Therapeutic and prophylactic agents and related compositions and methods

Abstract

The invention relates to methods of immunising a subject and / or of eliciting an immune response in a subject comprising administering one or more complexes comprising lipopolysaccharide and immunoglobulin, and methods of preventing or treating gram-negative bacterial infection and / or a disease or condition caused by or associated with a gram-negative bacterial infection. Complexes and compositions comprising such complexes, and the use of such complexes and compositions in the preparation of pharmaceutical compositions and medicaments are also disclosed.

Description

[0001] THERAPEUTIC AND PROPHYLACTIC AGENTS AND RELATED COMPOSITIONS AND METHODS

[0002] TECHNICAL FIELD

[0003] The invention relates to complexes comprising lipopolysaccharide and immunoglobulin, and methods of preventing or treating a gram-negative bacterial infection and / or a disease or condition caused by or associated with a gram-negative bacterial infection in a subject reliant on such complexes. Compositions comprising such complexes for use in such methods are also provided. The invention further relates to methods for and compositions for use in eliciting an immune response in a subject and / or in immunising a subject, for example to prevent or treat a gram-negative bacterial infection or a disease or condition caused by or associated with a gram-negative bacterial infection.

[0004] BACKGROUND OF THE INVENTION

[0005] The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field or in any particular jurisdiction.

[0006] It is well recognised that pathogenic microorganisms are causative of and / or associated with a wide variety of diseases and conditions and have a very substantial impact on both health and wellbeing, and on economic activities. Gram-negative bacteria are causative of or associated with a number of important infections, each of which is frequently exacerbated by the release of endotoxins from the bacterial membrane. Pneumonia, peritonitis, urinary tract infections, sepsis and bloodstream infections, persistent infections of wounds or surgical sites, and meningitis, are just some of the serious infections caused by gram-negative bacteria, such as but not limited to Escherichia coli, Serratia marcescens, Enterobacter cloacae, Salmonella spp., Pseudomonas aeruginosa, Proteus vulgaris and Klebsiella pneumoniae.

[0007] The availability of prophylactic treatment and / or rapid and effective treatment of infections of bacterial pathogens is of particular importance with the increasing prevalence of multidrug resistance in bacterial pathogens of humans, and of other animal species. Multidrug-resistant gram-negative bacteria, such as various Enterobacteriaceae, are particularly problematic, as there is a paucity of effective treatment agents for these organisms.

[0008] A representative example of a condition primarily caused by bacterial infection is mastitis, an inflammation of the mammary gland typically associated with intramammary infection. While mastitis is problematic in a number of mammalian species, including humans, the diagnosis and treatment of mastitis is of significant importance to the dairy industry. Indeed, mastitis is the costliest disease in dairy cattle. In large part, this cost is associated with the need to discard milk from cows undergoing treatment for mastitis for which a withholding period in which milk cannot be collected for consumption pertains, together with reduced milk production, including amongst cows with asymptomatic or subclinical mastitis. The cost of veterinary care of infected cows, and labour costs, are significant. Animal wellbeing is also a serious consideration. Important gram-negative bacterial pathogens associated with mastitis in cows include coliform bacteria such as E. coli, Enterobacter spp., Klebsiella spp., Serratia spp. and Citrobacter. Acute bovine mastitis, like a number of diseases or conditions associated with gram-negative bacterial infections, is caused by infection with the bacteria, but is in fact initiated by lipopolysaccharide (LPS) shed from the bacterial cell wall, and the resulting inflammatory cascade and LPS-mediated endotoxicity. Accordingly, current treatments for mastitis and other gram-negative bacterial infections must be cognisant of this risk of LPS-mediated endotoxicity associated with such infections, which can be initiated or exacerbated by bacteriocidal treatments such as some antibiotics. Here, there is a risk that any bacteria killed by the bacteriocidal agent will release still more LPS and thereby exacerbating endotoxicity.

[0009] Accordingly, the availability of robust and effective treatments, and particularly prophylactic treatments, for infections of pathogenic gram-negative bacteria (including those which reduce or avoid endotoxicity or which go at least some way to reducing or avoiding exacerbating endotoxicity) is of critical importance. There is a need to develop new and improved methods for preventing or treating gram-negative bacterial infections and / or the diseases or conditions they cause or with which they are associated.

[0010] The present invention broadly relates to methods of preventing or treating a gram-negative bacterial infection and / or a disease or condition caused by or associated with a gram-negative bacterial infection, and / or one or more methods of immunising a subject or eliciting an immune response in a subject, and / or one or more complexes, compositions, and agents useful in such methods.

[0011] SUMMARY OF THE INVENTION

[0012] In a first aspect, the invention relates to a method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is bound specifically to said lipopolysaccharide.

[0013] In another aspect, the invention relates to a method of immunising a subject in need thereof, the method comprising administering to the subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is bound specifically to said lipopolysaccharide.

[0014] In various examples, said immunoglobulin inhibits one or more biological activities of or associated with said lipopolysaccharide.

[0015] In one example, the complex is a complex as herein disclosed or is present in a composition as herein contemplated.

[0016] In various examples, the immune response is to said immunoglobulin and / or said lipopolysaccharide. For example, the immune response is to said lipopolysaccharide.

[0017] In one example, the immune response is induction of an antibody response.

[0018] In one example, the immune response is induction of a mucosal IgA response.

[0019] In one example, the immunisation is against the organism(s) from which said lipopolysaccharide is derived.

[0020] In one example, said immunoglobulin and / or said lipopolysaccharide comprises one or more exogenous antigens.

[0021] In one example, the complex comprises one or more antigens other than said immunoglobulin and said lipopolysaccharide.

[0022] In one example, the immune response is to said one or more exogenous antigens. In one example, the immunisation is against the organism(s) from which said one or more exogenous antigens is derived.

[0023] In one example, the immunoglobulin comprises, consists essentially of, or consists of immunoglobulin in or from colostrum.

[0024] In one example, the complex comprises two or more immunoglobulin molecules.

[0025] In one example, the complex comprises two or more different types of immunoglobulin.

[0026] In one example, the two or more different types of immunoglobulin are two or more classes or subclasses of immunoglobulin.

[0027] In one example, two or more of the immunoglobulins have differing binding affinity and / or differing binding specificity.

[0028] In various examples, said immunoglobulin comprises, consists essentially of, or consists of species-specific immunoglobulin. For example, said immunoglobulin comprises, consists essentially of, or consists of immunoglobulin from the species to which the complex is to be administered, or immunoglobulin modified so as to be recognised as 'self'. For example, when the subject is a human, in certain particularly contemplated examples the immunoglobulin comprises, consists essentially of, or consists of human immunoglobulin or humanised immunoglobulin.

[0029] In one example, the invention relates to a method of immunising a newborn subject, the method comprising administering to the newborn subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is bound specifically to said lipopolysaccharide.

[0030] In one example, the newborn subject is a mammalian subject 24 hours old or less.

[0031] In another aspect, the invention relates to a method of immunising a subject in need thereof or of eliciting an immune response in a subject, the method comprising administering to the subject one or more lipopolysaccharide-specific immunoglobulins together with lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

[0032] In one example, the administration of the one or more lipopolysaccharide-specific immunoglobulins is separate to the administraton of the lipopolysaccharide. For example, the administration of the one or more lipopolysaccharide-specific immunoglobulins preceeds the administration of the lipopolysaccharide.

[0033] In one example, the method comprises administering the one or more lipopolysaccharide-specific immunoglobulins in an amount sufficient to bind all of the lipopolysaccharide to be administered.

[0034] In one example, the method comprises administering a molar excess of lipopolysaccharidespecific immunoglobulin relative to lipopolysaccharide to be administered.

[0035] In one example, the subject to which the complex, composition, or vaccine as contemplated herein is administered is a newborn mammalian subject, such as a newborn ruminant mammalian subject.

[0036] In one example, the newborn mammalian subject is 24 hours or less old.

[0037] In one example, the newborn ruminant mammalian subject, for example a newborn bovine subject, is 24 hours or less old.

[0038] In one example, the newborn mammalian subject is capable of expressing one or more IgG- specific receptors in the gut mucosa.

[0039] In one example, the newborn mammalian subject comprises one or more gut epithelial cells expressing neonatal Fc receptor (FcRn). In one example, the subject to which the complex, composition, or vaccine as contemplated herein is administered is a mammalian subject with one or more gut epithelial cells expressing neonatal Fc receptor (FcRn).

[0040] In one example, the method comprises administering the complex, composition, or vaccine to a mammalian subject prior to downregulation of neonatal Fc receptor expression.

[0041] In one example, the method comprises administering the complex, composition, or vaccine to a mammalian subject prior to the mammalian subject undergoing gut closure.

[0042] In another aspect, the invention relates to a method of preventing or treating a gram-negative bacterial infection or a disease or condition caused by or associated with a gram-negative bacterial infection in a subject, the method comprising administering to the subject an effective amount of a complex as contemplated herein.

[0043] In one example, the method is a method of preventing or treating a disease or condition caused by or associated with a gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex as contemplated herein.

[0044] In another example, the method is a method of preventing or treating a gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex as contemplated herein.

[0045] In various examples, the complex is administered as a pharmaceutical composition as herein contemplated.

[0046] In one example, the prevention or treatment comprises prevention, treatment, or amelioration of one or more symptoms of or associated with bacterial infection with gram-negative bacteria.

[0047] In one example the effective amount is an amount effective to induce an immunological response to lipopolysaccharide. In one example, the effective amount is an amount effective to induce an immunological response to lipopolysaccharide, wherein administration of the effective amount does not induce one or more inflammatory responses, such as one or more inappropriate inflammatory responses.

[0048] In one example, the gram-negative bacteria is a bacteria selected from the group consisting of Escherichia spp., Pasteurella spp., Serratia spp., Klebsiella spp., Salmonella spp., and Campylobacter spp..

[0049] In one example, the gram negative bacteria is selected from the group consisting of E. albertii, E. coll, E. fergusonii, E. hermannii, E. ruysiae, and E. marmotae.

[0050] In one example, the gram negative bacteria is selected from the group consisting of P. aerogenes, P. anatis, P. avium, P. bettyae, P. caballi, P. canis, P. dagmatis, P. gallicida, P. gallinarum, P. granulomatis, P. langaaensis, P. lymphangitidis, P. mairii, P. multocida, P. oralis, P. pneumotropica, P. skyensis, P. stomatis, P. testudinis, P. trehalosi, P. ureae, and P. volantium.

[0051] In one example, the gram negative bacteria is selected from the group consisting of S. aquatilis, 5. entomophila, 5. ficaria, 5. fonticola, 5. glossinae, 5. grimesii, 5. liquefaciens, 5. marcescens, 5. myotis, 5. nematodiphila, 5. odorifera, 5. plymuthica, 5. proteamaculans, 5. quinivorans, 5. rubidaea, 5. symbiotica, 5. ureilytica, and S. vespertilionis.

[0052] In one example, the gram negative bacteria is selected from the group consisting of Klebsiella aerogenes, Klebsiella granulomatis, Klebsiella grimontii, Klebsiella huaxiensis, Klebsiella kielensis, Klebsiella michiganensis, Klebsiella milletis, Klebsiella oxytoca, Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella quasivariicola, Klebsiella senegalensis, Klebsiella steroids, and Klebsiella variicola.

[0053] In one example, the gram negative bacteria is selected from the group consisting of Salmonella bongori, Salmonella enterica, Salmonella enterica subsp. arizonae, Salmonella enterica subsp. diarizonae, Salmonella enterica subsp. enterica, Salmonella enterica subsp. houtenae, Salmonella enterica subsp. indica, and Salmonella enterica subsp. salamae.

[0054] In one example, the gram negative bacteria is a Camplyobacter species selected from the group consisting of C. canadensis, C. lari, C. volucris, C. armoricus, C. peloridis, C. novaezeelandiae, C. cuniculorum, C. avium, C. troglodytis, C. helveticus, C. upsaliensis, C. vulpis, C. insulaenigrae, C. bills, C. hepaticus, Campylobacter lari concheus, C. ornithocola, C. subantarcticus, C. jejuni, C. estrildidarum, C. aviculae, C. taeniopygiae, C. coll, Campylobacter hyointestinalis lawsonii, C. lanienae, C. magnus, C. iguaniorum, C. hyointestinalis, Campylobacter fetus testudinum, C. fetus, C. sputorum, C. gracilis, C. hominis, C. blaseri, C. geochelonis, C. portucalensis, C. corcagiensis, C. ureolyticus, C. rectus, C. massiliensis, C. showae, C. anatolicus, C. curvus, C. concisus, C. pinnipediorum, C. mucosalis, C. majalis, and C. suis.

[0055] In one example, the administration is of an amount effective to reduce endotoxicity associated with said bacterial infection.

[0056] In one example, the administration is of an amount effective to induce a mucosal IgA response.

[0057] In one example, the immunoglobulin comprises, consists essentially of, or consists of immunoglobulin from colostrum.

[0058] In one example, the immunoglobulin comprises, consists essentially of, or consists of IgG and / or IgA from colostrum.

[0059] In one example, the colostrum is bovine colostrum, including pooled bovine colostrum.

[0060] In one example, administration to the subject comprises parenteral administration.

[0061] In one example, administration to the subject comprises topical or oral administration, or administration to a mucosal tissue.

[0062] In one example, administration to the subject comprises instillation, for example intra-mammary instillation.

[0063] In one example, administration to the subject comprises administration ex vivo.

[0064] In one example, ex vivo administration comprises administration to a sample from the subject, followed by administration of at least some of the sample to the subject.

[0065] In another aspect, the invention relates to a method of preventing or treating mastitis in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex as contemplated herein.

[0066] In one example, the prevention or treatment comprises prevention, treatment, or amelioration of one or more symptoms of or associated with mastitis.

[0067] In a further aspect the invention relates to use of a complex as contemplated herein in the preparation of a medicament for eliciting an immune response in a subject.

[0068] In another aspect, the invention relates to use of a complex as contemplated herein in the preparation of a medicament for immunising a subject, for example immunising a subject against a gram-negative bacterial infection or against a disease or condition caused by or associated with a gramnegative bacterial infection. In a further aspect the invention relates to use of a complex as contemplated herein in the preparation of a medicament for use in preventing or treating a bacterial infection of gram-negative bacteria or of a disease or condition caused by or associated with a bacterial infection of gram-negative bacteria.

[0069] In one example, the medicament is a vaccine. In one example, the medicament is a vaccine formulated for oral administration, such as an oral vaccine.

[0070] In various examples, the method as described herein comprises administering to the subject an additional therapeutic agent.

[0071] In various examples, the additional therapeutic agent is an antibiotic selected from the group consisting of aminoglycosides, such as gentamicin, amikacin; beta-lactams; carbapenems, including imipenem, meropenem; cephalosporins, including cefotaxime, ceftazidime; chloramphenicols; fluorquinolones, such as ciprofloxacin, delafloxacin; fosfomycin; penicillins; polymyxins, such as colistin, polymyxin B; glycylcycline, such as tigecycline; sulphonamides, such as co-trimoxazole; tetracyclines, including doxycycline, eravacycline, minocycline, omadacycline; and ureidopenici Ilins, such as piperacillin; and any combination of two or more thereof, including combinations such as ceftolozane / tazobactam, ceftazidime / avibactam, meropenem / vaborbactam, and imipenem / cilastatin / relebactam.

[0072] In various examples, the disease or condition is selected from the group consisting of Brucellosis; Campylobacter infections; Cholera; Escherichia coli E. coli) infections; Haemophilus influenzae infections; Klebsiella infections; Proteus infections, Legionellosis, including Legionnaires' disease; Pertussis; Plague; Pseudomonas infections; Salmonella infections; sepsis, septic shock, Shigellosis; Tularemia; Porphyromonas gingivalis infections, Heligobacter infections and Typhoid fever.

[0073] In various examples, the protein capable of binding lipopolysaccharide is selected from the group consisting of Protein G, Protein A, a Protein A / G conjugate, a Protein G fusion protein, a Protein A fusion protein, a Protein A / G fusion protein, an immunoglobulin-binding fragment thereof, or any combination of two of more thereof.

[0074] In various examples, the complex comprising lipopolysaccharide comprises or is bound using one or more secondary antibodies, such as one or more species-specific secondary antibodies.

[0075] In other examples, the complex comprising immunoglobulin and lipopolysaccharide additionally comprises or is bound using a protein capable of binding lipopolysaccharide other than an antibody. Representative examples of such protein capable of binding lipopolysaccharides include, for example, Protein G, Protein A, a Protein A / G conjugate, a Protein G fusion protein, a Protein A fusion protein, a Protein A / G fusion protein, an immunoglobulin-binding fragment thereof, or any combination of two of more thereof.

[0076] In one example, the subject is human.

[0077] In one example, the subject is bovine, caprine, or ovine.

[0078] Any of the examples described above or elsewhere herein can relate to any of the aspects presented herein.

[0079] In another aspect the invention relates to a complex comprising immunoglobulin and lipopolysaccharide, wherein the immunoglobulin is capable of binding specifically to said lipopolysaccharide and inhibiting one or more biological activities of or associated with said lipopolysaccharide. In one example, the complex comprises immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is bound specifically to said lipopolysaccharide and is capable of inhibiting one or more biological activities of or associated with said lipopolysaccharide. For example, said immunoglobulin is bound specifically to said lipopolysaccharide and inhibits one or more biological activities of or associated with said lipopolysaccharide.

[0080] In one example, the lipopolysaccharide comprises Lipid A and one or more of said immunoglobulin binds specifically to said Lipid A. In one example, said immunoglobulin inhibits one or more biological activities of or associated with Lipid A.

[0081] In one example, the complex exhibits reduced TLR4 binding and / or agonism compared to equivalent lipopolysaccharide alone. For example, said immunoglobulin inhibits TLR4 binding and / or agonism by lipopolysaccharide.

[0082] In one example, the complex exhibits reduced endotoxicity compared to equivalent lipopolysaccharide alone. For example, said immunoglobulin inhibits endotoxicity elicited by said lipopolysaccharide.

[0083] In one example, the complex has a reduced ability to induce an inflammatory response, for example when administered to a subject. In one example, the complex has a reduce ability to induce an inappropriate inflammatory response, for example in a subject to which it is administered.

[0084] In one example, the complex does not induce an inflammatory response, for example when administered to a subject. In one example, the complex does not induce an inappropriate inflammatory response, for example in a subject to which it is administered.

[0085] In one example, said complex does not induce an inflammatory response when administered to a subject in which an inflammatory response is initiated when said lipopolysaccharide and / or said immunoglobulin are administered separately. For example, said complex does not induce an inflammatory response when administered to a subject in which an inflammatory response is or would be initiated when said lipopolysaccharide is administered separately. For example, said complex does not induce an inappropriate inflammatory response when administered to a subject in which an inappropriate inflammatory response is or would be initiated when said lipopolysaccharide is administered separately.

[0086] In one example, the complex is or has been isolated and / or purified from colostrum.

[0087] In one example, the complex is or has been isolated and / or purified from colostrum having an amount or concentration of IgG comparable to or exceeding the amount of IgA present in the colostrum.

[0088] In one example, the complex is formed in vitro.

[0089] In one example, the complex is formed in vitro from IgG purified or isolated from colostrum.

[0090] In one example, the complex comprises IgG purified or isolated from colostrum and is or has been formed in vitro by admixture with isolated, purified, synthetic, or recombinant lipopolysaccharide.

[0091] In one example, the complex is or has been isolated and / or purified from the systemic circulation of a mammal, such as a ruminant mammal.

[0092] In one example, said immunoglobulin comprises, consists essentially of, or consists of IgG.

[0093] In one example, said IgG comprises, consists essentially of, or consists of IgG having binding specificity to Lipid A.

[0094] In one example, said IgG comprises, consists essentially of, or consists of IgG having binding specificity to the lipopolysaccharide core. In one example, the complex consists essentially of or consists of immunoglobulin and lipopolysaccharide.

[0095] In one example, the complex comprises one or more antigens, such as one or more exogenous antigens other than said immunoglobulin and said lipopolysaccharide.

[0096] In one example, said immunoglobulin and / or said lipopolysaccharide comprises one or more exogenous antigens.

[0097] In one example, the complex comprises one or more biological ly-active moieties other than said immunoglobulin and said lipopolysaccharide.

[0098] In one example, the immunoglobulin comprises, consists essentially of, or consists of immunoglobulin from colostrum.

[0099] In one example, the immunoglobulin comprises, consists essentially of, or consists of IgG and IgA from colostrum.

[0100] In one example, the immunoglobulin comprises, consists essentially of, or consists of IgG from colostrum.

[0101] In one example, the colostrum is bovine colostrum, including pooled bovine colostrum.

[0102] In one example, the colostrum is a selected colostrum (that is, a colostrum selected for use in preparation or provision of complexes and / or immunoglobulins as contemplated herein) enriched in IgG. For example, the selected colostrum is enriched in IgG compared to IgA. For example, the amount and / or concentration of IgG relative to IgA, or the IgG:IgA ratio, present in the selected colostrum is high, for example, is higher than that of colostrum that is not selected.

[0103] In one example, said immunoglobulin comprises one or more immunoglobulins that has greater avidity to LPS derived from E. coli strain K12 LPS than to LPS derived from wild type E. coli.

[0104] In one example, said immunoglobulin comprises one or more immunoglobulins that has specificity to Lipid A. In one example, said immunoglobuling comprises one or more immunoglobulins that has specificity to the Lipid A / KDO glycolipid core of bacterial lipopolysaccharide.

