lu and Cerny (2002) J Immunol 2002 169:4920-7.
  • Natural antibodies may be produced by cells of the Ly-1 -bearing B cell lineage (Ly-1 B cells), found mainly in the peritoneum, but also present in spleen (see, for example, Reference 18 and references therein).
  • Cells classified as Bl B cells (CD5 ) are also considered to produce natural antibodies (see, for example, Baumgarth et al (2000) J Exp Med 192, 271- 280; Ochsenbein et al (1999) Science 286, 2156-2159; Macpherson et al (2000) Science 288, 2222-2226; Reid et al (1997) [reference 21]; Hardy et al (1994) [reference 19]).
  • Bl cells are distinguished from "conventional" B2 cells by features including a capacity for self renewal, the expression of germline Ig gene products, and a unique anatomical location (the peritoneum).
  • binding of the natural antibody to the test antigens detected may be detected using methods well known to those skilled in the art. For example, binding may be detected by western blotting of mixtures of test proteins on membrane, or if test protein is purified or in recombinant form, by ELISA
  • the term "natural antibody” incorporates antibody-like molecules that retain the binding specificity of a natural antibody.
  • the variable heavy (V H ) and variable light (V L ) domains of the antibody are involved in antigen recognition, a fact first recognised by early protease digestion experiments. Further confirmation was found by "humanisation" of rodent antibodies.
  • Variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parented antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • variable domains that antigenic specificity is conferred by variable domains and is independent of the constant domains is known from experiments involving the bacterial expression of antibody fragments, all containing one or more variable domains.
  • variable domains include Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); single-chain Fv (ScFv) molecules where the V H and V L partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci.
  • ScFv molecules we mean molecules wherein the V H and V L partner domains are linked via a flexible oligopeptide.
  • antibody fragments rather than whole antibodies
  • the smaller size of the fragments may lead to improved solubility and binding kinetics.
  • Effector functions of whole antibodies, such as complement binding, are removed.
  • Fab, Fv, ScFv and dAb antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.
  • ⁇ plasmon resonance techniques may be used to detect binding to a surface, for example a surface carrying an array.
  • Surface plasmon resonance techniques are described in, for example, Plant et al (1995) Analyt Biochem 226(2), 342-348.
  • Methods may make use of natural antibodies (or further molecules (which may be, for example, anti-IgG or anti-IgM antibodies) which bind to the natural antibodies) that are labelled, for example with a radioactive or fluorescent label. Label free methods (for example Deltadot) may also be used.
  • the method of the invention may further comprise the steps of (1) providing a test fragment or fraction derivable from the selected candidate molecule or sample; and (2) determining whether the test fragment or fraction binds to the natural antibody
  • the method of the invention may be performed in an iterative manner on progressively less "complex" samples; or may be used to identify the portion of an identified molecule that is necessary for natural antibody binding. The latter may be performed using recombinant test molecules.
  • test fragment or fraction derivable from the selected candidate molecule is also included anti-idiotopes, mimetics or mimotopes (or potential mimotopes) of the natural antibody-recognised epitope.
  • a monoclonal antibody directed to the binding site of the natural antibody may be as effective as the antigen itself as a vaccine.
  • the method if the invention may further comprise the step of testing the selected candidate as a vaccine component in a test animal, for example a rodent, for example a mouse.
  • the selected candidate (which may be a single molecule or a mixture of molecules) may be administered to a test animal.
  • the candidate may be administered to the test animal prior to exposure of the test animal to the target cell or organism (as a model for prophylactic vaccination) or after exposure of the test animal to the target cell or organism (as a model for therapeutic vaccination). Effects of the vaccination on susceptibility to subsequent challenge, or on response to an existing challenge may be measured, as well known to those skilled in the art. Examples of vaccination models are described, for example, in Example 1 and references cited therein.
  • Example 1 it may be informative to test the effect of the candidate in a test animal defective in IL-4 signalling; for example defective in IL-4 or the IL-4 receptor. Examples of such animals are described in Example 1. This may be done alongside or instead of testing in animals which are normal for IL-4 signalling.
  • a candidate useful as a vaccine may show a difference in response between the normal IL-4 signalling vs defective IL-4 signalling animals, for example a better response with normal IL-4 signalling than defective IL-4 signalling. It may also be informative similarly to test the effect of the candidate in a test animal (for example mouse) with a genetic defect or defects leading to loss of natural antibodies.
