WO2007085057A1 - A medical protocol - Google Patents

Abstract

Methods and compositions are disclosed, which employ an MBL modulator for treating or preventing an infection in a subject that is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment. In particular embodiments, the methods and compositions enhance the armamentarium for organizations charged with managing infectious diseases and subjects at risk of and/or exposed to highly transmissible and potentially debilitating or fatal infectious agents such as those causing epidemics or capable of being used in biowarfare.

Description

TITLE OF THE INVENTION

"A MEDICAL PROTOCOL"

FIELD OF THE INVENTION

[0001] This invention relates generally to the field of infectious diseases. More particularly, the present invention relates to medical protocols and products for treating or preventing an infectious disease in a subject. The invention encompasses treating or preventing infections where the distinguishing characteristics of the causative organism or organisms may not have been fully determined. Thus, the subject may be at risk of exposure to an infectious agent. Alternatively, the subject may have already been exposed to an infectious agent and may or may not yet be sick as a result of infection by the infectious agent. In particular embodiments, the subject is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment. In particular embodiments, the present invention employs polypeptides or agents that enhance the functional activity of pattern recognition molecules in order to modulate host defense systems. The present invention finds broad application in the field of infectious disease. For example, the present protocols will enhance the armamentarium for organizations charged with managing infectious diseases and subjects at risk of and/or exposed to highly transmissible and potentially debilitating or fatal infectious agents such as those causing epidemics or capable of being used in biowarfare.

[0002] Bibliographic details of publications referred to by author in this specification are also listed at the end of the specification.

BACKGROUND OF THE INVENTION

[0003] Immune-based therapeutics provide some of the most widely used pharmaceutical and veterinary agents. Vaccine technology has been particularly successful. For example, vaccines against tetanus, diphtheria, whooping cough, polio and small pox have been outstandingly effective. Vaccines traditionally use immuno-interactive components of the infectious agent to engender a recallable immune response that modulates the development of infections with the agent. Vaccines may comprise live attenuated pathogens, killed whole pathogens, purified sub-component vaccines or genetically engineered vaccines. More recently, the use of DNA vaccines has been recognized. However, there are still many infectious agents for which there are no effective therapeutic or prophylactic vaccines.

[0004] The problem is compounded by various factors such as environmental conditions and host-pathogen interaction factors. On the one hand, man is traveling more frequently by plane between countries increasing the risk and rate of transmission of human pathogens among the dispersed population. Highly contagious virus infections such as influenza viruses are likely to be widely dispersed through this route. On-going wars and other natural disasters do nothing to diminish the threat of epidemics through biological warfare or lack of infrastructure/sanitation. On the other hand, many pathogens are capable of rapid and/or bulk genetic changes via numerous different mechanisms that allow them to evade host defenses such as those provided by vaccination. For example, multi-drug resistant strains of Streptococcus, Staphylococcus, Pseudomonas, Entβrococcus and Mycobacterium spp. represent a significant problem by exhibiting resistance to currently available antibiotic therapies.

[0005] The immune system provides biological responses as a defense to infection by pathogens or other foreign molecules. These biological responses may be broadly divided into acquired and innate responses. The acquired immune response provides antigen specific immune responses, an antigen being any substance that is capable of reacting in or inducing an immune response. The more ancient innate system is activated by molecular patterns, such as the distribution of particular sugar groups on the surface of micro-organisms. The innate response is immediately effective and no previous encounter with the molecular pattern is required. The pattern recognition molecules of the innate system recognize a relatively small number of patterns that are conserved in a large number of micro-organisms. It is estimated that the human innate immune system can recognize approximately 10,000 different molecular patterns. The innate system may also play an important role in modulating the acquired immune response including aspects of these responses such as autoimmunity and inflammation that can themselves cause severe pathology. [0006] One of the most important pattern recognition proteins is mannose-binding lectin

(MBL), also called mannan-binding protein, which binds particularly well to mannose and N- acetylglucosamine sugars. MBL belongs to the collectin family of C-type lectin proteins which also comprises soluble proteins such as conglutinin and the surfactant proteins, SP-A and SP-D, and membrane bound collectins (CL-Ll, CL-L2 and CL-Pl). MBL is mainly produced in the liver and its expression there may be induced in response to growth hormone stimulation. MBL is also produced by cells of the monocyte-macrophage lineage. Collectin family proteins form trimers that assemble into larger oligomers. Each collectin polypeptide chain consists of four regions: a relatively short N- terminal region, a collagen-like region, an alpha-helical coiled-coil region, and a carbohydrate-binding region. The structural unit of human MBL comprises three polypeptide chains of 248 amino acids. The N-terminal collagen-like region forms a triple helix structure that is stabilized by disulphide bonds. The C-terminal portion comprises three separate polypeptide arms of 93 amino acids terminating in the globular binding (lectin) domain. The globular arms of MBL form oligomers of the structural unit and only higher order oligomers comprising at least four structural MBL units can carry out MBL effector functions. In particular, monomeric MBL cannot bind MBL associated serine proteases (MASPs) and cannot activate complement. The ability to activate complement is enhanced in higher order oligomers. [0007] When MBL binds to its molecular pattern a number of different effector functions may be activated. In particular, MBL mediates killing of a wide range of extracellular and intracellular pathogens via the complement pathway and/or via direct opsonization.

[0008] MBL is structurally similar to the complement component, CIq and can bind to Clq-like receptors as well as collectin receptors on the surface of phagocytes. In relation to direct opsonization, the binding of MBL to its molecular pattern facilitates phagocyte binding to the bound MBL via Clq-like or collectin receptors on the phagocyte.

[0009] The complement system is a major arm of the innate immune system although it is also used in acquired responses. The complement system involves a series of protein-protein interactions and proteolytic cleavages mediated by specific serine proteinases to produce C3 or C3-like convertases. C3 activates the so called complement cascade. The cascade produces, inter alia, inflammation, chemoattraction of phagocytes, attachment of antigens or molecular patterns to phagocytes, activation of arms of the acquired immune response, promotion of lysis of Gram-negative bacteria and non-self cells, and removal of immune complexes from the body. [0010] In the classical complement system, C3 convertase cleaves C3 to release C3b and an anaphylactic agent C3a. C3b binds covalently to the surface of micro-organisms in a process termed opsonization. Surface bound C3b is bound by phagocytes which cause destruction of the micro-organism by lysosomal enzymes. C3 convertase and C3b also combine to form C5 convertase. Cleavage of C5 by C5 convertase precipitates the release of the chemoattractant, C5a and a lytic C5b complex. C3a causes local inflammatory responses.

[0011] The lectin complement system is analogous to the classical system. The C3 convertase of the lectin complement system, C4b2a is generated by cleavage of C4 and C2 by CIq- associated protein, CIs or MASP-2. MASP-2 forms a complex with MBL or ficolin. Once MBL binds to the molecular pattern, MASP-2 catalyses the covalent attachment of C4b to the surface of micro- organism or cell bearing the molecular pattern. C2 binds to surface bound C4b which is cleaved by CIs or MASP-2 producing C4b2a. The alternative complement pathway is also analogous to the classical system but does not require a specific pattern recognition molecule or CIq to bind C3b/C4b to the surface of the micro-organism (Rooijakkers etal, Nature Immunology, 6(9):920-927, 2005).

[0012] The MBL gene (MBL2) is located on Chromosome 10 and comprises four exons. Polymorphisms in the MBL structural gene are quite frequent as indicated by a recent Australian study which showed that 30% of normal blood donors were heterozygous for structural gene mutations and a further 8% were homozygous or had double mutations in structural genes (Minchinton et ah, Scand. J. Immunol., 56(6):630-641, 2002). Using a stringent definition of MBL deficiency (mannan binding level < 0.5 μg/mL and C4 deposition < 0.3 U/μL) the group comprised 24% MBL deficient members (Eisen et al, Curr. Drug Targets, 5(l):89-105, 2004). Allelic variants causing low MBL levels are found in most ethnic groups investigated.

[0013] Allelic variants have been identified comprising one or more polymorphisms within the 5' promoter region and/or exon 1 that encodes the collagen-like domains of the N-terminal part of MBL. At least five point mutations have been identified in the promoter region at positions - 550, -427, -349, -336, and -221. The allele comprising a mutation at position -550 of the MBL structural gene is called H/L. The allele comprising a mutation at position -221 of the MBL structural gene is called XJY. At least three polymorphisms have been identified in exon 1 at codons 52, 54 and 57 encoding variant alleles D, B and C, respectively. The polymorphic alleles are collectively called "O" and the wild-type allele is called "A".

[0014] The consequence of these mutations appears to be lower levels of MBL in the case of promoter mutations. Regarding the structural mutations, the C allele appears to be non-functional while the D allele exhibits reduced mannan binding and reduced oligomer formation and complement activation. The B allele appears to result in low levels of MBL. [0015] MBL mutations such as those described above predispose for conditions such as failure to thrive in children, diarrhea and severe recurrent infections. MBL deficiency in humans is associated with an increased risk for severe bacterial infections (see Eisen et al., Clin. Infect. Dis., 37(ll):1496-1505, 2003;). In relation to meningococcal disease, see Hibberd et al, Lancet, 353(9158): 1049-1053, 1999 and pneumococcal infection, see Roy etal, Lancet., 359(9317):1569- 1573, 2002.

[0016] US Patent No. 6,562,784 in the name of Thiels et al. discusses MBL levels and infection rates and proposes the use of MBL in immunocompromised individuals to treat or prevent infections as well as in individuals at risk of becoming immunocompromised from a medical treatment. Patients with MBL levels below 0.5μg/mL were stated to be more susceptible to infection following chemotherapy. Thiels et al. suggest that subjects with clinically significant infections have lower MBL levels.

[0017] International Publication No. WO 2004/026330 in the name of K.U. Leuven Research and Development discloses the use of MBL or an MBL regulator to restore normal MBL levels in critically ill patients who were not previously immunocompromised. An MBL level of less than 0.5 μg/mL is stated to be a prognostic marker indicative for treatment with MBL for critically ill subjects,

[0018] Accordingly, the understanding in the art is that MBL deficiency is associated with poor prognosis in immunocompromised or critically ill subjects or in those individuals who may become immunocompromised by a medical treatment. The prior art therefore suggests that these subjects could be treated with MBL in order to correct or prevent MBL deficiency. SUMMARY OF THE INVENTION

[0019] In the work leading up to the present invention, it was discovered that immunocompetent subjects with severe infection who have higher levels of MBL have better outcomes as assessed in a clinical setting by Sequential Organ Failure Assessment (SOFA). The prior art suggests that MBL deficient subjects who are immunocompromised or critically ill would benefit from normal levels of MBL. However, even with normal levels the subject may still succumb to sepsis. It is therefore unexpected that subjects with higher levels of MBL have better outcomes.

[0020] Accordingly, in one aspect, the present invention provides methods for treating or preventing an infection in a subject, wherein the subject is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment. These methods generally comprise administering to the subject an effective amount of an MBL modulator selected from an MBL polypeptide or an agent from which an MBL polypeptide is producible. In accordance with this aspect of the invention, the MBL modulator is administered in order to achieve a hypersupplemented level of MBL in the subject. In some embodiments, the subject has at least normal levels of MBL prior to administration. In accordance with the present invention, even subjects exhibiting a normal level of MBL can receive clinical benefits from hypersupplemented MBL levels. Specifically, in some embodiments, hypersupplemented levels of MBL are used to prevent or reduce the risk of infection by an infectious organism. The method is particularly convenient where a subject is at risk from exposure to an infectious organism where no other effective medical treatment or prophylactic measure is available. In some embodiments, the subject protocol facilitates an improved prognosis. In an illustrative embodiment, the method improves the prognosis of subjects with sepsis, whether or not they have normal levels of MBL at the outset of sepsis. Suitably, supplementation with MBL does not itself modulate an acute phase response. In some embodiments the MBL modulator comprises MBL that is derived from plasma. In other embodiments, the MBL modulator comprises recombinantly produced MBL polypeptide.

[0021] In some embodiments, the methods further comprise testing the subject to determine the level of MBL in the subject prior or subsequent to administration of MBL modulator. Suitably, the methods comprise: assessing the MBL level and/or activity in a subject. In some embodiments, the methods further comprise characterizing the genotype or proteome of an infectious organism or antibodies or immune cells that are immuno-interactive with the organism in a sample in order to diagnose the infection. In further embodiments, the methods comprise: testing for MBL binding to the diagnosed infectious organism. In still further embodiments, the methods comprise assessing the genotype or allelic form or forms of MBL in the subject.