[0105] In one example, said immunoglobulin comprises one or more colostrum immunoglobulins that has greater specificity to the glycolipid core of bacterial LPS than immunoglobulin from a source other than colostrum.

[0106] In one example, said immunoglobulin comprises one or more immunoglobulins having high affinity to LPS. In one example, said immunoglobulin comprises one or more immunoglobulins having high intrinsic affinity to LPS.

[0107] In one example, said immunoglobuling comprises one or more immunoglobulins that has high affinity, for example high intrinsic affinity, to the Lipid A / KDO glycolipid core of bacterial lipopolysaccharide.

[0108] In one example, said immunoglobulin has high functional affinity to LPS.

[0109] In one example, said immunoglobulin comprises one or more immunoglobulins having high avidity to LPS.

[0110] In various examples, said immunoglobulin has sufficiently high affinity or avidity for LPS so as to be resistant to dissociation from LPS in the presence of a non-ionic detergent, such as a polysorbate such as a polyoxyethylene sorbitol (e.g. polysorbate 20, polysorbate 40) or a polyoxyethylene (20) sorbitan monoalkylated. In various examples, said immunoglobulin has sufficiently high affinity or avidity for LPS so as to be resistant to dissociation from LPS in the presence of urea, for example, in the presence of 0.5 M urea, in the presence of 1 M urea, in the presence of 1.5 M urea, or in the presence of 2 M urea.

[0111] In various examples, said immunoglobulin comprises, consists essentially of, or consists of species-specific immunoglobulin.

[0112] In one example, the complex is formed in vitro, for example from IgG purified or isolated from colostrum, such as a colostrum having an amount or concentration of IgG comparable to or exceeding the amount of IgA present in the colostrum.

[0113] In one example, the complex comprises IgG purified or isolated from colostrum and is or has been formed in vitro by admixture with isolated, purified, synthetic, or recombinant lipopolysaccharide.

[0114] In one example, the complex is a complex prepared essentially as set out herein in the Examples.

[0115] In one example, the complex is formed from at least partially purified immunoglobulin, from at least partially isolated immunoglobulin, from recombinant immunoglobulin, and / or from synthetic immunoglobulin, wherein the immunoglobulin is capable of binding to and / or reducing the endotoxic activity of LPS and / or reducing endotoxicity associated with LPS.

[0116] In one example, the immunoglobulin is capable of reducing the endotoxic activity of LPS.

[0117] In one example, the immunoglobulin is capable of reducing the endotoxicity associated with LPS.

[0118] In one example, the immunoglobulin is capable of binding to and / or reducing the endotoxic activity of and / or the endotoxicity associated with the lipopolysaccharide with which it is complexed. For example, the immunoglobulin is capable of binding to and reducing the endotoxic activity of the lipopolysaccharide with which it is complexed. In one example the immunoglobulin is capable of binding to and reducing the endotoxicity associated with the lipopolysaccharide with which it is complexed.

[0119] In one example, the complex is formed from at least partially purified lipopolysaccharide, from at least partially isolated lipopolysaccharide, from recombinant lipopolysaccharide, and / or from synthetic lipopolysaccharide.

[0120] In one example, the complex is formed from at least partially purified immunoglobulin, from at least partially isolated immunoglobulin, from recombinant immunoglobulin, and / or from synthetic immunoglobulin, and from at least partially purified lipopolysaccharide, from at least partially isolated lipopolysaccharide, from recombinant lipopolysaccharide, and / or from synthetic lipopolysaccharide.

[0121] In another aspect, the invention relates to a method of isolating, purifying, or preparing a complex as herein disclosed comprising immunoglobulin and lipopolysaccharide, the method comprising a. providing colostrum; b. contacting under nondenaturing conditions said colostrum with a capture reagent capable of binding to immunoglobulin; c. recovering the agent and / or the immunoglobulin; thereby providing said complex.

[0122] In another aspect, the invention relates to a method of isolating, purifying, or preparing a complex as herein disclosed comprising immunoglobulin and lipopolysaccharide, the method comprising a. providing colostrum; b. contacting under nondenaturing conditions said colostrum with a capture reagent capable of binding to lipopolysaccharide; c. recovering the agent and / or the lipopolysaccharide; thereby providing said complex. In another aspect, the invention relates to a method of isolating, purifying, or preparing a complex as herein disclosed comprising immunoglobulin and lipopolysaccharide, the method comprising a. providing a biological sample from a neonatal mammalian subject, wherein said neonatal mammalian subject has ingested colostrum; b. contacting under nondenaturing conditions said biological sample with a capture reagent capable of binding to immunoglobulin; c. recovering the agent and / or the immunoglobulin; thereby providing said complex.

[0123] In still another aspect, the invention relates to a method of isolating, purifying, or preparing a complex as herein disclosed comprising immunoglobulin and lipopolysaccharide, the method comprising a. providing a biological sample from a neonatal mammalian subject, wherein said neonatal mammalian subject has ingested colostrum; b. contacting under nondenaturing conditions said biological sample with a capture reagent capable of binding to lipopolysaccharide; c. recovering the agent and / or the lipopolysaccharide; thereby providing said complex.

[0124] In various examples, the capture reagent capable of binding immunoglobulin is an antibodybinding agent.

[0125] In various examples, the antibody-binding agent is an antibody or antibody fragment, an antibody binding protein or fragment thereof, or a combination thereof.

[0126] In various examples, the antibody-binding agent comprises an antibody or fragment thereof capable of selectively binding one or more components of the complex comprising immunoglobulin and lipopolysaccharide. In one example, the antibody binding agent comprises colostrum, milk, serum, or any combination thereof, comprising one or more antibodies.

[0127] In one example, the antibody binding agent is or comprises polyclonal antibodies.

[0128] In various examples, the antibody-binding agent is an antibody binding protein.

[0129] In various examples, the antibody-binding agent is selected from the group consisting of: an anti- Ig antibody or a fragment thereof, Protein A or an antibody-binding fragment thereof, Protein G or an antibody-binding fragment thereof, an anti IgA antibody or an IgA antibody-binding fragment thereof, or an IgA-binding reagent.

[0130] In one example, the antibody-binding agent is Concanavalin A.

[0131] In various examples, the capture reagent capable of binding LPS is or comprises an agent selected from the group consisting of: a molecular crowding agent, a polyethylene, a polyethylene glycol, and Polymyxin B.

[0132] In one example, the capture reagent capable of binding LPS is selected from the group consisting of a lectin, a protein capable of binding lipopolysaccharide, and a lipopolysaccharide binding compound.

[0133] In one example, the antibiotic is selected from the group consisting of cyclic peptide-comprising antibiotics.

[0134] In one example, the capture reagent capable of binding LPS comprises Polymyxin B.

[0135] In one example, the capture reagent capable of binding LPS comprises a molecular crowding agent.

[0136] In one example, the capture reagent capable of binding LPS comprises a polyethylene or a polyethylene glycol. In one example, the polyethylene glycol is PEG20.

[0137] In one example, the capture reagent capable of binding LPS comprises Polymyxin B and one or more agents selected from the group consisting of a polyethylene or a polyethylene glycol. In one specifically contemplated example, the capture reagent comprises Polymyxin B and PEG20.

[0138] In a further aspect the invention relates to a pharmaceutical composition comprising a complex as contemplated herein.

[0139] In one example, the pharmaceutical composition is for use in the prevention or treatment of a bacterial infection of gram-negative bacteria or of a disease or condition caused by or associated with a bacterial infection of gram-negative bacteria.

[0140] In one example, the pharmaceutical composition is for reducing endotoxicity associated with lipopolysaccharide present in a subject or a sample from a subject, wherein the pharmaceutical composition comprises a complex as contemplated herein.

[0141] In one example, the pharmaceutical composition is for reducing one or more inappropriate inflammatory responses associated with lipopolysaccharide present in a subject or a sample from a subject, wherein the pharmaceutical composition comprises a complex as contemplated herein.

[0142] Typically, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

[0143] In one example, the pharmaceutical composition comprises a complex as contemplated herein, wherein the immunoglobulin comprising said complex comprises, consists essentially of, or consists of IgG.

[0144] In one example, the immunoglobulin comprises, consists essentially of, or consists of IgG and / or IgA from colostrum.

[0145] In one example, the immunoglobulin comprises, consists essentially of, or consists of IgG from colostrum. In one example, the immunoglobulin comprises, consists essentially of, or consists of IgG from a selected colostrum, such as a colostrum in which the amount and / or concentration of IgG relative to IgA, or the IgG:IgA ratio, present in the selected colostrum is high, for example, is higher than that of colostrum that is not selected.

[0146] In one example, the colostrum is bovine colostrum, including pooled bovine colostrum.

[0147] In various examples, the pharmaceutical composition comprises one or more agents selected from the group consisting of: a glycoprotein, a pharmaceutically acceptable non-ionic detergent, a pharmaceutically acceptable molecular crowding agent, and a pharmaceutically acceptable capture reagent.

[0148] In one example, the glycoprotein is lactoferrin, for example bovine lactoferrin.

[0149] In various examples, the composition comprises, or the contacting the complex and / or the immunoglobulin and / or the lipopolysaccharide with the capture reagent is performed in the presence of, one or more pharmaceutically acceptable detergents. For example, the composition comprises one or more capture reagents and a buffer comprising one or more pharmaceutically acceptable detergents, such as one or more non-ionic detergents, for example a polysorbate such as a polyoxyethylene sorbitol (e.g. polysorbate 20, polysorbate 40) or a polyoxyethylene (20) sorbitan monoalkylated.

[0150] In one example, the pharmaceutical composition is a vaccine, such as a vaccine for oral administration. In one example the pharmaceutical composition is an oral vaccine composition capable of eliciting a mucosal immune response.

[0151] In one example the pharmaceutical composition is an oral vaccine composition capable on oral administration to a subject of eliciting an IgA response.

[0152] In one example, the oral vaccine composition is capable on oral administration to a subject of inducing or increasing the production of IgA antibodies, such as anti-LPS IgA antibodies.

[0153] In one example, the pharmaceutical composition comprises anti-LPS Ig, such as anti-LPS IgG, formulation for separate, sequential, or simultaneous administration with LPS.

[0154] Other aims, aspects, features and advantages of the present invention will become apparent from the description that follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred examples of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0155] BRIEF DESCRIPTION OF THE FIGURES

[0156] The invention is exemplified in the following non limiting embodiments and with reference to the accompanying figures, in which:

[0157] Figure 1 presents a graph showing Bovine colostrum attenuates endotoxicitv and contains IaG antibodies that recognize antigenic epitopes presented on Escherichia coli (E.coli) lipopolysaccharide (LPS). Bovine colostrum endotoxicity: (A) before (open bars) and after boiling (>98°C) for 10 mins (filled bars), measured by Limulus Amoebocyte Lysate (LAL) bioassay, data expressed as mean EU / mL ± SD of three separate bovine colostrum preparations, where * represents statistical (PcO.Ol) increase from bovine colostrum (not boiled). LPS detection (B) of K12-LPS (2.5pg / mL), employing milk powder (A) and three different pooled bovine colostrum preparations #1(»), #2 (■) or #3 (A) was measured by ELISA. Data is expressed as mean absorbance ± SD from six separate experiments, where # represents statistical (PcO.Ol) difference from milk powder. Utilizing an enriched IgG bovine colostrum preparation, IgG specificity (C) was assessed using E.coli K12-LPS (o), shed E.coli LPS (•) and lysates extracted from three (Streptococcus uberis [S.uberis, ■]. Staphylococcus aureus [S.auerus, ] and coagulase-negative staphylococci (CNS, ♦) gram-positive bacteria strains isolated from clinical acute mastitis milk, data expressed as mean absorbance ± SD from five separate experiments, where # represents statistical (PcO.OOl) difference of E.coli K12-LPS and shed E.coli LPS from gram-positive bacterial lysates.

[0158] Figure 2 presents a graph showing that soluble LPS / IoG antibody immune complexes are found in bovine colostrum. Soluble LPS / IgG immune complexes: (A) in five separate bovine colostrum concentrates (o), or fresh post- partum colostrum (•) were measured by ELISA. Data is expressed as mean absorbance ± SD from 4 replicate measures. Colostrum soluble LPS / IgG complex levels (B) calculated as ng / mL LPS equ. before (o) and after boiling (>98°C) for 10 mins (•). Data is expressed as mean absorbance ± SD, where # represents statistical significance (PcO.Ol) from boiled colostrum. Specificity of LPS / IgG immune complex detection (C) was measured by ELISA after preincubation with bovine lactoferrin (•) or skimmed powdered milk (o). Data is expressed as ng / mL LPS equ. ± SEM from four separate experiments, * represents statistical significance (PcO.Ol) from skimmed powdered milk values.

[0159] Figure 3 presents a graph showing the Immunoprecipitation of K12-LPS with bovine colostrum IgG antibodies reduces endotoxicitv capacity. Protein levels: (A) in resuspended immune precipitants of K12-LPS and a bovine colostrum IgG extract measured by absorbance280nm against BSA standards, data is expressed as mean mg / mL ± SEM of four separate experiments, with * denotes statistical (Pc0.05) an increase from baseline values. Immune precipitates (before [o] and after boiling (<98°C for 10 mins [•]) were assessed for LPS / IgG immune complexes and endotoxicity capacity. LPS / IgG immune complexes (B) were measured by ELISA, data was calculated as ng / mL LPS equ. and expressed as mean % control ± SEM from four separate experiments; * represents statistical (Pc0.05) increase from boiled values. Endotoxicity capacity (C) was measured using a Limulus Amoebocyte Lysate (LAL) bioassay and expressed as mean EU / mL ± SEM of 4 separate experiments, where * signifies statistical (Pc0.05) increases from not-boiled values.

[0160] Figure 4 presents a graph showing the Soluble milk LPS / IgG immune complex levels in early acute Escherichia coli (E. coli) mastitis parallel increases in endotoxicitv capacity and lactate dehydrogenase (LDH) activity. Soluble LPS / IgG immune complexes: (A) in 87 healthy (open bar) or 59 E.coli acute mastitis (fill bar) milk samples was measured by ELISA and expressed as mean ng / mL LPS equ. ± SEM; * represents statistical (PcO.Ol) increase from healthy milk LPS / IgG complex levels. Endotoxicity capacity (BI) of not-boiled (open bars) or boiled (98°C) for 10 mins (filled bars) in 87 healthy or 59 acute E.coli mastitis milk samples measured using Limulus Amoebocyte Lysate (LAL) bioassay and expressed as mean EU / mL ± SEM; * represents statistical (PcO.Ol) increase from not-boiled milk, whereas # signifies statistical (PcO.Ol) significance from healthy milk values. LDH activity (CI) in 87 healthy (open bar) or 59 E.coli acute mastitis (fill bar) milk samples was measured by enzyme kinetics and expressed as mean pmoles / min ± SEM; * represents statistical (PcO.Ol) increase from healthy milk soluble LPS / IgG levels. Linear regression plots of soluble LPS / IgG immune complex levels and endotoxicity capacity (BII) and LDH activity (CII) were generated showing regression equation together with R2values.

[0161] Figure 5 presents a graph showing milk from early acute Escherichia coli (E. coin mastitis IgG antibodies shows a reduced recognition for K12-LPS. Colostrum or acute E. coli mastitis milk IgG antibodies: (A) that recognize either K12-LPS (open bar) or WT-LPS (filled bar) measured by ELISA. Data is expressed as mean absorbance ± SEM; * denotes statistical (P<0.05) decrease from WT-LPS values. A frequency distribution plot (B) of ELISA absorbance values of the 59 milk samples for the recognition of either K12-LPS (dashed line) or WT-LPS (solid line).

[0162] Figure 6 presents a graph showing that IgG antibodies from bovine colostrum or representative acute Escherichia coli (E. coli) mastitis milk sample exhibit differential recognition and avidity for WT-LPS and K12-LP: . (A) IgG antibody extracts from bovine colostrum in the recognition of WT-LPS (o) or K12-LPS (□) or a representative acute mastitis milk sample in the recognition of WT-LPS (•) or K12-LPS (■) assessed by ELISA. Data is expressed as mean absorbance ± SEM of four separate experiments; * denotes no significant difference between colostrum IgG recognition of WT-LPS and K12-LPS, whereas # represents a statistical (PcO.Ol) difference in acute mastitis milk IgG antibodies in the recognition of WT-LPS and K12-LPS. Resistance to urea dissociation (B) of IgG antibodies extracts from colostrum bound to WT-LPS (o) and K12- LPS (□) or a representative acute mastitis milk sample bound to WT-LPS (•) or K12-LPS (■) were assessed by ELISA. Data is expressed as mean % control (no urea) ± SEM of six separate experiments; $ represents statistical (P<0.05) difference of colostrum bound to K12-LPS from the other values measured using 2 M urea.

[0163] Figure 7 presents a graph showing Soluble WT-LPS / IaG immune complexes with colostrum IgG antibodies reduce supernatant endotoxicitv capacity. Supernatant from immune precipitation of WT-LPS with IgG antibodies extracted from bovine colostrum (o) or a representative acute E. coli mastitis milk sample (•) was assessed for LPS / IgG immune complexes and endotoxicity capacity. LPS / IgG immune complexes the supernatant: (A) were measured by ELISA and expressed as mean ng / mL LPS equ. ± SEM of four separate experiments; *represents statistical (P<0.05) increase from baseline levels. Endotoxicity capacity in the supernatant (B) was measured using a Limulus Amoebocyte Lysate (LAL) bioassay and expressed as mean EU / mL ± SEM of four separate experiments; * denotes a statistical (P<0.05) decrease from immune complexes using WT-LPS with acute mastitis milk IgG antibodies.

[0164] Figure 8 presents two graphs showing soluble LPS / IaG immune complexes attenuate WT-LPS- induced pro-oxidants increases in milk granulocytes. N-acetyl cysteine inhibition of WT-LPS- induced reactive oxygen species (ROS) in H2DCFH loaded milk granulocytes: (A) monitored by oxidation of H2DCFH to DCF, and data expressed as mean fluorescent intensity (FI) after 20 mins ± SD of four separate experiments; *signifies (P<0.01) a decrease from WT-LPS alone. Promotion of ROS generation in H2DCFH loaded milk granulocytes in response to WT-LPS alone (■) or in the supernatant after immune precipitation of either bovine colostrum (o) or a representative acute E. coli mastitis milk (•) IgG antibodies with 25 ng / mL WT-LPS were expressed as mean ± SEM (B) change in FI over 20 mins for experiments and as (C) % of WT- LPS alone FI after 20 mins, from four separate experiments; # and * represents statistical (P<0.05) difference from supernatant values after WT-LPS immune precipitation with acute mastitis milk IgG antibodies and WT-LPS alone.

[0165] Figure 9 is a graph showing the presence and concentration of LPS / IgG complexes in plasma samples obtained from young calves, as described herein in Example 2.

[0166] Figure 10 presents two graphs showing Colostrum LPS-Ig complexes in samples sourced from NZ (Westland, WL) or US (Peptide Ignition, PI) : (A) LPS-IgG complexes in Colostrum preps and (B) LPS-IgA complexes in Colostrum preps, as discussed herein in Example 3.

[0167] Figure 11 presents three graphs showing LPS-Ig complexes present in blood samples collected from calves in the CMR (Figure 11A), WL (Figure 11B), and PI (Figure 11C) treatment groups as discussed herein in Example 4.

[0168] Figure 12 presents six graphs showing the amount of bacteria-specific anti-LPS IgG antibodies at days 0, 7, and 21 across the three treatment groups (CMR, Figures 12A and 12B; PI, Figures 12C and 12D; WL, Figures 12E and 12F), as discussed in Example 4 herein. Figure 13 presents a graph showing the average fold increase in the level of IgAs specific for LPS from 6 different bacteria in the saliva of calves from the WL (solid bars) and the PI (hatched bars) treatment groups, as discussed in Example 5 herein.

[0169] Figure 14 presents three graphs showing faecal anti LPS IgA levels (pg / ml, adjusted to lmg / ml protein) specific to three different gram-negative bacteria ( / (. pneumonia, Figure 14A; S. marcescens, Figure 14B; S. enterica, Figure 14C) across the 20 day trial as described in Example 6 herein.

[0170] Figure 15 presents three graphs showing faecal anti LPS IgG levels (pg / ml, adjusted to lmg / ml protein) specific to three different gram-negative bacteria ( / (. pneumonia, Figure 15A; S. marcescens, Figure 15B; S. enterica, Figure 15C) across the 20 day trial as described in Example 6 herein.

[0171] Figure 16 presents four graphs the levels of anti-LPS IgA levels specific to four different gram-negative bacteria (E. coli K12, Figure 16A; K. pneumonia, Figure 16B; S. enterica, Figure 16C; S. marcescens, Figure 16D) in plasma across the 20 day trial as described in Example 7 herein.

[0172] Figure 17 presents two graphs showing the endotoxin activity of in vitro generated LPS / IgG complexes (full bars) and heat denatured (boiled) samples (hatched bars), with the complexes comprising IgG derived from colostrum (Figure 17A), and IgG derived from serum (Figure 17B), as described in Example 8 herein.

[0173] Figure 18 presents two graphs showing the levels of LPS / IgG complexes in blood samples from calves to which in vitro generated LPS / IgG complexes (Figure 18A) or CMR (Figure 18B) were administered, as described in Example 8 herein.

[0174] Figure 19 presents two graphs showing the detection of LPS-specific IgA (Figure 19A) and LPS-specific IgG (Figure 19B) in the plasma of a calf to which in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was orally administered, as described in Example 8 herein.

