WO2017013603A1 - Adjuntive immunotherapy for the prevention or treatment of infectious disease - Google Patents

Abstract

A method of treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof in a mammal caused by a pathogen or potential pathogen, comprising administration of 1) an agent effective against the pathogen or potential pathogen and 2) an immunomodulatory or immunostimulatory agent.

Description

ADJUNTIVE IMMUNOTHERAPY FOR THE PREVENTION OR TREATMENT OF

INFECTIOUS DISEASE

FIELD OF THE INVENTION

The present invention relates to a method of treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof in a mammal caused by a pathogen or potential pathogen, comprising administration of an immunomodulatory or

immunostimulatory agent. BACKGROUND OF THE INVENTION

Immune responses are divided into two categories in vertebrate animals: innate and adaptive immunity. Upon infection, recognition of microorganisms is primarily mediated by a set of germline-encoded molecules on innate immune cells that are referred to as pattern recognition receptors (PRRs) (Medzhitov and Janeway Jr. (2007) Curr. Opin. Immunol. 9(1): 4-9). These pattern recognition receptors are expressed as either membrane-bound or soluble proteins that recognize invariant molecular structures, called pathogen-associated molecular patterns (PAMPs) (Janeway Jr. and Medzhitov (2002) Trends Microbiol. 8(10): 452-456). Pathogen-associated molecular patterns are unique, conserved, and essential microbial components, such as LPS, that are structurally different from host molecules (Medzhitov and Janeway Jr. (2007) Curr, Opin. Immunol. 9(1): 4-9; Janeway Jr. and

Medzhitov (2002) Trends Microbiol. 8(10): 452-456).

Innate immunity is the first line of antibody-independent defense against infections and, in many instances, can eliminate infectious agents. The principle effector cells of innate immunity are neutrophils, mononuclear phagocytes, natural killer (NK) cells, and mucosal epithelial cells. Neutrophils and macrophages express surface PRRs that recognize microbes in the blood and tissues, stimulate the ingestion (phagocytosis, e.g., mannose or opsonin receptors), and release chemoattractant molecules stimulating the recruitment of other phagocytes such as dendritic cells. Similarly, epithelial cells of mucosal sites also express PRRs that recognize microbes which stimulate an increase in lung epithelial barrier function, activate the expression of chemoattractant molecules and the expression of numerous antimicrobial products. For example, the induction of antimicrobial peptides (AMPs), defensins, and reactive oxygen species (ROS) are expressed into the airway lining fluid in response to epithelial recognition of microbes (Whitsett and Alenghat (2015) Nat. Immunol. 1 : 27-35; Rogan et al. (2006) Resp. Res. 7(29): 1-11 ; Hiemstra (2007) Exp. Lung Res. 33(10): 537-42; Kovach et al. (2012) J. Immunol. 189(1): 304-311). PRRs fall into several classes, for example, toll-like receptors (TLRs), C-type lectin receptors (CLRs), and nucleotide-binding oligomerization domain receptors (NOD-like receptors). In mammals, the toll-like receptors (TLRs) have been assigned numbers 1 to 11 (TLR1-TLR1 1 ). AH TLRs are membrane bound proteins with a single transmembrane domain. The cytoplasmic domains are approximately 200 amino adds and share similarity with the cytoplasmic domain of the IL-1 receptor. The extracellular domains of the Toll family of proteins are relatively large (about 550-980 amino acids) and may contain multiple ligand- binding sites. TLRs recognize extracellular or endosomai pathogen-associated molecular patterns (PA Ps). Pathogen-associated molecular patterns recognition by TLRs trigger the synthesis and secretion of cytokines and activation of other host defense programs that are necessary for innate or adaptive immune responses. Interaction of TLRs with their specific PA MP induces NF- Β signaling and the MAP kinase pathway and therefore the secretion of pro-inflammatory cytokines and co-stimulator molecules. Molecules released following TLR activation signal to other cells of the immune system making TLRs key elements of innate immunity and adaptive immunity.

Toll-like receptor 4 (TLR4), a classic PRR and member of the TLR superfamily, has been studied extensively in pathogen-mediated host responses and functions as a primary sensor to detect lipopolysaccharide (LPS), a component from the outer wall of Gram negative bacteria. A critical role for TLR4 in host resistance has been demonstrated with infectious challenge in C3H/HeJ mice (an inbred mouse strain that is hyporesponsive to LPS). In response to Gram negative bacterial challenge; these mice display impaired bacterial killing and a more susceptible phenotype. LPS and lipid A, the TLR4 receptor ligand of LPS, are potent stimulators of host defense systems. However, the profound pyrogenicity and lethal toxicity of LPS and lipid A have precluded their medical use (Johnson et al. (1994) Clin. Microbiol. Rev. 7(3): 277-289).

Since the discovery of LPS and is structure, intensive work has been performed to elucidate the moieties responsible for its immunostimulatory properties in attempt to create analogs that retain these favorable assets while reducing or eliminating toxicity.

Monophosphoryl lipid A (MPL) is an acylated diglucosamine derivative of lipid A and was the first toll-like receptor 4 (TLR4) agonist approved for use in humans. MPL adsorbed onto alum is incorporated in a recombinant hepatitis B vaccine (Fendrix, GlaxoSmithKline Vaccines, Rixensart, Belgium) and a human papillomavirus 16/18 virus-like particle vaccine (Cervarix, GlaxoSmithKline Vaccines), both for human use. Further research into structure- function relationships of TLR4-based vaccine adjuvants has led to the development of a novel class of synthetic lipid A mimetics known as aminoalkyl glucosaminide 4-phosphates (AGPs), disclosed in US patents 6,355,257 (Johnson et al.), 7,902, 159 (Johnson et al.), and Johnson et al. (1999) Bioorg. Med. Chem. Lett. 9: 2273-2278. Compounds of this class trigger TLR4 signaling with varying signaling specificity, potency, and safety attributes. Some AGPs have shown promise as vaccine adjuvants, for example, the aminoalkyl glucosaminide 4-phosphate CRX-529 has demonstrated an acceptable safety and efficacy profile and is incorporated the hepatitis B vaccine, SUPERVAX. Furthermore, some AGPs, such as CRX- 527 are also capable of eliciting nonspecific protection against a wide range of infectious pathogens (Cluff et al. (2005) Infect. Immun. 73: 3044-3052; Baldridge et al. (2002) J.