  • cell based responses may be assessed.
  • in vitro priming of CD8+ T cell responses using conventional methods, may be tested.
  • Natural antibody-test molecule complexes would be added to mononuclear cells derived from tissues or blood and CD8+ T cells generated in culture detected, as in e.g. Huang et al (2003) J Infect Dis 187:315-9, Tuettenberg et al (2003) Gene Ther 10:243- 50, Gnjatic et al (2002) Proc Natl Acad Sci USA 99: 11813-8
  • the response in the test animals may be measured to a vaccine preparation that contains a conventional adjuvant.
  • the response in the test animals is measured to a vaccine preparation that does not contain a conventional adjuvant.
  • adjuvants include Freund's complete or incomplete adjuvant; BCG; alum; Aquila's QS21 stimulon (Aquila Biotech, Worcester, MA, USA) which is derived from saponin; Quil A, another saponin-derived adjuvant; mycobacterial extracts (other than a candidate compound or sample) and synthetic bacterial cell wall mimics; and proprietory adjuvants such as Ribi's Detox (Superfos, Denmark).
  • the target organism is an intracellular pathogen or a cell infected with an intracellular pathogen.
  • the intracellular pathogen is a mycobacterium, for example Mycobacterium tuberculosis (causative agent of tuberculosis) or M. leprae (causative agent of leprosy).
  • the intracellular pathogen may be a Leishmania parasite (causative agent of various forms of leishmaniasis, for example L. donovani, L. infantum, L. major).
  • the target cell may be a cancer cell, for example a human cancer cell (or veterinary species cancer cell, for example feline or canine), or a cell of the host organism (for example human or veterinary species, for example feline or canine or poultry) infected with an intracellular pathogen.
  • the cell may be a virally-infected cell.
  • the cancer cell may be colorectal, melanoma, breast, prostate (these are favoured targets for current cancer vaccine studies using DNA based or cell based vaccination)
  • the viral infection may be, for example, HIV, Hepatitis C, papillomavirus.
  • the molecules tested may be all of the (for example) polypeptides of the target organism or cell, or may be a subset thereof, for example, polypeptides (or other molecules) believed to be exposed on the organism or cell surface.
  • a further aspect of the invention provides a kit of parts comprising a natural antibody and a test molecule or test population as defined in relation to the previous aspect of the invention.
  • a kit may be useful in carrying out a method of the invention.
  • the natural antibody may be as defined in relation to the previous aspect of the invention.
  • the kit may further comprise other reagents useful in carrying out a method of the invention, for example reagents useful in detecting the interaction of a test compound with a natural antibody.
  • a further aspect of the invention provides a candidate for an antigen for use in a vaccine against a target cell or organism identified or identifiable by the method of the invention, wherein the candidate is not hydrophilic acylated surface protein Bl (HASPB1).
  • HASPB1 hydrophilic acylated surface protein Bl
  • a further aspect of the invention provides a molecule or sample derivable from a target cell or organism and capable of binding to a natural antibody, or a candidate as defined in the preceding aspect of the invention, for use in medicine.
  • a further aspect of the invention provides the use of a molecule or sample or candidate as defined in relation to the previous aspect of the invention in the manufacture of a medicament for use as a vaccine against the target cell or organism.
  • a further aspect of the invention provides a method of treating a patient in need of vaccination against a target cell or organism wherein the patient is administered an effective amount of a molecule or sample or candidate as defined in relation to the preceding aspect of the invention.
  • the patient When the target cell is a cancer cell, the patient may be an individual with cancer or at risk of cancer due to, for example, family history or previous occurrence of cancer, or due to exposure to agents associated with increased risk of cancer (for example tobacco smoke).
  • the target cell is M. tuberculosis
  • the patient may have or be at risk of tuberculosis, for example through having HIV or AIDS, or through exposure to individuals at risk; for example a healthworker.
  • the patient may have or be at risk of leishmaniasis by living in or travelling to countries where leishmaniasis transmission occurs, or through immunosuppression due to HIV / AIDS or elective immunosuppression e.g. for transplantation.
