WO2014138290A1 - Compositions and methods for treating bacterial infections - Google Patents

Abstract

The present invention relates to compositions and methods of treating a patient who has been exposed to, or who is at risk of exposure to, a pathogen (e.g., a bacterial pathogen or a eukaryotic pathogen such as a fungus or a parasite), and more specifically to inhibiting fibrinolysis to complement host defenses that are mediated by T cells, antibodies, antigens or vaccines. The host defenses can be mediated by T cells or antibodies, or induced by antigens or a vaccine. For example, the invention features the use of a therapeutically effective amount of an agent that inhibits fibrinolysis and an antigen expressed by a pathogen; cells expressing the antigen; an antibody that specifically binds an epitope on the pathogen; or cells expressing such antibodies (e.g., in the preparation of a medicament or in the preparation of a medicament to treat a patient who has been exposed to, or who is at risk of exposure to, the pathogen).

Description

COMPOSITIONS AND METHODS FOR TREATING BACTERIAL INFECTIONS CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 61/772,916, which was filed March 5, 2013.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant number R01-AI071295 and grant number R01-AI061577 awarded by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for treating bacterial infections and more particularly to treatments that activate an adaptive immune response in a host and increase the level or stability of fibrin at the site of infection.

BACKGROUND

Bacterial infections represent major health hazards to humans. Diseases like typhoid, cholera, bubonic plague, and tuberculosis have killed millions of people over time. In recent decades, vaccines and antibiotics have proved effective in the developed world. However, the emergence of antibiotic-resistant bacterial strains has shaken the public's sense of safety in continued antibiotic use. Vaccines that provide protective, adaptive immunity against pathogens remain available against many common bacterial infections. However, outbreaks still occur. Moreover, Yersinia pestis, one of the most deadly human pathogens, remains endemic in rodent populations around the world and has been weaponized (Smiley, Expert Rev. Vaccines 7:209- 221, 2008). Thus, there remains a need for therapeutic and prophylactic approaches to combat bacterial infections.

There is an interesting link between bacterial infection and fibrin, the protein responsible for blood clotting. Fibrinogen, a soluble precursor to fibrin, circulates in plasma and is cleaved by thrombin to produce fibrin. This occurs upon damage to the vasculature, after which fibrin forms a polymerized mesh of protein fibers that help to plug the damaged blood vessels and stem blood loss. As the damaged tissue repairs, the fibrin clot undergoes fibrinolysis as plasminogen is activated by partial proteolysis, which is mediated in turn by proteins such as tissue plasminogen activator or urokinase (Rijken and Lijnen, J. Thromb. Haemost. 7:4-13, 2009). Many bacterial pathogens promote fibrinolysis by activating plasminogen. For example, certain streptococci express streptococcal streptokinase, staphylococci such as Staphylococcus aureus express staphylokinase, and Yersinia pestis expresses Pla. The Pla protein promotes fibrinolysis by activating host plasminogen and also inactivating alpha-2-antiplasmin, plasminogen activator inhibitor 1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI) (Beesley et al, J. Bacteriol. 94: 19-26, 1967; Sodeinde et al, Science 258: 1004-1007, 1992; Kukkonen et al, Mol. Microbiol. 40: 1097-1111, 2001; Haiko et al, J. Bacteriol. 192:4553-4561, 2010; Vails Seron et al, J. Thromb. Haemost. 8:2232-2240, 2010). It is generally believed that these bacterial mechanisms for degrading fibrin help bacteria evade physical barriers provided by fibrin within the host.

SUMMARY

The present invention is based, in part, on our work with the causative agent of plague {Yersinia pestis) and our studies indicating that inhibiting fibrinolysis complements host defenses that are mediated by T cells, antibodies, or vaccines. This finding supports a dual approach to treatment options for a wide variety of invasive human pathogens, including existing, emerging and drug-resistant pathogens. Accordingly, in one aspect, the invention features methods of treating a patient who has been exposed to, or who is at risk of exposure to, a pathogen {e.g., a bacterial pathogen). The methods can include a step of administering, to a patient (a) an antigen expressed by the pathogen, or a mimic of said antigen, or cells that express the antigen and (b) an agent that inhibits fibrinolysis. In any embodiment, one can administer a plurality of either or both types of agents. For example, one can administer one or more antigens; one or more cell types that respectively express two or more antigens; one or more agents that inhibit fibrinolysis; or combinations thereof. For example, one can administer one antigen and two agents that inhibit fibrinolysis; one cell type that expresses a plurality of antigens and one or two agents that inhibit fibrinolysis; two cell types, each of which express distinct antigens, and one or two agents that inhibit fibrinolysis; and so forth. In instances where T cells are already primed in the patient, perhaps from an earlier vaccine or from an earlier exposure to the bacteria, the agent that inhibits fibrinolysis can be administered without also administering the antigen or cells that express the antigen. Accordingly, in another aspect, the invention features methods that include the steps of: (a) determining whether a patient carries T cells primed to react to a pathogen of interest and (b) supplying, to the patient, an agent that inhibits fibrinolysis. The agent that inhibits fibrinolysis would then be administered to the patient (e.g., by a self-administration) when the patient is exposed to the pathogen or is entering a situation of high risk.

Any of the methods described herein can be expressed in terms of "use" of the present compositions (e.g., in the preparation of a medicament or in the preparation of a medicament for the treatment of a disease, condition or disorder associated with a pathogen (e.g. , a bacterial pathogen, a fungus, or parasite).