Endotoxin Res. 8: 453^158; Bazin et al. (2008) Bioorg. Med. Chem. Lett. 18: 5350-5354). One particular example is the use of CRX-527 in mouse models of infectious disease. From studies of cytokine induction and gene arrays, CRX-527 has a similar profile to that LPS, with LPS being a modestly stronger stimulant (US 8,629,1 16; Stover et al. (2004) J. Biol. Chem. 279: 4440-4449; Johnson et al. (1999) Bioorg. Med. Chem. Lett. 9: 2273-2278; Bowen et al. (2008) Sci. Signal. 5(211): ra13). The ether lipid analog of CRX-527 referred to as CRX-601 (Bazin et al. (2008) Bioorg. Med. Chem. Lett. 18: 5350-5354) retains CRX-527- like potency, but with an improved stability profile as disclosed in WO 2014141127 (Johnson et al., published 2014).

The development and approval of a non-specific anti-infective agent would help meet the ever present and growing need for new antimicrobial agents. New anitmicrobial agents and treatments are in need due to the growing number of microorganisms resistant to drug therapy, the re-emergence of previously deadly infectious diseases, and the emergence of new infectious diseases.

US patents 6,800,613 (Persing et al.), 7,902, 159 (Persing et al.) and 7,960,522 (Johnson et al.) disclose certain aminoalkyl glucosaminide 4-phosphate compounds and their use as vaccine adjuvants and immuno-effectors. Also disclosed therein is their use as stand-alone therapeutic agents, including, specifically, their use for the prevention or treatment of infectious disease. One such compound is the compound of US patent

7,960,522, claim 7, hereinafter referred to as "CRX-601". US patents 6,800,613 (Persing et al.), 7,902, 159 (Persing et al.) and 7,960,522 (Johnson et al.) disclose the use of AGPs in the absence of antigen for the prevention or therapeutic treatment of conditions such as infectious disease.

BRIEF SUMMARY OF THE INVENTION

Briefly, in one aspect, the present invention discloses a method of treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof in a mammal, in particular in humans, caused by a pathogen or potential pathogen, comprising administration of 1) an agent effective against the pathogen or potential pathogen and 2) an immunomodulatory or immunostimulatory agent. BRI EF DESCRIPTION OF THE DRAWI NGS

Fig. 1 illustrates the mortality rates of rabbits treated with ceftazidime (CAZ) with or without TLR4 agonist CRX601 in a model of Pseudomonas aeruginosa- induced pneumonia.

Fig. 2 illustrates the bacterial counts in the lungs of rabbits treated with ceftazidime (CAZ) with or without TLR4 agonist CRX- 601 in a model of Pseudomonas aeruginosa- induced pneumonia.

Fig. 3 illustrates the bacterial counts in the spleen of rabbits treated with ceftazidime (CAZ) with or without TLR4 agonist CRX- 601 in a model of Pseudomonas aeruginosa- induced pneumonia.

Fig. 4 illustrates the mortality rates of mice treated with TLR4 agonist CRX- 601 or vehicle only in a model of Pseudomonas

pneumonia.

Fig. 5 illustrates the bacterial counts in the lungs of mice treated with TLR4 agonist CRX- 601 or vehicle only in a model of Pseudomonas

pneumonia.

Fig. 6 illustrates the bacterial counts in the spleen of mice treated with TLR4 agonist CRX- 601 or vehicle only in a model of Pseudomonas

pneumonia.

DETAI LED DESCRIPTION OF THE I NVENTION

The present invention relates to a method of treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof in an individual who has or is at risk for developing such an infection. The invention described herein provides for a method of enhancing or augmenting the biological activity of anti- infective agents by the additional administration of an immunomodulatory or immunostimulatory agent. The

immunomodulatory or immunostimulatory agent comprises one or more ligands for one or more innate receptors. TLRs are a class of proteins that play a key role in the innate immune system. They are single membrane-spanning non-catalytic receptors that recognize structurally conserved molecules derived from microbes, also known as pathogen associated molecular patterns (PAMPs). These PAMPs are in turn recognized by antigen presenting cells (APC) and mucosal epithelial cells which in turn are activated and induce host defense mechanisms to help clear invading pathogens.

In certain embodiments the immunomodulatory or immunostimulatory agent is a TLR agonist. These immunostimulatory agonists include those know in the art for activating TLR2/1 , TLR2/6, TLR3, TLR4, TLR5, TLR9, or TLR7. In one embodiment, the

immunomodulatory or immunostimulatory agent is a TLR4 agonist. In a preferred embodiment, the TLR4 agonist is CRX-601. CRX-601 is a member of the aminoalkyl glucosaminide-4-phosphate (AGP) family known chemically as ω-aminoalkyl 2-amino-2- deoxy-4-phosphono-p-D-glucopyranosides. The AGPs are a class of synthetic lipid A mimetics that are structurally related to the major biologically active component in

monophosphoryl lipid A. In AGPs the reducing sugar has been replaced with an N-[(R))-3-n- alkanoyloxytetradeconoyl]aminoalkyl aglycon unit. Like other disaccharide lipid A derivatives, the AGP comprise six fatty acids for maximal biological activity, but unlike disaccharide derivatives the AGPs contain a conformationally flexible β-linked aglycon unit which permits energetically favored close packing of the six fatty acyl chains. Tight packing of six fatty acids in a hexagonal array is believed to play an essential role in the bioactivity of lipid A-like molecules. The AGP CRX-601 depicted here is described in detail in US 7,960,522 B2.

wherein (alkyl)„ is 5 and R₆ is C0₂H.

In another embodiment, CRX-601 is administered as a buffered aqueous formulation or formulated into liposomes, nanocapsuies, microparticies. lipid particles, vesicles, or the like. These compositions are used for the introduction of the immunomodulatory or immunostimulatory agent into suitable host cells/organisms. In particular, the compositions of the present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

The invention described herein provides for a method of enhancing or augmenting the biological activity of anti-infective agents by the additional administration of an

immunomodulatory agent. Although not limited by, herein we specifically exemplify the synergistic augmentation of biological effect (antimicrobial activity) of CRX-601 , a TLR4 agonist, in the presence of the anti-infective agent ceftazidime (CAZ).

Thus, the present invention provides for a method of treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof caused by a pathogens or potential pathogens in mammals, preferably humans. Examples of pathogens include viruses, bacteria, parasites, and fungus. In certain aspects, the pathogen is a virus. The virus can be from the Adenoviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxovirinae, Pneumovirinae, Picornaviridae, Poxyiridae, Retroviridae, or Togaviridae family of viruses. In a further embodiment the virus is the cause of an upper or lower respiratory infection. Representative medically relevant examples include, but are not limited to rhinovirus, coronavirus, parainfluenza virus, adenovirus, enterovirus, respiratory syncytial virus (RSV), bocavirus, influenza viruses, human metapneumovirus (hMPV),

orthomyxoviridae, cytomegalovirus, Epstein-Barr virus, herpes simplex virus, and

morbillivirus.