[0022] In some embodiments, the methods further comprise co-administering with the MBL modulator at least one additional agent that modulates or modifies the immune response or activity of MBL in the subject. In illustrative examples of this type, phagocytosis is enhanced in a ■ subject by co-administration of one or more of a hormone, cytokine, lymphokine, hematopoietic factor, chemokine, antibody or part thereof, co-stimulatory molecule and biological response modifier. Other functional activities which are modified in these embodiments include complement activation, opsonization, protein-, glycoprotein- or glycolipid-binding, and receptor binding. [0023] In some embodiments, the methods further comprise co-administering with the

MBL modulator at least one additional agent selected from anti-infective agents such as antimicrobials, antibiotics, antivirals, antifungals, anthelmintics, antiprotozoals or nematocides. In these embodiments, the additional agent is used to enhance the treatment or prevention of the infection. [0024] In another aspect, the present invention provides the use of an MBL modulator in the manufacture of a medicament for treating or preventing an infection in a subject that is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment. In some embodiments, the MBL modulator is formulated together with a pharmaceutically acceptable carrier, diluent and/or excipient. [0025] In still another aspect, the invention provides kits for use in treating or preventing an infection in a subject that is not immunocompromised or at risk of becoming immunocompromised. These kits generally comprise an MBL modulator, hi specific embodiments, the kits comprise an MBL polypeptide and optionally at least one MASP in a form suitable for storage and/or for local or systemic administration. In some embodiments, the kits include reagents for determining the level or activity of MBL polypeptide in the subject.

[0026] In some embodiments, the infection is caused by an infectious agent or microbe selected from a bacterium, fungus, virus, algae, parasite, (including ecto-or endo-parasites), prion, oomycetes, slime, moulds, nematodes, mycoplasma and the like, hi illustrative examples, the infectious agent is selected from one or more of the following orders, genera or species: Acinetobacter, Actinobacillus, Actinomycetes, Actinomyces, Aeromonas, Bacillus, Bacteroides, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Citrobacter, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Erysipelothrix, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Micrococcus, Moraxella, Morganella, Mycobacterium (tuberculosis), Nocardia, Neisseria, Pasteurella, Plesiomonas, Propionibacterium, Proteus, Providencia, Pseudomonas, Rhodococcus, Salmonella, Serratia, Shigella, Staphylococcus, Stenotrophomonas,

Streptococcus, Treponema, Vibrio (cholera) and Yersinia (plague), Adenoviridae, African swine fever- like viruses, Arenaviridae (such as viral haemorrhagic fevers, Lassa fever), Astroviridae (astroviruses) Bunyaviridae (La Crosse), Calicivirid (Norovirus), Coronaviridae (Corona virus), Filoviridae (such as Ebola virus, Marburg virus), Parvoviridae (B 19 virus), Flaviviridae (such as hepatitis C virus, Dengue viruses), Hepadnaviridae (such as hepatitis B virus, Deltavirus), Herpesviridae (herpes simplex virus, varicella zoster virus), Orthomyxoviridae (influenza virus) Papovaviridae (papilloma virus) Paramyxoviridae (such as human parainfluenza viruses, mumps virus, measles virus, human respiratory syncytial virus) , Nipah virus, Hendra virus, Picornaviridae (common cold virus,), Poxviridae (small pox virus, orf virus, monkey poxvirus) Reoviridae (rotavirus) Retroviridae (human immunodeficiency virus) Paroviridae (paroviruses) Papillomaviridae, (papillomaviruses) alphaviruses and Rhabdoviridae (rabies virus), Trypanosoma, Leishmania, Giardia, Trichomonas, Entamoeba, Naegleria, Acanthamoeba, Plasmodium, Toxoplasma, Cryptosporidium, Isospora, Balantidium, Schistosoma, Echinostoma, Fasciolopsis, Clonorchis, Fasciola, Opisthorchis and Paragonimus, Pseudophyllidea (e.g., Diphyllobothriuni) and Cyclophyllidea (e.g., Taenia). Pathogenic nematodes include species from the orders; Rhabditida (e.g., Strongyloides), Strongyliάa (e.g., Ancylostoma), Ascarida (e.g., Ascaris, Toxocara), Spirurida (e.g., Dracunculus, Brugia, Onchocerca, Wucheria), and Adenophorea (e.g., Trichuris and Trichinella), Prototheca and Ptiesteria, Absidia, Aspergillus, Blastomyces, Candida (yeast), Cladophialophera, Coccidioides, Cryptococcus, Cunninghamella, Fusarium, Histoplasma, Madurella, Malassezia, Microsporum, Mucor, Paecilomyces, Paracoccidioides, Penicillium, Pneumocystis, Pseudallescheria, Rhi∑opus, Rhodotorula, Scedosporium, Sporothrix, Trichophyton and Trichosporon. For the avoidance of doubt the infectious agent may be a known pathogen, an emerging or re-emerging pathogen or an organism which has never previously been identified as a pathogen in a particular subject. In some embodiments, the infectious agent is resistant to usual medicaments, e.g., resistant to at least one anti-infective agent. In some embodiments, the infectious agent is resistant to multiple anti-infective agents (e.g., multi-drug resistant).

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a graphical representation showing a significant negative correlation between MBL levels and sequential organ failure assessment (SOFA) score. The SOFA score on Day 3 of illness is plotted against patients MBL level (μg/mL) providing a significant (p=0.011) negative correlation (Pearson correlation = -0.194).

[0028] Figure 2 is an alignment of polypeptide sequences of MBL from human, chimpanzee, mouse, rat, cow and chicken. Identical residues are identified by the symbol "*". Conservative substitutions are identified by the symbol " : ". Semi-conserved substitute are identified by the symbol " . ".

DETAILED DESCRIPTION

J. Definitions

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. Each embodiment described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise. [0030] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "a cell" means one cell or more than one cell.

[0031] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0032] The term "agent" or "modulatory agent" includes a compound that induces a desired pharmacological and/or physiological effect. The term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "agent" is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular agents. The term "agent" includes a cell which is capable of producing and secreting the polypeptides referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes this polypeptide, hi illustrative examples of this type, the MBL- encoding nucleotide sequence is operably connected to a regulatory element in a nucleic acid construct. Thus, the term "agent" extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells. The term agent includes plasma or other blood products.

[0033] "Analogs" contemplated herein include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.

[0034] Reference herein to "bacteria" or "bacterial infection" includes any bacterial pathogen including emerging bacterial pathogen of vertebrates. Representative bacterial pathogens include without limitation species of: Acinetobacter, Actinobacillus, Actinomycetes, Actinomyces, Aeromonas, Bacillus, Bacteroides, Bordetella, Borrelia, Brucella (brucellosis), Burkholderia, Campylobacter, Citrobacter, Clostridium, Corynebacterium, Enter obacter, Enterococcus, Erysipelothrix, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Micrococcus, Moraxella, Morganella, Mycobacterium (tuberculosis), Nocardia, Neisseria Pasturella, Plesiomonas, Propionibacterium, Proteus, Providencia, Pseudomonas,

Rhodococcus, Salmonella, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Treponema, Vibrio (cholera) and Yersinia (plague).

[0035] By "biologically active portion" is meant a portion of a full-length polypeptide which portion retains the desired biological activity of the parent molecule. As used herein, the term "biologically active portion" includes deletion mutants and peptides, for example of at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350 contiguous amino acids (and every integer in between). One example of a biologically active portion is a form of the polypeptide without a signal or leader sequence. Portions of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a peptide or polypeptide of the invention with proteinases such as endoLys- C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Recombinant nucleic acid techniques can also be used to produce such portions. The biological activities of portions are tested in vivo and/or in vitro. A biological activity of MBL includes a function of its various domains. One domain is the collagen-like domain which binds specific serine proteinases such as, for example MASP-2. Thus, the ability to bind MASP-2 is a useful indicator of biological function. Another domain is the N-terminal portion of MBL, which binds to collectin receptors and/or Clq-like receptors. Also, the carbohydrate binding domain, which binds to molecular patterns in glycoproteins or glycolipids that are associated with or on the surface of pathogens or pathogen infected cells such as macrophages, CD4 lymphocytes etc. hi some embodiments, MBL activity is an ability to activate complement. In other embodiments, MBL activity includes the ability to opsonize pathogens or pathogen-infected cells. In still further embodiments, MBL biological activity is an ability to promote an inflammatory, immunomodulatory and/or a clinically beneficial response.

[0036] By "cell" is meant any suitable prokaryotic or eukaryotic cell. For ease of administration, a eukaryotic a cell is genetically compatible e.g., a syngeneic cell that is genetically identical to the subject or is genetically compatible with the subject to minimise any adverse immune response. In illustrative examples, liver cells are used.

[0037] By "co-administered," "co-administration" and the like is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the MBL polypeptide may be administered together with another agent in order to enhance its effects. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.

[0038] "Complementary" as used herein, refers to the capacity for precise pairing between two nucleobases of a polypeptide. For example, if a nucleobase at a certain position of a polypeptide is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the target nucleic acid molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. [0039] Throughout this specification, unless the context requires otherwise, the words

"comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0040] By "derivative" is meant a polypeptide that has been derived from the basic MBL sequence by modification, for example by conjugation or complexing with other chemical moieties or , by post-translational modification techniques as would be understood in the art. The term "derivative" also includes within its scope alterations that have been made to an MBL polypeptide including additions, or deletions that provide for functionally equivalent molecules. A functional derivative of a polynucleotide encoding an MBL polypeptide comprises a sequence of nucleotides having at least 60% similarity to the polynucleotide. A "part" or "portion" of a polynucleotide is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10-35 nucleotides including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600 nucleotides or nucleic acid molecules having any number of nucleotides within these values.

[0041] By "effective amount," is meant the administration of an amount of active agent to a subject, either in a single dose or as part of a series or slow release system, which is effective for prevention or treatment. The effective amount will vary depending upon the health and physical condition of the subject and the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. Notwithstanding the above, administration is effected to attain a hypersupplemented level of MBL in the subject, as described in more detail below. [0042] The terms "expression" or "gene expression" refer to either production of RNA message or translation of RNA message into proteins or polypeptides. By "expression vector" is meant any genetic element capable of directing the transcription of a polynucleotide contained within the vector and suitably the synthesis of a peptide or polypeptide encoded by the polynucleotide. Such expression vectors are known to practitioners in the art. [0043] The term "gene" as used herein refers to any and all discrete coding regions of the cell's genome, as well as associated non-coding and regulatory regions. The term is intended to mean the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression. In this regard, the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The DNA sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.

[0044] "Homolog" is used herein to denote a gene or its product which is related to another gene or product by decent from a common ancestral DNA sequence. [0045] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances as known to those of skill in the art.

[0046] The phrase "hybridizing specifically to" and the like refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0047] As used herein, a "hypersupplemented MBL level" is a level (including concentration or activity) of MBL which is significantly greater than the MBL level in a subject with normal MBL levels, In some embodiments, the significantly greater level represents an at least about 10%, 15% or 20% increase, or even an at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% increase above normal MBL levels. In other embodiments, the greater level of MBL in a subject equates to about an at least about 2, 3, 4, 5, 6, 7,8, 9 or 10-fold increase, or even about an at least 20, 30, 40, 50 or 100-fold increase in MBL. In some embodiments, a hypersupplemented MBL level is generally at least about 1 to about 15 μg/mL, usually at least about 1 to 10 μg/mL, at least about 1.5 to 7 μg/mL, at least about 1.5 to 5 μg/mL, at least about 1.5 to 4 μg/mL, at least about 1 to 2 μg/mL MBL in whole blood, serum or plasma, or other fluid sample. In some embodiments a hypersupplemented MBL level is assessed relative to the MBL level in a subject prior to administration of the MBL to the subject. In another embodiment, the level of MBL is assessed by measuring a functional activity of MBL (e.g., the ability to fix complement etc). The functional activity of an endogenous MBL molecule may also be predicted upon allele testing. Normal levels of MBL in blood/serum are generally quite low irrespective of genotype. Levels of circulating MBL, as assessed by different assays and reported as adult population means, medians and/or ranges, have been reported in previous studies of unselected Japanese, Caucasoid, African and Eskimo populations. Mannan binding assays indicate mean MBL levels of 3.97μg/mL and 4.48 μg/mL (range 0 - 15.73) in Caucasoids, as determined respectively by Aittoniemi et al (Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 105:617-622, 1997) and Aittoniemi et al.. (Clinical and Experimental Immunology, 116:505-508, 1999). Using a double antibody assay Caucasoids had a median MBL of 0.99 μg/mL (range 0-4.89) (Madsen et al, Immunogenetics, 40:37-44, 1994). Using the same assay in 1085 Japanese gave a mean of 1.72 μg/mL (SD +/- 1.15, range 0.07-6.40) (Terai et al, , Biochemical Medicine and Metabolic Biology, 50:111-119, 1993). Eskimos had median MBL levels of 2.98 μg/mL (range 0-8.24) (Madsen et al., 1994 (supra); Garred et al., European Journal of Immunogenetics,

19:403-412, 1992) and Africans a median MBL of 0.58 μg/mL (range 0-13.92) in one study (Garred et al, 1992 (supra)) and a median of 0.59 μg/mL (range 0 - 7.95) in another (Madsen et al, 1994 (supra)). Another normal African population tested using the double antibody assay, provided a range ofO - 4.05 μg/mL (Madsen et al, The Journal oflmmunology, 161:3169-3175, 1998). In aUK population, a Time Resolved Immunofluorometric Assay (TRIFMA) revealed MBL mean levels to be 1.22 μg/mL (SD +/- 1.05, median 1.08) (Crosdale et al, European Journal of Immunogenetics, 27: 111 -117, 2000) and with the same assay, the MBL range in a Danish Caucasoid population was 0.002 - 5.48 μg/mL (Steffensen et al, Journal of Immunological Methods, 241 :33-42, 2000). In a study conducted on Australian blood donors, mean MBL levels were determined (Minchinton et al, Scand. J. Immunol., 56(6):630-641, 2002). Mean MBL levels determined by double antibody, mannan-binding and C4 deposition assays in donors stratified according to MBL coding genotype are shown in the Table 4. For homozygous wild type subjects the mean MBL level was 2.66 μg/mL (range 0 - 7.90). In heterozygous donors the mean MBL level was 0.83 μg/mL (range 0 - 4.46). From the foregoing, it shall be understood that a "normal MBL level" may vary according to the subject that is treated and according to the assay used for measuring MBL levels. However, the term "hypersupplemented" will typically represent additional MBL that is administered to the subject, in order to achieve an MBL level that is at least 10% higher in the subject than the MBL level before the administration.