In embodiments of any of the present methods, one can administer one or more antibodies that specifically bind one or more pathogens (e.g., bacterial pathogens) or one or more cells or cell types expressing such antibodies rather than, or in addition to, administering antigen(s) or a cell or cell types that express the antigen. This approach may be desirable where antibody production in the patient is sub-optimal, e.g elderly or immunocompromised individual. Any of the present methods can also include a step of identifying a patient in need of treatment. Patients at risk of exposure to a bacterial pathogen include (i) patients who develop a non-healing infection that appears to be antibiotic-resistant; (ii) patients entering a hospital or other facility (e.g., a care facility such as a rehabilitation facility or nursing home) known to have an antibiotic-resistant bacteria in circulation; and (iii) persons (e.g. , military personnel) entering an area where some antibiotic-resistant bacteria is common. In all of these cases, the patient could be immunized with the antigen and then treated (when necessary) with an antifibrinolytic, whether that antifibrinolytic agent targets a compound within the patient in such a way that the agent facilitates clot breakdown or targets a compound the pathogen expresses to facilitate clot breakdown. Thus, while the types of agents described herein (i.e., an antigen, a cell expressing an antigen, an antibody that specifically binds a pathogen, a cell expressing such an antibody, and an agent that inhibits fibrinolysis) can be administered at the same time or in near succession (e.g., within minutes to hours), it is also the case that days, weeks, months, or even years may pass between the administration of an agent that elicits an immune response and administration of an agent that inhibits fibrinolysis. In determining a treatment protocol, one could also carry out a step to determine whether or not a patient has been exposed to a given pathogen previously (e.g. , by determining whether or not a patient has T cells mediating a cellular, acquired immune response). The first and second agents may be administered by the same or different routes of administration. Bacterial infections can occur where there is a break in the skin. For example, the skin's integrity can be disrupted by almost any sort of trauma, whether intentional, as in a surgical procedure, or unintentional, as in an accident or other mishap. Other breaches may be caused by ulceration, as can occur in patients who are bed-ridden, who have a cancer, or who have diabetes. Accordingly, one or more of the types of agents described herein can be formulated for topical administration at the site of an insult. For example, the agent(s) can be incorporated into a lotion, cream, gel, foam, or the like, or incorporated into a device that is designed to contact a wound, such as a dressing, bandage, splint, or the like. In other embodiments, a first agent (an antigen, a cell expressing an antigen, an antibody that specifically binds a pathogen, and/or a cell expressing such an antibody) and/or a second agent (that inhibits fibrinolysis) can be administered intravenously, intraperitoneally, subcutaneously, intradermally or intramuscularly. In other embodiments, the first and/or second agents can be administered to the eye, ear, or sinuses, and these routes are contemplated particularly for the treatment of pathogens that tend to affect these organs (e.g., H. influenzae and M. cattarrhalis) . In other embodiments, the first and/or second agents can be administered orally, for example to treat pathogens that tend to affect the gastrointestinal tract, or through inhalation or insufflation to treat, inter alia, pathogens that cause respiratory infections.

Patients amenable to treatment include human patients (including infants, children, teenagers, adults and elderly patients). However, the methods can also be carried out with veterinary patients (including domesticated animals, livestock, and animals kept in captivity).

The bacterial pathogen can be of any bacterial genus, including genus Yersinia,

Haemophilus, Salmonella, Streptococcus, Staphylococcus, Mycobacterium, Escherichia, Helicobacter, Moraxella, Mycoplasma, Neisseria, Proteus, Pseudomonas, or Borrelia.

Infections caused by eukaryotic pathogens or parasites can also be treated by the compositions and methods described herein. Non-limiting examples of eukaryotic pathogens are fungi, including Candida and Pneumocystis, or parasites, such as those in the genus Plasmodium.

The agent that inhibits fibrinolysis can be an agent that inhibits the expression or activity of a compound in either the host or pathogen that breaks down fibrin-based clots. For example, the agent can inhibit the expression or activity of plasminogen, plasmin, tissue -type plasminogen activator (tPA), or urokinase-type plasminogen activator. Alternatively, or in addition, one can upregulate the expression or activity of an agent in the host that inhibits fibrinolysis. For example, the agent can be one that increases the expression or activity of a plasminogen activator inhibitor (PAI; e.g., PAI-1), including PAI-1 itself, a biologically active fragment or variant thereof, or a nucleic acid sequence (e.g., in an expression vector) that encodes PAI-1 or a biologically active fragment or variant thereof. PAI-1 inhibits tP A and urokinase. Thus, increasing the expression or activity of PAI-1 inhibits the conversion of plasminogen to plasmin and, therefore, the breakdown of fibrin. In other embodiments, the agent that inhibits fibrinolysis can be one that increases the expression or activity of a thrombin-activatable fibrinolysis inhibitor (TAFI; also known as carboxypeptidase B2 (CPB2), carboxypeptidase U (CPU), or plasma carboxypeptidase B (pCPB)). TAFI removes residues from fibrin that are important for plasminogen to bind to and breakdown fibrin. Thus, when TAFI is more active, more fibrin remains within a clot. Accordingly, the agent that inhibits fibrinolysis can be TAFI, a biologically active fragment or variant thereof, or a nucleic acid sequence (e.g. , in an expression vector) that encodes TAFI or a biologically active fragment or variant thereof. Where the agent inhibits fibrinolysis by targeting a bacterial protein, the agent can be one that inhibits the expression or activity of a bacterial plasminogen-binding protein or plasminogen activator.

Regardless of the exact target, an inhibitor of fibrinolysis can be a nucleic acid construct (e.g., an antisense oligonucleotide, a microRNA, or an RNA that mediates RNAi (e.g. , an siRNA or shRNA where the intent is downregulation and a nucleic acid encoding a biologically active protein or a biologically active fragment or variant thereof where the intent is expression or upregulation of a target), a polypeptide (e.g., an antibody or receptor antagonist where the intent is downregulation or the polypeptide per se where the intent is expression or upregulation of a target), or a small molecule (e.g., a small organic compound or a salt or prodrug thereof).

In another aspect, the invention features a kit in which one or more of the component agents described herein are packaged together with instructions for use. For example, the invention features kits that include (a) an antigen expressed by a pathogen (e.g. , bacterial pathogen) and/or cells expressing the antigen; (b) an agent that inhibits fibrinolysis; and (c) instructions for use. Alternatively, or in addition, the kits can include an antibody that specifically binds a bacterial pathogen or cells expressing such antibodies together with an agent that inhibits fibrinolysis. The kits can further include materials useful in administering the contents to a patient (e.g., sterile gloves, an antiseptic, sterile pads or drapes, needles, syringes, tubing, sutures, dressings, and paraphernalia generally). A material or device for dressing or otherwise treating a bodily wound is itself an aspect of the present invention. The material or device will include a substrate and, in contact with the substrate (a) an antigen expressed by a pathogen (e.g., a bacterial pathogen) or cells expressing the antigen and/or (b) an agent that inhibits fibrinolysis. In other embodiments, the substrate can adhere to (a) antibodies that specifically bind the bacterial pathogen or cells expressing such antibodies; and/or (b) an agent that inhibits fibrinolysis. More generally, the invention features a drug delivery device that includes the two types of agents described herein (i.e., a first agent that mediates or constitutes a cellular immune response (e.g., an antigen expressed by a bacterial pathogen, a cell expressing the antigen, an antibody that specifically binds the antigen, or a cell expressing the antibody) and a second agent that inhibits fibrinolysis). The device can be a sterile container (e.g. a pouch, bag, or similar device for holding fluids for intravenous administration or a syringe for holding fluids for subcutaneous or intramuscular injection). The device can be an inhaler. The device can be a transdermal patch.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Fibrinogen contributes to innate defense against Y. pestis. (A) Survival of wild type C57BL/6 mice and RAG-deficient mice (RAG KO) after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27 (n=20-21 mice/group). (B) Survival (n=18-19 mice/group) and

(C) day 4 bacterial burden for fibrinogen-deficient mice (Fib KO) and littermate control fibrinogen-heterozygous mice (Fib Het) after intranasal challenge with 2xl0⁵ CFU KIM D27.