In yet a further aspect, the pathogen or potential pathogen being treated or protected against is a bacteria. Representative bacilli include, but are not limited to Hemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Helicobacter pylori, Salmonella enteritidis, and Salmonella typhi, Listeria, Staphylococcus, Streptococcus, Enterococcus, Actinobacteria and Clostridium Mycoplasma that lack cell walls and cannot be Gram stained, including those bacteria that are derived from such forms. In a further embodiment the bacteria is the cause of an upper or lower respiratory infection.

Representative medically relevant examples include, but are not limited to group A

Streptococcus (Streptococcus pyogenes), Haemophilus influenzae, Psuedomonas spp., Mycobacteria spp., Pasterurella spp., Pneumocystis jiroveci, Mycobacterium tuberculosis, Peptostreptococcus spp., Fusobacterium prevotella, Klebsiella pneumonia, Moraxella catarrhalis, Streptococcus pneumoniae, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Legionella pneumophila, Staphylococcus aureus, Corynebacterium

diphtheriae, Neisseria gonorrhoeae, Fusobacterium necrophorum, Bordetella pertussis, Treponema pallidum, Chlamydia trachomatis, Pseudomonas aeruginosa, Bacillus anthracis and Chlamydophila psittaci. Certain bacteria including but not limited to Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli are often associated with hospital-acquired (nosocomial) infections, and particularly related to mechanical ventilation or vascular catheterization of a patient.

In still another aspect, the pathogen or potential pathogen being treated or protected against is a fungus, such as members of the family Aspergillus, Candida, Crytpococus, Histoplasma, Coccidioides, Blastomyces, Pneumocystis, or Zygomyces. In still further embodiments a fungus includes, but is not limited to Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, or Pneumocystis carinii. The family zygomycetes includes Basidiobolales (Basidiobolaceae), Dimargaritales (Dimargaritaceae), Endogonales (Endogonaceae), Entomophthorales (Ancylistaceae, Completoriaceae, Entomophthoraceae, Meristacraceae, Neozygitaceae), Kickxellales (Kickxellaceae), Mortierellales (Mortierellaceae), Mucorales, and Zoopagales. The family Aspergillus includes, but is not limited to Aspergillus caesiellus, A. candidus, A. carneus, A. clavatus, A. deflectus, A. flavus, A. fumigatus, A. glaucus, A. nidulans, A. niger, A. ochraceus, A. oryzae, A. parasiticus, A. penicilloides, A. restrictus, A. sojae, A. sydowi, A. tamari, A. terreus, A. ustus, A. versicolor, and the like. The family Candida includes, but is not limited to Candida albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. milleri, C. oleophila, C. parapsilosis, C. tropicalis, C. utilis, and the like. In a further embodiement the fungus is the cause of an upper or lower respiratory infection. Representative medically relevant examples include, but are not limited to

Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Aspergillus spp., Candida spp., Candida albicans, Cryptococcus neoformans, Zygomycetes spp., Fusarium spp., Penicillium marneffii, Pseudallescheria boydii, Phialemonium obovatum, Pythium insidiosum, Absidia corymbifera, Enterocytozoon bieneus, Hormographiella aspergillata, Aspergillis spp., Histoplasma capsulatum, Pneumocystis jiroeci, Curvularia spp., Bipolaris spp., Exserohilum spp., Mucor spp., Rhizopus spp., Absidia spp., Cunninghamella spp., Metarrhizium anisopliae and Irpex lacteus.

In another embodiment, the pathogen or potential pathogen being treated or protected against is a NIAID Category A-C priority pathogens. Representative Category A Priority Pathogens include, but not limited to Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (plague), Variola major (smallpox), Francisella tularensis (tularemia), Arenaviruses, Bunyaviruses, Flaviruses, and Filoviruses.

Representative Category B Priority Pathogens include, but not limited to Burkholderia pseudomallei (melioidosis), Coxiella burnetii (Q fever), Brucella species (brucellosis), Burkholderia mallei (glanders), Chlamydia psittaci (Psittacosis), Typhus fever, Food- and waterborne pathogens (Diarrheagenic E.coli, Pathogenic Vibrios, Shigella species,

Salmonella, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica) , Caliciviruses, Hepatitis A, Cryptosporidium parvum, Cyclospora cayatanensis, Giardia lamblia, Entamoeba histolytica, Toxoplasma gondii, Naegleria fowleri, Balamuthia mandrillaris, Microsporidia, Mosquito-borne encephalitis viruses (West Nile virus, LaCrosse encephalitis, California encephalitis, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, Japanese encephalitis virus, St. Louis

encephalitis virus). Representative Category C Priority Pathogens include, but are not limited to, Nipah virus, Hendra virus, additional hantaviruses, Tickborne hemorrhagic fever viruses (Bunyaviruses and Flaviruses), Tickborne encephalitis complex flaviviruses

(Tickborne encephalitis viruses, European subtype Far Eastern subtype, Siberian subtype, Powassan/Deer Tick virus), Yellow fever virus, Tuberculosis, including drug-resistant TB, Influenza virus, Other Rickettsias, Rabies virus, Prions, Chikungunya virus, Coccidioides spp., Severe acute respiratory syndrome associated coronavirus (SARS-CoV), MERS-CoV, and other highly pathogenic human coronaviruses. For a list of Class A, B, and C

pathogens, see, for example

In another embodiment, the pathogen or potential pathogen being treated or protected against is a parasite. Examples of such parasites include, but are not limited to, Trypanosoma spp. (e.g., Trypanosoma, brucei, gambiense cruzi), Plasmodium spp. (e.g., Plasmodium falciparum), Giardia lamblia, Leishmania spp. (e.g., Leishmania donovani and mexicana amazonensis), Acanthamoeba, Balamuthia mandrillaris, Sarcocystis spp. (e.g., Sarcocysti bovihominis), Echinococcus spp. (e.g., Echinococcus granulosus) Toxoplasma gondii, Trichomonas spp.( e.g., Trichomonas vaginalis), Fasciola spp. (e.g., Fasciola hepatica), Fasciolopsis buski, Schistosoma spp.( e.g., Schistosoma mansoni), Trichinella spp. (e.g., Trichinella spiralis), Trichuris spp. (e.g., Trichuris trichiura), Opisthorchis spp. (e.g., Opisthorchis viverrini), Paragonimus spp. (e.g., Paragonimus westermani),

Ancylostoma duodenale, Ascaris spp. (e.g. Ascaris ascaris), Brugia spp. (e.g., Brugia malayi), Loa loa filarial, Toxocara (e.g., canis), Wuchereria bancrofti wherein the term spp. refers to aH species in that given genus.