[0048] The term "immunocompromised" as used herein refers to a subject with an innate, acquired, or induced inability to develop a normal immune response. An immunocompromised subject, therefore, has a weakened or impaired immune system relative to one of a normal subject. A subject with a weakened or impaired immune system has an "immunodeficiency" or "immunocompromised condition," which is associated with a primary or secondary deficiency, induced or non-induced, in one or more of the elements of the normal immune defense system. An immunocompromised condition is commonly due to a medical treatment, e.g., radiation therapy, chemotherapy or other immunosuppressing treatment, such as induced by treatment with steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine or rapamycin, in particular in relation to cancer treatment or the treatment or prevention of transplant rejection. Additionally, the immunocompromised condition may be due to an acquired immunodeficiency, such as AIDS, or leukemia, in particular neutropenia or other secondary immunodeficiencies or to an immunodeficiency due to some pathogenic infections or malnutrition. However, it will be understood that the phrase "risk of acquiring an immunocompromised condition resulting from a medical treatment" refers only to medical treatments that leads to or confers an immunocompromised condition, especially chemotherapy or other immunosuppressing treatment, such as induced by treatment with radiation, steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine or rapamycin. The presence of an immunocompromised condition in a subject can be diagnosed by any suitable technique known to persons of skill the art. Strong indicators that an immunocompromised condition may be present is when rare diseases occur or the subject gets ill from organisms that do not normally cause diseases, especially if the subject gets repeatedly infected. Other possibilities are typically considered, such as recently acquired infections — for example, HTV, hepatitis, tuberculosis, etc. Generally, however, definitive diagnoses are based on laboratory tests that determine the exact nature of the immunocompromised condition. Most tests are performed on blood samples. Blood contains antibodies, lymphocytes, phagocytes, and complement components — all of the major immune components that might cause immunodeficiency. A blood cell count will determine if the number of phagocytic cells or lymphocytes is below normal. Lower than normal counts of either of these two cell types correlates with an immunocompromised condition. The blood cells are also checked for their appearance. Occasionally, a subject may have normal cell counts, but the cells are structurally defective. If the lymphocyte cell count is low, further testing is usually conducted to determine whether any particular type of lymphocyte is lower than normal. A lymphocyte proliferation test may be conducted to determine if the lymphocytes can respond to stimuli. The failure to respond to stimulants correlates with an immunocompromised condition. Antibody levels and complement levels can also be determined for diagnosing the presence of an immunocompromised condition. However, it shall be understood that the methods of the present invention are not predicated upon diagnosing the absence of an immunocompromised condition in the subjects to be treated. The subject methods are useful for treating the general population, which mostly comprises normal or "immunocompetent" subjects that do not have immunocompromised conditions.

[0049] Reference herein to a "infectious agent," "infectious organism," "microbe" or "pathogen" includes any one or more species or subspecies of bacterium, fungus, virus, algae, parasite, (including ecto-or endo-parasites) prion, oomycetes, slime, moulds, nematodes, mycoplasma and the like. The present invention is particularly suited to treating or preventing mixed infections by more than one microbe. Pathogenic algae include Prototheca and Ptiesteria. Also includes within the scope of these terms are prion proteins causing conditions such as Creutzfeldt- Jakob disease. As the skilled artisan will appreciate, pathogenicity or the ability of a classically non-pathogenic agent to infect a subject and cause pathology can vary with the genotype and expression profile of the infectious agent, the host and the environment. Fungal pathogens include without limitation species of the following genera: Absidia, Acremonium, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida (yeast), Cladophialophera, Coccidioides, Cryptococcus, Cunninghamella, Curvularia, Epidermophyton, Exophiala, Exserohilum, Fonsecaea, Fusarium, Geotrichum, Histoplasma, Hortaea, Lacazia, Lasiodiplodia, Leptosphaeria, Madurella, Malassezia, Microsporum, Mucor, Neotestudina, Onychocola, Paecilomyces, Paracoccidioides, Penicillium, Phialophora, Piedraia, Piedra, Pityriasis, Pneumocystis, Pseudallescheria, Pyrenochaeta, Rhizomucor, Rhizopus, Rhodotorula, Scedosporium, Scopulariopsis, Scytalidium, Sporothrix, Trichophyton, Trichosporon and Zygomycete. Pathogenic conditions include any deleterious condition that develops as a result of infection with an infectious organism.

[0050] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated peptide" or an "isolated polypeptide" and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural environment, (e.g., blood, plasma etc) and from association with other components of the cell. Without limitation, an isolated polynucleotide, peptide, or polypeptide can refer to a native sequence that is isolated by purification or to a sequence that is produced by recombinant or synthetic means. The level of purity required may be determined in toxicity studies. The purified product may also be tested for contamination such as viral contamination prior to use. [0051] The term "MBL polypeptide" as used herein refers to MBL purified from natural sources or produced by synthesis or recombinant technologies, including biologically active fragments of full-length MBL polypeptides as well as variants and derivatives of those full-length polypeptides and biologically active fragments. This term encompasses molecules which exhibit no less than about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 99% of the functional activity of MBL in serum. The functional activity may be assessed by any suitable assay, including assays based on mannan or MBL binding, complement deposition or activation.

[0052] By "modulation" or "modulator" in relation to a particular molecule is meant directly or indirectly up-regulating or down-regulating the level or activity of the molecule.

[0053] By "pharmaceutically acceptable" carrier, excipient or diluent is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e. the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like. [0054] Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein is a salt, ester, amide, prodrug or derivative that this not biologically or otherwise undesirable.

[0055] Pathogenic "protozoa" include, without limitation, Trypanosoma, Leishmania, Giardia, Trichomonas, Entamoeba, Naegleria, Acanthamoeba, Plasmodium, Toxoplasma, Cryptosporidium, Isospora and Balantidium.

[0056] Larger pathogenic "parasites" include those from the phyla Cestoda (tapeworms), Nematoda and Trematoda (flukes). Pathogenic trematodes are, for example, species of the following genera; Schistosoma, Echinostoma, Fasciolopsis, Clonorchis, Fasciola, Opisthorchis and Paragonimus. Cestode pathogens include, without limitation, species from the following orders; Pseudophyllidea (e.g., Diphyllobothrium) and Cyclophyllidea (e.g., Taenia). Pathogenic nematodes include species from the orders; Rhabditida (e.g., Strongyloides), Strongylida (e.g., Ancylostomά), Ascarida (e.g., Ascaris, Toxocara), Spirurida (e.g., Dracunculus, Brugia, Onchocerca, Wucheria) and Adenophorea (e.g., Trichuris and Trichinella).

[0057] The terms "polynucleotide," "genetic material," "genetic forms," "nucleic acids" and "nucleotide sequence" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference nucleotide sequence whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides.

[0058] The terms "polypeptide," "proteinaceous molecule," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages. The term "polypeptide variant" refers to polypeptides which are distinguished from a reference polypeptide by the addition, deletion or substitution of at least one amino acid residue, hi certain embodiments, one or more amino acid residues of a reference polypeptide are replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide (conservative substitutions) as described hereinafter.

[0059] Reference herein to "derived from" means that the sample is obtained from a particular source but not necessarily directly from that source.

[0060] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, GIy, VaI, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.

[0061] "Similarity" refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table A below. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids Research 12, 387- 395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0062] Reference to "storage" herein refers to a period prior to use during which the polypeptides or agents are unused but retain their suitability for administration either directly or indirectly after suitable modification. [0063] "Stringency" as used herein refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the observed degree of complementarity between sequences. "Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Generally, stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe. It will be understood that a polynucleotide will hybridize to a target sequence under at least low stringency conditions, preferably under at least medium stringency conditions and more preferably under high stringency conditions. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.5 M to at least about 0.9 M salt for washing at 42° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 42° C. High stringency conditions include and encompass from at least about 31 % v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization at 42° C, and at least about 0.01 M to at least about 0.15 M salt for washing at 42° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, ImM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. Other stringent conditions are well known in the art. A skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (supra) at pages 2.10.1 to 2.10.16 and MOLECULAR CLONING. A LABORATORY MANUAL (Sambrook, et al, , eds.) (Cold Spring Harbor Press 1989) at sections 1.101 to 1.104.

[0064] "Subjects" contemplated in the present invention include any animal of commercial humanitarian or epidemiological interest including conveniently, primates, livestock animals (such as sheep, cows, horses, donkeys, pigs, fish and birds), laboratory test animals (such as mice, rabbits, guinea pigs and hamsters and the like), companion animals (such as dogs and cats), or captive wild animals. Avian species include poultry birds and caged avian species. In some embodiments the subject is a mammalian animal. In other embodiments, the subject is a human subject. The present composition and methods have applications in human and veterinary medicine, domestic or wild animal husbandry, cosmetic or aesthetic treatments for the skin after injury or surgery.

[0065] By "substantially complementary" it is meant that an oligonucleotide or a subsequence thereof is sufficiently complementary to hybridize with a target sequence. Accordingly, the nucleotide sequence of the oligonucleotide or subsequence need not reflect the exact complementary sequence of the target sequence. In a preferred embodiment, the oligonucleotide contains no mismatches and with the target sequence.

[0066] Reference herein to "a virus" includes any virus or viral pathogen or emerging viral pathogen. Viral families contemplated include Adenoviridae, African swine fever-like viruses, Arenaviridae (such as viral haemorrhagic fevers, Lassa fever), Astroviridae (astroviruses) Bunyaviridae (La Crosse), Caliciviridae (Norovirus), Coronaviridae (Corona virus), Filoviridae (such as Ebola virus, Marburg virus), Parvoviridae (B 19 virus), Flaviviridae (such as hepatitis C virus, Dengue viruses), Hepadnaviridae (such as hepatitis B virus, Deltavirus), Herpesviridae (herpes simplex virus, varicella zoster virus), Orthomyxoviridae (influenza virus) Papovaviridae (papilloma virus) Paramyxoviridae (such as human parainfluenza viruses, mumps virus, measles virus, human respiratory syncytial virus, Nipah virus, Hendra virus), Picornaviridae (common cold virus),

Poxviridae (small pox virus, orf virus, monkey poxvirus) Reoviridae (rotavirus) Retroviridae (human immunodeficiency virus) Paroviridae (paroviruses) Papillomaviridae, (papillomaviruses) alphaviruses and Rhabdoviridae (rabies virus).

2. MBL modulators [0067] The present invention provides methods for treating or preventing an infection, including administering to a subject that is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment, an effective amount of an MBL modulator selected from an MBL polypeptide or an agent from which an MBL polypeptide is producible. MBL polypeptide species homologs (orthologs) are highly conserved sharing at least about 60% amino acid sequence identity. In accordance with the present invention, an MBL polypeptide encompasses any naturally-occurring MBL polypeptide from any animal including human species as well as their biologically active portions and variants or derivatives of these, as defined herein. The protein and nucleotide sequences of MBL may be found in publicly available databases such as the electronically searchable databases of the National Centre for Biotechnology Information. Accession No. AAV80468, for example, provides an illustrative protein sequence of human MBL (set out in SEQ ID NO: 2). Accession No. AY826184 (set out in SEQ ID NO: 1 and 2) provides an illustrative nucleotide (cDNA) sequences encoding human MBL. Nucleic acid and amino acid sequences from mouse, rat, cow, chicken and a range of non-human primates such as chimpanzees are as set forth in SEQ ID NO: 4 to SEQ ID NO: 13 (see Table 1).

[0068] The polypeptides may be produced by any convenient method such as by purifying the polypeptide from naturally-occurring reservoirs including blood, serum and plasma. Methods of purification include precipitation, agglutination, fractionation, size exclusion, affinity chromatography, ion exchange chromatography, filtration, dialysis and separation. MBL is conveniently prepared from blood products as described in International Publication No. WO 03/090774 and by Laursen et al, Biochem. Soc. Trans., 31 (Pt 4):758-762, 2003. The identity and purity of derived polypeptide is determined for example by SDS-polyacrylamide electrophoresis, or chromatographically, such as by high performance liquid chromatography (HPLC). Purification is conveniently achieved using procedures such as gel filtration, ion exchange chromatography, affinity chromatography including hydrophobic interaction chromatography or immunochromatography. The level of purity of the isolated protein may be conveniently determined chromatographically, by sequencing, SDS-PAGE analysis etc.