[0175] Figure 20 presents two graphs showing the detection of LPS-specific IgA in faecal samples (Figure 20A) and in saliva samples (Figure 20B) from a calf to which in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was orally administered, as described in Example 8 herein.

[0176] Figure 21 presents two graphs showing the detection of LPS-specific IgA in faecal samples (Figure 21A) and in saliva samples (Figure 21B) of a calf to which a bolus of in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was orally administered, as described in Example 8 herein.

[0177] DETAILED DESCRIPTION

[0178] The present invention generally relates to agents, compositions, and methods for preventing or treating a gram-negative bacterial infection and / or of preventing or treating a disease or condition associated with the presence of gram-negative bacteria in a subject.

[0179] The presence in a subject of LPS from gram-negative bacteria has long been recognised as being responsible for endotoxicity associated with gram-negative bacterial infection, and as such an important contributor to symptoms of and indeed the severity of many diseases or conditions associated with such infections.

[0180] The present invention further relates to a complex comprising immunoglobulin and lipopolysaccharide (LPS) in which immunoglobulin is bound specifically to said lipopolysaccharide and is capable of attenuating, for example is capable of inhibiting, one or more biological activities of or associated with said lipopolysaccharide.

[0181] As described and exemplified herein, the inventors have identified and isolated such complexes from bovine colostrum and from the systemic circulation of neonatal bovine, and in characterising these complexes have established they are effective in reducing endotoxicity and inflammatory responses usually associated with exposure to lipopolysaccharides from gram-negative bacteria.

[0182] The present invention in various aspects also relates to methods of using such complexes, for example to prevent or treat gram-negative bacterial infection, or to induce an immunological response in a subject, or to immunize or vaccinate a subject, by administering the complex and / or a composition comprising said complex, to a subject.

[0183] Importantly, the complexes, compositions, and methods enable the treatment and particularly the prophylactic treatment of a subject at risk or suspected of having a gram-negative bacterial infection, including a subject at risk of developing a disease associated with such gram-negative bacterial infections, while minimising the risk of LPS-mediated endotoxicity and / or one or more inflammatory responses associated with such infection or with LPS.

[0184] Additionally, in certain circumstances, by eliciting one or more beneficial immunological responses and / or mitigating one or more inflammatory responses to gram-negative bacterial infection, the complexes, compositions, and methods disclosed herein enable the use of existing therapies that are (in the absence of the present complexes, compositions and / or methods) currently contraindicated, such as bacteriocidal agents that risk exacerbating LPS-mediated endotoxicity.

[0185] The invention thus in certain aspects relates to the applicant's development of therapeutic complexes, compositions, and methods, such as complexes and compositions capable of, and methods for, eliciting an immune response in a subject, for example an immune response to gram-negative bacteria and / or to LPS, and / or complexes and compositions capable of, and methods for reducing one or more inflammatory responses to gram-negative bacterial infection and / or LPS, and / or complexes and compositions capable of, and methods for preventing or treating LPS-mediated endotoxicity.

[0186] Selected definitions

[0187] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0188] Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g. "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer or value is also specifically contemplated. The term "administering" as used herein refers to providing an amount, and typically a therapeutically effective amount, of an agent or composition to a subject using one or more methods of administering therapeutic agents as are known in the art. These methods in various examples comprise administering agents using oral, sublingual, intravenous, subcutaneous, transcutaneous, intramuscular, intracutaneous, intrathecal, epidural, intraocular, intracranial, inhalation, rectal, vaginal, and the like administration. In specifically contemplated examples such as those directed to the prevention or treatment of gram-negative bacterial infections of the gut, oral administration is particularly contemplated. In specifically contemplated examples such as those directed to the prevention or treatment of mastitis, administration by intramammary instillation is contemplated.

[0189] The term "and / or" can mean "and" or "or".

[0190] The terms "antibody" and "immunoglobulin" or "Ig" are generally used interchangeably herein, and refer to a glycoprotein produced by the immune system in response to the presence of an antigen.

[0191] The term "antigen" means a molecule having distinct surface features or epitopes capable of stimulating a specific immune response. Antibodies (immunoglobulins) are produced by the immune system in response to exposure to antigens. Antigens maybe proteins, carbohydrates or lipids, although only protein antigens are usually classified as immunogens because carbohydrates and lipids cannot easily elicit an immune response on their own.

[0192] The terms "comprise", "comprises", and "comprising" as used in this specification and claims are not to be interpreted in an exclusive or exhaustive sense, and mean "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprise", "comprises", or "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "including", "include" and "includes" are to be interpreted in the same manner.

[0193] The term "consisting essentially of" when used in this specification refers to the features stated and allows for the presence of other features that do not materially alter the basic characteristics of the features specified.

[0194] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0195] An "effective amount" as contemplated herein is an amount sufficient to effect beneficial or desired results, including clinical results. An effective amount can be administered in one or more administrations by various routes of administration. The effective amount will vary depending on, among other factors, the disease or condition indicated, the severity of the disease or condition, the age and relative health of the subject, the potency of the agent administered, the mode of administration and the treatment desired. A person skilled in the art will be able to determine appropriate dosages having regard to these any other relevant factors. It will be appreciated that the various methods of prevention or treatment contemplated herein will typically embody the administration of an effective amount of the one or more therapeutic agents, complexes or compositions disclosed herein.

[0196] The term "Immunoglobulin A" or "IgA" means a subclass of Ig that has an important role in the immune function of mucosal surfaces, including in the context of particularly contemplated examples herein, the udder of bovine cows.

[0197] The term "Immunoglobulin G" or "IgG" means a subclass of Ig that is mostly found in circulating bodily fluids such as blood and lymph.

[0198] The term "inhibit" and grammatical equivalents as used herein such as "inhibition" and "inhibiting" as used herein refer to an attenuation of or an at least partial reduction in the entity, process, activity, or object in respect of which these terms are used, such as an attenuation of or an at least partial reduction in, for example, a process or activity (for example, the at least partial reduction in the occurrence, onset, frequency, magnitude or duration of a process or activity), or an at least partial halting, delay, or amelioration of, for example, a process or activity. Accordingly, inhibition as used herein contemplates any degree of attenuation or reduction through to a total ablation or negation of the entity, process, activity, or object in respect of which this and related terms are used. In certain contexts, inhibition may be of an extant entity, process, activity, or object, such as of a process that has already begun. In other contexts, inhibition is used in a similar manner to "prevent", for example the inhibition of an entity, process, activity or object that does not yet exist or is not yet present or manifest.

[0199] The term "pharmaceutical composition" is defined herein to refer to a composition comprising at least one therapeutic agent to be administered to a subject in order to treat a particular disease or condition affecting the subject or to elicit a therapeutic or prophylactic response in the subject, together with one or more pharmaceutically acceptable carriers.

[0200] The term "pharmaceutically acceptable" is defined herein to refer to those compounds, carriers, materials, compositions and / or dosage forms, which are, within the scope of sound medical judgment, suitable for contact with the tissues a subject without excessive toxicity, irritation allergic response and other problem complications commensurate with a reasonable benefit / risk ratio.

[0201] The term "pharmaceutically acceptable carrier" refers to a carrier, such as but not limited to an excipient, diluent, adjuvant or vehicle, that may be administered to a subject and will usually be administered together with the therapeutic agent. Such carriers are well known in the art, and may be as simple as water or saline, or may in certain examples comprises a number of different excipients, diluents, adjuvants and / or vehicles as may be required, for example for effective formulation.

[0202] The term "prevent" and grammatical equivalents as used herein such as "prevention" and "preventing" as used herein refers to the halting of an entity, process, activity, or object in respect of which this term is used that is not yet extant, has not yet manifest, or has not yet begun. For example, prevention may be of, for example a bacterial infection, LPS-mediated endotoxicity, or an inflammatory response to LPS in a subject, that has not yet begun. In certain examples, such prevention is for a certain period of time - for example, for so long as the concentration of the complex and / or compositions as described herein is maintained above a certain threshold. It will be appreciated that in such examples the term "prevent" does not contemplate prevention in perpetuity.

[0203] The term "reverse" and grammatical equivalents as used herein such as "reversal" and "reversing" when used herein with reference to a gram-negative bacterial infection or a disease or condition associated with such infection refers to the return of the infection or of the disease or condition to a former or less developed state, including a state in which the infection, disease or condition is no longer present. The terms "remission" and "regression" are to be interpreted in a similar manner.

[0204] A "subject" as used herein is an animal, usually a mammal, including a mammalian companion animal, or a human. Representative companion animals include feline, equine, and canine. Representative agricultural animals include bovine, ovine, caprine, cervine, and porcine. Ruminant mammals are in certain examples particularly contemplated. Also particularly contemplated are subjects which are used commercially to produce milk, such as bovine, ovine, and caprine subjects.

[0205] The term "treating" or "treatment" and related terms such as "treat" as used herein broadly includes any kind of treatment activity in which some desired therapeutic effect is achieved, including the amelioration, diagnosis, cure, mitigation, delay of onset or of progression, or prevention of a disease or condition in a subject, or any activity that otherwise affects the structure or any function of the body of the subject, and includes a treatment relieving, reducing or alleviating at least one symptom or sequelae in a subject or effecting a delay of onset or of progression of a disease or condition or symptom or sequelae thereof. For example, treatment can be the diminishment of one or several symptoms or sequelae of a disease or disorder, or complete eradication of a disease or disorder. Within the meaning of the present disclosure, the term "treat" also denotes to arrest, delay the onset ( I . e. , the period prior to clinical manifestation of a disease) and / or reduce the risk of developing or worsening a disease. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment, for example, an increase in overall survival compared to a subject not receiving treatment as described herein, and / or an increase in progression-free survival compared to a subject not receiving treatment as described herein. The term "treating" can also mean an improvement in the condition of a subject having a disease or condition, e.g., one or more of a decrease in a marker for the disease or condition in a subject, a decrease or no substantial increase in the rate of disease progression in a subject, and an improvement in one or more physiological or metabolic responses or metrics in a subject (e.g., as compared to the one or more metric(s) in a subject having a similar disease or condition receiving no treatment or a different treatment, or as compared to the one or more metric(s) in the same subject prior to treatment). Prophylactic treatments, including those in which treatment is administered before one or more indicia or symptoms of a disease or condition manifest, are particularly contemplated.

[0206] Accordingly, the terms treat, treatment, and treating as used herein encompass preventing, inhibiting, or arresting the development of a gram-negative bacterial infection and / or of a disease or condition associated with such infection, and / or causing the amelioration, reduction, remission or regression of a gram-negative bacterial infection or a disease or condition associated with such infection or one or more symptoms, consequences, sequelae, or side effects thereof. These terms accordingly encompass preventing, inhibiting, or arresting the development of or ameliorating or mitigating LPS- induced endotoxicity and / or one or more inflammatory response to gram-negative bacterial infection and / or to LPS in a subject. Methods of assessing treatment, including methods of assessing amelioration, inhibition, arrest, reduction, remission and / or regression of disease states are known and will be apparent to a person skilled in the art.

[0207] The treatment of a gram-negative bacterial infection or a disease or condition associated with such infection, and / or the prevention, inhibition or reversal of processes associated with a gramnegative bacterial infection or a disease or condition associated with such infection may manifest in a number of ways. For example, in some examples the treatment, prevention, inhibition or reversal of processes such as for example LPS-mediated endotoxicity or an inflammatory response to LPS in a subject, leads to clearance of the infection, and / or leads to slowing of disease progression, and / or improved quality of life in subjects with the disease.

[0208] In various examples the prevention or treatment (including for example the inhibition or reversal) of processes associated with a gram-negative bacterial infection or a disease or condition associated with such infection manifests as decreased endotoxicity, and / or decreased infection (for example a reduction in the number of bacterial present in a subject). In another example, the prevention or treatment (including for example the inhibition or reversal) of processes associated with a gramnegative bacterial infection or a disease or condition associated with such infection manifests as one or more decreased inflammatory responses in a subject. In other examples the prevention or treatment (including for example the inhibition or reversal) of processes associated with a gram-negative bacterial infection or a disease or condition associated with such infection manifests as increased survival, or an increased rate of survival of a subject population.

[0209] Those skilled in the art will recognise, on reading this description, that various uses of such methods in the prevention or treatment of infections of gram-negative bacteria and / or a disease or condition caused by or associated with an infection of gram-negative bacteria, and particularly pathogenic gram-negative bacteria, are provided.

[0210] Accordingly, in one aspect, the invention relates to a method of preventing or treating a disease or condition caused by or associated with a gram-negative bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex comprising immunoglobulin and lipopolysaccharide, in which said immunoglobulin is bound specifically to said lipopolysaccharide and is capable of inhibiting one or more biological activities of or associated with said lipopolysaccharide.

[0211] Various aspects of the invention are described in further detail in the following subsections. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions included herein, will be preferred. Although methods and materials similar or equivalent to those described herein can be used in the practice of the invention, examples of suitable methods and materials are described below. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0212] Complexes

[0213] In one aspect, the invention relates to a complex comprising immunoglobulin and lipopolysaccharide, in which said immunoglobulin is bound specifically to said lipopolysaccharide and is capable of inhibiting one or more biological activities of or associated with said lipopolysaccharide.

[0214] Immunoglobulin

[0215] As described and exemplified herein, the complexes according to this disclosure comprise immunoglobulin capable of specifically binding to and inhibiting one or more biological activities of or associated with lipopolysaccharide.

[0216] The general structure, function, and classification of immunoglobulins is well-understood. As used herein the term "Immunoglobulin" or "Ig" means an antibody - a large Y-shaped protein produced in nature mainly by plasma cells and functioning in the immune system to recognise and neutralise pathogens such as pathogenic bacteria and viruses.

[0217] Immunoglobulins can be classified according to their structure and function, elements of which include their distribution within an organism, the manner in which they are induced, and the cells with which they interact.

[0218] Immunoglobulin A (IgA) is an isotype of Ig that has an important role in the immune function of mucosal surfaces, including the mucosa of the mammalian gastrointestinal tract, of the mammary gland such as the udder of ruminants such as bovine cows, the nasopharageal mucosa, and the like.

[0219] Immunoglobulin D (IgD) is an isotype of Ig reportedly co-expressed with IgM and reportedly involved in B cell activation. Immunoglobulin E (IgE) is an isotype of Ig primarily associated with allergic response and associated with immune responses to certain parasites such as protozoan parasites. IgE is reportedly essential to type I hypersensitivity and related diseases, including allergic rhinitis and atopic dermatitis.

[0220] Immunoglobulin G (IgG) is an Ig isotype that is mostly found in circulating bodily fluids such as blood and lymph.

[0221] Immunoglobulin M (IgM) is an isotype of Ig produced mainly in the spleen and is the first class of antibody to appear in response to initial exposure to an antigen.

[0222] At a very basic level, the structure of immunoglobulins comprises an antigen-binding domain that is highly variable, and conserved regions that are much less variable in which protein structures associated with conserved function, which will usually depend on isotype, reside.

[0223] The immunoglobulin present in the complexes contemplated herein bind specifically to and form a stable complex with lipopolysaccharide. As shown herein, the binding of the immunoglobulin to lipopolysaccharide inhibits one or more biological activities of or associated with the lipopolysaccharide, such as but not limited to endotoxicity.

[0224] Lipopolysaccharides

[0225] Lipopolysaccharides (LPS, often referred to as endotoxin) are carbohydrates present in the outer membrane of Gram-negative bacteria, generally comprising a O-antigen, an inner oligosaccharide core, and an outer core. LPS has been reported to replicate septic shock when experimentally administered to mammals including humans, and has been reported to be a major factor in gram-negative sepsis. Similarly, LPS has been reported to replicate symptoms of mastitis when experimentally administered.

[0226] The O-antigen is a repetitive glycan polymer attached to the core oligosaccharide and is the outermost component of LPS molecules. The composition of the O-antigen varies from species to species and strain to strain, with over 160 different O-antigen structures reported for E. coli strains alone. It has been reported that the O-antigen is the most variable portion of the LPS molecule, and most relevant to antigenic specificity.

[0227] The core oligosaccharide comprises sugars, commonly heptose and mannose derivatives such as 3-Deoxy-D-manno-oct-2-ulosonic acid (also known as keto-deoxyoctulosonate (KDO)), and often other non-carbohydrate components including amino acids or phosphates.

[0228] Lipid A is a hydrophobic fatty acid-containing molecule that anchors the LPS in the bacterial membrane. Typically, lipid A is a phosphorylated glucosamine disaccharide comprising multiple fatty acids. The particular composition of lipid A can differ across bacterial species and stains, though it is the most conserved component of LPS.

[0229] LPS has been reported to bind the CD14 / TLR4 / MD2 receptor complex in many animal cell types. From a health perspective, the CD14 / TLR4 / MD2 receptor-positive cells of particular interest include monocytes, dendritic cells, macrophages, and B cells, the binding of LPS to which promotes the secretion of pro-inflammatory cytokines, nitric oxide, and eicosanoids.

[0230] In certain examples, such as examples of the preparative methods disclosed herein, a capture reagent binds to LPS or to LPS / antibody complexes. In various examples, the capture reagent is selected from the group consisting of a lectin, a protein capable of binding lipopolysaccharide, and a lipopolysaccharide binding compound.

[0231] In certain examples, the capture reagent is an antibiotic, such as a cyclic peptide-comprising antibiotic. In one particularly contemplated example, the capture reagent comprises Polymyxin B. In one example, the capture reagent comprises a molecular crowding agent. In one example, the molecular crowding reagent is a polyethylene glycol.

[0232] In various examples, the capture reagent is a protein capable of binding lipopolysaccharide, such as lipopolysaccharide binding protein, a casein, lactoferrin, or an albumin such as bovine serum albumin, or a cholesterol.

[0233] In one example, the LPS and / or complexes comprising LPS is bound, for example, using as a binding reagent an immunoglobulin, such as a species-specific, anti-Ig secondary antibody. In another example, the LPS is bound using a protein capable of binding lipopolysaccharide, such as Protein G.

[0234] In one example, anti-LPS antibodies (and / or complexes comprising anti-LPS antibodies) are bound, for example, using species-specific, anti-Ig secondary antibodies. In another example, anti-LPS antibodies are bound using an immunoglobulin-binding protein, such as Protein G, Protein A, a Protein A / G conjugate, a Protein G fusion protein, a Protein A fusion protein, a Protein A / G fusion protein, an immunoglobulin-binding fragment thereof, or any combination of two of more thereof.

[0235] In one example, the capture reagent is Concanavalin A.

[0236] Lactoferrin

[0237] In one example, the capture reagent is lactoferrin.

[0238] In one example, the pharmaceutical composition comprises, and / or the complex or pharmaceutical composition is administered together with, lactoferrin.

[0239] Lactoferrin (LF) is a mammalian innate host-defence protein that exhibits anti-microbial activity. In adult bovine milk, LF occurs in small quantities ranging from 0.1 to 0.3 mg / mL. Structurally, LF is a single polypeptide that forms two homologous globular domains, one of which is heavily glycosylated. Bovine LF, in particular, contains mannose residues within this glycosylated domain. Furthermore, the globular configuration of LF also creates regions of positive charge amino acids that are expected, without wishing to be bound by any theory, to be capable of associating with and possibly binding to negatively charged moieties, such as negatively charged regions of proteins and other biological molecules such as LPS.

[0240] Specifically contemplated examples

[0241] 1. A complex comprising immunoglobulin and lipopolysaccharide, and wherein immunoglobulin is capable of binding specifically to said lipopolysaccharide and inhibiting one or more biological activities of or associated with said lipopolysaccharide.

[0242] 2. The complex according to numbered paragraph 1 wherein said lipopolysaccharide comprises Lipid A and one or more of said immunoglobulin binds specifically to said Lipid A.

[0243] 3. The complex according to numbered paragraph 1 or 2, wherein said complex exhibits reduced TLR4 binding and / or agonism compared to equivalent lipopolysaccharide alone.

[0244] 4. The complex according to any one of numbered paragraphs 1 to 3, wherein said complex exhibits reduced endotoxicity compared to equivalent lipopolysaccharide alone.

[0245] 5. The complex according to any one of numbered paragraphs 1 to 4, wherein said complex does not induce an inflammatory response. 6. The complex according to numbered paragraph 5 wherein said complex does not induce an inflammatory response when administered to a subject in which an inflammatory response is initiated when said lipopolysaccharide and / or said immunoglobulin are administered separately.

[0246] 7. The complex according to any one of numbered paragraphs 1 to 6 isolated and / or purified from colostrum.

[0247] 8. The complex according to any one of numbered paragraphs 1 to 7, wherein said immunoglobulin comprises, consists essentially of, or consists of IgG.

[0248] 9. The complex according to numbered paragraph 8, wherein said IgG comprises, consists essentially of, or consists of IgG having binding specificity to Lipid A.

[0249] 10. The complex according to any one of numbered paragraphs 1 to 9, wherein the complex consists essentially of or consists of immunoglobulin and lipopolysaccharide.

[0250] 11. The complex according to any one of numbered paragraphs 1 to 9, wherein said immunoglobulin and / or said lipopolysaccharide comprises one or more exogenous antigens.

[0251] 12. The complex according to any one of numbered paragraphs 1 to 9 or 11, wherein the complex comprises one or more antigens other than said immunoglobulin and said lipopolysaccharide.

[0252] 13. The complex according to any one of numbered paragraphs 1 to 9, 11, or 12, wherein the complex comprises one or more biolog ica I ly-active moieties other than said immunoglobulin and said lipopolysaccharide.