  • a further aspect of the invention provides a pharmaceutical composition or vaccine comprising or encoding a molecule or sample or candidate as defined in relation to the preceding aspect of the invention and a pharmaceutically acceptable carrier. It is preferred that the pharmaceutical composition or vaccine does not further comprise an adjuvant molecule .
  • the pharmaceutical composition or vaccine (for example a DNA vaccine) may incorporate an immunomodulator, for example a cytokine or cytokines.
  • the DNA vaccine may encode a cytokine or cytokines in addition to the antigen.
  • the DNA vaccine may be followed by boosting with repeated DNA vaccines, or in a heterologous prime/boost regime using a recombinant viral vector encoding the molecule or sample (see, for example, McShane (2002) Curr Opin Mol Ther 4, 23-27; Gilbert et al (2002) Vaccine 20, 1039-1045; Gonzalo et al (2002) Vaccine 20, 1226- 1231). DNA vaccines are also discussed in, for example, Hanke (2001) Curr Mol Med 1, 123-135; Kwissa (2003) J Mol Med 1, 91-101.
  • Suitable vectors or constructs which may be used to prepare a suitable recombinant polypeptide or polynucleotide will be known to those skilled in the art.
  • a polynucleotide capable of expressing the required polypeptide or polypeptides may be prepared using techniques well known to those skilled in the art, for example using databases of genomic sequences.
  • the polynucleotide is capable of expressing the polypeptide(s) in the patient.
  • the polypeptide(s) for example encoding a natural antibody- reactive antigen from the target cell or organism, may be expressed from any suitable polynucleotide (genetic construct) as is described below and delivered to the patient.
  • the genetic construct which expresses the polypeptide comprises the said polypeptide coding sequence operatively linked to a promoter which can express the transcribed polynucleotide (eg mRNA) molecule in a cell of the patient, which may be translated to synthesise the said polypeptide.
  • Suitable promoters will be known to those skilled in the art, and may include promoters for ubiquitously expressed, for example housekeeping genes or for tissue-selective genes, depending upon where it is desired to express the said polypeptide (for example, in dendritic cells or precursors thereof).
  • a dendritic cell or dendritic precursor cell-selective promoter is used, but this is not essential, particularly if delivery or uptake of the polynucleotide is targeted to the selected cells, eg dendritic cells or precursors.
  • Dendritic cell-selective promoters may include the CD83 or CD36 promoters.
  • DC dendritic cells
  • the nucleic acid sequence capable of expressing the polypeptide(s) is preferably operatively linked to regulatory elements necessary for expression of said sequence.
  • "Operatively linked” refers to juxtaposition such that the normal fumc _ tion of the components can be performed.
  • a coding sequence "op ⁇ - —-atively linked" to regulatory elements refers to a configuration wherein the--; -mni-icleic acid sequence encoding the antigen can be expressed under the cc--->r ⁇ -Lt ⁇ rol of the regulatory sequences.
  • regulatory sequences refers to nucleic acid sequences necessary _t c»r the expression of an operatively linked coding sequence in a partici l . ⁇ ar host organism.
  • the regulatory sequences which are suifca-- ⁇ --)l-e for eukaryotic cells are promotors, polyadenylation signals, and enhance
  • Vectors means a DNA molecule comprising a single strand, louble strand, circular or supercoiled DNA. Suitable vectors include retrcD ⁇ - ⁇ irases, adenoviruses, adeno-associated viruses, pox viruses and bacterial p lg». SEiiids. Retroviral vectors are retroviruses that replicate by randomly inte .g ⁇ aprating their genome into that of the host. Suitable retroviral vectors are d-ei-sczribed in WO 92/07573.
  • Adeno virus is a linear double-standard DNA Virus. Suitable ad—o-ano viral vectors are described in Rosenfeld et al, Science, 1991, Vol. 252, pauses ⁇ 432.
  • Adeno-associated viruses belong to the parvo virus fairzi ⁇ L ly and consist of a single strand DNA or about 4-6 KB.
  • Targeting the vaccine to specific cell populations may be achieved, for example, either by the site of injection, use of targeting vectors and delivery systems, or selective purification of such a cell population from the patient and ex vivo administration of the peptide or nucleic acid (for example dendritic cells may be sorted as described in Zhou et al (1995) Blood 86, 3295-3301; Roth et al (1996) Scand. J. Immunology 43, 646-651).