(D) Survival (n=15-16 mice/group) and (E) day 4 bacterial burden for RAG/fibrinogen-deficient mice (RAG/Fib KO) and littermate control RAG/fibrinogen-heterozygous mice (RAG/Fib Het) after intranasal challenge with 2xl0⁵ CFU KIM D27. (F) Survival of wild type C57BL/6 mice and RAG KO mice after intranasal challenge with 2xl0⁶ CFU Y. pestis strain D27-pLpxL (n=15 mice/group). (G) Survival (n= 18-20 mice/group) and (H) day 4 bacterial burden for Fib KO mice and littermate control Fib Het mice after intranasal challenge with 2xl0⁶ CFU D27-pLpxL. (I) Survival (n=22-25 mice/group) and (J) day 4 bacterial burden for RAG/Fib KO mice and littermate control RAG/Fib Het mice after intranasal challenge with 2xl0⁶ CFU D27-pLpxL. Data for all panels is pooled from 2-3 independent experiments. Figure 2. Fibrinogen is dispensable for antibody-mediated defense against Y. pestis. (A,D,E,H) Survival (n=19-20 mice/group) and (B,C,F,G) day 4 bacterial burden for wild type C57BL/6 mice, fibrinogen-deficient mice (Fib KO) and littermate control fibrinogen- heterozygous mice (Fib Het) after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27 followed one day later by intraperitoneal treatment with (A-C) 10 ug LcrV-specific mAb 7.3 (aLcrV), (E-G) 1 μg aLcrV, (D) 3 μg Fl-specific mAb F1-04-A-G1 (aFl), or (H) 0.3 μg aFl . Control groups received isotype-matched control mouse IgGl mAb. Data for all panels is pooled from 2-3 independent experiments.

Figure 3. Fibrinogen can be essential for T cell-mediated defense against Y. pestis.

(A,D) Survival and (B,C,E,F) day 4 bacterial burden for wild type C57BL/6 mice, fibrinogen- deficient mice (Fib KO) and littermate control fibrinogen-heterozygous mice (Fib Het) after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27. (A-C) The day prior to infection, mice received intravenous injections of cell culture media (control) or 5xl0⁶ cultured polyclonal Y. pestis-primed T cells. (D-F) Prior to infection, mice were immunized with YopE₆9-77 peptide or control ovalbumin peptide OVA₂57_264 (Ova). Data for panels A-C and D-F are pooled from 2 and 4 independent experiments, respectively (n=10 mice/group and 30-32 mice/group for A and D, respectively).

Figure 4. Neutrophils contribute to T cell-mediated defense against Y. pestis. (A,B) Fibrinogen-deficient mice (Fib KO) and littermate control fibrinogen-heterozygous mice (Fib Het) were immunized with YopE₆9_77 peptide or left unvaccinated (unvac control). The YopE- immunized mice were challenged with 2xl0⁵ CFU Y. pestis strain KIM D27 (D27) or left unchallenged (no D27). Four days after challenge, flow cytometry revealed (A) similar numbers of lung CD8 T cells staining with K^bYopE₆9-77 tetramer in Fib Het and Fib KO mice and

(B) significantly reduced numbers of lung neutrophils (CD1 lb⁺Ly6G⁺) in the D27-challenged Fib KO mice. (C) Wild type C57BL/6 mice were immunized with YopE₆9-77 peptide or received only cholera toxin adjuvant (CT), and then all were challenged with 2xl0⁵ CFU Y. pestis strain KIM D27 (n=20 mice/group). As indicated, mice received Ly6G-specific mAb 1 A8 (aLy6G) or isotype-matched control rat IgG2a mAb. Data for all panels is pooled from 2 independent experiments.

Figure 5. Fibrinogen reduces hemorrhagic pathology and increases neutrophil viability during immune defense against Y. pestis. Fibrinogen-heterozygous mice (Fib Het) and fibrinogen-deficient mice (Fib KO) were challenged with (A,C,D,F,G,I,J,L) 2xl0⁵ CFU Y. pestis strain KIM D27 or (Β,Ε,Η,Κ) 2xl0⁶ CFU Y. pestis strain D27-pLpxL and hepatic tissue was collected four days after initiating infections. Where indicated (C,F,I,L), mice were immunized with YopE₆9-77 prior to challenge. (A-F) Representative paraformaldehyde-fixed samples stained with hematoxylin and eosin stained sections (400x). Hemorrhagic pathology (collections of red blood cells; black arrows) was evident in Fib KO mice challenged with D27-pLpxL (E) and in the YopE-immunized Fib KO mice challenged with KIM D27 (F). (G-L) Representative fresh- frozen samples stained with anti-Fl to identify Y. pestis (green; white arrows), F4/80 to identify macrophages (blue), anti-Ly6G to identify neutrophils (red), and Hoescht dye to identify nuclei (white). The white bar depicts 50 μιη. (Μ,Ν) Scoring of lesion types for mice (n=5/group) described in A-F. The Material and Methods section describes the criteria used for assigning lesion types. Typical type 1 lesions are shown in B and C; a typical lesions of type 2 is shown in A; a typical type 3 lesion is shown in D; and typical type 4 lesions are shown in E and F. The graphs depict (M) the number of lesions and (N) the percentage of hemorrhagic lesions (type 3 and 4). Statistical significance was analyzed using Student's t-tests.

Figure 6. Fibrinogen-deficiency does not reduce infection-induced inflammation. Realtime PCR data showing levels of mRNA encoding TNFa, IFNy, CXCL-1 and lipocalin-2 in liver tissue collected four days after control or YopE-immunized mice were challenged with 2xl0⁵ CFU Y. pestis strain KIM D27 or 2xl0⁶ CFU Y. pestis strain D27-pLpxL. The data is presented as log 10 fold-change relative to uninfected wild type mice (n=13) and is pooled from 2-4 experiments (n=9-20 mice/group). Box and whisker plots show the maximum, minimum, median, 25^th percentile, and 75^th percentile. The dotted line depicts the mean value for uninfected wild type mice. Asterisks depict statistical comparisons of each group to the uninfected wild type mice using Student's t-tests (* p<0.01, ** pO.001, *** pO.0001). ns = not significant (p>0.05).

Figure 7. Kinetics of fibrin formation during Y. pestis infection. Hepatic levels of (A) bacterial CFU, (B) fibrin, (C) TF mRNA, (D) FXI mRNA, (E) TAFI mRNA, and (F) PAI-1 mRNA in wild type C57BL/6 mice at days 1-4 after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27 (solid symbols) or 2xl0⁶ CFU Y. pestis strain D27-pLpxL (open symbols). Data shown is the median and interquartile range for 14-15 mice per time point. The dashed line depicts the limit of detection (A,B) or the average value for na^'ive control mice (C-F). Data for all panels is pooled from 2 independent experiments. Asterisks depict statistical comparisons of D27 versus D27-pLpxL at day 4 (** pO.001, *** pO.0001).