[0069] In another approach, the polypeptides may be prepared by a procedure including the steps of: (a) preparing a construct comprising a polynucleotide sequence that encodes an MBL polypeptide and that is operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the MBL polypeptide; and (d) isolating the MBL polypeptide from the host cell. Recombinantly produced proteins can be modified for expression and secretion and their isolation facilitated with affinity or purification tags, such as the FLAG epitope, maltose binding protein, glutathione-S-transferase, thioredoxin, and the like. In illustrative examples, the nucleotide sequence encodes at least a biologically active portion of the sequence set forth in any one of SEQ ID NO: 2, 5, 7, 9, 11 and 13. Recombinant polypeptides can be prepared using standard protocols as described for example in Sambrook, et al, (1989, supra), in particular Sections 16 and 17; Ausubel et al, (1994, supra), in particular Chapters 10 and 16; and Coligan et al, CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. For example, recombinant MBL produced in various host cells has been derived by Vorup- Jensen et al, Int. Immunopharmacol., l(4):677-687, 2001. [0070] Alternatively, the subject polypeptides may be synthesized by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard {supra) and in Roberge et al. (Science, 269:202, 1995).

[0071] In specific embodiments, the MBL polypeptide is in oligomeric form and thus defines MBL oligomers having a size distribution substantially identical to the size distribution of MBL in serum, for example a size distribution profile at least 50% identical to the size distribution profile of MBL in serum. By identical is meant that at least 50% of the oligomers has an apparent molecular weight higher than 200 kDa, when analysed by SDS-PAGE and/or western blot. In specific embodiments, the size distribution profile is at least 75% identical to the size distribution profile of MBL in serum, such as at least 90% identical to the size distribution profile of MBL in serum, and more preferred at least 95% identical to the size distribution profile of MBL in serum. [0072] When purifying from an MBL source initially having another size distribution profile (e.g., recombinant MBL sources) it is desirable that the affinity chromatography used to purify from the MBL source favors purification of oligomers having an apparent molecular weight higher than 200 kDa. This can be obtained, for example, using a carbohydrate-derivatized matrix having substantially no affinity to subunits and/or dimers of MBL. Suitably, the carbohydrate-derivatized matrix has affinity for substantially only tetrameric, pentameric anchor hexameric MBLs. Illustrative matrices of this type are disclosed in U.S. Pat. No. 6,562,784.

[0073] The functional activity of MBL polypeptide may be determined for example by any suitable assay including assays based on mannan or MBL binding, complement deposition or activation. In one non-limiting example, the functionality or functional activity of MBL is estimated by its capacity to form an MBL/MASP complex leading to activation of the complement system. When C4 is cleaved by MBL/MASP an active thiol-ester is exposed and C4 becomes covalently attached to nearby nucleophilic groups. A substantial part of the C4b will thus become attached to the coated plastic well and may be detected by anti-C4 antibody. An illustrative assay of this type comprises: 1) coating microlitre wells with 1 mg mannan in 100 mL buffer; 2) blocking with Tween- 20; 3) applying test samples, e.g. diluted MBL preparations 4) applying MBL deficient serum (this leads to the formation of the MBL/MASP complex); alternatively the MBL and the MBL deficient serum may be mixed before application with the microlitre wells; 5) applying purified complement factor C4 at 5 mg/mL; 6) incubating for one hour at 37° C; 7) applying Europium-labeled anti-C4 antibody; 8) applying enhancement solution and 9) reading the Europium by time resolved fluorometry. Between each step the plate is incubated at room temperature and washed, except between step 8 and 9. Estimation by ELISA may be carried out similarly, e.g. by applying biotin- labeled anti-C4 in step 7; 8) applying alkaline phosphatase-labelled avidin; 9) applying substrate, and 10) reading the color intensity.

[0074] The functionality may be expressed as the specific activity of MBL, such as 1 unit of MBL activity per ng MBL. A non-functional MBL may be defined as MBL having a specific activity less than 50% of plasma MBL specific activity, such as less than 25% of plasma MBL specific activity wherein the plasma MBL is purified from an individual not suffering from any MBL mutations, in particular the reference plasma MBL is plasma pool LJ 6.57 28/04/97.

[0075] The MBL polypeptide includes all biologically active naturally occurring forms of MBL as well as biologically active portions thereof, and variants or derivatives of these. Biological activity as used herein refers broadly to the ability of MBL polypeptide to treat or prevent an infection. Biologically active portions of MBL polypeptide include without limitation parts of the amino acid sequence set out in any one of SEQ ID NO : 2, 5 , 7, 9, 11 and 13. A biologically active portion of a full-length MBL polypeptide may comprise, for example, at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120 or 150, or even at least about 200, 220, 240 or 248 contiguous amino acid residues, or almost up to the total number of amino acids present in a full-length MBL polypeptide. Suitably, the portion is a "biologically-active portion" having no less than about 10%, 20%, 30%, 40% 50%, 60%, 70%, 80%, 90%, 99% of the activity of the full-length MBL polypeptide from which it is derived. Suitable biologically active portions include soluble forms of the polypeptide without a leader or signal peptide. [0076] MBL polypeptides includes "variant" polypeptides that are distinguished from a naturally-occurring MBL polypeptide or from a biologically active portion thereof by the addition, deletion and/or substitution of at least one amino acid residue. Thus, variants include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is, they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Chimeric or fusion polypeptides are produced by standard recombinant DNA techniques. In one example, polynucleotide fragments coding for different polypeptide sequence are ligated together using one of several conventional techniques. Alternatively fusion genes can be synthesised by DNA synthesisers. Variants may be generated which exhibit, for example, altered glycosylation, acetylation or phosphorylation patterns. They may have altered re-folding properties or three-dimensional structure and oligomer formation characteristics. Biologically active variants of a native MBL polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a MBL polypeptide may differ from that polypeptide generally by as much 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. MBL polypeptides may be chimeric or fusion polypeptides designed to have binding properties of different MBL polypeptides (for example species homo logs, analogs or allelic variants) or modified MBL polypeptides (derivatives, parts etc).

[0077] An MBL polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a MBL polypeptide can be prepared by mutations in the encoding nucleic acid sequence. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA 82:488-492), Kunkel et al. (Methods in Enzymol. 154:367-382, 1987), U.S. Pat. No. 4,873,192, Watson, J. D. et al. ("Molecular Biology of the Gene", Fourth Edition, Beηjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do or do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.). For example deletion of all or part of the collagen-like domains will produce an MBL variant which is functionally inactive in relation to its serine proteinase binding characteristics, but retains its carbohydrate binding characteristics. Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of MBL polypeptides. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify MBL polypeptide variants such as those with particular or enhanced mannose binding behaviour (Arkin and Yourvan Proc. Natl. Acad. Sci. USA 89:7811-7815, 1992; Delgrave et al. Protein Engineering 6:327- 331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below. [0078] Variant MBL polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to the parent MBL amino acid sequence. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows: [0079] Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

[0080] Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

[0081] Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine). [0082] Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan. [0083] Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

[0084] This description also characterises certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al. (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington DC; and by Gonnet et al, Science 256(5062); 1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

[0085] The degree of attraction or repulsion required for classification as polar or non- polar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.

[0086] Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table A. TABLE A

AMINO ACID SUB-CLASSIFICATION

[0087] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional MBL polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in Table B below under the heading of exemplary substitutions. More preferred substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. TABLE B

EXEMPLARY AND PREFERRED AMINO ACID SUBSTITUTIONS

[0088] Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).

[0089] Thus, a predicted non-essential amino acid residue in an MBL polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of an MBL polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.

[0090] Accordingly, the present invention also contemplates variants of the naturally- occurring isolated MBL polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to a parent MBL polypeptide sequence as, for example, set forth in SEQ ID NO: 2. Desirably, variants will have at least 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to a reference MBL polypeptide sequence as, for example, set forth in SEQ ID NO: 2, 5, 7 and 9. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids but which retain the properties of the parent MBL polypeptide are contemplated. MBL polypeptides also include polypeptides that are encoded by polynucleotides that hybridise under stringency conditions as defined herein, especially high stringency conditions, to MBL polynucleotide sequences, or the non-coding strand thereof.

[0091] hi some embodiments, variant polypeptides differ from an MBL sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in SEQ ID NO: 2, 5, 7 and 9 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment the sequences should be aligned for maximum similarity. "Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a non-essential residue or a conservative substitution. Naturally-occurring MBL polypeptides contain a significant number of structural characteristics in common with each other as for example depicted in Figure 2. This alignment shows positions which are amenable to conservative substitution and other that accommodate non-conservative substitutions.

[0092] A position which is amenable to non-conservative substitutions can be altered without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. A position which is non amenable to conservative or non-conservative alterations is a position that, when altered from the wild-type sequence of an MBL polypeptide of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.

[0093] In another embodiment, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 98% or more similarity to a corresponding sequence of an MBL polypeptide as, for example, set forth in any one of SEQ ID NO 2, 5, 7, and 9 and has the desired biological activity of an MBL polypeptide.

[0094] Derivatives are similar to variants but are derived from a parent MBL molecule by synthetic, recombinant or other chemical or biochemical methods. Portions or parts, comprise at least about 10 and preferably at least 20 or 30 or at least about 40 amino acids. Analogs include but are not limited to side-chain modifications incorporating unnatural amino acids or their derivatives during synthesis and the use of cross-linking agents or other methods which impose conformational constraints. Mimics are useful to provide the molecular interactions of the reference molecule with added advantages of for example ease of manufacture, stability, activity. In relation to genetic molecules (ie those comprising nucleic acids) the terms portions, derivatives and variants have mutatis mutandis, meanings analogous to those ascribed to these forms in relation to polypeptide molecules.

[0095] Another useful group of compounds are analogs and mimics (mimetics) of MBP. These molecules retain the desired biological activity of a reference MBL polypeptide and may also possess additional characteristics which improve their efficacy, such as exhibiting a longer half-life in vivo or alternatively which are, for example, readily synthesized or readily taken up by skin cells. A peptide mimetic or mimic has some chemical similarity to the parent molecule e.g., MBL, but agonizes its activity. A peptide mimic may be a peptide-containing molecule which mimics elements of protein secondary structure (as described for example in Johnson et al "Peptide Turn Mimetics" in Biotechnology and Pharmacy, Pezzuto et al, Eds., Chapman and Hall, New York, 1993). Non-peptide "small molecules" are often preferred for many in vivo pharmaceutical applications and accordingly mimetics may be designed for pharmaceutical use. Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a particular property, particularly where a lead compound has already been identified. As a first step, residues critical for enhancing healing are identified and this framework used as a pharmacophore. The structure may then be modeled using computational and other analyses. Alternatively, the three dimensional structure of the agent may be known in which case further agents may be designed along the same lines.

[0096] Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs. This term also does not exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell. [0097] Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

[0098] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0099] The carboxyl group may be modified by carbodimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide. [0100] Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenoI and other mercurials; carbamoylation with cyanate at alkaline pH.

[0101] Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. [0102] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

[0103] Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy- 5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.

[0104] MBL is a glycoprotein and variants of the carbohydrate residues attached to MBL are also contemplated. Sugar chains may for example be modified enzymatically and then tested for binding to known substrates. [0105] Agents from which MBL polypeptide is producible conveniently include without limitation cells from which MBL polypeptide is produced or polynucleotides encoding MBL polypeptides. In some embodiments, the agent is an autologous cell derived from the subject to be treated or a syngeneic cell. In some embodiments, the cell is genetically modified in order to secrete MBL polypeptide. Other cells, such as liver cells and monocytes or macrophages secrete MBL polypeptide naturally. In other embodiments, the cell is a genetically modified cell capable of producing MBL polypeptide. In still further embodiments, the cell is a stem cell.

[0106] In a preferred embodiment, the agent is expressed from transplanted cells or graft tissue. For example, MBL levels are conveniently enhanced via transplantation of cells expressing the subject agent, such as for example liver cells or tissues or liver progenitor cells. Progenitor cells may, for example, be purified using density gradient centrifugation to harvest cells of the correct buoyant density. Such cells may be propagated in vitro and/or transferred to a recipient, or, they can be transfected with genetic material expressing the subject agents and subsequently transferred to a recipient. [0107] Recombinant methods for producing genetically modified cells from which MBL polypeptide is producible are described for example in Vorup-Jensen et al. {supra). Essentially, a polynucleotide encoding a MBL polypeptide is engineered within an expression construct or shuttle vector and operably linked to a regulatory element (e.g., a promoter) that is operable in the cell in which it is desired to express the polynucleotide. The promoter may be inducible or constitutive, and, optionally, tissue-specific. The promoter may be, for example, viral or mammalian in origin. In some embodiments, a nucleic acid construct is used in which the promoter-polynucleotide cassette (and any other desired sequences) is flanked by regions that promote homologous recombination at a desired site within the genome of a subject, thus providing for intra-chromosomal expression of the polynucleotide. See e.g., Koller and Smithies (1989, Proc. Natl. Acad. Sci. USA, 86:8932-8935). In other embodiments, the nucleic acid construct that is delivered remains episomal and induces an endogenous and otherwise silent gene. Generally, a selective marker gene such as an antibiotic resistance marker gene is employed to facilitate selection of appropriately modified cells. In some embodiments, the polynucleotide (cDNA) is selected (amplified) or modified by removal of sequences encoding signal sequences to facilitate secretion of a soluble or mature MBL polypeptide. [0108] In other embodiments, the MBL modulator is attached or otherwise associated with a medical device including contraceptive device or material such as, for example, a stent, artificial replacement part (joint, valve etc), suture, condom, synthetic skin or a cream, gel or dressing. In some embodiments, the polypeptide or agents are applied to, attached to or otherwise associated with a medical or other device, tissue or composition. For example, in some embodiments, the MBL modulator is associated with a dressing. In others embodiments, it is associated with a suture, graft, substitute skin or other composition which is applied to the subject.