  • targeting vectors may comprise a tissue- or tumour- selective promoter which directs expression of the antigen at a suitable place.
  • the genetic construct can be DNA or RNA it is preferred if it is DNA.
  • the genetic construct is adapted for delivery to a human cell (or, if appropriate to a cell of a veterinary species, for example a feline, canine or poultry cell).
  • the constructs of the invention may be introduced into the cells by any convenient method, for example methods involving retroviruses, so that the construct is inserted into the genome of the (dividing) cell.
  • Targeted retroviruses are available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into pre-existing viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy).
  • retroviral vectors are lentiviral vectors such as those described in Verma & Somia (1997) Nature 389, 239-242. It will be appreciated that retroviral methods, such as those described below, may only be suitable when the cell is a dividing cell. For example, in Kuriyama et al (1991) Cell Struc. and Func. 16, 503-510 purified retroviruses are administered. Retroviral DNA constructs which encode the desired polypeptide(s) may be made using methods well known in the art. To produce active retrovirus from such a construct it is usual to use an ecotropic psi2 packaging cell line grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS foetal calf serum
  • Transfection of the cell line is conveniently by calcium phosphate co-precipitation, and stable transformants are selected by addition of G418 to a final concentration of 1 mg/ml (assuming the retroviral construct contains a neo gene). Independent colonies are isolated and expanded and the culture supernatant removed, filtered through a 0.45 ⁇ m pore-size filter and stored at -70 C.
  • the retrovirus it is convenient to inject directly retroviral supernatant to which 10 ⁇ g/ml Polybrene has been added. The injection may be made into the area in which the target cells are present, for example subcutaneously.
  • a polycation-antibody complex is formed with the DNA construct or other genetic construct of the invention, wherein the antibody is specific for either wild-type adenovirus or a variant adenovirus in which a new epitope has been introduced which binds the antibody.
  • the polycation moiety binds the DNA via electrostatic interactions with the phosphate backbone.
  • the adenovirus because it contains unaltered fibre and penton proteins, is internalised into the cell and carries into the cell with it the DNA construct of the invention. It is preferred if the polycation is polylysine.
  • the DNA may also be delivered by adenovirus wherein it is present within the adenovirus particle, for example, as described below.
  • a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells is employed. This is accomplished by conjugating the iron- transport protein transferrin to polycations that bind nucleic acids. Human transferrin, or the chicken homologue conalbumin, or combinations thereof is covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell.
  • the transferrin-polycation molecules form electrophoretically stable complexes with DNA constructs or other genetic constructs of the invention independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs).
  • complexes of transferrin-polycation and the DNA constructs or other genetic constructs of the invention are supplied to the target cells, a high level of expression from the construct in the cells is expected.
  • High-efficiency receptor-mediated delivery of the DNA constructs or other genetic constructs of the invention using the endosome-disruption activity of defective or chemically inactivated adenovirus particles produced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci. USA 89, 6094-6098 may also be used.
  • This approach appears to rely on the fact that adenoviruses are adapted to allow release of their DNA from an endosome without passage through the lysosome, and in the presence of, for example transferrin linked to the DNA construct or other genetic construct of the invention, the construct is taken up by the cell by the same route as the adenovirus particle.
  • This approach has the advantages that there is no need to use complex retroviral constructs; there is no permanent modification of the genome as occurs with retroviral infection; and the targeted expression system is coupled with a targeted delivery system, thus reducing toxicity to other cell types.
  • Non- viral approaches to gene therapy are described in Ledley (1995) Human Gene TJierapy 6, 1129-1144.
  • Alternative targeted delivery systems are also known such as the modified adenovirus system described in WO 94/10323 wherein, typically, the DNA is carried within the adenovirus, or adenovirus-like, particle.
  • Michael et al (1995) Gene Therapy 2, 660-668 describes modification of adenovirus to add a cell- selective moiety into a fibre protein.
  • a further aspect of the invention provides a virus or virus-like particle comprising a genetic construct encoding a candidate molecule of the invention.
  • suitable viruses or virus-like particles include HSV, AAV, vaccinia, lentivirus and parvovirus.