[0253] 14. A method of isolating, purifying, or preparing a complex comprising immunoglobulin and lipopolysaccharide, the method comprising a. providing colostrum; b. contacting under nondenaturing conditions said colostrum with a capture reagent capable of binding to immunoglobulin; c. recovering the agent and / or the immunoglobulin; thereby providing said complex.

[0254] 15. A method of isolating, purifying, or preparing a complex comprising immunoglobulin and lipopolysaccharide, the method comprising a. providing colostrum; b. contacting under nondenaturing conditions said colostrum with a capture reagent capable of binding to lipopolysaccharide; c. recovering the agent and / or the lipopolysaccharide; thereby providing said complex.

[0255] 16. The method according to numbered paragraph 14 or 15 additionally comprising combining said complex with one or more carriers, such as one or more agriculturally or pharmaceutically acceptable carriers. 17. A pharmaceutical composition comprising a complex according to any one of numbered paragraphs 1 to 13 or a complex isolated, purified, or prepared in a method according to any one of numbered paragraphs 14 to 16.

[0256] 18. The pharmaceutical composition according to numbered paragraph 17, wherein the immunoglobulin comprises, consists essentially of, or consists of IgG and / or IgA.

[0257] 19. The pharmaceutical composition according to numbered paragraph 17 or 18, wherein the immunoglobulin comprises, consists essentially of, or consists of IgG and / or IgA from colostrum.

[0258] 20. A method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

[0259] 21. A method of immunising a subject in need thereof, the method comprising administering to the subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

[0260] 22. The method according to numbered paragraph 20 or 21, wherein the complex is a complex according to any one of numbered paragraphs 1 to 13 or is present in a pharmaceutical composition according to any one of numbered paragraphs 17 to 19.

[0261] 23. The method according to numbered paragraph 20 wherein the immune response is to said immunoglobulin and / or said lipopolysaccharide.

[0262] 24. The method according to numbered paragraph 21 wherein the immunisation is against the organism(s) from which said lipopolysaccharide is derived.

[0263] 25. The method according to numbered paragraph 20 or numbered paragraph 21, wherein said immunoglobulin and / or said lipopolysaccharide comprises one or more exogenous antigens.

[0264] 26. The method according to any preceding numbered paragraph, wherein the complex comprises one or more antigens other than said immunoglobulin and said lipopolysaccharide.

[0265] 27. The method according to numbered paragraph 25 or 26 wherein the immune response is to said one or more exogenous antigens.

[0266] 28. The method according to any one of numbered paragraphs 25 to 27 wherein the immunisation is against the organism(s) from which said one or more exogenous antigens is derived.

[0267] 29. The method according to any one of numbered paragraphs 20 to 28 wherein the immunoglobulin comprises, consists essentially of, or consists of immunoglobulin in or from colostrum.

[0268] 30. The method according to any one of numbered paragraphs 20 to 29 wherein the complex comprises two or more immunoglobulin molecules.

[0269] 31. The method according to any one of numbered paragraphs 20 to 30 wherein the complex comprises two or more different types of immunoglobulin.

[0270] 32. The method according to numbered paragraph 31 wherein the two or more different types of immunoglobulin are two or more classes or subclasses of immunoglobulin. The method according to any one of numbered paragraphs 20 to 32 wherein two or more of the immunoglobulins have differing binding affinity and / or differing binding specificity. A method of preventing or treating a bacterial infection or a disease or condition caused by or associated with a gram-negative bacterial infection, the method comprising administering to a subject a complex according to any one of numbered paragraphs 1 to 13 or a pharmaceutical composition according to any one of numbered paragraphs 17 to 19. The method of numbered paragraph 34, wherein the bacterial infection is a gram-negative bacterial infection. The method according to any one of numbered paragraphs 34 to 35, wherein the lipopolysaccharide comprises one or more bacterial lipopolysaccharides. The method according to any one of numbered paragraphs 34 to 36, wherein the treating or preventing comprises treatment, prevention, or amelioration of one or more symptoms of or associated with bacterial infection. The method according to any one of numbered paragraphs 34 to 37, wherein the bacterial infection is causative of or associated with mastitis. The method according to any one of numbered paragraphs 35 to 38, wherein the gram-negative bacteria is a bacteria selected from the group consisting of Escherichia spp., Pasteurella spp., Serratia spp., Klebsiella spp., Salmonella spp., and Campylobacter spp.. The method according to any one of numbered paragraphs 35 to 39, wherein the administration is of an amount effective to reduce endotoxicity associated with said bacterial infection. The method according to any one of numbered paragraphs 20 to 40, wherein the immunoglobulin comprises, consists essentially of, or consists of immunoglobulin from colostrum. The method according to any one of numbered paragraphs 20 to 41, wherein the immunoglobulin comprises, consists essentially of, or consists of IgG and / or IgA from colostrum. The method according to numbered paragraph 41 or 42, wherein the colostrum is bovine colostrum, including pooled bovine colostrum. The method according to any one of numbered paragraphs 20 to 43, wherein administration to the subject comprises parenteral administration. The method according to any one of numbered paragraphs 20 to 44, wherein administration to the subject comprises topical or oral administration, or administration to a mucosal tissue. The method according to any one of numbered paragraphs 20 to 45, wherein administration to the subject comprises instillation, for example intra-mammary instillation. The method according to any one of numbered paragraphs 20 to 46, wherein administration to the subject comprises administration ex vivo. 48. The method according to numbered paragraph 47, wherein ex vivo administration comprises administration to a sample from the subject, followed by administration of at least some of the sample to the subject.

[0271] 49. The method according to any one of any one of numbered paragraphs 20 to 48, wherein administration of the complex does not elicit an inflammatory response in the subject.

[0272] 50. A complex according to any one of numbered paragraphs 1 to 13 or a pharmaceutical composition according to numbered paragraph 17 or 19, for use in or when used in eliciting an immune response in a subject in need thereof or in immunising a subject in need thereof.

[0273] 51. A complex according to any one of numbered paragraphs 1 to 13 or a pharmaceutical composition according to numbered paragraph 17 or 19, for use in or when used in the treatment or prevention of a microbial infection or of a disease or condition caused by or associated with a microbial infection.

[0274] 52. A complex according to any one of numbered paragraphs 1 to 13 or a pharmaceutical composition according to numbered paragraph 17 or 19, for use in or when used in the treatment or prevention of a bacterial infection of gram-negative bacteria or of a disease or condition caused by or associated with a bacterial infection of gram-negative bacteria.

[0275] 53. Use of a complex comprising immunoglobulin and lipopolysaccharide in the preparation of a medicament for use in eliciting an immune response in a subject in need thereof or in immunising a subject in need thereof, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

[0276] 54. Use of a complex comprising immunoglobulin and lipopolysaccharide in the preparation of a medicament for use in treating or preventing a microbial infection or a disease or condition caused by or associated with a microbial infection, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

[0277] 55. Use of a complex comprising immunoglobulin and lipopolysaccharide in the preparation of a medicament for use in treating or preventing a bacterial infection or a disease or condition caused by or associated with a bacterial infection of gram-negative bacteria, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

[0278] 56. The use according to numbered paragraph 53 or 55, wherein the complex is a complex according to any one of numbered paragraphs 1 to 13.

[0279] 57. A method of treating or preventing a disease or condition caused by or associated with a microbial infection in a subject in need thereof, or of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex according to any one of numbered paragraphs 1 to 13 or a pharmaceutical composition according to numbered paragraph 17 or 19.

[0280] 58. A method of treating or preventing a disease or condition caused by or associated with a bacterial infection in a subject in need thereof, or of treating or preventing a bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex according to any one of numbered paragraphs 1 to 13 or a pharmaceutical composition according to numbered paragraph 17 or 19.

[0281] 59. The method according to numbered paragraph 57 or 58, wherein the method comprises administering to the subject an additional therapeutic agent.

[0282] 60. The method according to numbered paragraph 59, wherein the additional therapeutic agent is an antibiotic selected from the group consisting of aminoglycosides, such as gentamicin, amikacin; beta-lactams; carbapenems, including imipenem, meropenem; cephalosporins, including cefotaxime, ceftazidime; chloramphenicols; fluorquinolones, such as ciprofloxacin, delafloxacin; fosfomycin; penicillins; polymyxins, such as colistin, polymyxin B; glycylcycline, such as tigecycline; sulphonamides, such as co-trimoxazole; tetracyclines, including doxycycline, eravacycline, minocycline, omadacycline; and ureidopenici Ilins, such as piperacillin; and any combination of two or more thereof, including combinations such as ceftolozane / tazobactam, ceftazidime / avibactam, meropenem / vaborbactam, and imipenem / cilastatin / relebactam.

[0283] Additional agents and activities

[0284] While examples of the methods and compositions contemplated herein employ antibodies, including species-specific antibodies, other agents capable of binding to LPS / Ig-containing complexes in which an antibody is present are also suitable for use. Specifically contemplated examples include Protein A, Protein G, and Protein A / G, each of which have been used extensively for the binding to and / or purification of antibodies. While full length Protein A or Protein G can be used, in the context of antibody binding a truncated recombinant form is typically used.

[0285] Protein A (SpA) in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene. SpA is a 42 kDa protein comprising several regions with different functions: The signal sequence (S region) in the N-terminal part is followed by four or five highly homologous immunoglobulin G (IgG)- binding domains in tandem (the E, D, A, B, and C regions). The C-terminal region, also referred to as the X region, has two domains: (i) a repeat region XR, consisting of variable repeats with mostly octapeptide structures, and (ii) the XC region, consisting of a conserved sequence including an LPXTG- binding motif, which confers anchoring to the cell wall. It has been reported that SpA interacts with human IgG by binding to the Fc part of the immunoglobulin, and further that SpA can bind to other host structures, such as the von Willebrand factor to promote adhesion to platelets.

[0286] Protein G originates in group C and G Streptococcal bacteria. The native protein is a 56-kDa or 58-kDa polypeptide with multiple binding sites for immunoglobulins as well as a binding site for albumin. The most commonly used recombinant form of the protein is a truncated version that has the albumin binding site removed but that retains the IgG-binding capabilities of the native molecule. Protein G binds to antibodies through the heavy chains in the region of the Fc fragment, but at a different site than that of Protein A. Protein A and Protein G exhibit different antibody binding specificities and affinities, and thus offer options in binding and purifying antibodies, depending on the type of antibody desired.

[0287] A chimeric fusion protein consisting of the combination of Protein A and Protein G, called Protein A / G, merges the advantages of both protein specificities into one molecule. As a consequence, Protein A / G is particularly contemplated as a reagent capable of binding LPS / Ig complexes as disclosed herein.

[0288] Since most IgG type antibodies bind to Protein A, G, or A / G via their Fc fragments on the heavy chains, the antigen binding sites at the ends of the Fab fragments remain open to interact with antigens. As a consequence, binding of antigen-bound IgG antibodies (i.e., IgG antibody:antigen complexes) is readily achieved using these proteins.

[0289] In certain examples of methods described herein, detectably-labelled reagents, such as detectably-labelled Protein G, Protein A, and Protein A / G, typically being recombinant forms of each, are used. These are readily available from commercial suppliers. For example, horseradish peroxidase conjugated Protein G (HRP-Protein G, cat# M00090) can be sourced from GenScript (NJ, USA), with HRP-Protein A (cat# M00089) also being available from this supplier. ThermoFisher Scientific supplies a range of recombinant Protein A / G and Protein G conjugates suitable for the detectable binding of antibodies, including peroxidase- or alkaline phosphatase-conjugated Protein A / G and Protein G, with biotinylated conjugates also being available from the same supplier, while fluorescein conjugated Protein G is available, for example from Rockland Immunochemicals Inc., PA, USA.

[0290] In various examples, the complex comprises one or more antigens, such as one or more exogenous antigens other than said immunoglobulin and said lipopolysaccharide.

[0291] In one example, one or more of said immunoglobulin comprises one or more exogenous antigens, such as an antigen or epitope from protein other than an immunoglobulin. In one example, the exogenous antigen is from an organism to which an immunological response, such as immunity, is desirable elicited. In one example, the exogenous antigen is from the same bacterial species or strain as the lipopolysaccharide comprising the complex is from. In one example, the exogenous antigen is from an organism other than that from which the lipopolysaccharide comprising the complex is from.

[0292] As exemplified herein, representative complexes as contemplated herein have been identified in and isolated from bovine colostrum.

[0293] Thus, in certain examples, the therapeutic agent is, the composition comprises, or the treatment comprises the administration of, a complex as herein contemplated from colostrum.

[0294] Colostrum

[0295] Colostrum is produced by most mammals just prior to giving birth. It is a nutrient- and bioactiverich 'first milk', shown in various species to comprise immune cells, immunoglobulins, growth factors and cytokines. Other bioactives, including lactoferrin, lysozyme, lactoperoxidase, and anti-inflammatory bioactives have also been shown to be present in colostrum.

[0296] Without wishing to be bound by any theory, the inventors believe that as exemplified herein in the Examples, complexes comprising colostrum immunoglobulins bound to LPS do not exhibit the degree of endotoxicity usually observed with an equivalent amount of (non-complexed) LPS, and that such complexes are effective in removing and / or attenuating and / or masking the endotoxic activity associated with LPS. While bovine colostrum has been extensively characterized herein in the Examples, the inventors believe, again without wishing to be bound by any theory, that any source of colostrum will comprise Ig / LPS complexes as herein contemplated.

[0297] Particularly convenient sources of colostrum include those from dairy animals, such as caprine, ovine, camelids, equine, and bovine animals.

[0298] Administration and formulation

[0299] It will be apparent to a person skilled in the art that the formulation of the complexes, agents, and / or compositions described herein will depend on the method of administration. For example, the complexes, agents, and / or compositions described herein are in certain examples formulated as creams, lotions, tablets, capsules, pellets, dispersible powders, granules, suppositories, syrups, elixirs, lozenges, injectable solutions, sterile aqueous or non-aqueous solutions, suspension or emulsions, patches and the like.

[0300] For example, in various examples the complexes, agents, and / or compositions described herein are formulated as tablets, capsules, pellets, dispersible powders, granules, solutions, syrups, suspensions or emulsions.

[0301] In exemplary examples, the complexes, agents, and / or compositions described herein are formulated for instillation, for example intramammary instillation.

[0302] In other examples, the complexes, agents, and / or compositions described herein are formulated as a solid dosage form, such as tablets, capsules or pellets.

[0303] The formulations that are suitable for a particular method of administration will be apparent to those skilled in the art.

[0304] As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the complexes, agents, and / or compositions described herein, care should be taken to ensure that the activity of the agent or composition is not destroyed in the process and that the agent or composition is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the agent or composition by means known in the art, such as, for example, microencapsulation. Similarly, the route of administration chosen should be such that the agent or composition reaches its site of action.

[0305] Those skilled in the art may readily determine appropriate formulations for the complexes, agents, and / or compositions described herein using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art, and examples of preferred pH ranges for specific complexes, agents, and / or compositions described herein are provided in the Examples. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing complexes, agents, such as methionine or sulphite, and metal chelators such as EDTA.

[0306] The complexes, agents, and / or compositions described herein may be prepared in parenteral dosage forms, including those suitable for intramammary, intravenous, intrathecal, and intracerebral or epidural delivery. The pharmaceutical forms suitable for injectable use include sterile injectable solutions, suspensions, or dispersions, instillable solutions, suspensions, or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable or instillable solutions, suspensions, or dispersions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi.

[0307] The solvent or dispersion medium for the injectable solution, suspension, or dispersion may contain any of the conventional solvent or carrier systems for compound actives, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include agents to adjust osmolality, for example, sugars or sodium chloride. In various examples, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable or instillable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable or instillable use may be delivered by any appropriate route including intramammary, intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion.

[0308] Sterile injectable solutions are prepared by incorporating the active agent in the required amount in the appropriate solvent with various of the other ingredients such as those enumerated above, as required, followed by filtered sterilization. Generally, dispersions or suspensions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable or instillable solutions, preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-fi Itered solution of the active ingredient plus any additional desired ingredients.

[0309] Other pharmaceutical forms include oral and enteral formulations, in which the active agent may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active agent may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active agent in such therapeutically useful compositions is such that a suitable dosage will be obtained.

[0310] The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of Wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active agent(s) may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active agent to specific regions of the gut.

[0311] Liquid formulations may also be administered enterally via a stomach or oesophageal tube.

[0312] Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible and for specific diseases or infections is specifically contemplated for the complexes, agents, and / or compositions described herein to be administered intramammarily, topically, intranasally, intravaginally, intraocularly and the like. Other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions, are also contemplated.

[0313] The complexes, agents, and / or compositions described herein may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases. The complexes, agents, and / or compositions may also be administered using a nebuliser.

[0314] Pharmaceutically acceptable vehicles and / or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0315] In certain circumstances, for example for use in the treatment of particular subjects, it is especially advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle. The specification for the dosage unit forms contemplated herein are dictated by and directly dependent on (a) the characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

[0316] As mentioned above the principal active agent may be compounded for convenient and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active agent in amounts ranging from 0.25 pg to about 2000 mg or more. Expressed in proportions, the active compound may be present in from about 0.25 pg to about 2000 mg / mL of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

[0317] The term "therapeutically effective amount" as used herein refers to a dose of an agent or composition sufficient to provide a concentration high enough to effect the desired result. For example, in certain examples a therapeutically effect amount is a dose of an agent described herein sufficient to result in one or more of the following; the treatment, prevention, inhibition or reversal of a gramnegative bacterial infection and / or of a disease or condition caused by or associated with such infection, including one or more symptoms thereof or process associated therewith, such as LPS-mediated endotoxicity.

[0318] The therapeutically effective amount of an agent will in certain examples be affected by a number of factors and can be adjusted based on these factors. For example, the therapeutically effective dose may be affected by the bodyweight of the subject, metabolic capacity and synergy between combinations of actives administered. The dose that can be administered to a subject may also be affected by other factors such as interactions with other medicines that the subject is taking and severity of / ability to tolerate any side effects of the complexes, agents, and / or compositions administered.

[0319] In some examples, the desired result to be achieved by the therapeutically effective amount comprises the slowing of disease progression, an improvement in the quality of life of the subject and / or an increased rate of survival.

[0320] In various examples, the desired result to be achieved by the therapeutically effective amount is the amelioration of one or more symptoms associated with a gram-negative bacterial infection and / or of a disease or condition associated with such infection, such as sepsis, organ failure, and the like.

[0321] Also contemplated herein is a pharmaceutical composition comprising a therapeutically effective amount of an agent as hereinbefore defined, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent.

[0322] The term "composition" is intended to include the formulation of an active ingredient with a carrier, such as an encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.

[0323] The complexes, agents, and / or compositions described herein are in certain examples administered individually, and in other examples are administered in combination, either with other complexes, agents, and / or compositions as described herein, or with other therapeutic agents, or both. Such combinations may allow for separate, sequential or simultaneous administration of the complexes, agents, and / or compositions as herein described with the other active ingredient(s). The combinations may be provided in the form of a pharmaceutical composition.

[0324] For example, in some examples two or more complexes or other agents described herein are administered to a subject together. When administering combinations of complexes or agents to a subject, the dose of each individual complex or agent may be less than the therapeutically effective amount such that the combined dose of the two or more complexes or agents is equal to or greater than the therapeutically effective amount.

[0325] Furthermore, in some examples one or more complexes described herein is administered to a subject together with one or more additional agents. The additional agents may be, for example, agents that treat, inhibit and / or reverse a gram-negative bacterial infection and / or of a disease or condition associated with such infection, and / or causing the amelioration, reduction, remission or regression of a gram-negative bacterial infection or a disease or condition associated with such infection or one or more symptoms or side effects thereof, or ameliorate, reduce or reverse one or more processes associated with such infections or disease, such as the immunological response to gram-negative bacterial infection.

[0326] In certain examples, the complexes, agents, and / or compositions contemplated herein are administered together with one or more immunomodulators.

[0327] In certain examples, the complexes, agents, compositions, and methods described herein enable the use of therapies and / or therapeutic regimens that may otherwise be contraindicated, for example due to an increased risk of liberating LPS and attendant endotoxicity, such as that that may occur when bacteriocidal agents such as some antibiotics are used. In such examples, the complexes, agents, compositions, and methods disclosed herein can be utilised in combination with, for example, the administration of one or more antibiotics effective to treat gram-negative bacterial infections. For example, particularly contemplated therapeutic methods comprises administration of one or more agents selected from the group consisting of aminoglycosides, such as gentamicin, amikacin; beta- lactams; carbapenems, including imipenem, meropenem; cephalosporins, including cefotaxime, ceftazidime; chloramphenicols; fluorquinolones, such as ciprofloxacin, delafloxacin; fosfomycin; penicillins; polymyxins, such as colistin, polymyxin B; glycylcycline, such as tigecycline; sulphonamides, such as co-trimoxazole; tetracyclines, including doxycycline, eravacycline, minocycline, omadacycline; and ureidopenicillins, such as piperacillin; and any combination of two or more thereof, including combinations such as ceftolozane / tazobactam, ceftazidime / avibactam, meropenem / vaborbactam, and imipenem / cilastatin / relebactam.

[0328] In some examples the one or more additional agents may for example be agents that reduce side effects associated with the complexes, agents, or compositions described herein.

[0329] When one or more complexes described herein are administered in combination with one or more additional agents, the dose of each complex or agent administered may be less than the dose that would be administered if each complex or agent was administered separately.