3. Combination therapy

[0109] The present invention encompasses co-administration of one or more additional agents in concert with the MBL modulator. It will be understood that, in embodiments comprising administration of combinations of an MBL modulator with other agents, the dosage of the MBL modulator may on its own comprise an effective amount and additional agent(s) may further augment the therapeutic or prophylactic benefit to the patient. Alternatively, the combination of the MBL modulator and the additional agent(s) may together comprise an effective amount for preventing or treating the infection. It will also be understood that effective amounts may be defined in the context of particular treatment regimens, including, e.g., timing and number of administrations, modes of administrations, formulations, etc.

[0110] In some embodiments, the additional agent encompasses one or more anti-infective drugs inclusive of antimicrobials, which include without limitation compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc. and thus include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals. Anti-infective drugs also include within their scope anthelmintics and nematocides. Illustrative antibiotics include quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin; gemifloxacin; and garenoxacin), tetracyclines, glycylcyclines and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline; linezolide, eperozolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), β-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin), ketolides (e.g., telithromycin, cethromycin), coumermycins, lincosamides (e.g., clindamycin, lincomycin) and chloramphenicol.

[0111] Illustrative antivirals include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine.

[0112] Non-limiting examples of amebicides or antiprotozoals include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. Anthelmintics can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and thiabendazole. Illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafme hydrochloride. Non-limiting examples of antimalarials include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. Antituberculotics include but are not restricted to clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate. [0113] In some embodiments, the combination therapy includes co-administration of the

MBL modulator with at least one additional agent that enhances the immune response of the subject (e.g., a response that includes an activity selected from phagocytosis, complement activation, opsonisation, protein-, glycoprotein- or glycolipid-binding, and receptor binding. Agents of this type are suitably selected from selected from a hormone, cytokine, lymphokine, haematopoietic factor, chemokine, antibody or part thereof, co-stimulatory molecule and biological response modifier. In some embodiments, the additional agent is cytokine such as for example IL-2, G-CSF, GM-CSF, IL-7, EL-12, IL-3, IFNγ. In some embodiments, the agent is IL-2.

4. Formulations

[0114] The MBL modulator and optionally the additional agents mentioned above (also referred to herein as "active agents" or "actives") are formulated in pharmaceutical compositions which are prepared according to conventional pharmaceutical compounding techniques, See, for example, Remingtons Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration.

[0115] Administration of the active agents is performed by any convenient local or systemic manner including, without limitation intravenously (where water soluble), respiratorialy, nasopharyngealy, intravesicaly, intraocularly, intreperitoneally, subcutaneously, cutaneously, sublingually, vaginaly, nasally, topically, transdermally, intradermally, intramuscularly, intrathecaly, intracerebraly, orally, rectaly, or via patch or implant.

[0116] For oral administration, the active agents can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent a convenient oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See, for example, International Patent Publication No. WO 96/11698. Agents may be protected from digestion by complexing the agent with a composition to render it resistance to digestion or by packaging the agent in a resistant carrier such as a liposome, emulsion or other polymeric material. US Patent No. 5,391,377 for example describes lipid compositions for oral delivery. [0117] For parenteral (including intraocular, intravesical etc) administration, the active agents may be dissolved in a pharmaceutical carrier and administered as either a solution of a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecaly, they may also be dissolved in cerebrospinal fluid.

[0118] For transmucosal, transdermal or transcleral administration, penetrants appropriate to the barrier are used. For example, detergents, bile salts and fusidic acid derivatives are used for transmucosal delivery. For inhalation, various delivery systems are known such as dry powder aerosols, liquid delivery systems, air jet nebulisers, propellant systems and the like. Polymeric lenses or coatings comprising cationic emulsions may be employed for oral, injectable or dermal delivery. Various formulations may be adopted to provide the required biodistribution and pharmacokinetic characteristics. Biodegradable polymers capable of sustained delivery are particularly preferred. The use of lipid monolayers or bilayers in liposome or liposome like formulations are known in the art, for example, the use of stealth liposomes and/or micelles. See, for example, Remington's, US Patent No. 6,110,490 & 6,096,716. [0119] The pharmaceutical compositions may be formulated in any suitable carrier, diluent or excipient. For example, the active agents can be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non-ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.

[0120] Alternatively, the active agents can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which is also preferred for the practice of the present invention. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifϊers, isotonic saline, and pyrogen-free water. [0121] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0122] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, eg. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0123] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0124] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0125] Dosage forms of the active agents may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of an active compound of the invention may be achieved by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, controlled release may be achieved by using other polymer matrices, liposomes and/or microspheres.

[0126] The compositions of the present invention may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In such a case, the particles of the formulation may advantageously have diameters of less than 50 micrometers, suitably less than 10 micrometers.

[0127] In some embodiments, the actives are administered by topical or intradermal application of a gel, powder, film, aerosol, foam, colloid, polymer sealant, patch, emulsion, liquid or suspension effective to deliver the active agents, for example to a suitable site, e.g., skin, membrane, burn site, wound bed and/or surrounding tissue. The agents and compositions can be formulated into a wide variety of carriers. For example, the active agents are formulated together with carriers such as microparticles, gels, bioactive foams, synthetic skin preparations, liquids and creams. Microparticles may for example be microspheres, microcapsules, liposomes and the like adapted for slow release of agents over time. Such particles are conveniently biodegradable or biocompatible. In addition bioadhesive molecules such as bioadhesive peptides or polymers are conveniently used to bind polypeptides, cells, polynucleotides etc of the present invention to the dermal injury site and enhance their effectiveness. Film forming compositions and bioadhesive polymers are described in US Patent No. 6, 103,266. Film forming compositions for topical use and delivery of active agents are described for example in US Patent No. 6,797,262. Intradermal delivery of polypeptides is described in particular in published US Patent Application No. 20040073160.

[0128] An emulsion is a composition comprising more than one phase where at least one of the phases consist of finely divided phase domains (such as, for example, particles or droplets) distributed throughout a continuous phase domain. An emulsion is formed, for example, when two immiscible liquids such as oil and water are sufficiently well mixed. The finely divided domains are generally referred to as dispersed or discontinuous phase domains. The dispersion may be further defined in terms of the size of the dispersed domains. Micro emulsions are particularly useful emulsions due to their thermodynamic stability and optical properties. A micro emulsion comprises dispersed domains having a diameter in the order of 10-6 M. A micro emulsion includes, for example a strict water in oil (or oil in water) micro emulsion, a bicontinuous monophase, a micellar solution or a swollen micellar solution.

[0129] Micro emulsions are widely used and are generally favoured for their optical properties or ability to be absorbed by the skin or to cross membranes including biological membranes. Micro emulsions also form useful drug delivery systems, for example, including those which provide some level of protection of the active agent or provide prolonged release capabilities. In particular applications, the micro emulsion includes a solution in which solute molecules may be dispersed.

[0130] Various techniques are available for the production of micro emulsions. In general, micro emulsions are produced by emulsifying components under conditions including typically sufficient force or the required temperature to generate the required dispersion level, conductivity, viscosity, percolativity or other dispersion characteristics.

[0131] Micro emulsion formation can be assessed using scattering and spectroscopic techniques such as neutron scattering, time-average scattering, quasi-electric light scattering i.e., high- resolution ultrasonic spectroscopy or photon correlation spectroscopy. The partition coefficients of micro emulsions may also be measured chromatographically. The selection of particular formulations is based on a number of different paradigms depending upon the desired application. Illustrative paradigms include the hydrophilic-lipophilic balance, the phase-inversion temperature, or the cohesive-energy ratio. Micro emulsions may be formulated using a wide range immiscible liquids and other additional agents. Thermodynamically stable micro emulsions systems include those which are biocompatible. For example, oils suitable for use in forming a micro emulsion include vegetable oils, synthetic or natural triglycerides, fatty acid esters such as isopropyl myristate or ethyl oleate, and phospholipids such as lecithin or lysolecithin. Other organic liquids including, but not limited to, benzene, tetrahydrofuran, and n-methyl pyrrolidone, or halogenated hydrocarbons, such as methylene chloride, or chloroform may also be used as the oil component of the micro emulsion. The proportion of oil or mixture of oils used in a micro emulsion is typically in the range about 10 and 60% by volume. Surfactants and co-surfactants are employed to enhance emulsion formation by altering the interfacial tension between phases and to enhance emulsion stability. Examples include anionic surfactants such as fatty acid soaps, acyl sulfates, or acyl sulfosuccinates; cationic surfactants, such as alkyl primary, secondary, tertiary, or quaternary amines; nonionic surfactants, for example, sorbitan esters or polyethoxylated esters of acyl acids, copolymers of polyethylene oxide and polypropylene oxide. The content of the surfactant or surfactants in a micro emulsion, can range, for example, from between about 0.1 to 60% by volume. Co-surfactants include aliphatic alcohols. Shorter chain alcohols, such as ethanol, are particularly preferred to more toxic, longer chain alcohols for use as co- surfactants. Alcohol content may range, for example, from about 0 to about 30% by volume in the micro emulsion. Solvents or other agents may also be employed to enhance emulsion formation or stability. Other agents may be introduced to provide functions such as pH, ionic content, polymerisation, smell, sterility, colour, viscosity etc. Micro emulsions may also be generated using any suitable synthetic plastic or polymeric, monomeric or hybrid colloidal material.

[0132] By whatever route or combination of routes, the active agent is administered in a therapeutically effective amount. As disclosed herein, higher MBL levels are associated with lower SOFA scores indicative of resistance to infection (see Figure 1). The mean SOFA score of patients who died due to sepsis was 8 in the SOFA validation study (Moreno et al, Intensive Care Med., 25(7):686-696, 1999).

[0133] The active agents may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the infection being treated, whether a recurrence of the condition is considered likely, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., active agents may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable. [0134] In embodiments relating to combination therapy, the MBL modulator and the additional agent may be provided in effective amounts to treat an infection in the subject or to augment the capacity to prevent or impede the development of a future infection. This process may involve administering the MBL modulator separately, simultaneously or sequentially with the additional agent. In some embodiments, this may be achieved by administering a single composition or pharmacological formulation that includes both agents, or by administering two separate compositions or formulations at the same time, wherein one composition includes the MBL modulator and the other, the additional agent. In other embodiments, the treatment with the MBL modulator may precede or follow the treatment with the additional agent by intervals ranging from minutes to days. In embodiments where the MBL modulator is applied separately to the additional agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the MBL modulator would still be able to exert an effect on the subject's immune system, in particular, to enhance a subject's capacity to mount an innate immune response against an infectious agent. In such instances, it is contemplated that one would administer both agents within about 1-12 hours of each other and, more suitably, within about 2-6 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several hours (2, 3, 4, 5, 6 or 7) to several days (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0135] It is conceivable that more than one administration of either the MBL modulator or additional agent will be desired. Various combinations may be employed, where the MBL modulator is "A" and the additional agent is "B", as exemplified below:

[0136] A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B.

[0137] Other combinations are contemplated. Again, both agents are delivered to a subject in a combined amount effective to enhance a subject's capacity to treat an infection, or inhibit or prevent an infection from occurring. [0138] The present invention also provides kits comprising MBL polypeptide or an agent from which MBL polypeptide is producible. Kits are contemplated which retain MBL polypeptides or MBL producing agents in a form suitable for subsequent administration. Thus, the polypeptides or agents may be stored separately or together with components of compositions which render the polypeptides or agents suitable for administration. In one example, a MBL polypeptide is separately stored in a kit in freeze-dried form and reconstituted prior to use with an aqueous buffer stored in a separate compartment in the kit. Other components optionally include gels, creams, ointments, powders, films, aerosols, emulsions, sealants, dressings and the like suitable for administration. In other embodiments, the components are sterilized. In yet other embodiment, the components of the kit further comprise preservatives, anti-bacterial anti-fungal agents etc.