  • Immunoliposomes are especially useful in targeting to cell types which over-express a cell surface protein for which antibodies are available, as is possible with dendritic cells or precursors, for example using antibodies to CD1, CD 14 or CD83 (or other dendritic cell or precursor cell surface molecule, as indicated above).
  • MPB-PE N-[4-(p-maleimidophenyl)butyryl]- phosphatidylethanolamine
  • MPB-PE N-[4-(p-maleimidophenyl)butyryl]- phosphatidylethanolamine
  • MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface.
  • the liposome is conveniently loaded with the DNA or other genetic construct of the invention for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 ⁇ m and 0.2 ⁇ m pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min.
  • Freshly prepared MPB-PE- liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight.
  • the immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min.
  • Immunoliposomes may be injected, for example intraperitoneally or directly into a site where the target cells are present, for example subcutaneously.
  • Preferred vectors include lentivirus vectors and adenoviral vectors, for example vectors similar to those described in Foxwell et al (2000) Ann Rheum Dis 59 Suppl 1, 154-59 or Bondeson et al (2000) J Rheumatol 27(9), 2078-2089. It will be appreciated that it may be desirable to be able to regulate temporally expression of the polypeptide(s) (for example antigen and possibly also cytokine(s)) in the cell.
  • polypeptide(s) for example antigen and possibly also cytokine(s)
  • expression of the polypeptide(s) is directly or indirectly (see below) under the control of a promoter that may be regulated, for example by the concentration of a small molecule that may be administered to the patient when it is desired to activate or repress (depending upon whether the small molecule effects activation or repression of the said promoter) expression of the polypeptide. It will be appreciated that this may be of particular benefit if the expression construct is stable ie capable of expressing the polypeptide
  • a preferred construct of the invention may comprise a regulatable promoter.
  • regulatable promoters include those referred to in the following papers: Rivera et al (1999) Proc Natl Acad Sci USA 96(15), 8657-62 (control by rapamycin, an orally bioavailable drug, using two separate adenovirus or adeno-associated virus (AAV) vectors, one encoding an inducible human growth hormone (hGH) target gene, and the other a bipartite rapamycin-regulated transcription factor); Magari et al (1997) J Clin Invest 100(11), 2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6), 613-22 (review of adeno-associated viral vectors); Bohl et al (1998) Blood 92(5), 1512-7 (
  • Tetracycline - inducible vectors may also be used. These are activated by a relatively -non toxic antibiotic that has been shown to be useful for regulating expression in mammalian cell cultures. Also, steroid-based inducers may be useful especially since the steroid receptor complex enters the nucleus where the DNA vector must be segregated prior to transcription.
  • This system may be further improved by regulating the expression at two levels, for example by using a tissue-selective promoter and a promoter controlled by an exogenous inducer/repressor, for example a small molecule inducer, as discussed above and known to those skilled in the art.
  • a tissue-selective promoter and a promoter controlled by an exogenous inducer/repressor, for example a small molecule inducer, as discussed above and known to those skilled in the art.
  • an exogenous inducer/repressor for example a small molecule inducer
  • one level of regulation may involve linking the appropriate polypeptide- encoding gene to an inducible promoter whilst a further level of regulation entails using a tissue-selective promoter to drive the gene encoding the requisite inducible transcription factor (which controls expression of the polypeptide (for example antigen)-encoding gene from the inducible promoter).
  • Control may further be improved by cell- type- specific targeting of the genetic construct.
  • the methods or constructs of the invention may be evaluated in, for example, dendritic cells generated in vitro, as known to those skilled in the art, before evaluation in whole animals.
  • Such delivery / expression systems may be tested for polypeptide expression in rodent models, for example after gene transfer / DNA vaccination / infection with viral vectors.
  • the genetic constructs of the invention can be prepared using methods well known in the art.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include tlie step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Formulations suitable for parenteral ackri stration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • Particularly useful administration routes include parenteral, oral, imtramuscular, intradermal and subcutaneous.
  • FIG. 1 IL-4 is required for vaccination against leishmaniasis a
  • IL-4 a and b Spleen cells from individual BALB/c mice (a) and B ALB.
  • IL-4 ⁇ ' mice immunized with rHASPBI (circles) or OVA (squares) were cultured with (closed symbols) or without (open symbols) rHASPBI and assayed for IFN ⁇ -producing CD8 + T cells, c, CTL activity of BALB/c derived (circles) and B ALB.