Figure 8. Fibrin contributes to innate and T cell-mediated defense against Y. pestis. (A) Survival for FXI-deficient mice (FXI KO), control het-TF mice (mTF^+/"fiTF⁺), and low-TF mice (mTF ^{~ ~}hTF⁺) after intranasal challenge with 2xl0⁶ CFU Y. pestis strain D27-pLpxL (p=0.008 for low-TF versus het-TF; n=9-10 mice/group). (B) Survival for YopE-immunized FXI KO, het-TF and low-TF mice after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27 (p=0.003; for low-TF versus het-TF; n=6-8 mice/group). (C) Survival for control C57BL/6 mice and coumadin-treated C57BL/6 mice after intranasal challenge with 2xl0⁶ CFU Y. pestis strain D27-pLpxL (p=0.007; n=15 mice/group). (D) Survival for YopE-immunized C57BL/6 mice and coumadin-treated YopE-immunized C57BL/6 mice after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27 (p=0.046; n=15 mice/group). (E) Survival for wild type C57BL/6 mice and PAI-l/TAFI-deficient mice (P/T KO) after intranasal challenge with 2xl0⁶ CFU Y. pestis strain D27-pLpxL (p=0.005; n=20-58 mice/group). (F) Survival for YopE- immunized wild type C57BL/6 mice and YopE-immunized P/T KO mice after intranasal challenge with 2xl0⁵ CFU Y. pestis strain KIM D27 (p=0.03; n=23-25 mice/group). Data for all panels is pooled from 2-4.

Figure 9. C57BL6 mice survive challenge with lethal doses of Y. pestis that lack the plasminogen-activating activity of the gene encoding Pla. (Left-hand panel) % survival following intranasal challenge with Y. pestis strain C092 (a wild type strain). (Center panel) % survival following intranasal challenge with Y. pestis strain C092 APla, in which the

plasminogen activator encoded by Pla is lacking. (Right-hand panel) % survival following intranasal challenge with Y. pestis strain C092 Pla-D206A, in which the plasminogen activator encoded by Pla is lacking.

DETAILED DESCRIPTION

As noted above, innate defense against pathogens can be fibrin-dependent, and many pathogens have evolved the capacity to degrade fibrin. Our data demonstrate that adaptive defense mediated by T cells or antibodies can also be fibrin-dependent and that reducing the capacity of pathogens to degrade fibrin can improve adaptive defense. Thus, an individual (a term we use synonymously with "patient" or "subject") who is at risk of illness caused by a bacterial pathogen can be treated with one or more agents that activate an adaptive immune response and one or more agents that increase and/or stabilize fibrin levels in the individual.

Activating an adaptive immune response: An adaptive immune response can be induced by active immunization or passive immunization. Where active immunization is used, one would administer to the patient an agent (e.g., an antigen) that elicits an immune response. The administration can be carried out, for example, by any method known in the art as a vaccination, and the antigen can be selected depending on the illness one wishes to treat or guard against. For example, where a patient is to be hospitalized for a prolonged time, the antigen can be one that elicits an immune response against an antibiotic-resistant bacterium (e.g., methicillin-resistant Staphylococcus aureus). In some embodiments, the antigen will be administered intramuscularly or intravenously and may be administered together with a suitable adjuvant. Oral vaccination methods can also be employed. In other embodiments, active immunization can be achieved by administering a cell that expresses an antigen. Regardless of the exact route of administration or formulation, multiple antigens can be administered. Where the treatment is directed to patients who have been exposed to Yersinia pestis (or who are at risk of such exposure), the antigen can be a YopE protein or an antigenic fragment thereof (e.g., YopE₆9-77). Other Yersinia pestis antigens that can be used include attenuated strains like Yersinia pestis strain D27-pLpxL or Yersinia pestis strain C092 Pla-D206A.

Passive immunization can be achieved by administering antibodies that specifically bind an antigen expressed by a bacterial pathogen or cells that express such antibodies. Antibodies suitable for passive immunization include LcrV-specific mAb 7.3 (aLcrV), Fl -specific mAb Fl- 04-A-G1 and Pla-specific mAb. Other antibodies or antisera may also be used. The antibodies can be tetrameric antibodies or single-chain antibodies (scFv); the antibodies can be antigen- binding fragments of tetrameric antibodies; and the antibodies can be human, humanized, or chimeric antibodies. One of ordinary skill in the art will recognize that other configurations, for example, diabodies or bi-specific antibodies can also be administered in the context of the present methods.

Agents that inhibit fibrinolysis: Agents that can be administered as described herein to inhibit fibrinolysis can target a protein expressed by the patient (e.g. , normally or naturally expressed by the patient) or expressed by the invading pathogen (e.g., normally or naturally expressed by the invading pathogen). Further, the agents themselves can be naturally or non- naturally occurring (e.g., a naturally occurring nucleic acid or protein or a biologically active fragment or other variant thereof). Generally, treatments for nonsurgical clinical conditions in which fibrin formation is poor tend to focus on replacing fibrin or a component of the clotting cascade. On the other hand, treatments for perioperative bleeding are more frequently achieved by inhibiting plasmin. The most widely used plasmin inhibitor for reducing perioperative bleeding, Trasylol™ (aprotinin), has been administered by injection to reduce bleeding during complex surgery. Although there has been some controversy regarding the safety of aprotinin, it is a plasmin inhibitor, and aprotinin or a biologically active fragment or variant thereof, can be employed in the methods described herein by physicians in consultation with their patients, particularly in the event of an infection with a high mortality rate. Aprotinin is also associated with desirable secondary characteristics relating to the inhibition of serine proteases other than plasmin. By inhibiting proteases involved in inflammation, aprotinin can inhibit inflammation as well. Alternatively, an inhibitor of fibrinolysis may be a small molecule drug like ε- aminocaproic acid or tranexamic acid or serine protease inhibitors having antifibrinolytic activity. In yet another embodiment, the inhibitor may be, or may be derived from, peptide sequences of substrates of bacterial plasminogen activators. Inhibitors may be isolated using a screen for inhibitor of protease activity of bacterial plasminogen activator(s) or plasmin, using a detectably modified substrate such as a chromogenic substrate. Additional inhibitors are described in Okada, Chem. Pharm. Bull. (Tokyo), 48(12): 1964, 2000 (synthesized plasmin and plasma kallikrein inhibitors, Trans-(4-aminomethylcyclohexanecarbonyl)-Tyr(0-Pic)-octylamide and Trans-(4-aminomethylcyclohexanecarbonyl)-Tyr(0-2-Pyrim)-4-carboxyanilide); Okada, Bioorg. Med. Chem. Lett. 10(19):2217, 2000 (synthesized plasmin and plasma kallikrein inhibitors, Trans-(4-aminomethylcyclohexanecarbonyl)-Tyr(0-Pic)-octylamide and Trans-(4- aminomethylcyclohexanecarbonyl)-Tyr(0-2-Pyrim)-4-carboxyanilide); Fish, J. Med. Chem. 50(10):2341, 2007 (Selective urokinase-type plasminogen activator inhibitor, l-(7- sulfonamidoisoquinolinyl) guanidines) ; Zhu, Mol. Cancer Ther. 6(4): 1348, 2007 (inhibitor of urokinase-type plasminogen activator, 4-oxazolidinone analogues UK122); Agarkov, Bioorg. Med. Chem. Lett. 18(1) :427, 2007 (small peptide substrates to inhibit Y. pestis plasminogen activator Pla); Yun, J. Cell Mol. Med. 13(10):4146-53, 2009 (polyphosphates and omptins); Teno, Bioorg. Med. Chem. Lett. 21(21):6305, 2011 (Lysine-nitrile derivatives); Swedberg, Chembiochem. 13(3):336, 2012; Sun, Proc. Natl. Acad. Sci. USA 109(9):3469, 2012