5. Treatment or prevention of infections

[0139] The present invention encompasses the use of an MBL modulator selected from an MBL polypeptide or an agent from which a MBL polypeptide is producible in the manufacture of a medicament for the treatment or prevention of an infection in a subject that is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from medical treatment. In some embodiments, therefore, the medicament is administered to immunocompetent subjects having an existing infection, which may be known or unknown and which may be sensitive or resistant to treatment with an anti-infective agent. In other embodiments, the medicament is administered for the prevention of an infection in immunocompetent subjects at risk of exposure to an infectious agent. Accordingly, these applications may be useful in the context of a known risk of infection in order to prevent the infection or to treat an early asymptomatic infection. A known risk of infection may include the presence of infected individuals within a population (e.g., a meningitis outbreak in a population); known exposure or suspected exposure to one or more biological warfare agents (e.g., a drug-resistant microbial agent used as a biological weapon); a subject with burns; a subject with a wound or a subject undergoing surgery and other risk factors; or any combination of two or more of the above. In some embodiments, particularly in the case of biowarfare or epidemics, exemplary subjects include doctors, nurses and soldiers as well as members of general population who are at risk of exposure to the infectious agent. In other embodiments, particularly in the case of burns, the subject is suitably selected from firemen, kitchen personnel or other individuals exposed or at risk of exposure to burn-causing agents or environments. In still other embodiments, particularly in the case of wounds (especially open wounds), the subject is suitably selected from soldiers and policemen, security personnel or any other individual exposed or at risk of exposure to weapons assaults. In still other embodiments, particularly in the case of surgery, the subject is suitably selected from any individual having or at risk of having a surgical procedure including peritoneal, pericardial, obstetric, gynecological, neurosurgical, arthroscopic, orthopedic, plastic, reconstructive, muscle, or tendon surgery. In other embodiments, the medicament is for the prevention or treatment of infection in a subject where the subject has been exposed to an infectious agent but is not exhibiting any symptoms of infection by a pathogenic organism. In yet other embodiments, the medicament is for treatment of an infection where the subject has been exposed to a pathogenic organism and is exhibiting one or more symptoms of infection. Symptoms of infection with a pathogenic organism would include for example one or more of fever, rash and malaise.

6. Methods of determining the level or activity of MBL in a subject

[0140] In some embodiments, the methods of the invention further comprise: assessing the MBL level and/or functional activity in a subject. MBL protein concentration, functional activity and genotype may be determined by a wide range of techniques known to those of skill in the art. Reference may be made in particular to Current Protocols in Molecular Biology (Ausubel et ah, eds.) (John Wiley & Sons, Lie, 1995); Molecular Cloning. A Laboratory Manual (Sambrook et ah, eds.) (Cold Spring Harbour Press, 1989); Monoclonal Antibodies: A Laboratory Manual (Harlow and Lane eds.) (Cold Spring Harbour Press, 1988); Dieffenbach et al, PCR Primer: A Laboratory Manual, 1995). [0141] The concentration of MBL or MASP in a blood or other sample from a subject may be determined using an immuno assay such as an enzyme linked immunosorbent assay (ELISA) assay, a double antibody assay, a time resolved immunofluorometric assay (TRIFMA), an indirect labeled antibody technique radioimmunoassay or fluorescent activator cell sorts (FACS), nephelometry or the like, (see for example, Wild D. "The Immunoassay Handbook" Nature Publishing Group, 2001).

[0142] Antibodies or parts thereof may be used which are specific for different forms or parts of MBL in monomeric, dimeric or oligomeric form. Methods for making polyclonal or monoclonal antibodies are known. Reference may be made to Current Protocols in Molecular Biology {supra) and particularly Unit 11.6 therein. Methods for making monoclonal antibodies are known to those of skill in the art for example as disclosed in Kohler and Milstein (Nature 256:495-499, 1975). Fragments of antibodies include for example Fv, scFv, Fab, Fab' and F(ab')2 fragments may be preferred for particular applications.

[0143] Briefly, an ELISA assay typically uses an immobilized antibody to capture the protein to be detected. The same or different antibody, which is detectably labeled is then used to detect bound protein after a washing step to remove unbound sample. The detectable label may be an enzyme or other detectable marker such as a fluorochrome, bioluminescent molecule or colloidal particle, such as gold, as described above.

[0144] Quantitative RAPID assays would also be conveniently used to monitor MBL levels. These assays use capillary flow of sample components in chromatographic devices to avoid at least some washing and pipetting steps generally used in ELISA assays. Reference to a "chromatographic device" includes a device of any solid, semi-solid, matrix or gel material which is known in the art for facilitating or supporting chromatographic flow or separation. Quantitative assays are slightly more complex than qualitative tests and require an understanding of the kinetics of the reaction and behaviour of the detectable marker under control and test conditions.

[0145] Genotyping typically uses oligonucleotide probes or primers to distinguish between genotypes as described herein. [0146] Alternatively, MBL levels and functional activity can be measured using functional assays such as measuring the ability of MBL to activate complement, as described herein. An assay for active MASP is described in WO 03/090774 based on the use of substrates derived from the MASP cleavage site on the C4 protein.

[0147] In some embodiments, MBL function is measured by C4b deposition. For these assays, typically serum is incubated in mannan-coated microtitre plates, as described for example in Section 2. After removal of serum, exogenous C4 or MBL deficient serum is added and any MBL- MASP-2 complex bound to the mannan surface cleaves C4 to C4b. The amount of C4b deposition is determined using an anti-human C4 monoclonal antibody.

[0148] The infectious organism which causes the infection in a subject carries molecular patterns detected by pattern recognition molecules of the host. For intracellular pathogens, molecular patterns are present on the surface of the infected cell. Alternatively, pattern recognition molecules directly or indirectly block or reduce infection or re-infection after host cell lysis or exit.

[0149] In some embodiments, the subject medical protocol includes steps for distinguishing between pathogenic infectious agents in a subject or sample. [0150] Assays to distinguish between infectious organisms include the detection of proteinaceous molecules, generally with protein-binding molecules such as antibodies or nucleic acid based protein binding molecules. The principals and conduct of such binding assays are well known to those of skill in the art. Alternatively, the genotypes of subtypes, genera, species, subspecies and strains are distinguished on the basis of their unique genetic sequences using oligonucleotide probe and/or primer DNA based assays. Whatever the format of the assay, product data are processed and compared with pre-existing data in order to assess the characteristics of the infectious organism and/or its ability to bind to MBL including variants, derivatives and/or parts thereof, and, if possible, to provide a diagnosis and/or a report. Conveniently, the processing steps are performed by a programmable computer. [0151] An oligonucleotide is typically rather short in length, generally from about 8 to 30 nucleotides, more preferably from about 10 to 20 nucleotides and still more preferably from about 11 to 17 nucleotides. The term can refer to molecules of any length and longer oligonucleotides of 70 to 100 nucleotides are also expressly contemplated. Oligonucleotides may be prepared using any suitable method, such as, for example, the phosphotriester method as described in an article by Narang et al., Methods Enzymol., 68:90, 1979) and U.S. Patent No. 4,356,270. Alternatively, the phosphodiester method (as described in Brown et al., Methods Enzymol., 68:109, 1979) may be used for such preparation. In some embodiments, the oligonucleotides are synthesised according to the method disclosed in U.S. Patent No. 5,424,186 (Fodor et al). This method uses lithographic techniques to synthesise a plurality of different oligonucleotides at precisely known locations on a substrate surface. Alternatively, oligonucleotides may be generated using PCR-based methods such as those described by Antson et al, Nucleic Acid Research, 28(12):e58, 2000. These PCR-based methods are particularly suitable for generating longer oligonucleotides, of typically more than 100 nucleotides. Oligonucleotides may be generated by careful chemical synthesis or using supported PCR based methods such as those generally directed by Antson et al, 2000 (supra). [0152] The distinguishing steps typically involve oligonucleotide amplification reactions which produce many polynucleotide copies of a particular target polynucleotide. If the target polynucleotide is single-stranded, complementary sequences may be produced in the reaction. The reaction is typically a polymerase chain reaction (PCR) or a similar reaction that uses a polymerase to copy a polynucleotide such as transcription mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA) and reverse transcription polymerase chain reaction (RT-PCR). A double stranded region formed through the hybridisation of an oligonucleotide to a single-stranded form of the polynucleotide is required to prime (start) the reaction. In some embodiments, the distinguishing step includes a biochemical reaction using a ligase or similar enzyme that covalently links two oligonucleotides or two oligonucleotide sub-sequences, such as a ligase chain reaction (LCA). Ligase enzymes ligate the two oligonucleotides or oligonucleotide sub-sequences when they hybridise at adjacent sites in the target polynucleotide. Alternatively, if the two oligonucleotides or hybridise at sites that are one or more nucleic acid residues apart, i.e. they are not adjacent, then the single stranded region between the double stranded regions is converted to a double stranded region by using a polymerase, and the ligase enzyme then links the adjacent oligonucleotides to form a continuous double stranded region. Thus, different infections agents and MBL genotypes may be distinguished.

[0153] The selection of oligonucleotides and sets of oligonucleotides as probes and primers is routine in the art. Methods and algorithms for identifying sequences are known to those with general knowledge in the area of bioinformatics and molecular systematics and include sequence alignment tools, dot matrix methods and database searching methods such as the Basic Local Alignment Sequence Tool (BLAST) and Flexible Sequence Similarity Searching (FASTA) programs. The binding properties of oligonucleotides, including degenerate oligonucleotides are also routinely identified using algorithms which are widely used and available (see for example, Jarman, Bioinformatics, 20(10): 1644- 1645, 2004).

[0154] Nucleic acid based tests are conveniently performed using an oligonucleotide array which is a substrate having oligonucleotides with different known sequences deposited at discrete known locations associated with its surface. For example, the substrate can be in the form of a two dimensional substrate as described in U.S. Patent No. 5,424,186. Such substrate may be used to synthesise two-dimensional spatially addressed oligonucleotide (matrix) arrays. Alternatively, the substrate may be tubular or in the form of a microsphere or bead connected to the surface of an optic fibre as, for example, disclosed by Chee et al. in WO 00/39587. Oligonucleotide arrays have at least two different features and a density of at least 400 features per cm2. In certain embodiments, the arrays can have a density of about 500, at least one thousand, at least 10 thousand, at least 100 thousand, at least one million or at least 10 million features per cm2. For example, the substrate may be silicon or glass and can have the thickness of a glass microscope slide or a glass cover slip, or may be composed of other synthetic polymers. Substrates that are transparent to light are useful when the method of performing an assay on the substrate involves optical detection.

[0155] In some embodiments, and to facilitate detection and analysis, oligonucleotides are a detectably and/or attachably modified. Any detectable or attachable modification system may be employed such as those reviewed in Syvanen Anne-Christine, Nature Genetics, 2:930-940, 2001; or as described in MOLECULAR CLONING. A LABORATORY MANUAL (supra) (Appendix 9-

Detection systems, Appendix 10- DNA array technology). For example, biotin or digoxigenin labelled oligonucleotides may be attached to anti-biotin or anti-digoxigenin antibodies, respectively. Detection may be via conjugated enzyme systems such as horseradish peroxidase or alkaline phosphatase to generate a colour change reaction.

[0156] The detectable modification is conveniently selected from: a non target polynucleotide nucleic acid sequence, a chromogen, a catalyst, an enzyme, a dye such as an infrared dye, flurochrome, a chemiluminescent, bioluminescent or phosphorescent moiety, a lanthanide ion, a radioisotope or a visual label such as gold or silver nanoparticles. Fluorescent dyes are particularly well established however, this is a rapidly moving field and the present invention is in no way limited to the use of any particular detectable modification. In some embodiments distinguishable fluorophores, dyes or particles are used to facilitate combinatorial analyses.

[0157] Detectable molecules that are only detectable when the oligonucleotide hybridises to a target sequence are contemplated. An example of such a detectably modified oligonucleotide is a molecular beacon. Molecular beacons comprise a fluorescent label which is quenched by association with a quenching molecule when the probe is unhybridized and separated from the quencher and therefore active when the probe is hybridised. These oligonucleotides are particularly useful a probes as unhybridized probe does not need to be removed in order to detect a signal.

[0158] In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Especially preferred labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In one embodiment in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide. Hybridisation is detected by incubation with streptavidin-reporter molecules.

[0159] Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially available fluorescent labels include, for example, fluorescein phosphoramidites such as Fluoreprime (Pharmacia), Fluoredite (Millipore) and FAM (Applied Biosystems International). Radioactive reporter molecules include, for example, 32P, which can be detected by X-ray or phosphoimager techniques.

[0160] The attachable modification is selected to conveniently immobilise or otherwise attach an oligonucleotide to a substrate such as, for example, an array comprising a membrane, wafer, pin, chip, microparticle, nanoparticle such as microspheres or any other bead like particle. The modification may permit direct or indirect binding or immobilisation to the substrate. In other embodiments, the oligonucleotide may be modified to facilitate amplification. In one illustrative embodiment, the oligonucleotide sequence comprises RNA and further comprises a substrate for amplification with bacteriophage Φβ replicase such as Marek's disease virus type 1 (MDV-I) RNA (Tyagi et al, Proc. Natl. Acad. Sci. USA, 93:5395-5400, 1996). In another embodiment, another substrate incorporated into an oligonucleotide is the substrate for DNA synthesis by phage Φ29 DNA polymerase. The presence of linker sequences between primer sequences facilitate circularisation of oligonucleotides after ligation of the primer pair sequences. Thus, the oligonucleotide may form padlock oligonucleotide stably catenated to a target sequence. Circularised padlock probes may be amplified by rolling circle amplification (RCA) with Φ29 DNA polymerase (Baner et al., Nucleic Acids Research, 26(22):5073-5078, 1996). This en∑yme has a high strand displacement activity which can release the circularised oligonucleotides from the target sequences which will fall off the target sequences if a nearby free end is introduced into the target sequence. Alternatively, amplification may be achieved by two-primer hyperbranched-RCA with Vent DNA polymerase.