[0330] In some examples when one or more complexes described herein are administered in combination with one or more additional agents, the dose of each complex or agent administered may be greater than the dose that would be administered if each complex or agent was administered separately. For example, this may be the case if the one or more additional agents leads to reduced side effects, allowing a greater dose to be tolerated.

[0331] It will be appreciated that the likelihood that prevention and / or treatment of a gram-negative bacterial infection or a disease or condition caused by or associated with a gram-negative bacterial infection is successful will usually be heavily time dependent. For prophylactic treatments, desirably administration of a complex or composition as herein contemplated is carried out prior to exposure to and / or infection of the subject by the bacteria. For other treatment, the speed with which an effective amount of a therapeutic agent can be administered to a subject in need thereof can be of critical importance to treatment efficacy. Indeed, early diagnosis or detection of disease will in many examples allow for rapid treatment, and in certain circumstances preventative treatment. In other circumstances, disease prevalence, such as the identification of the presence of disease-causing bacteria in a particular locus, environment, or population, supports prophylactic treatment.

[0332] Diagnosis of disease and monitoring treatment

[0333] In certain examples however, even when disease-causing bacteria has been identified as being present in a population or environment, there may be a reluctance to prophylactically treat one or more subjects not yet manifesting infection or symptoms of infection. Bovine mastitis is one such disease, where prophylactic treatment of all individuals in a herd would in many cases lead to reduced productivity and is thus generally avoided if possible.

[0334] Accordingly, in certain examples, the rapid detection of bacterial infection and / or the discrimination between exposure to gram negative bacteria and the acquisition of acquired immunity by an individual, such as a newborn mammal who has ingested colostrum, using the complexes, agents, compositions, and / or methods as herein described is particularly contemplated. For example, detection of LPS / Ig complexes in the systemic circulation of a subject, for example of a neonatal bovine subject, with no apparent bacterial infection and / or no apparent disease or condition caused by or associated with bacterial infection is anticipated by the applicant to be informative in determining whether such a subject is at reduced risk of infection or disease, for example due to acquired immunity mediated by exposure to LPS / Ig complexes as herein disclosed present in colostrum. Without wishing to be bound by any theory, the inventors believe a rapid prophylactic approach such as this is likely to reduce the overall LPS endotoxicity and disease impact, and in turn will facilitate or enable the subject's own immune system to respond.

[0335] Advantageously, the diagnosis of a disease or condition in a subject or the identification of a subject at increased risk of having or developing an infection or a disease or condition, wherein the disease or condition is caused by or associated with the presence of a gram-negative bacteria, will in certain examples be useful to inform and / or monitor the therapeutic approach or regimen used to treat (including to control, reverse, mitigate and / or prevent) such a disease or condition in the subject.

[0336] Thus, in certain examples the therapeutic methods disclosed herein involve or are informed by information provided by one or more diagnostic methods, such as a method that directly or indirectly determines the presence or absence of LPS / Ig complexes in a sample, such as a sample obtained from a subject to be or being treated.

[0337] For example, the LPS / Ig complexes, or the individual components thereof, can be detected using a variety of assays, such as the endotoxicity assays exemplified herein, or an immunological assay, such as ELISA as herein exemplified, or Western Blots, Lateral Flow tests and Biosensors. ELISA tests have the advantage of being comparatively fast and accurate and the tests are amenable to high throughput and automation. Lateral flow tests are advantageously employed where there is no expectation of having ready access to the laboratory equipment necessary for ELISA or other methods, such as in workplace or public event screening and the like. The rapid provision of results from lateral flow tests also enable rapid diagnoses, which in turn can ensure better treatment decisions and / or health outcomes, and can frequently avoid the need for time-consuming or costly sample logistics and laboratory analysis.

[0338] Gram-negative bacterial and bacterial pathogens

[0339] The complexes, compositions, methods and related aspects disclosed herein are useful in the prevention and / or treatment of gram-negative bacterial infections and finds particular application in the treatment of gram-negative bacterial pathogens and as a result the treatment and / or prevention of diseases or conditions caused by or associated with gram-negative bacterial infections.

[0340] Gram-negative bacteria present one of the world's most significant public health problems due to their high resistance to antibiotics. These microorganisms have significant clinical importance in hospitals because they put patients present in the intensive care unit (ICU) at high risk and lead to high morbidity and mortality. Two large groups of gram-negative bacteria, the Enterobacteriaceae and the non-fermenters, are responsible for most clinically relevant isolates. Other gram-negative organisms of clinical relevance exist, including but not limited to Neisseria, Haemophilus spp., Helicobacter pylori, and Chlamydia trachomatis. Gram-negative bacteria suitable for treatment using the methods and compositions described herein thus include the Enterobacteriaceae, Pasteurellaceae and Aeromonas groups.

[0341] Enterobacteriaceae

[0342] Enterobacteriaceae are a heterogeneous group widely dispersed in nature. They account for about 80% of gram-negative isolates with a myriad of disease-causing species in humans and other animals, including urinary tract infections, pneumonia, diarrhoea, meningitis, sepsis, endotoxic shock, and many others. Species of particular concern and which frequently affect humans include Escherichia, Proteus, Enterobacter, Klebsiella, Citrobacter, Yersinia, Shigella, and Salmonella, among others.

[0343] Characteristics of Enterobacteriaceae typically employed in laboratory characterization include their being bacilli, non-sporulated, and having variable motility, are able to grow in the presence and absence of oxygen, are able to ferment glucose, are cytochrome oxidase negative, and can reduce nitrate to nitrite.

[0344] Non-Fermenters

[0345] The non-fermenter gram-negative bacilli are usually encountered in the clinic less frequently than Enterobacteriaceae. However, they are clinically relevant since they cause severe, fatal infections, especially in a hospital environment. They also cause opportunistic diseases in ICU patients who undergo invasive procedures. The main non-fermenter gram-negative microorganisms that cause human disease are Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia pseudomallei, Stenotrophomonas spp., Alcaligenes spp., and Moraxella spp. These are characterised by being aerobic and non-sporulated, are incapable of fermenting sugars and instead use sugars via oxidative metabolism.

[0346] Gram-negative bacteria can cause serious infections and are able to reach almost all systems in the subject organism, including the digestive system, nervous system, urinary system, and bloodstream. These microorganisms readily colonize the intestines, airways, and skin, thereby favouring the spread to other parts of the organism, especially in immunocompromised individuals. Gram-negative bacteria cause infections including pneumonia, peritonitis (inflammation of the membrane that lines the abdominal cavity), urinary tract infections, bloodstream infections including sepsis, wound or surgical site infections, and meningitis.

[0347] Nosocomial infections of the lower respiratory tract are particularly challenging to treat. This is because the pathogenic gram-negative bacteria typically involved are not only responsible for a good portion of these infections, they are non-responsive to antibiotic therapy due to the high resistance rates and the poor penetration of drugs into the lung parenchyma.

[0348] Another major concern is gastroenteritis caused by Enterobacteriaceae, particularly Shigella spp., Salmonella spp., and enteropathogenic E. coli. Gastroenteritis affects millions of people worldwide and is generally related to a lack of sanitation. Bacterial meningitis - a potentially fatal disease if not treated in time - is likewise a major concern both in the community and in the hospital environment. Urinary tract infections are also common, especially in young women. However, these infections became a problem with the widespread emergence of multidrug-resistant bacteria. Bacteremia is an important complication of these infections because of the acquisition of resistance.

[0349] Gram-negative pathogenic bacterial infections suitable for treatment using the methods and related aspects disclosed herein include the following: Brucellosis; Campylobacter infections; Cholera; Escherichia coli E. coli) infections; Haemophilus influenzae infections; Klebsiella infections; Mastitis; Proteus infections, Legionellosis, including Legionnaires' disease; Pertussis; Plague; Pseudomonas infections; Salmonella infections; Shigellosis; Tularemia; Porphyromonas gingivalis infections, Heligobacter infections and Typhoid fever.

[0350] Certain gram-negative bacteria of particular clinical concern are discussed below.

[0351] Brucella infections, frequently referred to as Brucellosis, are sometimes asymptomatic but most commonly result in acute illness, with symptoms including fever, arthralgia, headache, malaise, anorexia, constipation, respiratory tract symptoms and hepatosplenomegaly observed. If not adequately treated, chronic and persistent infections in joints, bone, liver or spleen can result. Brucellosis is often seen in New Zealand in those working with livestock, while internationally the ingestion of unpasteurised goat's cheese is the most common risk factor. Campylobacter bacteria, usually Campylobacter jejuni, cause inflammation of the colon (colitis) that results in fever and diarrhoea. These bacteria are a common cause of infectious diarrhoea and are notifiable diseases / diseases of public health interest in a number of countries.

[0352] In many countries, all strains of Vibrio cholerae (the causative pathogen of Cholera) are notifiable despite only isolates 01 and 0139 having the potential to produce cholera toxin being associated with clinical cholera. Cholera is characterised by vomiting and potentially severe diarrhoea, which may lead to profound dehydration and death. Methods to treat Vibrio cholera are thus of significant interest, particularly in countries in which this pathogen is endemic or widespread.

[0353] Many strains of E. coli are harmless and / or non-pathogenic, being part of the normal gut microflora of mammals including humans. However, pathogenic strains of E. coli are frequently the causative agent of enteric disease and associated diarrhoea or dysentery, while other pathotypes cause extra-intestinal infections such as urinary tract infections or meningitis.

[0354] Legionella bacteria are ubiquitous in many environments, particularly in soil and aquatic environments, and Legionella infections are a common cause of pneumonia. Most cases in New Zealand are caused by L. longbeachae and L. pneumophila. Legionellosis is more common in older people, smokers, chronic disease sufferers and the immunocompromised. The most common clinical manifestation of Legionellosis reported worldwide is Legionnaires' disease, but non-pneumonic disease (such as Pontiac fever, an acute febrile illness usually accompanied by cough), and extrapulmonary disease, which usually manifest as infection of the skin, joints, pericardium or other organs, are thought to be often clinically un- or mis-diagnosed, and thus may be underreported.

[0355] Sepsis and septic shock are life-threatening conditions caused by one or more unregulated host responses to infection. Pathogenic gram-negative bacteria, including E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloaceae, Proteus mirabilis, and Pseudominas aeruginosa, are common causative agents of sepsis and septic shock. Sepsis most commonly occurs as a result of infections in the urinary tract, the lungs, or the peritoneum, but infections in other organs including skin, the soft tissue, and the CNS are observed. Septic shock is usually considered to be a subset of sepsis in which profound abnormalities in the circulatory system, in cellular processes or in metabolic pathways are observed and are associated with an increased risk of mortality.

[0356] In principle, any disease or condition associated with a gram-negative bacterial source of LPS is amenable to treatment using the complexes, compositions, methods and associated aspects disclosed herein, although the prevention of pathogenic bacterial infections and associated diseases such as those discussed above is a particular focus of the present invention.

[0357] Particularly contemplated herein are prophylactic complexes, agents, methods, and compositions comprising or utilising LPS / Ig complexes in which the immunoglobulin is bound to LPS and inhibits one or more biological activities of or associated with said LPS, and in particularly contemplated examples inhibits or reduces the endotoxic activity of gram-negative bacterial lipopolysaccharides, as described herein.

[0358] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples. EXAMPLES

[0359] Example 1: Characterisation of LPS / antibody complex in colostrum samples

[0360] This example presents the characterization of lipopolysaccharide (LPS) / IgG complexes present in bovine colostrum, including the elucidation of the LPS / IgG complexes' ability to attenuate the endotoxicity usually associated with LPS.

[0361] Briefly, post-partum colostrum or milk samples during early acute Escherichia coli mastitis were used to identify, obtain, and characterize LPS / IgG immune complexes and explore how the milk IgG repertoire influences the endotoxic properties of these immune complexes. Selective detection of LPS alone or in the form of LPS / IgG immune complexes enabled the identification of LPS-specific IgG antibodies within the diverse polyclonal antibody repertoire of bovine colostrum.

[0362] Characterization of these LPS / IgG antibody complexes obtained from colostrum established the attenuation of endotoxicity- that is, an absence of endotoxicity usually associated with LPS. In contrast, LPS / IgG complex obtained from mastitis milk samples did not show attenuation, as elevated milk LPS / IgG complex levels during mastitis paralleled endotoxicity capacity and severity of inflammation.

[0363] Mastitis milk samples also demonstrated a reduced ability to bind to antigens within the E. coli LPS glycolipid core (K12-LPS) compared to endotoxin / LPS shed from wild-type E. coli (WT-LPS). Further analysis, using IgG extracts from colostrum and a representative acute mastitis milk sample, revealed a reduced sensitivity for the recognition of K12-LPS by acute mastitis milk IgG compared to colostrum IgG. Moreover, colostrum was shown to contain IgG antibodies that displayed strong avidity for K12- LPS. In addition, soluble WT-LPS / IgG immune complexes generated using colostrum IgG antibodies reduce LPS endotoxicity and attenuate LPS-induced reactive oxygen species within milk granulocytes. This was not observed when using mastitis milk IgG antibodies.

[0364] These novel findings show how the heterogeneity within the IgG repertoire secreted into the bovine mammary gland influences the formation of LPS / IgG complexes that sequentially modulate LPS endotoxic properties to deliver the appropriate physiological (e.g., immune protection of the neonatal calf) and pathological (combat E. coli driven acute mastitis) outcomes.

[0365] Methods

[0366] Bovine milk samples

[0367] Source and selection of milk samples.

[0368] (a) Healthy: Milk from healthy mid-lactating Holstein-Friesian cows was sourced from five separate commercial dairy farms (Manawatu, New Zealand).

[0369] All cows produced milk with a < 100,000 somatic cell count (SCC) / mL as determined by routine herd testing and displayed no symptoms of mastitis. In addition, standard microbiology testing of the milk showed no bacteria growth.

[0370] (b) Acute Escherichia coli mastitis: Milk samples from lactating cows during the early stages of acute coliform mastitis were collected from a dairy farm (Holstein-Friesian herd) with an outbreak of acute mastitis during early lactation. Acute mastitis was diagnosed "on-farm" using clinical and mammary gland assessments. Foremilk testing revealed physical changes to milk. Milk was collected within 24 h from the onset of acute mastitis. Samples for which the presence of E. coli was verified using standard microbiology testing followed by Matrix-Assisted Laser Desorption Ionization Time Of Flight Mass Spectrometry (MALDI-TOF MS) analysis were selected.

[0371] (c) Ethics: Collection and use of milk from healthy cows only required approval from dairy farmers (as samples were collected during their routine milking). No additional ethical consideration was required. Ethical approval for the collection and analysis of acute mastitis milk samples was obtained from Animals Ethics Committee, Massey University / Te Kunenga ki Purehuroa, Palmerston North, NZ. Study entitled "Evaluating an ELISA for identifying whether acute mastitis is caused by E. coli": Ref #AEC 22 / 41, approved 19 / 08 / 2022.

[0372] Milk collection and biochemical indices. fa) Collection: Milk was processed by centrifugation at 6000 x g for 5 mins at 10 °C. The fat-free milk was then frozen -20 °C for future laboratory analysis.

[0373] (b) Endotoxicitv capacity: Milk endotoxicity was assessed using ToxinSensor™ chromogenic LAL endotoxin assay kit (GeneScript USA Inc., Piscataway, NJ USA) as per manufacturer's instructions.

[0374] Milk samples were either boiled (>98 °C) for 10 mins or not-boiled, then diluted in endotoxin-free water prior to assessing endotoxicity capacity. Data was measured against an E. coli endotoxin standard (0.1-5 EU / mL) and expressed as EU / mL.

[0375] (c) Lactate dehydrogenase (LDH) activity: Milk LDH activity was measured using standard kinetic enzyme assay (8, 9). Briefly, diluted milk samples (2.5%, v / v} in 30 mM Tris / HCI buffer pH 8.5 containing 1 mM EDTA (plus or minus 1 mM oxalic acid) were added to 4 mM sodium pyruvate and 0.15 mM NADH diluted in the same buffer and the change in absorbance 340 nm was monitored over 10 mins at 21 °C. Milk LDH activity was calculated from the specific difference in absorbance (minus oxalic acid values) over 10 mins and expressed as pmoles / min.

[0376] ELISA measurement of LPS and LPS / IgG immune complexes

[0377] Basic ELISA format.

[0378] The development of ELISAs involved generating a polymyxin B matrix that maximizes LPS capture, similar to what has been reported by others (10, 11, 12). Here, Polymyxin B sulphate; 50 pg / mL (Fort Richards Laboratories, Auckland, NZ) dissolved in 0.01 M phosphate buffered saline (PBS, pH 7.4) containing 0.1% (v / v) polyethylene glycol biphenol A epichlorohydrin copolymer (PEG-20, Sigma- Aldrich, Merck Pty. Ltd., Auckland, NZ) coated microplates were used as the capture matrix.

[0379] After washing plates with PBS pH 7.4, samples were added to wells and incubated for 90 mins at 37 °C. (I) For LPS; LPS samples or bacterial lysates were diluted in 0.1 M sodium citrate buffer, pH 5.4 containing 0.025% v / v) Triton X100 and incubated for 30 mins at RT. (ii) For LPS / IgG immune complex; bovine colostrum or milk samples were diluted in 0.1 M Tris / HCI buffer, pH 8.4 containing 10 mM EDTA and incubated o / n at 4 °C before transfer to Polymyxin B coated microplates and incubation for 60 mins. Plates were then washed in PBS, pH 7.4 containing 0.1% (v / v) Tween-20 (PBST), followed by blocking non-specific binding sites with 2% (w / v) bovine serum albumin (BSA, fatty acid-free, pH Scientific Ltd., Auckland, NZ) diluted in PBS, pH 7.4 for 30 mins at room temperature (RT), which was then discarded, and plates patted dry on absorbance pads. Next (I) For LPS; colostrum or raw milk (from healthy or acute E. coli mastitis cows) containing IgG antibodies or IgG antibody preparations were diluted in PBS, pH 7.4, added to wells and incubated for 60 mins at 37 °C, followed by washing with PBST. Detection of LPS / IgG antibody complexes was achieved using HRP-conjugates of Streptococcus species protein G or sheep anti-bovine IgG (Invitrogen, Thermo-Fisher, Auckland, NZ), which was added to wells and incubated for 45 mins at 37 °C. The microplate was washed with PBST, and peroxidase activity was measured using 3,3',5,5'-Tetramethylbenzidine (TMB) solution (Invitrogen, Thermo-Fisher, Auckland, NZ) for 15 mins at RT. The reaction was then stopped with 10% (v / v) sulphuric acid and the absorbance read at 450 nm and 650 nm in a SpectroMax plate reader (Molecular Devices, Bio-Strategy, Auckland, NZ). Furthermore, specificity of LPS / IgG immune complex capture by polymyxin B was verified by preincubating samples with 1- 200 pg / mL (final concentrations) bovine lactoferrin for 30 mins at RT prior to measurement by ELISA.

[0380] Calculation of ELISA results.

[0381] (a) For LPS: data was calculated and expressed as differences between 450-650 nm minus background absorbance for HRP-conjugate solutions only.

[0382] (b) For LPS / IgG immune complexes: Dilutions of a selected bovine colostrum concentrate that showed an ELISA absorbance range between 0.1 and 1.0, was used to calculate the LPS endotoxicity equivalence against an E.coli LPS type that was consistently identified (by MALDI-TOF MS) in acute mastitis milk collected for this study. A reproducible standard curve was generated that displayed colostrum dilutions as EU / mL (ng / mL) LPS equivalence. Linear regression analysis of best fit plot (R2>0.97) of colostrum standards between 2.2 - 300 ng / mL LPS equivalence was used to calculate and express LPS / IgG immune complex levels as ng / mL LPS equivalents in unknown milk samples.

[0383] Source of colostrum IgG antibodies and colostrum LPS / IgG immune complexes.

[0384] Bovine colostrum was collected from 2 different sources. (A) Colostrum powder concentrates containing known amounts of IgG antibodies were sourced from New Zealand and 2 other geographical origins; (I) Westland colostrum; 20% total IgG (Westland Milk Products, Hokitika, NZ), (ii) Launchpad 18 colostrum; 18% total IgG (AgriVantage, Chemiplas (NZ) Limited, NZ., (iii) NZ pure colostrum; 20% total IgG (NZ Pure Health, Auckland, NZ), (iv) Coiexan Premium grade colostrum; 60% total IgG (Colostrum Biotec GmbH, Konigsbrunn, Germany), (v) Peptide Ignition colostrum; 20% total IgG (TBR Labs, Spanish Fork, UT, U.S.A.). These colostrum preparations were resuspended in deionized water (filtered through 0.22 um syringe filter) with stirring overnight at 4 °C, to obtain soluble colostrum preparations containing approximately 10 mg / mL total IgG (based upon supplied manufacturer's information). (B) Fresh colostrum was collected aseptically from post-partum cows at the first milking from five individual cows and chilled on ice. Fat-free colostrum was made by centrifugation (3500 x g, 10 min, 10 °C) and filtration through gauze. All colostrum preparations were frozen at -20 °C and not refrozen once thawed.

[0385] Sources of bacterial antigens.