By "agent effective against said pathogen or potential pathogen", is meant any agent known or thought to be effective in treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof, caused by the pathogen or potential pathogen. Such agents include, for example, antibiotics, antifungals, antiparasitics, and antivirals.

Representative classes of antibiotics and examples of drugs include, but are not limited to, the class of antibiotics known as β-lactams, including for example, penicillins, amoxicillin, flucloxacillin and cephalosporins, for example such as loracarbef, ceftazidime, cefalexin, cefotetan, and cefmetazole. Like the other β-lactam antibiotics, the

cephalosporin's core structure comprises a beta-lactam ring. Cephalosporins (which may be taken herein to include cephamycins) are generally classified into one of five "generations." So-called first generation cephalosporins may include cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole. Second generation cephalosporins may include cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam. Third generation cephalosporins may include cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone,

cefoperazone, ceftazidime. Fourth generation cephalosporins may include, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome. Fifth generation cephalsporins may include ceftobiprole and ceftaroline. Other cephalsporins may include cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetime, ceftioxide.

Other representative classes of antibiotics and examples of drugs include the class of antibiotics known as aminoglycosides, including for example, streptomycin, neomycin, kanamycin, and paromomycin., the class of antibiotics known as chloroamphenicol, exemplified by chloroamphenicolthe class of antibiotics known as glycopeptides, including for example, vancomycin and teicoplanin; the class of antibiotics known as ansamycins, including for example, geldanamycin, rifamycin, and napthomycin; te class of antibiotics known as streptogramins, including for example, pristinamycin IIA and pristinamycin IA; the class of antibiotics known as sulfonamides, including for example, prontosil, sulfanilamide, sulfadiazine, sulfisoxazole; the class of antibiotics known as tetracyclines, including for example, tetracycline, doxycycline, limecycline, and oxytetracycline; the class of antibiotics known as macrolides, including for example, erythromycin, clarithromycin, and azithromycin; the class of antibiotics known as oxazolidinones, including for example, linezolid, posizolid, tedizolid, and cycloserine;the class of antibiotics known as quinolones, including for example, ciprofloxacin, levofolxacin, and trovafloacin; the class of antibiotics known as lipopeptides, including for example, daptomycin, surfactin, and mupriocin.

Representative classes of antifungals include, but are not limited to, the class of antifungals known as azoles including for example, ketoconazole, miconazole, clotrimazole, econazole fluconazole, itraconazole, voriconazole, and posaconazole. The class of antifungals known as polyenes, includes for example, nystatin, pimaricin, amphotericin B and amphotericin, includes lipid formulations of amphotericine B, and amphotericine B deoxycholate. The class of antifungals known as allylamines, includes for example naftifine and terbinafine. The class of antifungals known as echinocandins, includes for example, caspofungin, micafungin and anidulafungin. Also included herein are other antifungals agents including but not limited to flucytosine.

Representative classes of antivirals include, but are not limited to, the class of antivirals known as adamantane antivirals, including for example, amantadine and rimantadine. The class of antivirals known as neuraminidase inhibitors includes for example, oseltamivir, zanamivir, and peramivir. The class of antivirals known as nucleoside reverse transcriptase inhibitors includes for example, entecavir, Iamivudine, adefovir, didanosine, tenofovir, abacavir, Iamivudine, zidovudine, stavudine, emtricitabine, zalcitabine, teibivudine, and didanosine. The class of antivirals known as integrase strand transfer inhibitors includes for example, raltegravir. dolutegravir, and elvitegravir. The class of antivirals known as non- nucleoside reverse transcriptase inhibitors includes for example, nevirapine, efavirenz, etravirine, rilpivirine, deiavirdine, and nevirapine. The class of antivirals known as protease inhibitors includes for example, simeprevir, boceprevir, telaprevir, fosamprenavir, darunavir ritonavir, tipanavir, atazanavir, neifinavir, amprenavir, and the like. The class of antivirals known as purine nucleoside including for example, ribavirin, valacyclovir, acyclovir, famciclovir, ganciclovir, valgancyclovir, and cidofovir. The class of antivirals known as chemokine receptor antagonists includes for example, maraviroc. The class of antivirals known as antiviral interferons includes for example peginterferon alfa-2a and peginterferon aifa-2b. Further examples included herein are combinations of antivirals such as

abacavir/lamivudine (Epzicom©), dasabuvir/ombitasvir/paritaprevir/ritonavir (Viekira Pak®), emtricitabine/tenofovir (Truvada®), ledipasvir/sofosbuvir (Harvoni®),

abacavir/doSutegravir/!amivudine (Triumeq®), and the like.

The present invention requires the administration of both 1) an agent effective against the pathogen or potential pathogen and 2) an immunomodulatory or

immunostimulatory agent. The two required agents may be administered concurrently but concurrent administration is not required. What is required is that both agents must be administered during the treatment.

In the method of this invention, the agent effective against the pathogen or potential pathogen can be administered in its normal or suggested manners, in its normal or suggested frequencies, and in its normal or suggested doses. However, due to the synergy resulting in the present invention, the agent effective against the pathogen or potential pathogen may also be administered at a doses and/or frequencies less than its normal or suggested doses or frequencies.

Most animal models are used for the purpose of generating results that can be extrapolated to human diseases. In drug development, rodent studies are part of routine preclinical studies that determine progression to clinical studies in humans. Mice are the most commonly used species in biomedical research. The advantages of mice (relative to other species) as the choice for animal models include cost, size, public acceptance, availability of reagents, rapid generation time, and ease of genetic manipulation. However, a problem with this approach for the study of inflammation is that rodents are highly resilient to most models of induced inflammation compared with humans (Warren et al. (2010) J. Infect. Dis. 201 (2): 223-232; Warren et al. (2015) Proc. Natl. Acad. Sci. U S A. 1 12(4): E345)