(streptokinase inhibitors, compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG- 102487)); Soupe, J. Med. Chem. 56(3):820, 2013 (new cyclic plasmin inhibitors); and US patent Application Publication 2009/0069248, which is incorporated herein by reference. These peptides or inhibitors may further be chemically modified for increasing specificity, improved pharmaco-kinetics, decreased toxicity, increased bioavailability, irreversible or covalent binding, or other desirable properties.

As noted above, an agent administered in the context of the present invention can be naturally or non-naturally occurring. Many of the previously recognized plasmin inhibitors that are naturally occurring and useful in the present methods have been assigned to a class of serine protease inhibitors known as "standard" or Laskowski-mechanism inhibitors (Laskowski, Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 324: 1477, 2000; Laskowski and Kato, Ann. Rev. Biochem. 49:593, 1980). A prominent standard-mechanism inhibitor is aprotinin, as discussed above. Other Kunitz-type inhibitors include amyloid precursor-like protein 2, Kunitz domain (see Petersen et al., FEBS Lett. 53:338, 1997), bikunin (see Gebhard and Hochstrasser In Proteinase Inhibitors, Barret and Salvesen, Eds., Elsevier, Amsterdam, p. 389, 1986), Kunitz- type inhibitor 2 (see Kelaria et al., J. Biol. Chem. 272: 12209, 1997), Leucaena-type trypsin inhibitor (see Oliva et al., Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 64:1477, 2000), nexin 2, Kunitz domain (see Petersen et al., FEBS Lett. 53:338, 1994), TdPi tryptase inhibitor (see Paesen et al., J. Mol. Biol. 368:1172, 2007), textilinin-1 (see Flight et al. , Br. J. Haematol. 207: 145, 2009), tissue factor pathway inhibitor-1, unit 1, and tissue factor pathway inhibitor-2, unit 1 (see Petersen et al., Eur. J. Biochem. 310:235, 1996 and Kong et al., Biochem. Biophys. Res. Commun. 324: 1179, 2004, respectively), DX-1000 (see Markland et al., Biochemistry

35:8045, 1996), and KD1-L17R (see Bajaj et al, J. Biol. Chem. 286:4329, 2011). DX-1000 inhibits plasmin with a of 0.087 nM and is remarkably stable at a range of pH, temperature and redox reaction conditions. A second-generation PEGylated variant of DX-1000 has been developed to increase serum half-life. That variant, and any PEGylated variant of a plasmin inhibitor, can be used in the present methods and included in the kits of the invention. Amino acid substitutions have also been made and may also be made to improve the characteristics of a plasmin inhibitor. For example, when TFPI-2 is mutated to include a Leu to Arg amino acid substitution (producing the inhibitor KD1-L17R), a more potent inhibitor results.

Kazal-type inhibitors useful in the present methods include infestin, domains 3 and 4 (Campos et al, Insect Biochem. Mol. Biol. 32:991, 2002), which is one of the most potent, Aedes aegypti trypsin inhibitor (see Wantanabe et al., Biochimie 92:933, 2010), Bdellin (see Kim et al., J. Biochem. 130:431, 2001), bikazin salivary inhibitor (see Hochstrasser, Hoppe-Seyler's Z. Physiol. Chem., 356: 1659, 1974), and chicken liver trypsin inhibitor (see Kubiak et al., J. Biol. Macromol. 45: 194, 2009). While the invention is not so limited, the Ki of a given inhibitor (e.g., a plasmin protein inhibitor) can be at least or about 0.05-2.0 nM. There is also a useful subtillison inhibitor- like protein known as plasminostreptin (see Kakinuma et al. , J. Biol. Chem. 253: 1529, 1978).

As noted, agents useful in the present invention, whether to inhibit agents that promote fibrinolysis or to inhibit agents that would otherwise inhibit fibrinolysis, include nucleic acids, polypeptides, and small molecules (chemical entities or compounds). The synthetic basic PI residue analogue ONO-3307 (4-sulfamoyl phenyl-4-guanidinobenzoate methanesulfonate) inhibits plasmin about as well as thrombin, trypsin, and pKLK inhibit plasmin and is useful in the methods and kits described herein. Also useful are peptide chloromethyl ketone inhibitors such as D-Ile-Phe-Lys-CH₂C1 as described by Okada et al. (Chem. Pharm. Bull. 36:1289, 1988; see also Tsuda et al, Chem. Pharm. Bull. 37:3108, 1989) and the compounds described by Teno et al. (Chem. Pharm. Bull. 39:2340, 1991 and Chem. Pharm. Bull. 41:1079, 1993). These same investigators have developed other useful inhibitors that target the S1-S2' pockets of plasmin. 4-aminomethylcyclo-hexanecarbonyl was used as the PI basic residue analogue (see Okada et al, Chem. Pharm. Bull. 48: 1964, 2000; and Okada et al, Chem. Pharm. Bull. 48: 184, 2000). Xue and Seto used a different approach to generate useful plasmin inhibitors; they reacted a PI cyclohexanone group with the active-site serine to form a reversible hemiketal (see Xue and Seto, Bioorg. Med. Chem. 14:8467, 2006; Xue and Seto, J. Org. Chem. 70:8309, 2005; Xue and Seto, J. Med. Chem. 48:6908, 2005; and Abato et al, J. Org. Chem. 67:1184, 2002). These plasmin inhibitors are useful in the context of the present methods. Other useful plasmin inhibitors, such as CU-2010, have peptide-like properties (Kietrich et al., Anesthesiology