[0386] (a) Purified LPS: E. coll K12-LPS (K12-LPS) was purchased InvivoGen (San Diego, CA, USA.). Lyophilized LPS was resuspended in endotoxin-free deionized water (w / v) of 5 mg / mL and frozen at -20 °C until use. (b) Shed endotoxin / LPS sources: Agar / MacConkey bacterial cultures of milk collected from cows with clinical mastitis (using standard microbiological techniques) followed by identification using MALDI-TOF MS analysis, E.coli, Serratia marcescens, Klebsiella oxytoca, and Pseudomonas aeruginosa, were sub-cultured into liquid broth (LB) (Fort Richards Laboratories Ltd., Auckland, NZ) and grown for 48 h at 37 °C, followed by centrifugation (3500 x g 10 mins, RT) and the pellet resuspended in 0.01 M PBS, pH 7.4 to an absorbance 600 nm of ~ 1. Shed endotoxin / LPS (13) were generated by heating the resuspended bacteria at 80 °C for 30 mins and then sonicated for 1 min at RT, followed by removal of bacterial debris by centrifugation (4000 x g, 10 mins, RT). Samples were frozen at -20 °C until use. The amount of endotoxin / LPS present in the supernatant was determined using the LAL bioassay and calculated in EU / mL or pg / mL. (c) Whole bacteria lysates: Whole bacteria lysates were prepared using microfluidization. Briefly, gram-positive bacteria {Streptococcus uberis, Staphylococcus aureus, coagulase-negative staphylococci) isolated from clinical mastitis milk samples and characterized by MALDI-TOF MS, were grown in liquid broth for 48 h at 37 °C. Bacteria were separated by centrifugation, resuspended in PBS, pH 7.4 and heated at 80 °C for 10 mins. The suspension was then subjected to high pressure homogenization (microfluidization) to generate bacteria antigens. Protein concentration was then measured using a BCA protein assay (Pierce, Thermo-Fisher, Auckland, NZ) and expressed as pg / mL. Samples were frozen at -20 °C until use.

[0387] Milk IgG antibody / E. coll LPS interactions

[0388] E. coli LPS recognition by acute mastitis milk IgG antibodies.

[0389] To explore the differences in IgG specificity within acute mastitis milk samples, doses of K12-LPS and E.coli wild-type (WT)-LPS that showed similar absorbance values when detected by colostrum in the ELISA (i.e., positive control) were selected and used. K12-LPS (2.5 pg / ml) and WT-LPS (100 ng / mL) were incubated with polymyxin-B coated microplates for 90 mins at 37 °C. After washing the plates with PBST and blocking non-specific binding with 2% BSA / PBS, acute mastitis milk (diluted 1: 10 in PBS, pH 7.4) or colostrum preparation containing only IgG antibodies (diluted 1 mg / mL in PBS, pH 7.4) were added to wells and incubated for 60 mins at 37 °C. The ELISA was then completed as described above. Data was calculated as absorbance 450-650nm and shown as bar chart or a histogram depicting the frequency of the absorbance values for K12-LPS or WT-LPS recognition in each acute mastitis milk sample analyzed.

[0390] Evaluation of colostrum and acute E. coli acute mastitis milk IgG antibodies.

[0391] (a) Enrichment of milk IgG antibodies. A modified salting-out technique described by Skalka and colleagues (14) was used to enrich IgG antibodies in (I) a selected bovine colostrum preparation that contained IgG antibodies and undetectable levels of IgA and IgM antibodies, and (ii) a representative acute E. coli mastitis milk sample. Both milk samples contained IgG antibodies that recognized a range of LPS epitopes. After the initial removal of milk fat by centrifugation and gauze filtration, milk casein was precipitated out by acidification; lowering the pH to 4.5 with hydrochloric acid, followed by centrifugation and filtration. Contaminating LPS compounds (including associated proteins) were then removed from milk preparations using a purafix ET-R syringe filter (Filtrox-AG, St. Gallen, Switzerland), followed by neutralizing the milk pH to 7.4 with sodium hydroxide. Milk IgG antibodies were precipitated with ammonium sulphate (final saturation ~ 45%) for 16 h on ice, the pellet after centrifugation was resuspended in 10 mM PBS, pH 7.4 and dialyzed using cellulose membrane, MWCO 14KDa (Sigma- Aldrich, Merck Pty. Ltd., Auckland, NZ) against PBS (4 changes of buffer) and then passed through protein concentrators MWCO 30 KDa (Thermo-Fisher, Auckland, NZ). Protein content was measured using a BCA protein assay (Pierce, Thermo-Fisher, Auckland, NZ). Milk enriched IgG preparations were adjusted to 10 mg / mL with PBS using absorbance 280 nm and extinction coefficient for IgG with comparison to bovine IgG standards. Qualitative analysis of milk IgG antibody preparations (nonreduced and reduced conditions) by polyacrylamide gel electrophoresis, using standard methodologies, were used to ensure the presence of IgG, no detection of IgA or IgM, using appropriate Ig standards.

[0392] (b) Milk IgG antibody titer. In these experiments, again, doses of K12-LPS and WT-LPS that showed similar absorbance values when detected by IgG antibodies within colostrum were selected and used. K12-LPS (2.5 pg / mL) and WT-LPS (100 ng / mL) were incubated with polymyxin-B coated microplates for 60 mins at 37 °C. After washing the plates with PBST and blocking non-specific binding with 2% BSA / PBS, colostrum, and acute mastitis milk IgG extracts (0.0045-5 mg / mL) diluted in PBS, pH 7.4 were added to wells and incubated for 60 mins at 37 °C. The ELISA was then completed as described above using HRP-protein G to detect LPS / IgG complexes. Data were calculated as absorbance 450-650nm.

[0393] (c) Avidity of LPS / IgG antibody interaction. Colostrum and acute mastitis milk IgG antibodies dilutions that achieved an absorbance 450-650 nm of ~ 0.5 for the recognition of either K12-LPS (2.5 pg / mL) or WT-LPS (100 ng / mL) by ELISA was used to examine the avidity of LPS / IgG antibody interaction (15). Briefly, the LPS / IgG antibody complexes formed in the assays were incubated with 0.1- 3 M urea (diluted in PBS and adjusted to pH 7.4) for 30 mins at 37 °C. Plates were then washed with PBST, and the ELISA completed as described above using HRP-protein G to detect LPS / IgG complexes. Data were calculated as absorbance 450-650 nm and expressed as % of control (no urea).

[0394] Generation of LPS / IgG immune complexes.

[0395] (a) Immune precipitation of colostrum IgG antibodies and K12-LPS. Optimization IP experiments measuring protein content (i.e., change in absorbance 280 nm) in resuspended immune precipitates, after a 2 h incubation at 37 °C, followed by centrifugation at 6000 x g, 10 mins, RT, identified LPS / IgG antibody equivalence using 100 ng / mL K12-LPS and 1 mg / mL colostrum IgG extract. Subsequent immune precipitation experiments were performed using K12-LPS (3.1- 1000 ng / mL) and 1 mg / mL colostrum IgG antibody extract. Immune precipitants were resuspended in endotoxin-free deionized water and assessed for (I) protein content using absorbance 280 nm and BSA standards, (ii) LPS / IgG immune complex levels measured by ELISA as described above, and (iii) endotoxicity capacity using LAL bioassay and E. coll LPS standards (see above).

[0396] (b) Immune precipitation of milk IgG antibodies and WT-LPS. This approach was used to generate soluble LPS / IgG immune complexes. Prior to immunoprecipitation experiments, the amounts of colostrum and acute mastitis milk IgG extracts were optimized to achieve similar absorbance values in the ELISA for the recognition of WT-LPS (100 ng / mL). Using a modified method described by Fossati and colleagues (16), IgG antibody extracts of colostrum (0.2 mg / mL) and acute mastitis milk (1 mg / mL) were incubated with 3.1-200 ng / mL for 1 h at 37 °C, followed by centrifugation at 6000 x g for 10 mins, RT. Collected supernatants were assessed for LPS / IgG immune complex levels and then applied to evaluate (i) WT-LPS endotoxicity using LAL assay (as described above) and (ii) changes in reactive oxygen species (ROS) in milk granulocytes.

[0397] Milk granulocytes

[0398] Isolation of bovine milk granulocytes.

[0399] Milk granulocytes were isolated using a modified method described by Mehrzad and co-worker (17). Briefly, pooled raw milk collected from healthy cows within 2 h of milking was diluted 1: 1 in ice- cold 10 mM PBS, pH 7.2 and centrifuged at 600 x g for 10 mins, 10 °C. Milk supernatant and milk fat were removed, and the pellet resuspended in PBS and washed by stepwise centrifugation at 300 x g and 200 x g for 10 mins, 4 °C. The cell pellet was resuspended in PBS (containing ImM CaCl2 and MgCl2 and 0.5 mg / ml gelatine) adjusted to 2xl06cells / mL and stored on ice for maximum of 2 h. Quality I viability of milk granulocytes were assessed using microscopy and trypan blue staining. Only milk granulocyte preparations showing minimal cell debris and viability > 90% were used to assess oxidative status in milk granulocytes.

[0400] Monitoring milk granulocyte oxidative status.

[0401] Changes in milk granulocyte oxidative status (ROS generation) were monitored using 2', 7'- dichlorodihydrofluorescein diacetate (H2DCFDA) based on a method reported by Varricchi and colleagues (18). H2DCFDA (100 pM final concentration) was incubated with milk granulocytes (~ 2xl06cells / mL in PBS buffer) in the dark for 30 mins at 37 °C. Granulocytes were washed with PBS buffer and resuspended to ~ 2xl06cell / mL in the PBS buffer plus a subthreshold dose (20pM) Fe2+SO4 to facilitate any changes in pro-oxidative status (19). Changes in cellular fluorescent intensity (AFI); excitation 492nm, emission 527nm, were monitored over 30 mins at 37 °C in a SpectroMax M5 plate reader (Molecular Devices, San Jose, CA, USA). Data were calculated as AFI minus background (PBS buffer only) and expressed as AFI or AFI after 20 mins. The following experiments were performed within 2 h of preparing the H2DCFDA- loaded milk granulocytes; (i) direct addition of WT-LPS (3.1-1000 ng / mL final concentrations) to H2DCFDA loaded granulocytes, (ii) preincubation of H2DCFDA loaded granulocytes with N- acetylcysteine (0.5-10 mM final concentrations) for 30 mins in the dark at 37 °C prior to monitoring AFI after 20 mins in the absence or presence of 200 ng / mL WT-LPS, and (iii) IP supernatants (final dilution 1 : 10), WT-LPS (3.1-200 ng / mL, final concentrations) or PBS buffer were added to H2DCFDA loaded milk granulocytes and the AFI was monitored over 20 mins.

[0402] Statistical analysis

[0403] Data were analyzed using Microsoft Excel Analysis ToolPak statistical software. Results using milk indices, specific milk LPS IgG antibodies alone or in combination with LPS, and immune precipitation variables are expressed as mean ± standard deviation (SD) and / or standard error of the mean (SEM). Statistical significance for the comparison between these data variables was assessed using (i) two-way ANOVA, with a significant result (P<0.01) in this test followed by Tukey post-ftoc analysis set at P<0.01, 95% confidence level or (ii) paired Student's t-tests, set at confidence 95%, P<0.05. Linear regression analysis between soluble LPS / IgG immune complex levels and the two independent variables endotoxicity, or LDH activity, was set at P<0.01 with confidence level of 95%.

[0404] Results

[0405] Bovine colostrum attenuates LPS endotoxicity and contains IgG antibodies that recognize a wide repertoire of LPS antigenic epitopes

[0406] The attenuation of LPS endotoxicity by milk components is essential to minimizing harm to the mammary gland (20). Here we assessed endotoxicity capacity (Figure 1A), using the LAL bioassay, of five different bovine colostrum concentrates (2.5 mg / mL). The endotoxicity capacity detected in these colostrum preparations was highly variable ranging from 0 - 396 EU / mL (mean EU / mL ± SD 53.68±25.62), which increased ~ 7-fold (mean EU / mL ± SD 383.34 ± 97.72, P<0.01) when the colostrum preparations were boiled, indicating the presence of heat sensitive LPS / protein complexes.

[0407] The presence of colostrum IgG antibodies that specifically recognize the conserved LPS lipid A / KDO domain (which contains the endotoxic properties) to form LPS / IgG immune complexes able to mask endotoxic properties was investigated. Using a LPS source, K12-LPS (an E. coli mutant bacterium LPS that contains the lipid A / KDO glycolipid core but lack species specific O-antigens), the presence of a nti- K12-LPS IgG antibodies in bovine colostrum samples from three different geographical locations, Europe, U.S.A., New Zealand was examined (Figure IB). Here a modified ELISA that optimized the interaction between the phosphate group within the LPS glycolipid core and polymyxin B (10, 11, 12) was used to evaluate IgG antibody titer for the K12-LPS. A dose-dependent increase in absorbance using colostrum preparations from three different geological origins was observed. Colostrum dilutions of 0.1 mg / mL (total IgG) showed mean absorbances (± SD) of 0.57±0.05, 0.24±0.04 and 0.24±0.06 for colostrum preparations #1, #2, and #3, respectively. In contrast, an equivalent dilution of a pooled freeze-dried milk preparation from healthy lactating cows showed a mean absorbance of 0.01±0.00. Furthermore, the IgG antibodies present in colostrum preparation #1 displayed a 2.3-fold increased specificity (PcO.Ol) for K12-LPS compared the other colostrum preparations.

[0408] A bovine colostrum preparation (enriched in IgG antibodies) was then used to examine the IgG specificity for the recognition of LPS epitopes (Figure 1C). This particular colostrum preparation (~ 0.2 mg / mL total IgG content) was found to contain IgG antibodies that recognized both K12-LPS in a dosedependent manner (18 ng / mL - 5 pg / mL), and shed LPS extracted from WT-LPS epitopes (1 ng / mL - 1 pg / mL). This bovine colostrum preparation contained IgG antibodies that specifically recognized a 150 ng / mL dose of WT-LPS (mean absorbance ± SD of 0.68±0.09) with a ~ 7-fold greater sensitivity (PcO.001) than for the equivalent dose of K12-LPS (mean absorbance ± SD of 0.10±0.03). Moreover, this colostrum IgG preparation showed differential specificity and sensitivity for the recognition of shed endotoxin / LPS compounds from other gram-negative bacteria; Serratia spp., Klebsiella spp. and Pseudomonas spp. (data not shown).

[0409] In contrast, antigens present in whole bacterial lysates prepared from gram-positive bacteria primarily causing clinical mastitis; Streptococcus uberis, Staphylococcus aureus and coagulase-negative staphylococci, were not detected (Figure 1C). A 5pg / mL dose of the three lysates showed mean absorbances ± SD of 0.09±0.01, 0.014±0.02 and 0.01±0.01, respectively. However, binding to antigens present in all three gram-positive bacterial lysates in a direct ELISA format using the same amount of colostrum IgG antibodies was verified (data not shown).

[0410] Bovine colostrum contains IgG antibodies that support the formation of LPS / IgG immune complexes to attenuate endotoxicity capacity

[0411] A modified ELISA format specifically designed to detect soluble LPS / IgG immune complexes was established. Measurement of soluble LPS / IgG immune complexes (Figure 2A) in five separate bovine colostrum powder concentrates (adjusted to 1 mg / mL total IgG) showed absorbance variations ranging between 0.11 ± 0.02 to 0.78 ± 0.11, mean absorbance ± SD (4 replicates). Soluble LPS / IgG immune complex levels were also detected in fresh post-partum colostrum collected aseptically from five individual cows. A 1:5 dilution of these colostrum samples showed absorbance values ranging from 0.11 ± 0.05 to 0.39 ± 0.07, mean absorbance ± SD (4 replicates).

[0412] The identification of endogenous soluble LPS / IgG complexes in bovine colostrum enabled the development of a standard curve to express soluble LPS / IgG immune complexes as ng / mL LPS equivalence. A representative standard curved (Figure 2B) showed mean absorbances ± SD of between 10 (0.27±0.08) and 300 (0.86±0.16) ng / mL LPS equ., respectively. Boiling (>98 °C for 10 mins) the colostrum abolished (PcO.Ol) detection of LPS / IgG immune complexes (mean absorbances ± SD of 0.03±0.01 and 0.03±0.01 for 10 and 300 ng / mL endotoxicity equivalents, respectively.

[0413] Specificity of soluble LPS / IgG immune complex was assessed using bovine lactoferrin (Figure 2C), which binds to the LPS glycolipid core (21) preventing the immune complex being captured by polymyxin B in the ELISA. Preincubation of 2.5 mg / mL of selected bovine colostrum preparation with bovine lactoferrin for 30 mins at RT prior to ELISA caused a dose-dependent inhibitory effect on detection of soluble LPS / IgG complexes. A dose of 5 pg / mL bovine lactoferrin caused ~ 50 % inhibition (PcO.001) compared to an equivalent amount (w / v) of skim milk power (mean ng / mL LPS equ. 40.21±1.43 vs. 74.24±17.90, lactoferrin vs. skim milk).

[0414] Taken together, these experiments demonstrate the presence of soluble LPS / IgG complexes in bovine colostrum. Next, immune precipitation of K12-LPS (i.e., the LPS functional glycolipid core) with an IgG antibody extract of colostrum was performed to assess LPS endotoxicity masking capability (Figure 3). Preparative analysis (data not shown) of the colostrum IgG extract, by ELISA, showed no detectable LPS / IgG immune complexes or free LPS. Immune precipitation of K12-LPS (1-1000 ng / mL) together with 1 mg / mL colostrum IgG antibody extract showed a bell-shaped curve of precipitated protein (Figure 3A) with equivalence achieved using 100 ng / mL K12-LPS. This was significantly (P<0.01) increased from baseline protein levels, mean mg / mL ± SD, 0.21 ± 0.02 vs. 0.07 ± 0.00, 10 ng / mL K12-LPS vs. buffer, respectively.

[0415] Measurement of LPS / IgG immune complexes (Figure 3B) in resuspended precipitants (not boiled) also showed a bell-shaped curve, with equivalence reached using 100 ng / mL K12-LPS. This was also significantly (P<0.05) greater than baseline levels; absorbance mean ± SD of 0.51 ± 0.3 vs. 0.199 ± 0.04, 100 ng / K12-LPS vs. baseline, respectively. Heat denaturation significantly (P<0.05) reduced the ability to detect LPS / IgG immune complexes; absorbance mean ± SD of 0.22 ± 0.04 for 100 ng / mL K12-LPS. Endotoxicity capacity, assessed by LAL bioassay, showed no overall changes in resuspended immune precipitants (Figure 3C). Heat treatment, however, caused a marginal increase in endotoxicity capacity. Precipitation of colostrum IgG antibodies with 50 or 100 ng / mL K12-LPS (mean EU / mL ± SD 224.68±109.73 vs. 278.55±188.55, respectively) exhibited significantly (P<0.05) higher endotoxicity capacity once boiled than found in not boiled immune complexes (mean EU / mL ± SD of 139.26±36.34 vs. 137.5±37.89, respectively).

[0416] Soluble milk LPS / IgG immune complex levels detected in early acute E. coll mastitis correlate with increased milk endotoxicity capacity and LDH activity

[0417] The level of soluble LPS / IgG immune complexes (Figure 4A) in milk collected from cows during early acute E. coli mastitis (n=59 separate milk samples) was variable (mean ng / mL LPS equivalence ± SD of 41.32±73.10) but significantly (P<0.01) higher than the levels measured in healthy cow's milk (n= 87 separate milk samples, mean ng / mL LPS equivalence ± SD of 1.21±4.19). Assessment of endotoxicity capacity (Figure 4BI) in the same set of milk samples revealed large variations within both the healthy and acute mastitis milk samples. Milk endotoxicity capacity within milk samples was again variable in both healthy cows (mean EU / mL ± SD of 28.35±23.45) and acute mastitis milk (mean EU / mL ± SD of 98.25±243.58). Milk endotoxicity capacity was, however, significantly (P<0.01) higher in acute mastitis milk samples than in healthy cow milk samples. Heat denaturation of milk proteins significantly (P<0.01) increased the endotoxicity capacity of all milk samples evaluated (healthy cows showed a mean EU / mL ± SD of 159.21±202.65 compared to acute E. coli mastitis, mean EU / mL ± SD of 488.41±1085.43) thereby verifying the presence of endotoxicity masking protein complexes.

[0418] Interestingly, there was no significant (P=0.139) difference in the overall endotoxicity ability exhibited by boiled milk from healthy cows and acute mastitis milk samples (not boiled). However, once boiled, the endotoxicity capacity measured in acute E. coli mastitis milk became 3-fold higher than that found in healthy boiled milk samples. Moreover, a linear correlation (R2=65.5 %, P<0.01) between endotoxicity capacity and soluble LPS / IgG complex levels in acute mastitis milk samples (Figure 4BII) was observed. To further elucidate the relationship between soluble milk LPS / IgG immune complex levels and endotoxin-driven inflammation, milk LDH activity (a known biomarker of acute mastitis severity

[0022] ) was measured. Acute E. coli milk samples showed a mean ± SD LDH activity of 3.51±3.69 pmoles / min (Figure 4CI). This was, as expected, significantly (P<0.01) higher than the milk LDH activity measured in healthy cows (0.09±0.15 pmoles / min). Moreover, a linear correlation between milk LDH activity and soluble milk LPS / IgG immune complex levels (R2=69.4%, P<0.01) collected during early E. coli mastitis (Figure 4CII) was seen.

[0419] IgG antibody specificity for the LPS glycolipid core was reduced in acute E. coli mastitis milk

[0420] The applicants believe, without wishing to be bound by any theory, that a linear correlation between soluble LPS / IgG immune complex levels and the severity of mastitis during the initial stages of acute E. coli mastitis suggests a reduced ability to attenuate endotoxicity and the possibility of a difference in IgG specificity for epitopes within the LPS glycolipid core between colostrum and milk collected during acute mastitis.