A commonly used assay to assess novel pathways of inflammation consists of challenging mice with lipopolysaccharide (LPS), which activates cells through Toll-like receptor 4 (TLR4). Most wild-type mice are highly resilient to challenge with LPS. The dose of LPS used in most in vivo studies is 1-25 mg/kg (Glode et al. (1976) Infect. Immun. 14(3): 626-630; McCuskey et al. (1984) Infect. Immun. 45(1): 278-280; Schaedler et. al. (1961) J. Exp. Med. 113(113): 559-570; Reynolds et al. (2002) J. Endotoxin Res. 8(4): 307-314). This dose is -1 ,000,000 times the 2-4 ng/kg dose of LPS used in human volunteer studies to induce fever and cytokines (van der Poll and Lowry In: Tellado et. al. (1995) Prog. Surg. Basel, Karger 200: 18-32) and -1000-10,000 times the dose required to induce severe disease with shock in humans (Taveira da Silva et al. (1993) N. Engl. J. Med. 328(20): 1457- 1460; Sauter and Wolfensberger (1980) Lancet 2(8199): 852-853). Interestingly, compared to a dose of 2 ng/kg in human volunteers, a dose which induces cytokines and fever, rodents elicit a similar levels of cytokines at a dose of 500 ng/kg, however the mouse physiology is unaffected (Copeland et al. (2005) Clin. Diagn. Lab. Immunol. 12(1): 60-67). Despite these findings, the doses given to mice in NSR experiments with AGPs, such as CRX-527, having LPS like potency, range from -100-1000 times higher than the 500 ng/kg dose required to elicit cytokines levels that cause fever in humans. In Example 4, a fully tolerable dose of 1 mg/kg dose of CRX-601 was used in mice to test the ability of CRX-601 to induce NSR against P. ae/xgv^'nosa-induced pneumonia. We found that CRX-601 , when used at this dose level had a measurable effect on survival, lung bacterial count, and dissemination of the bacteria to the spleen. However, a 1 mg/kg dose of CRX-601 is -500,000 time the tolerable dose of LPS in humans and -2000 times the dose required to elicit cytokines levels in mice that cause fever in humans.

In contrast to rodents, the threshold pyrogenic dose for both rabbits and man has been shown to be nearly equivalent for several bacterially sourced endotoxins, when adjusted for weight (Greisman and Hornick (1969) Proc. Soc. Exp. Bio. Med. 131 : 1154- 1158). In Example 2, we initially determined the maximum tolerable dose (MTD), defined here as, the highest dose that does not induce a fever in New Zealand White (NZW) rabbits. Proximal doses to the MTD were then used in NZW rabbits to test the ability of CRX-601 to induce NSR against P. aeruginosa- induced pneumonia. In Example 3, interestingly, we found that CRX-601 , when used at these clinically relevant dose levels had no measurable effect on local lung bacterial counts or in the dissemination of the bacteria to the spleen. Furthermore, CRX-601 showed no advantageous effect on survival. In light of these findings, we were surprised to find that at clinically relevant doses, the additional administration of CRX-601 to antibiotic ceftazidime treatment resulted in a significant and synergistic augmentation of antimicrobial activity compared to ceftazidime treatment alone. The antibiotic, ceftazidime, is used as a standard of care antimicrobial for the treatment of P. aeruginosa infections.

Potent AGPs, such as CRX-527 and CRX-601 , have a similar potency and inflammatory response as endotoxin. As such, the choice of preclinical species is of paramount importance to determine the viability of progression to clinical studies in humans. As previously mentioned, rodents are highly resilient to most models of induced inflammation compared with humans, thus, complicating the extrapolation of experimental results to human diseases (Warren (2010) J. Infect. Dis. 201 (2): 223-232). In contrast to rodents, the threshold pyrogenic dose for both rabbits and man has been shown to be nearly equivalent for several bacterially sourced endotoxins, when adjusted for weight (Greisman and Hornick (1969) Proc. Soc. Exp. Bio. Med. 131 : 1154-1158). Furthermore, rabbits are generally used to test biologicals and to standardize endotoxin and other pyrogenic agents prior to use in humans. Thus, rabbits represent a rational species in which to measure the therapeutic ef f i cacy/to I e ra b i I i ty of inflammatory agents.

In the method of this invention, the immunomodulatory or immunostimulatory agent may be administered from 1 to 7 times per week for the duration of treatment with the agent effective against the pathogen or potential pathogen. In another embodiment the

immunomodulatory or immunostimulatory agent is administered from 1 to 3 times per week for the duration of treatment with the agent effective against the pathogen or potential pathogen. In a preferred embodiment the immunomodulatory or immunostimulatory agent is administered from 1 to 2 times per week for the duration of treatment with the agent effective against the pathogen or potential pathogen. The immunomodulatory or immunostimulatory agent, for example CRX-601 can be administered, for example, at a dose of 0.001 ng/kg to 1000 ng/kg. In another embodiment, CRX-601 is administered at a dose of 0.01 ng/kg to 100 ng/kg. In still yet another embodiment, CRX-601 is administered at a dose of 0.1 ng/kg to 100 ng/kg. In a preferred embodiment, CRX-601 is administered at a dose of 0.1 ng/kg to 10 ng/kg. In another preferred embodiment, CRX-601 is administered at a dose of 1 ng/kg to 100 ng/kg. In still yet another preferred embodiment, CRX-601 is administered at a dose of 1 ng/kg to 10ng/kg.

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of certain embodiments and are not intended as limitations of the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. Example 1

Materials and Methods P. aeruginosa- Induced Pneumonia Rabbit Model

Animals. Immunocompetent female New Zealand White rabbits (body weight, 2.8 to 3 kg) were bred and housed at the Zootechnical Center (University of Burgundy, France) in accordance with current European Institute of Health guidelines. They were placed in individual cages and provided ad libitum with water and food. Animals were randomly assigned to a control group or to a treatment regimen (TLR4 agonist, CRX-601 or antibiotic, ceftazidime).

Bacterial strains and antibiotics. Pseudomonas aeruginosa PA14 strain was used in this experiment. Minimal inhibitory concentration (MIC) was 4 mg/L for ceftazidime. A preliminary in vitro study was performed in order to standardize the inoculum sizes.

Preparation of the inoculum. Before each animal experiment, the PA14 strain from one frozen aliquot was cultured on Mueller-Hinton agar plates (MH, Biomerieux, France) and incubated for 24 hours at 37°C. Five colonies were inoculated into 9 ml_ of Brain Heart Infusion broth (BHI, Biomerieux, France), incubated for 6 hours at 37°C under agitation (200 rpm) and then cultured on MH agar plates for 18h at 37°C. This culture was diluted in physiological saline to obtain the desired concentration (8.5 Log10 or 9 Log10 colony-forming units (CFU)/ml_). No adjuvant was used. Viable bacterial counts were determined using optical density measurements in reference to a standard curve and then confirmed by using successive dilution cultures.

Administration of ceftazidime. A 2 day treatment of ceftazidime (CAZ) was started 5h after bacterial challenge. It was delivered through the central venous catheter with changing infusion rates obtained by a computer-controlled electric pump. One type of administration of CAZ was simulated in animals: the human equivalent of continuous infusion (cCAZ) at a daily dose of 4g. For this method of delivery, the target concentration of serum CAZ was approximately 30mg/L (the administered daily dose was 200 mg/kg/day).