[0161] Optionally, the methods may further comprise characterising the genotype or proteome of infectious agents or characterise or quantifying antibodies or immune cells that are immuno-interactive with those agents in a sample from the subject in order to diagnose the infectious organism. In still further other embodiments, the protocols comprise; (d) testing for MBL binding to the infectious organism diagnosed in c). In other embodiments, the methods comprise (e) assessing the genotype or allelic form or forms of MBL in the subject.

[0162] In other embodiments, the subject protocol includes testing an isolated infectious organism for its ability to bind to MBL in binding assays known to those of skill in the art.

EXPERIMENTAL

METHODS

PATIENTS AND METHODS

[0163] The Royal Brisbane and Women's Hospital is a 750-bed tertiary referral centre serving the northern region of Brisbane, Queensland, Australia. Over the period 19/12/2001 to

27/6/2003, sequential patients with proven sepsis due to blood stream infection were approached for consent for involvement in the study. A smaller number of patients with pneumonia were also included. Only patients with definitive evidence of infection were screened for enrolment into the study. Patient diagnoses that were included in the study were; blood stream infection (at least one isolate of a pathogenic bacteria or yeast from blood cultures), community-acquired pneumonia (lobar or bronchopneumonic infiltrate on chest X-ray [CXR] in patient with neutrophil leukocytosis) or bacterial meningitis (polymorph pleocytosis in cerebrospinal fluid with positive culture). Patients with skin contaminants grown in blood culture were excluded. Results from patients with admission diagnoses of pneumonia were also reviewed and the clinical investigators verified the presence of CXR changes. MBL parameters had been previously measured in 236 Queensland blood donors who were used as healthy controls to define a normal population (Minchinton et al., Scand. J. Immunol., 40(l):37-44, 1994). The Royal Brisbane Hospital Human Research Ethics Committee and the Queensland Guardianship Administration Tribunal gave approval for the hospital study.

[0164] Information on blood stream isolates was collected, in some instances multiple pathogenic isolates were present in the same specimen. In patients with severe community acquired pneumonia, respiratory pathogens grown from blood cultures were identified as the cause of the pneumonic illness. Clinical data was acquired by investigators blinded to MBL results. The clinical investigator, who reviewed microbiological results for all possible patients, was blinded to all MBL genotyping and phenotyping results. Demographic information was collected on all patients. Apache II scores (Knaus et al., Crit. Care Med., 13(10):818-829, 1985) were calculated on all case patients in the first 24 hours of their presentation with sepsis. Sequential organ failure assessment (SOFA) scores (Moreno et al, 1999 (supra)), were calculated over the first week of illness. Survival was measured at 28 days post presentation of sepsis.

FUNCTIONAL COMPLEMENT (C4B) DEPOSIΉON ASSAY. [0165] This assay demonstrates deposition of C4b following activation of MBL by binding with solid-phase purified mannan. This method was originally described for detection of C3b and C3bi (Super et al, Lancet., 2(8674): 1236-1239, 1989) and modified for C4b deposition (Valdimarsson et al., Scand. J. Immunol., 48(2):116-123, 1998). Using serum dilutions of 1:20 or higher, the C4 deposition assay provides a highly significant correlation between deposition of C4 and C3bi and MBL levels determined in a mannan-binding ELISA. There is no activation of either the classical or alternative complement pathways using solid-phase coupled mannan and no correlation between MBL levels and anti-mannan antibodies in blood donor samples (Super et al, Clinical and Experimental Immunology, 79:144-150, 1990). MBL deficient serum is added to enable complement activation. Details of this method were also previously described (Minchinton et al, 2002(supra)). One μl of MBL standard (AntibodyShop) was arbitrarily assigned one unit of C4 deposition activity. Between run CV for the assay at 1 :25 was 9.4%.

MANNAN-BINDING ASSAY

[0166] MBL level was quantified in μg/mL for each sample using a mannan-binding assay based on the method of Ho lmskov (Ho Imskov et al, Glycobiology, 3(2): 147-153, 1993) except that biotinylated HYB 131-01 monoclonal anti-MBL (AntibodyShop, Grusbakken 8, DK-2820 Gentofte, Denmark) was used to tag bound MBL, This method was previously described in detail (Minchinton et al, 2002 (supra)). Between run coefficient of variation (CV) was 6.1% at 1:25 and 8.8% at 1:100.

MBL GENOTYPING [0167] DNA was extracted from buffy coats using the QIAamp DNA kit (QIAGEN,

Clifton Hill, Australia). Five polymorphisms in the MBL2 promoter (-550 WL, -221 XIY) and the first exon (codons 52, 54 and 57) were genotyped using the polymerase chain reaction and sequence- specific primers as previously described (Minchinton et al, 2002 (supra); Mullighan et al, Scand. J. Immunol, 51(2):111-122, 2000).

RESULTS

PATIENTS

[0168] A total of 195 patients with documented blood stream infection or community- acquired pneumonia were enrolled in the study as cases. These consisted of 166 patients with documented blood stream infection, 35 with pneumonia and 12 with bacterial meningitis. Seven of the pneumonia patients and 11 of the 12 patients with meningitis were bacteremic. The median age was 61 (range 16-95 years), 110 patients were male and 85 female. Case patients with mean arterial pressures of <70mm Hg were regarded as having septic shock. Using this definition, 114 patients had septic shock and 81 had sepsis alone. Two hundred and thirty-six healthy blood donor controls had previously had their MBL status documented (Minchinton et al, 2002 (supra)) and these acted as healthy controls. The median age of this group was 45 (18-70) and the male to female ratio 1.11:1. MBL DEFICIENCY IN CASE AND CONTROL GROUPS

[0169] As shown in Table 2, C4 deposition was significantly lower in case subjects with blood stream infection and pneumonia than in healthy controls. C4 deposition was closely correlated with MBL levels in the cases studied herein (Pearson's coefficient R=O.673, p<0.001). While there was a trend to lower MBL levels in case subjects these were not significantly lower than in controls.

INFLUENCE OF MBL LEVEL ON SEPTIC SHOCK, SOFA SCORES AND ASSOCIATIONS WITH

SURVIVAL

[0170] As shown in Table 3, MBL deficient patients with septic shock had significantly lower C4 deposition than those who were not shocked. MBL levels also trended lower in these MBL deficient, septic shock cases. Amongst MBL sufficient patients, no similar differences were seen in MBL function or levels between the shocked and normotensive septic groups. Clinical indices and outcomes were compared in MBL deficient and sufficient sepsis patients. MBL deficiency was significantly associated with higher SOFA scores at Day 3 with non-significant trends present to higher Apache II and Day 0 and 5 SOFA scores. The median day 3 SOFA scores in non-survivors was 5 (compared with 2 in survivors p=0.03). MBL function was significantly different in patients with SOFA scores above and below 5 at day 3 (0.13 vs. 0.21 U/μl; p<0.02). The original SOFA score validation study showed that the mean SOFA score was 8 in those who died and 4 in survivors (Moreno et al, 1999 {supra)). Using these breakpoints to analyse MBL activity in the patients it was clear that there were significant differences in both MBL level and function in those with SOFA scores predictive of outcome in critical illness. For example, at day 3, MBL levels (1.37 vs. 0.51 μg/mL, p<0.01) and function (0.22 vs. 0.01 U/μl, p<0.001) were higher in patients with SOFA scores of <8 compared with those with SOFA scores of 8+. Patients with day 3 SOFA scores of 4 or less had higher MBL function (0.21 vs. 0.13 U/μl, p<0.02) and there was a trend to higher MBL levels (1.40 vs. 0.85 μg/mL, p=0.07) than in those with SOFA scores of >4. Multivariate analysis showed that MBL function remained a significant predictor of day 3 SOFA scores <8 (p<0.02) when age and white cell counts were taken into account.

NEGATIVE CORRELATION BETWEEN HIGHER MBL LEVEL AND LOWER SOFA SCORE INDICATIVE OF RESISTANCE TO INFECTION

[0171] Higher MBL levels were associated with resistance to infection. When MBL levels in patients were plotted against SOFA scores on days 0 to 5 of the study a negative correlation (range - 0.104 to -0.194) between MBL levels and SOFA score was observed. This was statistically significant on Day 1 (p=0.028) and Day 3 (p=0.011). In an illustrative embodiment, the data for Day 3 are presented graphically in Figure 1. DISCUSSION

[0172] This is the first study of the influence of MBL level on the development of sepsis to include patients purely on the basis of severe, documented infection and to assess MBL functional activity functional as well as quantitative level. In this regard, the association between MBL functional deficit and severe infection susceptibility appears to be reliably estimated. Patients with blood stream infection and pneumonia showed markedly reduced ability to activate complement through the MBL pathway compared with healthy controls. Among MBL deficient patients, lower levels of C4 function were present in patients with septic shock. MBL deficient patients also had more severe shock as shown by higher SOFA scores at Day 3 with Apache II and SOFA Day 0 and 5 also trending higher. There was no higher than expected carriage of MBL variant structural alleles or promoter haplotypes in the sepsis patients studied. This reflects the wide range of MBL activity within AJO patients (Minchinton et al, 2002 {supra)) and the small number of O/O patients in the study.

[0173] The finding of significantly reduced MBL function in patients with sepsis does not appear to be due to the impact of the inflammatory cascade in this group (Dean et al, Journal of Clinical Immunology, 4:346-352, 2005).

[0174] There was no consistent acute phase response, either positive or negative, in MBL activity. Patients with low MBL levels or MBL induced C4 deposition mostly maintained those levels. Patients with MBL2 variant alleles who were MBL deficient at the onset of their sepsis were unable to increase either their MBL level or function into the normal range. This lack of normalisation of MBL levels in A/O patients under infective stress has previously been reported in paediatric oncology patients (Neth et al, Lancet, 358(9282):614-618, 1999). A recent report from a mouse model shows that levels of both MBL and C4 cleavage were significantly decreased after development of sepsis (Windbichler et al, Infect. Immun., 72(9):5247-5252, 2004). Complement components are added to the C4 deposition assay used here, ruling out sepsis mediated complement consumption. Lower C4 deposition was seen in MBL deficient patients with septic shock compared with those with sepsis and no shock. This same relationship was not seen among MBL sufficient septic patients further suggesting the C4 deficiency is predisposing to and not merely reflecting shock.

[0175] The inclusion criteria of proven, mostly bloodstream, infection, has allowed a direct observation of the association between MBL deficiency and sepsis due to different bacteria. S. pneumoniae sepsis was strongly associated with MBL deficiency withJower C4 levels than other patients with all bacteraemias, gram positive bacteraemia and non-bacteraemic pneumonia (the majority of which will be pneumococcal). E. coli sepsis was also associated with MBL functional deficiency.

[0176] Other clinical studies support the association between MBL deficiency and systemic inflammatory response syndrome (SIRS). Amongst Danish patients with SIRS, MBL variant alleles were found more frequently in those with documented sepsis (Garred et al, J. Infect. Dis., 188(9):1394-1403, 2003). On the basis of predicted MBL levels associated with MBL2 genotypes it appeared that patients with the lowest MBL levels were most likely to develop septic shock with a fatal outcome (Garred et al, 2003 {supra)). Patients who were homozygous for MBL variant alleles were shown to be predisposed to invasive pneumococcal infection in a case controlled study of Caucasian patients (Roy et al, 2002 {supra)). Neither of these studies directly correlated MBL level or functional assays with the risk of sepsis. In patients with organ failure in a paediatric intensive care unit, MBL deficiency, as indicated by carriage of variant MBL2 alleles or low MBL levels, was significantly associated with the development of SIRS. This applied whether SIRS arose as a result of infection or non-infective causes. The intensity of the inflammatory response to infection was also associated with MBL deficiency. Increasing proportions of patients with localised infection, sepsis and septic shock had MBL deficiency (Fidler et al, Intensive Care Med., 30(7):1438-1445, 2004). MBL levels were also studied in a group of ICU patients randomised to treatment with intensive or conventional insulin regimens. While admission MBL levels were normal overall, low MBL levels were associated with death in those patients conventionally treated with insulin. Multivariate analysis showed that the insulin regimen, (Hansen et al, J. Clin. Endocrinol. Metab., 88(3): 1082-1088, 2003) but not baseline MBL levels, predicted survival.

[0177] MBL appears to be an important modulator of the inflammatory cascade. Proinflammatory cytokines are needed for an effective immune response to infectious disease. MBL has been shown enhance production of tumor necrosis factor (Huffnagle et al, J. Neurovirol., 5(1):76- 81, 1999) by mononuclear cells bound to micro-organisms, preventing disseminated infection. Septic shock, however, results from dysregulated production of TNF. In an animal model of MBL deficiency, MBL-null mice had reduced early levels of proinflammatory cytokines in S. aureus sepsis but higher levels than control wild-type mice after 24 hours (Shi era/., J. Exp. Med., 199(10):1379-1390, 2004). Production of TNF and IL-6 by monocytes incubated with N. meningitidis was decreased by the presence of MBL in a dose dependent fashion (Jack et al., The Journal of Infectious Disease,

184:1152-1162, 2001). Gram-negative lipopolysaccharide containing mannose heteropolymers has the potential for causing the most profound shock in experimental animals. MBL binds most avidly to mannose heteropolymer containing LPS (Zhao et al, Blood, 100(9):3233-3239, 2002).