[0421] IgG antibody specificity in colostrum and acute E.coli mastitis milk to recognize the LPS glycolipid core was evaluated (Figure 5A). The mean absorbance ± SD for the recognition of 100 ng / mL WT-LPS or 2.5 pg / mL K12-LPS was 1.19±0.11 and l.ll±0.13, respectively, when using colostrum antibodies (P=0.107). In contrast, the IgG specificity for WT-LPS (100 ng / mL) within acute mastitis milk was significant (P<0.05) higher than for K12-LPS (mean absorbance ± SD of 1.25±0.54 and 0.68±0.62, respectively).

[0422] Moreover, frequency analysis (Figure 5B) of ELISA absorbance data revealed a high percentage (77 %) of acute E. coli mastitis milk samples containing IgG antibodies that bound to K12-LPS epitopes with an absorbance value < 1, although a small number (18 %) did contain IgG antibodies that bound to K12-LPS with an absorbance value >1.5. In contrast, the majority (77 %) of IgG antibodies present in acute mastitis milk samples bound to WT-LPS with an absorbance value > 1, with 35% of milk samples containing IgG antibodies that bound to WT-LPS with an absorbance value >1.5.

[0423] To elucidate how variations in the milk IgG antibody repertoire influences the interaction with epitopes within the LPS glycolipid core, IgG antibody extracts from (i) a colostrum preparation enriched in IgG and (ii) a selected acute mastitis milk sample that contained IgG antibodies displaying a 72% lower specificity for K12-LPS epitopes compared to WT-LPS: mean absorbance 0.45 vs. 1.58, respectively, were used. Preparative analysis of the extracts verified undetectable levels of LPS / IgG immune complexes and free LPS. The colostrum IgG antibody titer for K12-LPS and WT-LPS were similar with no significant differences observed (Figure 6A). However, IgG antibodies within the acute mastitis milk extract showed a significant (PcO.001) difference in the antibody titer for the recognition of K12- LPS and WT-LPS. Acute mastitis milk IgG extract (1.25 mg / mL) contained antibodies that showed a ~ 36% reduction in specificity for K12-LPS compared to WT-LPS; mean absorbance ± SD, 0.74 ± 0.01 vs. 1.16 ± 0.05, respectively.

[0424] In addition, assessment of the binding strength between LPS and IgG antibodies within the milk IgG extracts was examined (Figure 6B). Here, the amount of colostrum and acute E. coli mastitis milk IgG extracts were adjusted to exhibit similar absorbance values (~ 0.5) when binding to both WT-LPS (100 ng / mL) and K12-LPS (2.5 pg / mL) in an ELISA. These assay parameters were then applied to determining the strength of the LPS-IgG interaction in the presence of the dissociating reagent urea. IgG antibodies extracted from acute mastitis milk when bound to either WT-LPS or K12-LPS in the ELISA showed similar dissociation profiles using 0.5 - 3 M urea. The urea dissociation of WT-LPS bound to colostrum IgG antibodies showed a similar urea resistant profile to those of LPS bound to acute mastitis milk IgG antibodies. However, a significant (P<0.05) ~ 23% increased resistance to 2M urea dissociation was observed in ELISAs where colostrum IgG antibodies were bound to K12-LPS (see Figure 6B, open circles). These findings showed that the IgG antibody repertoire within acute mastitis milk displays a lower specificity for the LPS glycolipid core than those within colostrum. Additionally, the avidity between colostrum IgG antibodies that specifically interact with K12-LPS is stronger than IgG antibodies bound to WT-LPS.

[0425] Bovine colostrum IgG antibody repertoire supports LPS endotoxicity masking and attenuation of LPS-induced pro-oxidants within milk granulocytes

[0426] The influence of IgG antibodies within colostrum and acute E.coli mastitis milk on LPS endotoxic properties was then investigated using soluble LPS / IgG immune complexes generated using WT-LPS.

[0427] To optimize the amounts of milk IgG extracts required to specifically recognize WT-LPS for these experiments, preparative experiments identified that 0.2 mg / mL colostrum and 1 mg / mL acute mastitis milk IgG extracts showed similar absorbance values in the ELISA for the recognition of 100 ng / mL WT- LPS. Immune precipitation of WT-LPS with milk IgG extracts showed the presence of soluble LPS / IgG immune complexes in the supernatant (Figure 7A). Increasing WT-LPS doses with the fixed amounts of IgG extracts generated a bell-shaped curve that was similar when using either IgG antibody preparation. A 25 ng / mL dose of WT-LPS generated soluble LPS / IgG immune complex levels with a mean ng / mL LPS equ. ± SD of 47.92±20.15 and 33.51±12.03 for colostrum IgG antibodies, and acute mastitis milk IgG antibodies, respectively.

[0428] These immune precipitant supernatants were then used to evaluate endotoxic properties. WT-LPS alone (Figure 7B) exhibited a dose-dependent increase in endotoxicity capacity, with 100 ng / mL WT- LPS showing a mean EU / mL ± SD of 108.31±2.46. Soluble LPS / IgG immune complexes generated using WT-LPS and acute mastitis milk IgG had no significant impact on LPS endotoxicity capacity and paralleled the dose-dependent increases observed with WT-LPS alone. In contrast, soluble LPS / IgG immune complexes present in the supernatant after precipitation with WT-LPS and colostrum IgG antibodies showed a significant (P<0.05) drop in endotoxicity capacity using 25 and 50 ng / mL WT-LPS (mean EU / mL ± SD of 36.97±12.18 and 42.12±25.41, respectively) when compared to soluble immune complexes formed using acute mastitis milk IgG antibodies (mean EU / mL of 56.54±11.70 vs. 79.17±14.0, respectively).

[0429] The influence of soluble LPS / IgG immune complexes on the ability of LPS to evoke ROS within milk granulocytes was then investigated (Figure 8). LPS can increase the pro-oxidants within milk granulocytes to favor oxidative burst and the initiation of the acute inflammatory response (17). These experiments showed that 3.1-200 ng / mL WT-LPS caused a dose-dependent increase in ROS generation over 20 mins at 37 °C (data not shown). Preincubation of milk granulocytes with the antioxidant N- a cetylcysteine (NAC) for 30 mins at 37 °C prior to the addition of 200 ng / mL LPS caused a dosedependent inhibition of ROS generation over 20 mins (Figure 8A). Preincubation of 2 mM NAC prior to LPS stimulation caused ~ 50 % (PcO.Ol) decrease in ROS generation after 20 mins (mean FI ± SD of 721.2±73.10 vs. 1352.0±91.61, 2mM NAC vs. WT-LPS alone, respectively). Supernatant containing the soluble LPS / IgG immune complexes after immune precipitation of WT- LPS with colostrum IgG antibodies caused a dose-dependent attenuation of LPS-induced changes in ROS over 20 mins. This, however, was not observed in the soluble immune complexes using IgG antibodies present within the acute mastitis milk extract. Using 25 ng / mL WT-LPS (Figure 8B), the soluble LPS / IgG immune complex generated using colostrum IgG caused a significant (P<0.05) reduction in ROS generation (minus background) after 20 mins (mean FI ± SD of 165.01± 122.20) when compared to the increases observed when using the supernatant after immune precipitation with acute mastitis milk IgG antibodies (mean FI ± SD of 237.54±104.51) or WT-LPS alone (mean FI ± SD of 241.92±143.71).

[0430] Expression of data as % changes in ROS generation after 20 mins (Figure 8C) revealed a significant (P<0.05) decrease in supernatants containing LPS / IgG immune complexes using 25 and 50 ng / mL WT-LPS and colostrum IgG antibodies (mean FI20 mins ± SD of 75.70±21.31 and 64.32±28.41, 25 and 50 ng / mL, respectively) compared to equivalent soluble immune complexes using acute mastitis milk IgG (mean FI20 mins ± SD of 109.01±13.52 and 109.81±6.52, 25 and 50 ng / mL, respectively) or WT-LPS alone (mean FI20 mins ± SD of 101.33±7.82 and 102.60±15.01, respectively).

[0431] Discussion

[0432] The experiments described here used different polymyxin B-based LPS capture ELISA formats to investigate the recognition and binding of LPS by milk IgG antibodies and to characterise LPS / IgG complexes to gain an insight into how milk IgG antibody specificity influences the efficacy of soluble LPS / IgG complexes to mask LPS endotoxicity.

[0433] An important prophylactic property of colostrum IgG antibodies in the neonatal calf is to mask the endotoxicity capacity of invading pathogens and support maturing innate and adaptive immunity (3, 20). Here, a measurable amount of LPS endotoxicity was observed in five pooled colostrum powder concentrates. The endotoxicity capacity significantly increased once the colostrum was boiled, indicating the presence of heat sensitive proteins that effectively "mask" the endotoxicity of LPS. Without wishing to be bound by any theory, the applicants believe this small amount of endotoxicity in combination with the masked LPS may play a role in the development of appropriate immune responses, for example in ruminant mammals. Bovine milk contains a variety of proteins capable of masking LPS, for example, both casein (23) and lactoferrin (21), also found in colostrum, demonstrate endotoxin sequestering properties in milk.

[0434] The identification here for the first time of soluble LPS / IgG immune complexes in pooled colostrum concentrates and in fresh colostrum collected during the first milking of post-partum cows, together with immune precipitation of K12-LPS (LPS glycolipid core) with an enriched colostrum IgG preparation to abolish endotoxicity capacity, supports a role for IgG in masking LPS endotoxicity. The presence of colostrum IgG antibodies that have high specificity to the LPS glycolipid core supports the applicant's view that the formation of LPS / IgG immune complexes contributes to or is responsible for in minimizing endotoxicity caused by gram-negative bacteria in the calf.

[0435] Milk collected from the early stages of acute E. coli mastitis showed an elevation in LDH activity and LPS endotoxicity with a correlation of ~ 70% to each other, indicating parallel increases in endotoxicity and severity of inflammation. Milk endotoxicity capacity from healthy cows was a lot lower, although heat treatment elevated endotoxicity to within levels observed physiologically in acute mastitis milk. This highlights the importance of milk "masking" proteins, such as casein, lactoferrin and mucosal anti-LPS IgA antibodies to sequester LPS in healthy cows (1, 2). Milk proteins, especially casein, however, are quickly degraded by inflammatory proteases during acute E. coli mastitis causing a release of LPS that augments endotoxin-driven inflammation (24). IgG antibody recruitment into the mammary gland simultaneously occurs with the onset of acute mastitis and correlates with LDH activity (22). An increase in soluble LPS / IgG levels was detected in milk samples collected during acute E. coli mastitis as reported above, but minimal levels were detected in milk from healthy cows. The positive correlation between soluble milk LPS / IgG complex levels with LDH activity and endotoxicity capacity observed here strongly suggests that IgG antibodies targeting LPS during the initial stages of acute mastitis do not attenuate LPS endotoxicity capacity. Moreover, over 90 % of acute mastitis milk samples displayed a weak recognition for the LPS glycolipid core (i.e., K12- LPS), whereas the IgG antibody recognition of epitopes within WT-LPS in the acute mastitis milk samples, although diverse, was higher than that of the LPS glycolipid core. This suggests that IgG antibodies recruited during acute mastitis may (like specificity of IgG antibodies generated through E. coli vaccination to control clinical mastitis [4, 5, 25]), contain a reduced relative level of IgG antibodies that recognize the LPS core.

[0436] The data presented herein show that changes in milk IgG specificity for LPS epitopes generate LPS / IgG immune complexes with distinct functional properties. During the onset of clinical E. coli mastitis, the ability of the host to respond to the invading E. coli bacteria and associated toxins is essential for activating and coordinating downstream immune processes (6, 7) that facilitate bacterial and endotoxin clearance resulting in recovery. Without wishing to be bound by any theory, the applicants posit milk IgG antibodies that directly bind to the LPS core with high affinity may passively interfere with the ability of the host to activate the appropriate immune response. Immune precipitation of IgG extracts of colostrum and milk collected during the initial stages of acute E. coli mastitis with WT-LPS or K12-LPS generated soluble LPS / IgG immune complexes that displayed similar recognition and avidity profiles for WT-LPS, but distinct profiles for the endotoxic glycolipid core. LPS compounds can elicit a range of biological actions independent of their endotoxic properties, and hence it is feasible that the masking by milk IgG antibodies will not necessarily prevent the activation of ROS generation in milk granulocytes or other biological properties. Soluble milk LPS / IgG complexes collected during the initial stages of acute mastitis correlated with the severity of inflammation. IgG antibodies extracted from an acute mastitis milk sample had negligible effects on both endotoxicity and LPS-induced reactive oxygen species (ROS) in milk granulocytes. In contrast, colostrum IgG antibodies were herein shown to form soluble LPS / IgG immune complexes that ameliorated LPS endotoxicity capacity and ROS generation in milk granulocytes.

[0437] In conclusion, the experiments characterising milk IgG antibodies and LPS / IgG complexes described here show that the specificity of IgG antibodies secreted into the bovine mammary gland (either post-partum or during an E. coli infection) influences the endotoxic properties of soluble LPS / IgG immune complexes, in turn leading to the coordinated support of appropriate innate-adaptive immune responses essential for health and recovery.

[0438] This in turn strongly supports the use of LPS / IgG complexes in the methods and compositions as herein contemplated, including for example in methods of eliciting an immune response in a subject or of vaccinating a subject using such complexes.

[0439] Example 2. Detection of LPS / IgG complexes in plasma of young calves

[0440] This example presents an investigation of the presence of lipopolysaccharide (LPS) / IgG complexes in the plasma of young calves.

[0441] Methods

[0442] Plasma was obtained from EDTA blood taken from young calves by centrifugation. Plasma was obtained from four young calves and diluted 1:5 in buffer for testing. The presence and / or concentration of LPS / IgG complexes was assessed using the ELISA method described above in Example 1.

[0443] A serial dilution of a standard (based on the presence of LPS / IgG complexes in colostrum) was included in the assay. Results and Discussion:

[0444] As can be seen in Figure 9, LPS / IgG complexes were detected in plasma from young calves. Furthermore, the concentration LPS / IgG complexes of the 1:5 dilution of calf plasma correspond to approximately a 0.08% concentration of the colostrum standard.

[0445] These data suggest that LPS / IgG complexes can be transported from orally supplied colostrum into the systemic circulation in young calves. These data also show that the systemic circulation provides a readily accessible source from which LPS / IgG complexes can be obtained.

[0446] Notably, this Example shows that as the circulating LPS / IgG complexes did not induce systemic inflammatory responses in the calves from which the plasma samples were obtained, the ability of the colostrum-derived LPS / IgG complexes to attenuate or completely mask LPS-associated endotoxicity is maintained during and after transport into and within the systemic circulation.

[0447] Example 3. Effect of intestinal mucosal transfer of colostrum LPS immune complexes in newborn calves fed colostrum

[0448] This example presents an investigation of whether colostrum-derived LPS / Ig immune complexes can be transferred across the intestinal mucosa into the circulation of newborn calves and to profile the appearance of blood anti-LPS Ig in the calf.

[0449] Methods

[0450] Trial 1 protocol

[0451] 9 pregant cows were selected. The calves were removed immediately after birth (prior to suckling colostrum) and randomized into 3 groups of 3 calves with different treatment regimes.

[0452] Group 1: fed on day 1 with milk replacer (CMR)

[0453] Group 2: fed on day 1 with Westland colostrum (WL)

[0454] Group 3: fed on day 1 with Peptide Ignition colostrum (PI)

[0455] All calves were fed on days 2 - 4 with transitional milk containing predominantly IgA with little IgG (obtained from cows 2 - 5 days after calving), then with milk replacer.

[0456] Various biological fluid samples (blood, saliva, faecal material) were collected at specific times over a 21-day period, as follows:

[0457] Blood samples: 0, 6, 12, 24, and 48 hours, and 4, 7, 14, and 20 days;

[0458] Saliva and faeces: 0, and 6 hours, and 2, 4, 7, 10, 12, 14, 16. 18, and 20 days.

[0459] The samples were analysed as follows.

[0460] • Plasma: Total IgG, LPS specific IgG, LPS / Ig complex

[0461] • Saliva: LPS specific IgA and IgG

[0462] • Faeces: LPS specific IgA and IgG

[0463] Accordingly, this example investigated: i. total blood IgG

[0464] II. Systemic anti-LPS (core) IgG antibodies (blood) and mucosal slgA and IgG antibodies (saliva I faeces) in calf. ill. blood LPS / endotoxin levels (masked vs. unmasked levels) and stress / inflammatory status. iv. health status of calf.

[0465] Results

[0466] Characterisation of WL and PI colostrum, and CMR

[0467] As can be seen in Figure 10 there are significant differences in the amount and composition of LPS / Ig complexes verified for WL colostrum compared to PI colostrum. Notably, WL colostrum had significantly more LPS / IgG complexes than PI colostrum (see Figure 10A), and the ratio of LPS / IgG:LPS / IgA was substantially higher in WL colostrum compared to PI colostrum. Notably, the levels of LPS / IgA complexes were similar in WL and PI colostrums (see Figure 10B).

[0468] IgG and LPS / Ig complexes were not present in CMR (data not shown).

[0469] Reactivity and quantitative differences in the representation of specific anti-LPS IgG levels from WL and PI colostrum to LPS from a range of different bacteria were observed (data not shown).

[0470] These data show that high levels of LPS / IgG complexes were found in WL colostrum, but not in PI colostrum. This Example supports the view that colostrums vary in their IgG content, and in their capacity to form LPS / IgG complexes.

[0471] Example 4. Levels of specific IgG and LPS / Ig complexes in calf plasma

[0472] This example presents an investigation of the detection of LPS-specific IgG and of LPS / Ig complexes in the circulation of ruminant subjects.

[0473] Method

[0474] Subjects, samples and analyses were as described in Example 3 above.

[0475] Results

[0476] Plasma levels of LPS-Ig complexes in calves fed CMR, or WL or PI colostrum

[0477] The amount of LPS-Ig complexes present in blood samples collected from the three trial groups is shown in Figure 11. As can be seen in Figure 11A, effectively no LPS / Ig complexes were identified in the CMR subjects.

[0478] Substantial differences in the amounts of LPS / IgG over the 20-day trial between WL and PI groups were observed (compare Figure 11B and Figure 11C). Notably, the level of LPS / IgG complexes in the blood of the WL group was maintained over the trial period. In contrast, the level of LPS / IgG in the blood of the PI group decreased over time.

[0479] Collectively, the applicant infers from this data that, due to low levels of LPS / IgG complexes in PI colostrum (see Example 3 above), complexes found in PI calves were formed with LPS from commensal bacteria.

[0480] Large differences in LPS / IgA complexes are evident between WL and PI calves (compare Figure 11B and Figure 11C). The high levels of LPS / IgA in PI calves may be indicative of the induction of a more LPS tolerogenic environment. Further, the data presented here suggests that LPS / IgA complexes in transitional milk are not transported into circulation.

[0481] Plasma anti LPS IgG levels

[0482] Figure 12 presents the amount of bacteria-specific anti-LPS IgG antibodies at days 0, 7, and 21 across the three treatment groups (CMR, Figures 12A and 12B; PI, Figures 12C and 12D; WL, Figures 12E and 12F). Differences between PI and WL (as exemplified on LPS from S. marcescens and S. enterica, see Figure 12C and 12D vs Figures 12E and 12F) is believed, without wishing to be bound by any theory, to be indicative of a higher active induction of IgG production in the WL animals. No induction of an anti-LPS IgG response was seen in the CMR group (Figure 12A, Figure 12B).

[0483] The data presented in this Example support the applicant's view that LPS / Ig complexes found in blood of WL group are likely derived from WL colostrum.

[0484] Complexes found in blood from PI group are likely formed in the gut by the interaction of Ig with LPS from commensal bacteria (due to very low levels of LPS / IgG complexes in PI colostrum). This shows that complexes can be readily formed in the intestine when colostrum antibodies and antigens are present. LPS / IgA complex levels were high in PI calves, but not in WL calves. Without wishing to be bound by any theory, the applicant believes that this is indicative of the induction of a more LPS tolerogenic environment in the PI calves.

[0485] Compellingly, robust induction of LPS-specific IgG generation was observed in the WL group, but induction of LPS-specific IgG generation was much lower in the PI group.

[0486] Example 5. Levels of LPS-specific IgA levels in calf saliva

[0487] This example presents an investigation of levels of LPS-specific IgA levels in calf saliva.

[0488] Method

[0489] Subjects, samples and analyses were as described in Example 3 above.

[0490] Results

[0491] Saliva levels of LPS-IgA complexes in calves fed CMR, or WL or PI colostrum

[0492] Figure 13 presents the average fold increase in the level of IgAs specific for LPS from 6 different bacteria in the saliva of calves from the WL and the PI groups.

[0493] As can be seen in Figure 13, the generation of LPS-specific IgA responses was induced in saliva of WL calves but was not evident or was substantially lower in saliva samples from PI calves. A 3.74- fold increase by day 21 was seen in saliva from the WL group, while no increase in LPS-specific IgA was seen in the PI group.

[0494] These data show that high levels of LPS-specific IgA were induced following oral administration of the WL colostrum high in LPS / IgG complexes, but not of PI colostrum in which LPS / IgG complexes were much lower or absent. This Example provides strong evidence of an oral vaccination effect following oral administration of LPS / IgG complexes, as established by the increased LPS-specific IgA antibody levels in saliva of newborn calves.