  • IL-4 ' ' mice were transferred into BALB/c recipients and parasite burden in spleen (d) and liver (e) determined on day 56 pi. * P ⁇ 0.05
  • IL-4 secretion is upstream of IL-12 production a, IL-4 and b, IL-12 response of spleen cells from BALB/c (black and white bars ) and BNLB.IL ⁇ 4R ⁇ / ⁇ (hatched and grey bars). Naive mice (black and hatched bars), mice injected with rHASPBI (white and grey bars).
  • CDllb+ phagocytes are responsible for primary IL-4 secretion a, IL-4 secretion by spleen cells separated by MACS from control BALB/c mice (black bars) and BALB/c.IL-4R (-/- (hatched bars) and BALB/c (white bars) and B ALB JL-4R / ⁇ - (grey bars) mice injected with rHASPBI.
  • IL-4 secretion requires immune complexes a, IL-4 response of spleen cells from BALB/c mice (black bars) or BALB.SCID mice (white bars) injected with rHASPBI with or without serum transfer, b, IL-4 response of BALB.SCID mice reconstituted with serum from wildtype or IgM-deficient BALB/c mice, c and d, The frequency of peritoneal (black bars) or splenic (white bars) B cells secreting IgM (c) and IgG (d) antibodies recognizing HASPB 1 or OVA. e, IL-4 response to rHASPBI in SCID mice reconstituted with serum from wildtype CBA/J or mutant CBA/N mice. , IL-4 response in SCID mice injected with OVA in the presence or absence of anti-OVA antibodies. * P ⁇ 0.05, ** P ⁇ 0.01.
  • Cytokine secretion and CD8+ T cell priming is complement dependent a and b
  • Secretion of IL-4 (a) and IL-12p70 (b) was measured in BALB/c mice depleted of complement with cobra venom factor, c and d, IL-4 (c) and IL-12p70 (d) secretion in B6 mice and B6.Clqa ' ⁇ mice in response to rHASPBI.
  • CD8 + T cells are essential for long-term vaccine-induced resistance against intracellular pathogens.
  • natural antibodies acting in concert with complement provide endogenous adjuvant activity for the generation of protective CD8 + T cells following vaccination against visceral leishmaniasis.
  • IL-4 was shown to be critical for the priming of vaccine- specific CD8 T cells, and we defined the primary source of IL-4 as a CDl lb + phagocyte.
  • IL-4 secretion was not observed in antibody deficient mice, and could be reconstituted with serum from normal mice but not mice lacking B-l B cells (xid mice).
  • no IL-4 response or CD8 + T-cell priming was seen in Clqa " " mice.
  • Vaccination against visceral leishmaniasis requires IL-4-dependent priming of CD8 + T cells
  • HASPB 1 hydrophilic acylated surface protein Bl
  • CD8 + T cell priming we assayed HASPB 1 -specific CTL activity. CTL able to kill HASPB 1 -expressing target cells were readily expanded from vaccinated BALB/c but not BALB.IL-4 ' ' mice (Fig 2c). Finally, we examined the protective capacity of these CD8 + T cells using an adoptive transfer approach. CD8 + T cells from vaccinated BALB/c but not BALB JL-4 ' ' ' mice could transfer protection in both the spleen (Fig 2d) and liver (Fig 2e) of infected BALB/c recipients. Together these experiments indicated for the first time a critical requirement for IL-4 in CD8 + T cell-dependent vaccination against leishmaniasis.
  • CD lib phagocytes are the primary source of IL-4 rHASPBI induced rapid IL-12p40 and IL-12p70 production in CD1 lc hi DC 3.
  • BALB/c and BALB.ZL- R / ⁇ mice were used to determine whether injection of rHASPBI resulted in both IL-4 and IL-12 secretion.
  • BALBJZ- R ⁇ _/" mice provided a means of dissecting any temporal relationships between these cytokines.
  • An increase in the frequency of IL-4 secreting cells was observed in both strains injected with rHASPBI (Fig 3d).
  • BALB/c mice had an increased frequency of IL-12p70 secreting cells, BALB JL-4R ' ⁇ mice did not (Fig 3b).