110: 123, 2009; Katz et al., Chem. Biol. 8: 1107, 2001; Szabo et al., J. Thorac. Cardiovasc. Surg. 139: 181, 2010; and Szabo et al., J. Thorac. Cardiovasc. Surg. 139:732, 2010). The plasmin inhibitor can also be a peptide aldehyde transition-state analogue such as KM(0₂)YR-H; a bee venom serine protease inhibitor (Choo et al. PLoS One., 2012; a plasmin inhibitor isolated from Russell's viper venom; or an aloe vera protein that inhibits the cleavage of human fibrin(ogen) by plasmin. Lysine and lysine analogues are also useful in inhibiting factors expressed by the host that mediate fibrinolysis. Tranexamic acid (Lysteda™, Cyklokapron™ in the U.S.) is a synthetic derivative of lysine that has been used to help prevent excessive blood loss during surgery and in various other medical conditions. It is an antifibrino lytic that binds specific sites on both plasminogen and plasmin to competitively inhibit plasminogen activation. An older analogue, ε-aminocaproic acid, is another useful lysine analogue. More generally, studies have shown that C-terminal lysine residues within group A streptococcal GAPDH may be important in the plasmin binding activities of this molecule (Winram et al., In: Genetics of streptococci, enterococci and lactococci, Ferretti et al, Eds., Dev. Biol. Stand. Basel, Karger, 85: 199-202, 1995). Accordingly, inhibiting the expression or activity of bacterial GAPDH or administering a small molecule therapeutic that interferes with the ability of GAPDH to bind plasmin provide a means for inhibiting fibrinolysis upon bacterial infection. Tranexamic acid works through such a mechanism.

Inhibiting a factor expressed by the patient has certain advantages, as therapies directed to the patient can be applied more universally than therapies directed to a particular bacterium. Nevertheless, the invention encompasses methods in which fibrinolysis is inhibited by targeting a factor within the invading pathogen that would otherwise promote fibrinolysis. Thus, one can administer, for example, an agent that inhibits a bacterial plasminogen activator {e.g. , streptokinase, staphylokinase, Pla, OmpT/PgtE, as described further below) or an agent that inhibits a surface binding protein for plamin(ogen). Plasminogen receptors that can be targeted include SDH/Pir, enolase, PAM, OspA/OspC, and HP-NAP.

The majority of group A streptococci that cause human disease secrete streptokinase, a plasminogen activator that complexes with human plasminogen. This induces a conformational change in the plasminogen molecule that enables the resulting complex to acquire plasminogen activator activity. The streptokinase-plasminogen complex, unlike the host plasminogen activators tPA and uPA, is not inhibited by host protease inhibitors and thus represents an efficient way to generate plasmin in human plasma and, thereby, degrade fibrin. Agents that inhibit streptokinase (or analogous enzymes expressed by other pathogens) can therefore be used to inhibit the pathogen's ability to degrade fibrin. For example, in the context of the present methods, one could administer a neutralizing specific anti-streptokinase antibody. Group A streptococci can also express high affinity surface binding molecules that are capable of binding plasmin such that it can no longer be regulated by host regulators such as a2 antiplasmin.

Accordingly, these surface binding molecules are also suitable targets in the context of the present invention. Various molecules produced by group A streptococci can also bind fibrinogen and can be targeted. These include M and M-related proteins, which are antiphagocytic, and others. Some group A isolates express an antiphagocytic M protein (PAM) that binds to Glu- plasminogen directly. More specifically, PAMs are expressed by some M serotypes of group A streptococci and certain group C and G isolates.

Where the invading bacterium is Yersinia pestis, one can inhibit fibrinolysis by inhibiting the expression or activity of the pla gene or its product.

The agents described herein can be administered to patients by delivery techniques known in the art. The formulation and administration of an antigen, for example, or of any other agent {e.g., antigen-expressing cells) meant to elicit an immune response {e.g., production of CD8⁺ T cells) can be carried out as described in Robinson and Amara {Nature Medicine 11:S25, 2005). For example, one can administer a live-attenuated bacterial pathogen, a replication- competent or replication-defective live-vectored vaccine, a DNA-based vaccine, or a

heterologous prime-boost. One of ordinary skill in the art could also consult, for example, Plotkin et al. {Vaccines, Elsevier Inc., 4^th Edition, 2004) and numerous other texts concerning vaccine preparation, adjuvants, and administration. The formulation and administration of an agent that inhibits fibrinolysis can be as previously described for currently known agents {e.g., tranexamic acid and aprotinin). For example, anti-fibrino lytic agents can be dissolved or suspended in sterile isotonic solutions for intravenous administration. For aprotinin, each mL can contain 10,000 KU (Kallikrein Inhibitor Units) (1.4 mg/mL) and can be administered according to the manufacturer's instructions. Aprotinin or any other inhibitor can be given with an initial "test" dose, followed by a loading dose (given over time; e.g., over 20-30 minutes), a "pump prime" dose, and a constant infusion dose. Transexamic acid has been formulated for oral administration at a dosage of 650 mg (currently prescribed as two 650 mg tablets taken three times daily for a maximum of five days for heavy menstrual bleeding). One of ordinary skill in the art will understand the techniques and processes for determining an effective dosage and route of administration in the context of the present methods.

Patients amenable to treatment: Patients amenable to treatment include humans or other mammals who have been exposed to or who are at risk of exposure to invasive human pathogens that promote fibrinolysis. These pathogens may express plasmin(ogen) receptors and/or produce plasminogen activators. Plasmin(ogen) receptors have been found on the surfaces of both gram positive and gram negative bacteria as well as on mycoplasma. These include bacteria of the genus Borrelia, Escherichia, Haemophilus, Helicobacter, Moraxella, Mycoplasma, Neisseria, Proteus, Pseudomonas, Salmonella, Staphylococcus, Streptococcus and Yersinia. Accordingly, the present methods can be used in connection with these bacteria (with the agent that inhibits fibrinolysis being one that binds to and inhibits a plasmin(ogen) receptor or a plasminogen activator) and, more specifically, to treat illness caused by B. burgdorferi, E. coli, H influenzae, H. pylori, M. catarrhalis, M. fermentans, N. gonorrhoeae, N. meningitidis, P. mirabilis,

P. aeruginosa, S. enteritidis, S. aureus, and group A, C, and G streptococci. The present methods can also be directed to Yersinia pestis.