Pharmacokinetic (PK) analysis for ceftazidime. For each animal, the concentrations of antibiotics in serum were determined on iterative blood samples obtained through the central venous catheter. CAZ concentrations were determined by a disc plate bioassay with Proteus mirabilis ATCC 21 100 as the indicator organisms. The limit of detection was 0.5 mg/L. Standard curves were established with solutions in serum. The serum samples were diluted in serum to ensure that their concentrations would be within the range of those on the standard curve. The standard samples were assayed for each experiment and

concentrations were assayed in duplicate. Administration of TLR4 agonist CRX-601. Animals received a single prophylaxis dose of TLR4 agonist (CRX-601) 24h prior to bacterial challenge. The aqueous formulation of the compound was administered intravenously to one group of animals at the dose of 5 ng/kg. The liposomal formulation was administered intravenously but at doses of 200 ng/kg or 2000 ng/kg.

Experimental pneumonia model in rabbits. Installation of double central venous catheters and production of bacteremic pneumonia in immunocompetent rabbits were performed as previously described (Piroth et al. (1999) Antimicrob. Agents Chemother. 43: 2484-92; Croisier et al. (2002) J. Antimicrob. Chemother. 50: 349-60). Briefly, 48h after jugular catheterization, bacterial pneumonia was induced by endobronchial challenge with 0.5 ml_ saline containing 8.5 or 9 Log10 CFU/mL of the PA14 strain.

Bacterial counts. Rabbits that survived over the experiment period (48 hrs) were anaesthetized and sacrificed 2h after the end of antibiotic infusion to avoid any carry-over effect. Rabbits that died within the first 48h were also included in the analysis. The spleen and pulmonary lobes were weighed and homogenized in 5 ml_ sterile saline. Bacteria were counted in a sample of this crude homogenate by plating 10-fold dilutions on cetrimide agar plates (Biomerieux, France) and incubating for 24 hours at 37°C. Bacterial concentrations in each pulmonary lobe and in the spleen were calculated after adjusting for weight. The lower limit of detection was 1 Iog10 CFU/g. For each rabbit, the mean pulmonary bacterial concentration was calculated according to each lobar bacterial concentration with lobar weight (expressed as CFU/g).

Statistical Analysis. Statistical analysis was performed used SAS version 9.3.

P. aeruginosa- Induced Pneumonia Mouse Model

Animals. Specific pathogen free male BALB/c mice from Jackson Labs at 7-9 weeks old and weighing approximately 18-24 grams were used throughout. Animals were allowed access to food and water ad libitum. Ten animals total were used per treatment arm. All procedures were performed in accordance with protocols approved by the GSK Institutional Animal Care and Use Committee, and met or exceeded the standards of the American Association for the Accreditation of Laboratory Animal Care (AAALAC), the United States Department of Health and Human Services and all local and federal animal welfare laws.

Bacterial strains and antibiotics. Pseudomonas aeruginosa PA01 was utilized in this study. Minimal inhibitory concentration (MIC) was 4 mg/L for ceftazidime. A preliminary in vitro study was performed in order to standardize the inoculum sizes.

Preparation of the inoculum. A frozen stock of Pseudomonas aeruginosa PA01 was thawed and subcultured into Brain Heart Infusion (BHI) broth (Becton Dickinson, Sparks MD) by adding 100uL to 50 mL broth and placing in a shaking incubator (approximately 120 RPM) at 37°C for approximately 18 h. This overnight broth culture was washed three times by centrifuging at 5000 RPM and 37°C for 5 minutes to pellet the bacteria, removing the supernatant, reconstituting in sterile saline, and repeating the process two additional times. After the final wash, the supernatant was removed, and the bacterial pellet was reconstituted with 30 ml_ of sterile saline. A 1000 μΙ aliquot of the reconstituted bacterial suspension was added to a new flask of 50 ml_ BHI and placed in the shaking incubator as above for 3 h to obtain a fresh subculture in the logarithmic growth phase. After 3h, this subculture was washed once as above and reconstituted with 10 mL sterile saline. To obtain the final stock for infecting, this fresh reconstituted bacterial suspension was diluted 1 : 1.7 for the first study and 1 :2 for the second study using sterile saline. An aliquot of the infecting stock was serial diluted and plated (see below) to determine the final concentration. For bacterial

enumeration of all Pseudomonas aeruginosa samples, serial 1 : 10 dilutions were performed and aliquots were subcultured onto trypticase soy agar plates supplemented with 5 % sheep blood (blood agar; BBL, Becton Dickinson, Sparks, MD). Plates were incubated at 37°C for 24 h prior to evaluation.

Administration of TLR4 agonist CRX-601. Animals received a single intravenous prophylaxis dose of CRX-601 , liposomal formulation, at a dose of 1 mg/kg 24h prior to bacterial challenge. Additional groups of animals received vehicle only and served as infected vehicle treated controls (VTC).

Experimental pneumonia model in mice. All mice were infected via intranasal inhalation as follows: The mice were anesthetized with Isoflurane (5%) and oxygen. Using the final bacterial suspension at room temperature, mice were infected intranasally by placing 50 μΙ_ on the nares and allowing the mice to inhale the inoculum. The final administered inoculum was 7.1 log 10 CFU/mouse.

Bacterial counts. Animals were euthanized at 48 hours post infection. Lungs and kidneys were excised aseptically and homogenized in 1 mL PBS using a laboratory blender (Stomacher 80, Seward Ltd., and Worthing, UK). For enumeration of viable bacteria, samples were serial diluted tenfold in saline and plated in triplicates of 20 μ\- onto blood agar plates by a modified Miles Misra technique using the Hamilton liquid handling system. The colonies were counted following overnight incubation at 37°C. The lower limit of

quantification was 1.2 Iog10 CFU/g tissue.

Statistical Analysis. The outcome measures for comparison of treatments was survival and the number of bacteria isolated from lung and spleens (log 10 CFU/g tissue) at the conclusion of the study. Lung and spleen results are presented as group means with standard deviations. Statistical analysis was performed using a two-tailed Students t-Test, and a p value of < 0.05 was considered significant. Example 2

CRX-601 PYROGENICITY TESTING IN NZW RABBITS

Purpose. The purpose of this study was twofold, 1) To evaluate the dose at which the test article causes a febrile reaction, when administered as an intravenous injection to New Zealand White rabbits and 2) To determine the doses to be used in the P.

pneumonia rabbit model using the data from 1 above.

Test Article Preparation. There were two test articles and one control used in this study. Test article 1 : CRX-601 liposomal was administered at three doses, consisting of 5000, 1000, or 200 ng/kg via marginal ear vein. Test article 2: CRX-601 aqueous was administered at one dose consisting of 2.5 ng/kg via marginal ear vein. The vehicle control article: HEPES-Saline buffer was administered in a single dose via marginal ear vein.