  • Phagocyte production of IL-4 requires natural antibodies
  • rHASPBI Phagocyte production of IL-4 requires natural antibodies
  • rHASPBI Phagocyte production of IL-4 requires natural antibodies
  • IL-4 secretion in response to rHASPBI was restored (Fig 5a).
  • This experiment further excluded the possibility that T cells, B cells and CD4 + NKT acted as a primary source of IL-4, and indicated a role for serum antibody.
  • IL-4 secretion was absent in B cell-deficient B6. ⁇ MT mice, but restored following serum transfer (data not shown).
  • HASPB and HASPA bind immunoglobulins of several classes from normal human sera taken from normal individuals with no previous exposure to leishmaniasis. Immune complex-mediated complement activation induces IL-4 secretion
  • IL-4 directly and indirectly plays a major role in CD4+ T cell differentiation 42, B cell activation 52 and in the regulation of macrophage antimicrobial function 53.
  • Our study suggests that the potential involvement of IL-4 in some previously reported complement-dependent effects 54, 55 now needs to be carefully addressed.
  • HASPB 1 The structural features of HASPB 1 that impart recognition by natural antibodies are at present unknown, but it is notable that HASPB 1 (together with other members of the HASP family) contains multiple repeats 23, a feature shared with other targets of natural antibody recognition 57. Although we have focused in this study on the cytokine requirements for CD8 + T cell responses, our data showing that IgG natural antibodies recognize rHASPBI suggest that delivery of this antigen into the MHC class I processing pathways may occur via FcR mediated uptake 9-11.
  • BALB/c mice were obtained from Tuck and Co. (Battlesbridge, UK) and CBA J mice from Charles River (Margate, UK).
  • BALB.IL-4 'A and BALB .IL-4R ⁇ mice ss, B6.Clqa ' mice 33, BALB JgM A 30, BALB.SCID and B6. ⁇ MT mice were kept under barrier conditions with sterile food and water ad libitum.
  • L. donovani amastigotes (strain LV9) were isolated from infected hamsters as previously described 59. Mice were infected with 2xl0 7 amastigotes i.v. by the lateral tail vein.
  • Recombinant HASPB 1 (rHASPBI) was either purified as described 3 or by reverse phase HPLC (purity: 97%; endotoxin level: 0.17 EU / mg rHASPBI; Dictagene, Lausanne, Switzerland). Mice were vaccinated with rHASPB 1 or OVA (Sigma Aldrich, Poole, UK) and infected 3 weeks later with 2x 10 7 amastigotes as described 3.
  • Spleen cells from rHASPBI- or OVA- vaccinated mice were cultured overnight with or without rHASPBI and rIL-2, and stained with Tricolor labeled anti-CD8 + (clone CT-CD8 ⁇ ; Caltag, Towcester, UK) and R-PE labeled anti-IFN ⁇ (clone BVD6-24G2; Serotec, Oxford, UK) 3. Isotype controls were used to set statistical markers. Samples were analyzed using a FACScan (Becton Dickinson, Mountain View, CA) and Cell Quest software. 200,000 cells were analyzed for each individual mouse.
  • CTL-assay Target cells were generated by transfecting P815 cells with the pCIneo vector, bearing the open reading frame of HASPB 1 23, or with the vector alone. Stable transfectants were produced by selection with G418 (1 mg/ml; Sigma- Aldrich). HASPB 1 expression was verified by FACS analysis. For the generation of effector CD8 + T cells, splenocytes from rHASPBI or OVA vaccinated mice were cultured with 30 ⁇ g/ml rHASPBI.
  • rhIL-2 (20U/ml) was added to the cultures and at day 7 cells were harvested, washed and CD8+ cells purified by MACS using anti- CD8 ⁇ microbeads (Miltenyi Biotec, Bergisch, Germany). CTL activity was measured in a 4h LDH release assay (Boehringer Mannheim, Lewes, UK), and results were read at 492 nm with an ELISA reader (Molecular Devices, Menlo Park, CA). Data are expressed as percentage specific lysis defined as (experimental lysis-spontaneous lysis)/ (maximum lysis- spontaneous lysis) x 100. Total spontaneous release was calculated as the sum of spontaneous release by both target and effector cells.
  • CD8+ T cells from rHASPBI- or OVA- vaccinated mice were isolated by MACS.
  • CD4 + cell contamination ranged from 1-2.5% of the transferred population.