Generally speaking, agents that inhibit fibrinolysis with or without an antigen or antibody may be administered after infection of the subject with Y. pestis, B. burgdorferi, E. coli, H.

influenzae, H. pylori, M. catarrhalis, M. fermentans, N. gonorrhoeae, N. meningitidis, P.

mirabilis, P. aeruginosa, S. enteritidis, S. aureus, and group A, C, and G streptococci. However, the antigen may be administered prophylactically in individuals at risk of infection with these bacteria, and the agents that inhibit fibrinolysis and/or an antibody may be administered after infection. The agents that inhibit fibrinolysis, and an antigen or antibody may be administered simultaneously or separately. Simultaneous administration may be performed using same or different route of administration. For example, the antigen may be administered parenterally and agents that inhibit fibrinolysis may be administered orally. If they are administered separately, optionally, the antigen may be administered in a susceptible population either after or before their administration may be separated by 12 hr, 24 hr, 48 hr, 1 week, one month, 3 months, 6 months, one year or more. Formulations, Kits, Dressings, and Devices: Any of the agents described herein and any combination thereof can be packaged in kit- form with instructions for use or incorporated into a dressing in such a way as to come into contact with a wound a patient has received. In addition to inclusion in dressings {e.g., bandages), antifibrino lytic agents can be formulated as lotions for topical administration, or delivered by inhalers or sprays for the treatment of skin, tissue and respiratory diseases caused by or aggravated by bacterial infection, including dermatitis, fasciitis, sinusitis and pneumonia. The invention also features eye, ear, or nasal drops for providing the first and/or second agent, optionally with an agent for ear infections (otitis media), eye infections (conjunctivitis), and sinusitis caused by H. influenzae or M. catarrhalis. Creams, foams, lotions, gels, and solutions can also be formulated for vaginal administration. These formulations can include, for example, an antigen from N. gonorrhoaea, a cell expressing such an antigen, an antibody directed to N. gonorrhoaea, a cell expressing such an antibody, and/or an

antifibrinolytic agent.

Dosages and Routes of Administration: The agents that inhibit fibrinolysis described above may be administered by topically or systemically. Topical administration may be performed using lotions, sprays, creams, gels, eye drops, ear drops, enemas etc. Systemic administration may include oral or injectable forms.

Oral dosage forms may be solid dosage forms like capsules, tablets, pills, powders, and granules that may be formulated as immediate release, delayed release or orally disintegrating dosage forms. Oral dosage forms may also be liquid dosage forms like pharmaceutically acceptable emulsions, solution, suspension, syrup and elixirs. Such compositions may also comprise adjuvants.

The injectable dosage forms for parenteral administration may be in the form of aqueous or non-aqueous solutions or suspensions, which may optionally be formulated as sterile powders or granules that need reconstitution prior to administration. The reconstitution may be performed using one or more pharmacologically acceptable of the carriers or diluents. The compounds may be dissolved in water, a suitable buffer, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil or a mixture thereof.

The dosage forms that include antigens may include adjuvants that are widely known in the pharmaceutical art. For administration, the agents described herein can be admixed with pharmacologically acceptable excipients like lactose, sucrose, starch powder, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,

polyvinylpyrrolidone, polyvinyl alcohol, sodium citrate, or magnesium or calcium carbonate or bicarbonate, water, a buffer, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil or a mixture thereof. Additional pharmacologically acceptable agents like wetting agents, solvants, solvates, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents may also be present. They may be then tableted or encapsulated for convenient administration or prepared as a cream, gel, solution, suspension, powder.

EXAMPLES

The studies described below demonstrate that fibrin is an essential component of T cell- mediated defense against plague but can be dispensable for antibody-mediated defense.

Depleting fibrin, either by genetic or pharmacologic means, abrogates innate and T cell-mediated defense in mice challenged intranasally with Y. pestis. The fibrin-deficient mice displayed reduced survival, increased bacterial burden, and exacerbated hemorrhagic pathology. They also showed fewer neutrophils within infected lung tissue and reduced neutrophil viability at sites of liver infection. Depletion of neutrophils from wild type mice weakened T cell-mediated defense against plague. The data suggest that T cells combat plague in conjunction with neutrophils, which require help from fibrin in order to withstand Y. pestis encounters and effectively clear bacteria.

The extreme virulence of Y. pestis results primarily from the capacity of this gram negative bacterium to overwhelm the innate immune defense mechanisms of the host. A number of distinct virulence mechanisms have been established, including inhibition of phagocytosis, suppression of oxidative burst, and the induction of apoptosis (Viboud and Bliska, Annu. Rev. Microbiol. 59:69-89, 2005). Y. pestis also evades innate immunity by surrounding itself with an anti-phagocytic capsule (Du et al, Infect. Immun. 70: 1453-1460, 2002) and producing a tetra- acylated form of LPS that antagonizes host recognition by TLR4 (Montminy et al., Nat.

Immunol. 7: 1066-1073, 2006; Telepnev et al. , J. Infect. Dis. 200: 1694-1702, 2009; and Knirel and Anisimov, Acta Naturae 4:46-58, 2012). In mouse model of bubonic plague, Pla-deficient Y. pestis grow to high titer at the peripheral injection site but typically fail to attain high titers in draining lymph nodes and distal organs (Sebbane et al., Proc. Natl. Acad. Sci. USA 103:5526-5530, 2006; Sodeinde et al., Science 258: 1004-1007, 1992; Welkos et al, Microb. Pathog. 23:211-223, 1997), suggesting that Pla facilitates the digestion of fibrin matrices at peripheral sites of infection, thereby disrupting physical barriers that impede bacterial dissemination (Titball and Oyston, Nat. Med. 13:253-254, 2007; Esmon et al., J. Thromb. Haemost. 9 Suppl 1:182-188, 2011). Consistent with that possibility, Pla-deficient strains regain high levels of virulence when injected subcutaneously into fibrinogen-deficient mice, which lack the capacity to produce fibrin matrices (Degen et al., J. Thromb. Haemost. 5 Suppl 1 :24-31, 2007).

In addition to facilitating dissemination from peripheral tissue, Pla, plasminogen and fibrin(ogen) also impact the nature of inflammatory cell accumulations at sites of Y. pestis infection. Inoculation of Pla-deficient Y. pestis promotes the formation of neutrophil-rich lesions, whereas inoculation of wild type strains leads to the formation of lesions that contain few inflammatory cells (Sebbane et al., Proc. Natl. Acad. Sci. USA 103:5526-5530, 2006;

Sodeinde et al, Science 258: 1004-1007, 1992; Degen et al., J. Thromb. Haemost. 5 Suppl 1 :24- 31, 2007; Flick et al., J. Clin. Invest. 113: 1596-1606, 2004). These studies suggest that Pla- mediated fibrinolysis may facilitate Y. pestis dissemination by reducing the accumulation and/or activation of inflammatory cells with the capacity to destroy Y. pestis bacteria (Degen et al. , J. Thromb. Haemost. 5 Suppl 1 :24-31, 2007; Flick et al. , J. Clin. Invest. 113: 1596-1606, 2004).

The studies described here aimed to dissociate impacts of fibrin on innate, antibody, and T cell-mediated defense using mouse models of septic pneumonic plague. The findings demonstrate that fibrin can be essential for both innate and T cell-mediated defense but dispensable for antibody-mediated defense. In these models, fibrin does not appear to act solely as a spatial cue for the accumulation of inflammatory cells but, rather, seems to restrict bacterial growth and help leukocytes survive encounters with Y. pestis bacteria.