Pyrogenicity Testing. The animals were assigned three animals per group. Body temperature was measured to the nearest 0.1 °C using rectal probes that have been calibrated to assure an accuracy of ±0.1 °C and have been tested to determine that a maximum reading will be reached in less than five minutes. The probe was inserted at least 7.5 cm into the rectum of the test rabbit and kept in place for at least two to three minutes before the temperature was read and recorded. On Day 1 , rectal temperature was measured 30 minutes prior to dosing (Pre-Dose) and then at 60 ± 5 minutes, 90 ± 5 minutes, 120 ± 5 minutes, 150 ± 5 minutes, and 180 ± 5 minutes after dose administration. On Days 2, 3, and 4, temperatures were determined 24 ± 1 hour (Day 2), 48 ± 2 hours (Day 3), and 72 ± 2 hour (Day 4) from the time of dose administration. A temperature increase of 0.5°C or greater for three consecutive measurements was considered an adverse pyrogenic response.

Results. Test article 1 , CRX-601 liposomal, was non-pyrogenic up to 1 ,000 ng/kg delivered via the marginal ear vein. Additional testing revealed an adverse pyrogenic response at 2,000 ng/kg. Test article 2, CRX-601 aqueous, was non-pyrogenic up to 2.5 ng/kg delivered via the marginal ear vein. Additional testing revealed an adverse pyrogenic response at 5 ng/kg.

Dose Rational. The lowest observable adverse effect level (LOAEL) was the dose chosen for each of the two CRX-601 formulations. The LOAEL was used to ensure target engagement and to represent dose proximal to the maximum clinical dose. The LOAEL for CRX-601 liposomal was 2,000 ng/kg. The LOAEL for CRX-601 aqueous was 5 ng/kg. EXAMPLE 3

CRX-601 DEMONSTRATES SYNERGISTIC ANTI-INFECTIVE ACTIVITY AS ADJUNTIVE THERAPY AGAINST PSEUDOMONAS AERUGINOSA-\NDUCED PNEUMONIA IN RABBIT MODEL BUT NO ANTI-INFECTIVE ACTIVITY AS A STAND-ALONE AGENT.

Previous studies in the current rabbit model have demonstrated an effective reduction in bacterial count in the lung and spleen from a human simulated PK of continuous therapeutic infusion of ceftazidime (Croisier, et al. (2008) Int. J. Antimicrob. Agents 32(6): 494-8). In this study, the efficacy of TLR4 agonist CRX-601 was tested as two different formulations, liposomal (DOPC-Cholesterol) and aqueous (HEPES-Saline). The test dose for each formulation represents their respective lowest observable adverse effect level

(LOAEL).

The primary study goal was to determine the efficacy of TLR4 agonist CRX-601 as a stand-alone anti-infective agent at clinically relevant doses in a rabbit model of pseudomonal pneumonia induced by P. aeruginosa (PA14). In addition, we also tested CRX-601 as adjunctive therapy with the antibiotic ceftazidime. Efficacy parameters were assessed 48h after PA14 induced pneumonia. Efficacy parameters were the following; % survival, residual bacteria in lung, and dissemination of bacteria to the spleen. A total of six (6) rabbits were used per treatment arm and animals were infected (challenged) with an inoculum at 9.0 +/- 0.1 Log10 CFU/mL via endobronchial intubation. For the analysis of bacterial load counts, animals that died prior to the 48h end point as well as those that survive the 48h were used.

Survival rates. Survival for CRX-601 (liposomal 2,000 ng/kg or aqueous 5 ng/kg), as a stand-alone treatment, was not statistically different from the vehicle control group; 50%, 60%, and 50%, respectively. In comparison, we observed 100% survival in animals treated with ceftazidime alone or ceftazidime with CRX-601 (both liposomal and aqueous groups), Fig 1.

Bacterial counts in lung and spleen. Lung bacterial counts are depicted in Fig 2 and spleen bacterial counts in Fig 3. The bacterial counts in the lung and spleen of animals treated with CRX-601 (liposomal or aqueous) as a stand-alone treatment, were not statistically different from the vehicle control group. Ceftazidime (only) treatment significantly decreased lung bacterial 3.3 Log10 CFU/g, p < .05. However, ceftazidime (only) treatment did not significantly impact the dissemination of bacteria to the spleen (1.3 Log10 CFU/g reduction, p > .05). Interestingly, the additional administration of CRX-601 , (liposomal or aqueous), to ceftazidime resulted in significant decreases in bacterial in both the lung and spleen. In animals treated with CRX-601 (aqueous 5 ng/kg) plus ceftazidime, bacterial counts were reduced by 3.5 Log10 CFU/g in the lung and 1.9 Log10 CFU/g in the spleen (both values significant, p < .05). The greatest reduction in bacteria was observed by the additional administration of CRX-601 (liposomal 2,000 ng/kg) to ceftazidime. Bacterial counts were reduced by 3.9 Log10 CFU/g in the lung and 2.3 Log10 CFU/g in the spleen (both significant, p < .05). EXAMPLE 4

CRX-601 AS A STAND-ALONE ANTI-INFECTIVE AGENT IS EFFECTIVE AGAINST PSEUDOMONAS AERUGINOSA-\NDUCED PNEUMONIA IN MOUSE MODEL

The purpose of the study was to measure the impact of CRX-601 liposomal (DOPC- Cholesterol) as a stand-alone agent in a mouse model of P.

pneumonia. In brief, mice (10 animals per treatment arm) were intravenously dose with either CRX- 601 (1 mg/kg) or buffered saline and 24 hours later intranasally inoculated with 50 μ\- of Pseudomonas aeruginosa (strain PA01) at 7.1 Log10 CFU/mL. The survival of the animals was recorded over 48h at which time the residual bacterial concentration in the lungs and spleen were measured. For the analysis of bacterial load counts, animals that died prior to the 48h end point were not used.

Survival rates. Only a 30% survival rate was observed at 48 hours for the vehicle control mice, whereas, the survival rate of mice pre-treated with CRX-601 (alone) was improved to 80%, p < .05 (Fig. 4).

Bacterial counts in lung and spleen. In mice, treatment with CRX-601 (liposomal), as a stand-alone agent, significantly reduced the bacterial count in both the lung and spleen. The bacterial count was reduced by in the lungs by 2.9 Log10 CFU/g in the lung, p < .01 (Fig 5) and 3.8 Log10 CFU/g, p < .001 in the spleen (Fig 6).