[0495] Example 6. Levels of LPS- specific IgA and IgG levels in calf faeces indicate effect of oral vaccination

[0496] This example presents an investigation of the levels of LPS-specific IgA and IgG in the faeces of calves to which different colostrums had been administered.

[0497] Methods

[0498] Subjects, samples and analyses were as described in Example 3 above.

[0499] Results

[0500] Figure 14 presents faecal anti LPS IgA levels (pg / ml, adjusted to lmg / ml protein) specific to three different gram negative bacteria ( / (. pneumonia, Figure 14A; S. marcescens, Figure 14B; S. enterica, Figure 14C) across the 20 day trial. As can be seen in Figure 14, an induction of faecal LPS-specific IgA responses from approximately day 13 is seen in the WL group but not in the PI group . Furthermore, the data suggests there was an early induction at around day 7 for S. marcescens and S. enterica LPS in the WL group, but not PI groups.

[0501] Figure 15 presents faecal anti LPS IgG levels (pg / ml, adjusted to lmg / ml protein) specific to three different gram negative bacteria ( / (. pneumonia, Figure 15A; S. marcescens, Figure 15B; S. enterica, Figure 15C) across the 20 day trial. As can be seen in Figure 15, no evidence of an induction of faecal LPS specific IgG was evident in the PI group, with the observed levels consistent with passive supply.

[0502] Figures 14 and 15 suggest that there was passive transfer of LPS-specific IgA and IgG with a peak at 12h (WL) and at 2 days (PI). A second peak for specific LPS IgA and IgG production (except for Klebsiella LPS) only in WL (not in PI or MR) calves was observed, showing an early (from days 4 to 10) and specific induction of a mucosal anti LPS IgA and IgG response.

[0503] These data show there was an induction of LPS-specific IgA and IgG responses in WL but not PI calves from days 14 to 21.

[0504] Without wishing to be bound by any theory, the applicant believes that the absence of the generation of mucosal anti-LPS responses in PI calves may indicate that a more tolerogenic immune environment was induced, possibly due to the relatively high levels of LPS / IgA complexes present in this colostrum.

[0505] Example 7. Levels of LPS- specific IgA levels in calf plasma indicate effect of oral vaccination

[0506] This example presents an investigation of LPS-specific IgA in plasma samples from calves to which different colostrums had been administered.

[0507] Methods

[0508] Subjects, samples and analyses were as described in Example 3 above.

[0509] Results

[0510] Figure 16 presents the levels of anti-LPS IgA levels specific to four different gram negative bacteria (E. coll K12, Figure 16A; K. pneumonia, Figure 16B; S. enterica, Figure 16C; S. marcescens, Figure 16D) in plasma across the 20 day trial. As can be seen in Figure 16, passive transfer of LPS-specific IgA into plasma peaked at 12h - 2d. PI calves had higher levels than WL calves. There then follows a decline in plasma LPS-specific IgA which was greater in PI than WL calves. By day 10, the LPS-specific IgA levels in WL calves were higher than those of PI calves. This was consistent for each bacteria.

[0511] The maintenance or indeed increase in the level of LPS-specific IgA in the plasma of WL calves evidences an induction of LPS-specific IgA responses from days 7 to 21. This was not observed in PI calves.

[0512] Again, without wishing to be bound by any theory, the applicant believes that the absence of any generation of mucosal anti-LPS responses in PI calves may indicate a more tolerogenic immune environment possibly induced by relatively high levels of LPS / IgA complexes.

[0513] These data show that high levels of LPS-specific IgA were induced and present in the circulation following oral administration of the WL colostrum high in LPS / IgG complexes, but not of PI colostrum in which LPS / IgG complexes were much lower or absent. This Example provides strong evidence of an oral vaccination effect following oral administration of LPS / IgG complexes, as established by the increased LPS-specific IgA antibody levels in blood of newborn calves.

[0514] Example 8. Oral vaccination of newborn calves with in vitro produced antigen / immune complexes.

[0515] This example presents an investigation of the endotoxicity associated with, and the effect of oral administration to a newborn calf of, in vitro generated LPS / IgG immune complexes.

[0516] Methods

[0517] Bovine colostrum specifically enriched for IgG, serum IgG, and LPS derived from mastitis E. coll were used as starting materials. Immunoprecipitation equivalence (IP) was established to obtain IgG / LPS complexes that could be detected by ELISA.

[0518] IP was performed using ratios of enriched IgG preparations and E. coll LPS for 1 hr at 37 °C and then overnight at 4 °C. Centrifugation of IP mixture was then carried out at 6000 xg for 10 mins, 10 °C, to pellet complexes. The supernatant was discarded and the pellet gently washed with PBS to remove any excess LPS and IgG. The pellets were then air dried for 30 mins at RT. The pellet was resuspended in PBS ~ 100 mg / mL (w / v). The resuspended pellets were pooled and then sonicated for 5 mins in an ice water bath.

[0519] The final mixture was assessed for LPS / IgG complexes and endotoxin activity (via LAL assay). Heat denaturation of the mixtures was also performed by boiling, and the endotoxic activity of the heat- denatured samples was assessed, again via LAL assay.

[0520] The in vitro LPS / IgG complexes (500 ml milk replacer supplemented with 5mg / ml LPS / IgG complexes) were given orally to newborn calves.

[0521] Results and Discussion:

[0522] Figure 17 presents the endotoxin activity of the in vitro generated LPS / IgG complexes (full bars) and the heat denatured (boiled) samples (hatched bars). Figure 17A presents the endotoxic activity of in vitro generated LPS / IgG complexes comprising IgG derived from colostrum, and Figure 17B presents the endotoxic activity of in vitro generated LPS / IgG complexes comprising IgG derived from serum.

[0523] As can be seen, in vitro formed LPS / IgG complexes comprising IgG derived from colostrum lack endotoxic activity. Notably, endotoxic activity of these complexes was restored after heat denaturation (compare Figure 17A hatched bars to solid bars).

[0524] Further, as shown in Figure 17B, LPS / IgG complexes comprising IgG derived from serum exhibited endotoxic activity, irrespective of whether they were heat denatured or not. Comparable levels of endotoxicity were observed (see Figure 17B, hatched bars vs solid bars).

[0525] In vivo testing of LPS / IgG complexes by oral administration to newborn calves did not result in any detrimental health effects (data not shown).

[0526] Figure 18 shows the levels of LPS / IgG complexes in blood samples from calves to which in vitro generated LPS / IgG complexes were administered. As can be seen in Figure 18A, LPS / IgG complexes were detected in blood samples, showing these complexes were transported into circulation when given to a newborn calf. In contrast, no such complexes were detected in a calf to which CMR was administered (see Figure 18B).

[0527] These data show that oral administration of in vitro generated LPS / IgG complexes comprising IgG derived from colostrum to newborn calves did not induce toxic shock or indeed any detrimental health effects.

[0528] Figure 19 presents data showing the detection of LPS-specific IgA (Figure 19A) and LPS-specific IgG (Figure 19B) in the plasma of a calf to which in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was orally administered. As can be seen in Figure 19A, by Day 10 there is a marked increase (compared to Day 1) in IgA levels specific for LPS from four different gram-negative bacteria (E. coli 5077, S. entericae, K, pneumoniae, S. marcescens). This is strongly supportive of a vaccination effect.

[0529] Similarly, as shown in Figure 19B by Day 10 there is a marked increase (compared to Day 1) in IgG levels specific for LPS from five different gram-negative bacteria (E. coli K12, E. coli 5077, S. entericae, K, pneumoniae, S. marcescens). This is again strongly supportive of a vaccination effect.

[0530] Figure 20 presents data showing the detection of LPS-specific IgA in faecal samples (Figure 20A) and in saliva samples (Figure 20B) from a calf to which in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was orally administered. As can be seen in Figure 20A, by Day 10 there was a marked increase (5.3 - 6.5 fold increase compared to Day 1) in faecal IgA levels specific for LPS from four different gram-negative bacteria (E. coli K12, E. coli 5077, S. entericae, and S. marcescens). This is strongly supportive of a vaccination effect. The peak at Day 2 is due to passive supply of IgA due to the feeding of transitional milk (CMR) containing IgA.

[0531] As can be seen in Figure 20B, by Day 13 there was a marked increase (2.1 - 3.4 fold increase compared to Day 1) in saliva IgA levels specific for LPS from four different gram-negative bacteria (E. coli K12, E. coli 5077, S. entericae, and S. marcescens) .

[0532] Oral vaccination of another calf with in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was performed. Administration of a bolus (comprising an equivalent amount of LPS / IgG complex to that administered to the first calf) of complex in PBS one hour prior to feeding with CMR was performed. Faecal and saliva samples were collected at days 1, 2, 4, 7, 10, and 13 as previously.

[0533] Figure 21 presents data showing the detection of LPS-specific IgA in faecal samples (Figure 21A) and in saliva samples (Figure 21B) from the calf to which the bolus of in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was orally administered. As can be seen in Figure 21A, by Day 13 there was a marked increase (2.9 - 4.6 fold increase compared to Day 1) in faecal IgA levels specific for LPS from four different gram-negative bacteria (E. coli K12, E. coli 5077, S. entericae, and S. marcescens). This is strongly supportive of a vaccination effect.

[0534] As can be seen in Figure 21B, by Day 13 there was a marked increase (2.4 - 4.0 fold increase compared to Day 1) in saliva IgA levels specific for LPS from four different gram-negative bacteria (E. coli K12, E. coli 5077, S. entericae, and S. marcescens) from this calf.

[0535] These data show that oral vaccination with a bolus dose of a composition comprising in vitro generated LPS / IgG complexes comprising IgG derived from colostrum was effective in inducing LPS- specific IgA responses, just as oral administration of comparable complexes when administered concomitantly with feeding was effective.

[0536] These data support the ability of in vitro generated LPS / IgG complexes generated using colostrum- derived IgG to traverse the gut mucosa into the circulation and elicit adaptive antibody immune responses (systemic and / or mucosal) in the subject to which they are administered, and are strongly supportive of a vaccination effect.

[0537] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[0538] Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[0539] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

[0540] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features.

[0541] Aspects of the invention have been described by way of example only, and it should be appreciated that variations, modifications and additions may be made without departing from the scope of the invention, for example when present the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

[0542] Publications.

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[0544] 2. Rainard P, Gilbert FB, Germon P. Immune defenses of the mammary gland epithelium of dairy ruminants. Front Immunol (2022) Vol 13 doi : 10.3389 / fimmu.2022.1031785.

[0545] 3. Lopez AJ, Heinrichs AJ. The importance of colostrum in the newborn dairy calf: Invited review. J Dairy Sc. (2022) 105:2733-2749. doi : 10.3168 / jds.2020-20114.

[0546] 4. Rainard P, Gilbert FB, Germon P, Foucras G. A critical appraisal of mastitis vaccines for dairy cows: Invited review. J Dairy Sci (2021) 104: 10427-10448. doi.org / 10.3168 / jds-2021-20434.

[0547] 5. Vangroenweghe F, Duchateau L, Burvenich C. J-5 Escherichia coli vaccination does not influence severity of an Escherichia coli intramammary challenge in primiparous cows. J Dairy Sci (2019) 103:6692-6697. doi.10.3168 / jds.2019-17799.

[0548] 6. Goulart DB, Mellata M. Escherichia coli mastitis in dairy cattle: Etiology, diagnosis, and treatment challenges. Front. Micro (2022) Vol 13. doi : 10.3389 / fmicb.2022.928346.

[0549] 7. Zaatout N. An overview on mastitis-associated Escherichia coir. Pathogenicity, host immunity and the use of alternative therapies. Micro Res (2022) 256: 126960. doi. org / 10.1016 / j.micres.2021.126960.

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[0553] 11. Blais BW, Bosley J, Martinez-Perez A, Popela M. Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli Oll i and 026. J Micro Meth (2006) 65:468-475. doi: 10.1016 / j.mimet.2005.09.004.

[0554] 12. Martinez-Sernandez V, Orbegozo-Medina RA, Romaris F, Paniagua E, Ubeira FMN. Usefulness of

[0555] ELISA methods for assessing LPS interactions with proteins and peptides. PLOS ONE (2016). doi.10.1371 / journal. pone.0156530.

[0556] 13. Marvin HJP, Ter Beest MBA, Withold B. Release of outer membrane fragments from wild- type

[0557] Escherichia coli and from several E. coli lipopolysaccharide mutants by EDTA and heat shock treatments. J Bacteriology (1989) 171(10) :5262-5267. doi : 10.1128 / jb.l71.10.5262- 5267.1989.

[0558] 14. Skalka V, Shakhno N, Ecer J, Curda L. Separation of immunoglobulin from colostrum using methods based on salting-out techniques. Czech J Food Sci (2017) 35(3) :259-266. doi : 10.17221 / 10 / 2017-CIFS.

[0559] 15. Pedersen GK, Hoschler K, Solbak SMO, Bredholt G, Pathirana RD, et al. Serum IgG titres, but not avidity, correlates with neutralizing antibody response after H5N1 vaccination. Vaccine (2014) 32:4550- 4557. doi : 10.1016 / j. vaccine.2014.06.009.

[0560] 16. Fossati G, Bucknail RC, Edwards SW. Insoluble and soluble immune complexes activate neutrophils by distinct mechanisms: change in functional responses induced by priming with cytokines. Ann Rheum Dis (2002) 61 : 13-19, doi : 10.1136 / ard.61.1.13. 17. Mehrzad J, Dosogne H, Meyer E, Heyneman R, Burvenich C. Respiratory burst activity of blood and milk neutrophils in dairy cows during different stages of lactation. J Dairy Res (2001) 68 :399-415. doi:01.1017 / 50022029901005039.

[0561] 18. Varricchi G, Modestino L, Poto R, Cristinziano L, Gentile L, et al. Neutrophil extracellular traps, and neutrophil-derived mediators as possible biomarkers in bronchial asthma. Clin Exp Med (2022) 22:285-300. doi : 10.1007 / sl0238-021-00750-8.

[0562] 19. LeBel CP, Ischiropoulos H, Bondy. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formulation and oxidative stress. Chem Res Toxicol (1992) 5:227-231. doi: 10.1021 / tx00026a012.

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[0564] 21. Kell DB, Heyden EL, Pretorius E. The biology of lactoferrin, an iron-binding protein that can help defend against viruses and bacteria. Front Immunol (2020) Vol 11. doi: 10.3389 / fimmu.2020.01221.

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Claims

CLAIMS1. A method of eliciting an immune response in a subject in need thereof, the method comprising administering to the subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

2. A method of immunising a subject in need thereof, the method comprising administering to the subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

3. The method according to claim 1 or claim 2, wherein the administration is oral administration and / or the complex is present in a composition formulated for oral administration.

4. The method according to any one of claims 1 to 3, wherein: a) the lipopolysaccharide comprises Lipid A and one or more of said immunoglobulin binds specifically to said Lipid A; or b) the complex exhibits reduced TLR4 binding and / or agonism compared to equivalent lipopolysaccharide alone; or c) the complex exhibits reduced endotoxicity compared to equivalent lipopolysaccharide alone; or d) the complex does not induce an inflammatory response; or e) the complex does not induce an inflammatory response when administered to a subject in which an inflammatory response is initiated when said lipopolysaccharide and / or said immunoglobulin are administered separately; or f) the complex is or has been isolated and / or purified from colostrum; or g) the complex is or has been isolated and / or purified from colostrum having an amount or concentration of IgG comparable to or exceeding the amount of IgA present in the colostrum; h) the immunoglobulin comprises, consists essentially of, or consists of IgG; or i) the immunoglobulin comprises, consists essentially of, or consists of IgG having binding specificity to Lipid A; or j) the immunoglobulin comprises, consists essentially of, or consists of IgG from a colostrum having an amount or concentration of IgG comparable to or exceeding the amount of IgA present in the colostrum; or k) the immunoglobulin is capable of reducing the endotoxic activity of lipopolysaccharide; l) the immunoglobulin is capable of binding to and reducing the endotoxic activity of and / or the endotoxicity associated with the lipopolysaccharide with which it is complexed; m) the complex is formed in vitro from IgG purified or isolated from colostrum; n) the complex comprises IgG purified or isolated from colostrum and is or has been formed in vitro by admixture with isolated, purified, synthetic, or recombinant lipopolysaccharide;o) the complex consists essentially of or consists of immunoglobulin and lipopolysaccharide; or p) the immunoglobulin and / or the lipopolysaccharide comprises one or more exogenous antigens; or q) the complex comprises one or more antigens other than said immunoglobulin and said lipopolysaccharide; or r) the complex comprises one or more biological ly-active moieties other than said immunoglobulin and said lipopolysaccharide; or s) any two or more of any of a) to r) above.

5. The method according to claim 1 wherein the immune response is to said lipopolysaccharide.

6. The method according to claim 2 wherein the immunisation is against the organism(s) from which said lipopolysaccharide is derived.

7. The method according to any one of claims 1 to 6 wherein the immune response is or the immunisation comprises: a) an induction of IgA or an increase in IgA levels or activity; or b) an induction of LPS-specific IgA or an increase in LPS-specific IgA levels or activity; or c) an induction of a mucosal immune response; or d) an induction of a mucosal IgA response; or e) any two or more of any of a) to d) above.

8. The method according to any one of claims 1 to 7 wherein the subject is a newborn mammalian subject.

9. The method according to any one of claims 1 to 8 wherein the subject is a newborn mammalian subject 24 hours old or less, or a mammalian subject one or more gut epithelial cells of which express neonatal Fc receptor.

10. The method according to claim 8 or claim 9 wherein the administration is oral administration.

11. The method according to any one of claims 8 to 10 wherein the complex is formed in vitro.

12. The method according to any one of claims 1 to 11 wherein the complex comprises two or more different types of immunoglobulin.

13. The method according to claim 12 wherein the two or more different types of immunoglobulin are two or more classes or subclasses of immunoglobulin, or wherein two or more of the immunoglobulins have differing binding affinity and / or differing binding specificity.

14. A method of preventing or treating a bacterial infection or a disease or condition caused by or associated with a gram-negative bacterial infection, the method comprising administering to a subject a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

15. The method of claim 14, wherein the bacterial infection is a gram-negative bacterial infection.

16. The method according to any one of claims 14 to 15, wherein the lipopolysaccharide comprises one or more bacterial lipopolysaccharides.

17. The method according to any one of claims 14 to 16, wherein the treating or preventing comprises treatment, prevention, or amelioration of one or more symptoms of or associated with bacterial infection.

18. The method according to any one of claims 14 to 17, wherein the bacterial infection is causative of or associated with mastitis.

19. The method according to any one of claims 15 to 18, wherein the gram-negative bacteria is a bacteria selected from the group consisting of Escherichia spp., Pasteurella spp., Serratia spp., Klebsiella spp., Salmonella spp., and Campylobacter spp..

20. The method according to any one of claims 15 to 19, wherein the administration is of an amount effective to reduce endotoxicity associated with said bacterial infection.

21. The method according to any one of claims 1 to 20, wherein the immunoglobulin comprises, consists essentially of, or consists of IgG from colostrum including immunoglobulin at least partially isolate from or purified from colostrum.

22. The method according to claim 21, wherein the colostrum is bovine colostrum, including pooled bovine colostrum.

23. The method according to any one of claims 3 to 22, wherein administration to the subject comprises oral administration.

24. The method according to any one of any one of claims 1 to 23, wherein administration of the complex does not elicit an inflammatory response in the subject.

25. A complex comprising immunoglobulin and lipopolysaccharide wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide, or a pharmaceutical composition comprising said complex, for use in or when used in eliciting an immune response in a subject in need thereof or in immunising a subject in need thereof.

26. A complex comprising immunoglobulin and lipopolysaccharide wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide, or a pharmaceutical composition comprising said complex, for use in or when used in the treatment or prevention of a microbial infection or of a disease or condition caused by or associated with a microbial infection.

27. A complex comprising immunoglobulin and lipopolysaccharide wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide, or a pharmaceutical composition comprising said complex, for use in or when used in the treatment or prevention of a bacterial infection of gram-negative bacteria or of a disease or condition caused by or associated with a bacterial infection of gram-negative bacteria.

28. A pharmaceutical composition comprising a complex comprising immunoglobulin and lipopolysaccharide, wherein said immunoglobulin is capable of binding specifically to saidlipopolysaccharide, wherein the pharmaceutical composition is formulated for oral administration.

29. Use of a complex comprising immunoglobulin and lipopolysaccharide in the preparation of a medicament for use in eliciting an immune response in a subject in need thereof or in immunising a subject in need thereof, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

30. Use of a complex comprising immunoglobulin and lipopolysaccharide in the preparation of a medicament for use in treating or preventing a microbial infection or a disease or condition caused by or associated with a microbial infection, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

31. Use of a complex comprising immunoglobulin and lipopolysaccharide in the preparation of a medicament for use in treating or preventing a bacterial infection or a disease or condition caused by or associated with a bacterial infection of gram-negative bacteria, wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

32. The use according to any one of claims 29 to 31, wherein the complex is a complex as defined in claim 4.

33. A method of treating or preventing a disease or condition caused by or associated with a microbial infection in a subject in need thereof, or of treating or preventing a microbial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex or a pharmaceutical composition comprising a complex, wherein said complex comprises immunoglobulin and lipopolysaccharide and wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

34. A method of treating or preventing a disease or condition caused by or associated with a bacterial infection in a subject in need thereof, or of treating or preventing a bacterial infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a complex or a pharmaceutical composition comprising a complex, wherein said complex comprises immunoglobulin and lipopolysaccharide and wherein said immunoglobulin is capable of binding specifically to said lipopolysaccharide.

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