  • Each animal received 3-4 x 10 6 CD8+ cells i.v.
  • recipient mice were infected and splenic and hepatic parasite burden was assessed at day 50 p.i.. ELISPOT assays
  • Alkaline phosphataseconjugated anti-mouse IgM ( ⁇ chain-specific) and anti-mouse IgG (Sigma- Aldrich, Poole, UK) were used to detect HASPB 1 IgM and IgG antibodies respectively.
  • mice received 200 ⁇ l of dichloromethylene-biphosphonate of control liposomes i.v. one day prior to rHASPBI.
  • Clodronate (Roche Diagnostics GmbH, Mannheim, Germany) liposomes were prepared as described earlier ⁇ . Depletion was confi ⁇ ned by F4/80, MOMA-1 and ERTR-9 staining of spleen sections 64. Mast cells were depleted with lmg anti-c-kit antibody (ACK2 5; a gift from Dr. H.
  • CVF cobra venom factor
  • Optimal conditions for using natural antibodies to probe proteomes may be determined for rodent, human and or veterinary species natural antibodies. This may be done in relation to detection by natural antibodies of a sub-set of specific antigens in whole pathogen proteomes. Identified antigens may be tested in vivo, for example in mice. This is most straightforward if the identified antigen already been cloned and/or expressed.
  • Interleukin-4 acts at the locus of the antigen-presenting dendritic cell to counter-regulate cytotoxic CD8+ T-cell responses. Nat Med 7, 206-14 (2001).
  • Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells. JExp Med 192, 823-33 (2000). 44. Ben-Sasson, S.Z., Le Gros, G., Conrad, D.H., Finkelman, F.D. & Paul, W.E. Cross-linking Fc receptors stimulate splenic non-B, non-T cells to secrete interleukin 4 and other lymphokines. Proc Natl Acad Sci U S A 87, 1421-5 (1990).

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Abstract

La présente invention concerne un procédé de recherche systématique de candidat antigène destiné à un vaccin contre une cellule ou un organisme cible. Le procédé se décompose en plusieurs opérations. (1) On expose à un anticorps naturel une molécule test ou un échantillon pouvant dériver de la cellule ou de l'organisme. (2) On vérifie que la molécule test ou l'échantillon se lie à l'anticorps naturel. (3) On sélectionne comme candidat antigène destiné à un vaccin la molécule test ou l'échantillon qui se lie à l'anticorps naturel. La cellule cible ou l'organisme peut être une cellule cancéreuse ou un pathogène intracellulaire. L'antigène naturel peut être un antigène naturel humain ou de rongeur. Le procédé peut également comporter une opération par laquelle on essaie comme vaccin sur un modèle rongeur le candidat identifié.
PCT/GB2004/001266 2003-03-22 2004-03-22 Procede WO2004083374A2 (fr)

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WO1999007880A1 (fr) * 1997-08-08 1999-02-18 The Research Foundation Of State University Of New York IDENTIFICATION D'ALLERGENES ET D'ANTIGENES DE LYMPHOCYTES T HUMAINS $i(IN VITRO)

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Publication number Priority date Publication date Assignee Title
WO1999007880A1 (fr) * 1997-08-08 1999-02-18 The Research Foundation Of State University Of New York IDENTIFICATION D'ALLERGENES ET D'ANTIGENES DE LYMPHOCYTES T HUMAINS $i(IN VITRO)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
OCHSENBEIN A.F. ET AL.: "Control of early viral and bacterial distribution and disease by natural antibodies." SCIENCE, vol. 286, 10 December 1999 (1999-12-10), pages 2156-2159, XP002296202 *
OCHSENBEIN A.F. ET AL.: "Natural antibodies and complement link innate and acquired immunity." IMMUNOL. TODAY, vol. 21, no. 12, December 2000 (2000-12), pages 624-630, XP004225440 *
ST[GER S. ET AL.: "Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis." J. IMMUNOL., vol. 165, 2000, pages 7064-7071, XP002296035 cited in the application *
ST[GER S. ET AL.: "Natural antibodies and complement are endogenous adjuvants for vaccine-induced CD8+ T-cell responses." NATURE MEDICINE, vol. 9, no. 10, October 2003 (2003-10), pages 1287-1292, XP002296036 *

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