[0178] Both plasma derived (Laursen et al, 2003 {supra)) and recombinant MBL (Vorup- Jensen et al, 2001 {supra)) are being prepared for clinical trials. Elective MBL therapy could be used for bone marrow transplantation patients. Hypersupplementation of MBL levels will also be beneficial as MBL does not demonstrate predictable positive acute phase activity and as shown herein, high MBL levels predict low SOFA scores indicative of better outcomes in critical illness due to sepsis. A diagnostic kit that identifies MBL level and/or function could usefully guide MBL therapy. A kit measuring MBL level and/or function in subjects undergoing MBL therapy is also expressly contemplated. [0179] This study is the first to study patients selected for documented infection, mostly of the bloodstream, to show an association between MBL functional deficiency and sepsis. Of all the innate immune effector molecules, MBL is potentially best suited to study in the therapy of infectious diseases. Deficiency states are common and appear to predispose to a wide variety of severe bacterial and fungal infections causing sepsis. MBL binds to a wide variety of pathogens promoting their killing. Infusions of MBL are safe and have a reasonably long half-life (Valdimarsson et ah, Scand. J. Immunol., 59(l):97-102, 2004). Physicians managing infectious diseases are faced with major challenges from increasingly resistant pathogens such as penicillin resistant pneumococci and multiresistant gram negatives such as E. coli. A new immune based therapy would not be expected to select for antimicrobial resistant organisms and would be a welcome addition in this context.

[0180] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0181] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [0182] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

TABLE 1

SUMMARY OF SEQUENCE IDENTIFIERS

TABLE 2

MBL MEASUREMENTS IN SEPTIC PATIENTS AND CONTROL GROUPS

differs from healthy controls p<0.001

TABLE 3

MBL ACTIVITY AND CLINICAL OUTCOMES IN CASE PATIENTS

TABLE 4

MEAN MBL LEVELS IN HEALTHY AUSTRALIAN BLOOD DONORS

BIBLIOGRAPHY

Aittoniemi et al, Acta Pathologica, Microbiologica, et Immunologica Scandinavica, 105:617-622 1997;

Aittoniemi et al, Clinical and Experimental Immunology, 116:505-508, 1999;

Angus et al, Crit. Care Med., 29(7):1303-1310, 2001;

Ausubel et al, Curr. Pro. in MoI. Bio., John Wiley & Sons, Inc, 1995;

Baner et al, Nucleic Acids Research, 26(22):5073-5078, 1996;

Brown et al, Methods Enzymol., 68:109, 1979;

Christensen et al, Biochem. J., 354:481-484, 2001;

Crosdale et al , European Journal of Immuno genetics, 27: 111-117, 2000.

Dean et al, Journal of Clinical Immunology, 4:346-352, 2005

Dieffenbach et al, PCR Primer: A Laboratory Manual, 1995;

Eisen et al, Clin. Infect. Dis., 37(11): 1496-1505, 2003;

Eisen etal, Curr. Drug Targets, 5(l):89-105, 2004;

Fidler et al, Intensive Care Med., 30(7):1438-1445, 2004;

Finfer et al , Intensive Care Med., 30(4):589-596, 2004;

Garred et al, European Journal of Immunogenetics, 19:403-412, 1992;

Garred et al, J. Infect. Dis., 188(9):1394-1403, 2003;

Hansen et al, J. Clin. Endocrinol. Metab., 88(3):1082-1088, 2003;

Harlow and Lane, Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;

Hibberd et al, Lancet, 353(9158):1049-1053, 1999;

Eolmskov et al, Glycobiology, 3(2):147-153, 1993;

Huffnagle et al., J. Neurovirol., 5(1):76-81, 1999;

Jack et al, The Journal of Infectious Disease, 184:1152-1162, 2001; Jarman, Bioinformatics, 20(10):1644-1645, 2004; Knaus et al., Crit. Care Med., 13(10):818-829, 1985; Roller and Smithies, Proc. Natl. Acad. Sci. USA, 86:8932-8935, 1989; Lanrsen et al, Biochem. Soc. Trans., 31(Pt 4):758-762, 2003; Lipscombe et al., Hum. MoI. Genet., l(9):709-715, 1992; Madsen et al, Immunogenetics, 40:37-44, 1994; Madsen e^ β/., The Journal of Immunology, 161:3169-3175, 1998; Minchinton etal, Scand. J. Immuno.l, 40(l):37-44, 1994; Minchinton et al., Scand. J. Immunol., 56(6):630— 641, 2002; Minchinton et al, Scand. J. Immunol., 56(6):630-641, 2002; Moreno et al, Intensive Care Med., 25(7):686-696, 1999; Mullighan etal, Scand. J. Immunol., 51(2): 111-122, 2000; Narang et al, Methods Enzymol, 68:90, 1979; Neth et al, Lancet, 358(9282):614-618, 1999; Oberholzer et al, Shock, 16(2);83-96, 2001; Padkin et al, Crit. Care Med., 31(9):2332-2338, 2003; Roberge et al, Science, 269:202, 1995; Rooijakkers et al, Nature Immunology, 6(9):920-927, 2005; Roy et al, Lancet, 359(9317):1569-1573, 2002;

Sambrook et al, Molecular Cloning: A Laboratoy Manual, Cold Spring Harbour Press, 1989; Steffensen et al, Journal of Immunological Methods, 241:33-42, 2000; Super et al, Lancet., 2(8674):1236-1239, 1989; Super et al, Clinical and Experimental Immunology, 79:144-150, 1990; Sumiya et al, Lancet, 337(8757): 1569-1570, 1991;

Syv&ien Anne-Christine, Nature Genetics, 2:930-940, 2001; Terai et al. , Biochemical Medicine and Metabolic Biology, 50:111-119, 1993; Tyagi et al, Proc. Natl. Acad. Sci. USA, 93:5395-5400, 1996; Valdimarsson et al, Scand. J. Immunol., 48(2): 116-123, 1998; Valdimarsson et al, Scand. J. Immunol,, 59(1): 97- 102, 2004; Vorup-Jensen et al, Int. Immunopharmacol., l(4):677-687, 2001; Windbichler et al, Infect. Immun., 72(9):5247-5252, 2004; Zhao etal, Blood, 100(9):3233-3239, 2002.

Claims

WHAT IS CLAIMED IS:

1. A method for treating or preventing an infection in a subject, comprising administering to the subject an effective amount of an MBL modulator selected from an MBL polypeptide or an agent from which an MBL polypeptide is producible, wherein the subject is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment.

2. A method according to claim 1, wherein the administration achieves a hypersupplemented level of MBL polypeptide in the subject.

3. A method according to claim 2, wherein the hypersupplemented level represents an at least about 10% increase above the subject's normal MBL level.

4. A method according to claim 3, wherein the normal level is from about 0.5 to about 5 μg/mL in serum.

5. A method according to claim 2, wherein the hypersupplemented level is at least about 1 to about 15 μg/mL in serum.

6. A method according to claim 1, wherein the infection is an infection caused by a microbe.

7. A method according to claim 6, wherein the microbe is a bacterium.

8. A method according to claim 6, wherein the microbe is a fungus.

9. A method according to claim 6, wherein the microbe is a yeast.

10. A method according to claim 6, wherein the microbe is a virus.

11. A method according to claim 6, wherein the microbe is an algae.

12. A method according to claim 6, wherein the microbe is a parasite.

13. A method according to claim 6, wherein the microbe is a helminth.

14. A method according to claim 6, wherein the microbe is a nematode.

15. A method according to claim 6, wherein the microbe is a mycoplasma.

16. A method according to claim 6, wherein the microbe is resistant to at least one anti- infective agent.

17. A method according to claim 6, wherein the microbe is pathogenic.

18. A method according to claim 1, wherein the MBL modulator is an MBL polypeptide.

19. A method according to claim 18, wherein the MBL polypeptide is produced in a native host organism.

20. A method according to claim 19, wherein the native host organism is a human or human cell natively expressing the MBL polypeptide.

21. A method according to claim 18, wherein the MBL polypeptide is produced by a host organism not natively expressing an MBL polypeptide.

22. A method according to claim 18, wherein the MBL polypeptide is a recombinant polypeptide.

23. A method according to claim 18, wherein the MBL polypeptide is in oligomeric form.

24. A method according to claim 23, wherein the MBL oligomers have a size distribution substantially identical to the size distribution of MBL in serum.

25. A metho'd according to claim 18, wherein the MBL polypeptide is isolated by a method comprising at least one step involving affinity chromatography.

26. A method according to claim 25, wherein the affinity chromatography step isolates MBL tetramers, pentamers, hexamers or combinations thereof.

27. A method according to claim 18, wherein the MBL polypeptide is isolated from plasma.

28. A method according to claim 1, wherein the MBL modulator is a cell that produces and secretes an MBL polypeptide.

29. A method according to claim 28, wherein the cell is selected from liver cells, monocytes and macrophages.

30. A method according to claim 1, wherein the subject has existing infection.

31. A method according to claim 1 , wherein the subject is at risk of exposure to an infectious agent.

32. A method according to claim 31 , wherein the subject is at risk of exposure to infected individuals.

33. A method according to claim 31 , wherein the subject is at risk of exposure to one or more biological warfare agents.

34. A method according to claim 30 or claim 31, wherein the subject has or is at risk of exposure to a burn.

35. A method according to claim 30 or claim 31 , wherein the subject has or is at risk of exposure to a wound.

36. A method according to claim 30 or claim 31, wherein the subject is undergoing, has undergone or is at risk of undergoing surgery.

37. A method according to any one of claims 31 to 33, wherein the subject is selected from doctors, nurses and soldiers as well as members of general population who are at risk of exposure to the infectious agent.

38. A method according to claim 34, wherein the subject is selected from firemen, kitchen personnel or other individuals exposed or at risk of exposure to burn-causing agents or environments.

39. A method according to claim 35, wherein the subject is selected from soldiers policemen, security personnel or any other individual exposed or at risk of exposure to weapons assaults.

40. A method according to claim 36, wherein the subject is selected from an individual that is undergoing, has undergone or is at risk of undergoing a surgery selected from peritoneal, pericardial, obstetric, gynecological, neurosurgical, arthroscopic, orthopedic, plastic, reconstructive, muscle, or tendon surgery.

41. A method according to claim 31, wherein the subject has been exposed to an infectious agent but is not exhibiting any symptoms of infection.

42. A method according to claim 31, wherein the subject has been exposed to a pathogenic organism and is exhibiting one or more symptoms of infection.

43. A method according to claim I₅ further comprising co-administering at least one additional agent that modulates or modifies the immune response or activity of MBL in the subject.

44. A method according to claim 43, wherein the modification of the immune response comprises enhancing an activity in the subject, wherein the activity is selected from phagocytosis complement activation, opsonisation, protein-, glycoprotein- or glycolipid-binding and receptor binding.

45. A method according to claim 43, wherein the at least one additional agent is selected from one or more of a hormone, cytokine, lymphokine, haematopoietic factor, chemokine, antibody or part thereof, co-stimulatory molecule and biological response modifier.

46. A method according to claim 43, wherein the additional agent is IL-2.

47. A method according to claim 1, further comprising co-administering at least one anti-infective agent that is effective against the infection, wherein the anti-infective is selected from antimicrobials, antibiotics, antivirals, antifungals, anthelmintics, antiprotozoals and nematocides.

48. A method according to claim 1, further comprising testing the subject to determine the level of MBL in the subject prior or subsequent to administration of the MBL modulator.

49. A method according to claim 48, wherein the subject is tested by determining the MBL level and/or activity in the subject.

50. A method according to claim 49, wherein serum or plasma levels of MBL in the subject are determined by quantitative analysis.

51. A method according to claim 50, wherein the analysis comprises at least one of en2yme linked immunosorbent assay (ELISA), time-resolved immunofluorescent assay (TRIFMA), radioimmunoassay (RIA) or nephelometry.

52. A method according to claim 1, further comprising characterizing the genotype or proteome of an infectious organism or antibodies or immune cells that are immuno-interactive with the organism in a sample in order to diagnose the infection.

53. A method according to claim 52, further comprising testing for MBL binding to the diagnosed infectious organism.

54. Use of an MBL modulator in the manufacture of a medicament for treating or preventing an infection in a subject that is not immunocompromised or at risk of acquiring an immunocompromised condition resulting from a medical treatment.

55. A use according to claim 54, wherein the MBL modulator is in the form of a kit.

56. A use according to claim 55, wherein the kit comprises an MBL polypeptide and optionally at least one MASP in a form suitable for storage and/or for local or systemic administration.

57. A use according to claim 56, wherein the kit comprises reagents for determining the level or activity of MBL polypeptide in the subject.