Claims

WHAT IS CLAIMED IS: 1. A method of treating, ameliorating, preventing, or reducing infectious disease or the symptoms thereof in a mammal caused by a pathogen or potential pathogen, comprising administration of 1) an agent effective against the pathogen or potential pathogen and 2) an immunomodulatory or immunostimulatory agent.

2. The method of Claim 1 wherein said agent effective against the pathogen or potential pathogen is an antibiotic agent.

3. The method of Claim 2 wherein said antibiotic agent is of the class of antibiotics known as β-lactams, Aminoglycosides, Chloroamphenicol, Glycopeptides, Ansamycins,

Streptogramins, Sulfonamides, Tetracyclines, Macrolides, Oxazolidinones, Quinolones, or Lipopeptides.

4. The method of Claim 2 wherein said antibiotic agent is amoxicillin, levofoxacin, ciprofloxin, clindamycin, vancomycin, gentamycin, azithromycin, cefalexin, ceftazidime, streptomycin, geldanamycin, neomycin, kanamycin, prontosil, minocycline, erythromycin, linezolid, loracarbef, or mupirocin.

5. The method of Claim 1 wherein said agent effective against the pathogen or potential pathogen is an antifungal agent.

6. The method of Claim 5 wherein said antifungal agent is from the class of antifungals known as Azoles, Polyenes, Allylamines, or Echinocandins.

7. The method of Claim 5 wherein said antifungal agent is ketoconazole, fluconazole, nystatin, amphotericin B, naftifine, caspofungin, micafungin, or flucytosine

8. The method of Claim 1 wherein said agent effective against the pathogen or potential pathogen is an antiviral agent.

9. The method of Claim 8 wherein said antiviral agent is from the class of antivirals known as adamantane antivirals, neuraminidase inhibitors, nucleoside reverse transcriptase inhibitors, integrase strand transfer inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, purine nucleoside, chemokine receptor antagonists, or antiviral interferons.

10. The method of Claim 8 wherein said antiviral agent is amantadine, rimantadine oseltamivir, zanamivir, peramivir, entecavir, lamivudine, adefovir, tenofovir, dolutegravir nevirapine, efavirenz, boceprevir, telaprevir, ribavirin, valacyclovir, acyclovir, ganciclovir, valgancyclovir, or cidofovir.

11. The method of Claim 1 wherein said agent effective against the pathogen or potential pathogen is an antiparasitic agent.

12. The method of Claim 11 wherein said antiparasitic agent is chloroquine, primaquine doxycycline atovaquone/proguanil, metronidazole, tinidazole, ivermectin, metronidazole, tinidazole.or albendazole.

13. The method of any of Claims 1-12 wherein said immunomodulatory or

immunostimulatory agent is a TLR agonist including those know in the art for activating TLR2/1 , TLR2/6, TLR3, TLR4, TLR5, TLR9 and TLR7.

14. The method of Claim 13 wherein said immunomodulatory or immunostimulatory agent is a TLR4 agonist.

15. The method of Claim 14 wherein said immunomodulatory or immunostimulatory agent is a synthetic TLR4 agonist.

16. The method of Claim 15 wherein said immunomodulatory or immunostimulatory agent is a synthetic lipid A mimetic of the aminoalkyi glucosaminide-4-phosphate (AGP) family known chemically as ω-aminoalkyl 2-amino-2-deoxy-4-phosphono-p-D-glucopyranosides.

17. The method of Claim 16 wherein said immunomodulatory or immunostimulatory agent is

wherein the n in (CH₂)_n is 5 and R₆ is C0₂H or CH₂OP0₃H₂.

18. The method of Claim 17 wherein R₆ is C0₂H.

19. The method of Claim 18 wherein said immunomodulatory or immunostimulatory agent is administered at a dose of .001 ng/kg to 1000 ng/kg.

20. The method of Claim 18 wherein said immunomodulatory or immunostimulatory agent is administered at a dose of .01 ng/kg to 100 ng/kg.

21. The method of Claim 18 wherein said immunomodulatory or immunostimulatory agent is administered at a dose of .1 ng/kg to 100 ng/kg.

22. The method of Claim 18 wherein said immunomodulatory or immunostimulatory agent is administered at a dose of .1 ng/kg to 10 ng/kg.

23. The method of Claim 18 wherein said immunomodulatory or immunostimulatory agent is administered at a dose of 1 ng/kg to 100 ng/kg.

24. The method of Claim 18 wherein said immunomodulatory or immunostimulatory agent is administered at a dose of 1 ng/kg to 10 ng/kg.

25. A method for preventing or treating bacterial infection comprising administering a TLR4 agonist and a cephalosporin to a patient at risk of contracting or continuing bacterial infection.

26. The method of claim 25 wherein administration of the TLR4 agonist and cephalosporin decreases risk of dissemination of the bacteria from a prior infection site to a subsequent infection site within the patient when compared to treatment with the cephalosporin alone.

27. The method of claim 26 wherein the treatment reduces bacterial load at both infection sites when compared to treating with the cephalosporin alone.

28. The method of claims 26 or 27 wherein the prior infection site is the patient's lung.

29. The method of claim 26-28 wherein the subsequent infection site is the patient's spleen.

30. The method of claim 25 wherein the TLR4 agonist is free of endotoxin.

31. The method of claim 30 wherein the TLR4 agoinist is a synthetic disaccharide.

32. The method of claim 30 wherein the TLR4 agonist is an AGP.

33. The method of claim 30 wherein the AGP is CRX-601.

34. The method of any of claims 25 -33 wherein the cephalosporin is a first, second, third, fourth, or fifth generation cephalosporin.

35. The method of any of claims 25-33 wherein the cephalosporin is selected from the group of loracarbef, ceftazidime, cefalexin, cefotetan, and cefmetazol.

36. The method of any of claims 25-35 wherein the cephalosporin is anti-pseudomonal cephalosporin.

37. The method of claim 36 wherein the anti pseudomonal cephalosporin is selected form the group consisting of ceftazidime, cefoperzone, cefepime, cefpirome, ceftobiprole and ceftaroline.

38. The method of any of the proceeding claims wherein the infection is a nosocomial infection.

39. The method of any preceding claim wherein the pathogen associated with the infection is of the Pseudomonas, Staphylococcus, Escherichia, Klebsiella or Acinetobacter genera.

40. The method of any preceding claim wherein the pathogen associated with the infection is Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia or Acinetobacter baumannii.

41. The method of any of the preceding claims wherein the patient is or will be on mechanical ventilation and, additionally or alternatively, has or will receive a vascular catheter, such as a central venous catheter.

42. A composition for treating infection comprising a cephalosporin and a TLR4 agonist.