The materials and methods used in the studies below were as follows.

Mice: All animal studies were conducted in accordance with Trudeau Institute Animal Care and Use Committee guidelines. Experimental mice were bred in a specific-pathogen-free facility at Trudeau Institute. Breeder stocks of C57BL/6 wild type mice, B cell-deficient μΜΤ mice, and PAI-1 -deficient mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Breeder stocks of C57BL/6 fibrinogen-deficient mice (Suh et al, Genes Dev. 9:2020-2033, 1995), Factor XI (FXI)-deficient mice (Gailani et al, Blood Coagul. Fibrinolysis 8:134-144, 1997), and TAFI-deficient mice (Swaisgood et al, J. Clin. Invest. 110: 1275-1282, 2002) were supplied by Jay L. Degen, David Gailani and Edward F. Plow, respectively. PAI-l/TAFI- deficient mice were generated at Trudeau Institute (Luo et al, J. Immunol. 187: 1866-1876, 2011). Nigel Mackman supplied breeder stock of C57BL/6 mice with very low levels of TF activity (low-TF mice). These mice lack expression of mouse TF due to its inactivation by gene targeting and instead express a human TF transgene, which imparts low-level TF activity (mTF^"7" hTF⁺) (Parry et al., J. Clin. Invest. 101:560-569, 1998).

Low-TF mice were compared with littermates expressing human TF that were heterozygous for mouse TF (het-TF; mTF^+/"fiTF⁺). Experimental mice were matched for age and sex, and infected or vaccinated between 6 and 10 weeks of age. Where indicated, wild type mice were anticoagulated pharmacologically by supplementing drinking water with 2 mg/liter Coumadin [3-(a-acetonylbenzyl)-4-hydroxycoumarin; Sigma-Aldrich] beginning 3 days prior to infection with replenishment every 48 hours; this anticoagulant regimen reduces fibrin deposition in mice during infection (Mullarky et al, Infect. Immun. 73:3888-3895, 2005).

Bacterial infections: Y. pestis strain KIM D27 (pgm-negative, pCDl+, pPCP+, pMT+) and KIM D28 (pgm-negative, pCDl^", pPCP+, pMT+) were provided by Dr. Robert Brubaker (Michigan State University, East Lansing, MI). Attenuated strain D27-pLpxL was generated as described previously (Szaba et al, Infect. Immun. 77:4295-4304, 2009) by transforming strain KIM D27 with plasmid pLpxL, which was provided by Drs. Egil Lien and Jon Goguen

(University of Massachusetts Medical School, Worcester, MA). For infections, strain D27 was grown overnight at 26°C in Bacto heart infusion broth (BHI; Difco Laboratories, Detroit, MI) supplemented with 2.5 mM CaCl₂. Cultures were then diluted to an optical density of 0.1 at 620 nm, re-grown in the same media for 3-4 hours at 26°C, quantified by measuring the optical density, and resuspended in saline at the desired concentration. Infections were performed by applying 30 μΐ to the nares of mice that were lightly anesthetized with isoflurane. The number of bacteria in the inoculating dose was confirmed by plating on BHI agar. The median lethal dose for strain KIM D27, as calculated also by the method of Reed and Muench {Am. J. Hyg. 27:493- 497, 1938), is approximately lxl 0⁴ CFU when grown and administered as described above. Strain D27-pLpxL was grown and administered as described for strain KIM D27, except the broth was supplemented with 100 μg/ml ampicillin (Szaba et al, Infect. Immun. 77:4295-4304, 2009). The median lethal dose for strain D27-pLpxL is greater than lxlO⁷ CFU when administered via the intranasal route (Szaba et al, Infect. Immun. 77:4295-4304, 2009).

Measurements of survival and bacterial burden: Mice were monitored at least once daily after initiating infection. Unresponsive or recumbent animals were considered moribund and euthanized. To measure bacterial burden, tissues were collected from mice that were euthanized by carbon dioxide narcosis. The number of viable bacteria in lung, spleen, and liver were measured by homogenizing tissues in saline, plating serial dilutions on BHI agar, and counting CFU after 48 hours growth at 26°C.

Immunotherapy: Hybridoma clones F1-04-A-G1 and 7.3 producing Fl - and LcrV- specific mAb, respectively, were described previously (Anderson et al., Am. J. Trop. Med. Hyg. 56:471-473, 1997; Hill et al, Infect. Immun. 65:4476-4482, 1997; Kummer et al, Vaccine

26:6901-6907, 2008; Lin et al, Vaccine 29:357-362, 2010). The mAb produced by these hybridomas were purified using protein G agarose. They contained less than 2.2 units per mg endotoxin as measured by Limulus Amebocyte Lysate assay. For passive immunotherapy, mice were treated with the indicated doses of mAb diluted in phosphate-buffered saline and administered intraperitoneally. Control mice received equivalent doses of isotype-matched non- protective LcrV-specific mAb (mouse IgGl; clone 26-2).

Adoptive T cell transfers: Cells for adoptive transfer were harvested from μΜΤ mice that had been immunized with D27-pLpxL (2xl0⁶ CFU) and rested for 60 days (Szaba et al, Infect. Immun. 77:4295-4304, 2009). Cells were isolated from spleens, restimulated in vitro in bulk culture with mitomycin c-treated naive splenocytes as antigen presenting cells and heat-killed Y. pestis strain KIM D27 grown at 37°C as antigen (Lin et al., J. Immunol. 187:897-904, 2011). Two days after initiation of culture, an equal volume of medium containing 40 units/ml recombinant human interleukin-2 was added. The cultures were replenished with interleukin-2- containing medium every other day. After two weeks of culture, cells were harvested, counted and injected intravenously into recipient mice (5xl0⁶ viable cells/mouse), which were challenged with Y. pestis the following day.

Immunizations with YopE and ovalbumin peptides: Peptide ΥορΕ₆9_₇₇ (H₂N- SVIGFIQRM-OH) and control ovalbumin peptide OVA₂₅₇-₂64 (H₂N-SIINFEKL-OH) were synthesized and purified (>95%) by New England Peptide (Gardner, MA). Mice were lightly anesthetized by isoflurane and immunized intranasally with a 15 μΐ saline solution containing 10 μg peptide and 1 μg cholera toxin (List Biological Laboratory, Campbell, CA). Mice were immunized on days 0, 7, and 21, and challenged with Y. pestis strain D27 on day 37. When indicated, neutrophils were depleted in vivo by intraperitoneal injection of 0.2 mg of Ly6G- specific mAb (clone 1A8; BioXcell) on days 36, 38, 40 and 42; control mice received injections of isotype-matched rat IgG2a mAb (BioXcell).

Flow cytometry: Preparation of lung cells (Parent et ah, Infect. Immun. 73:7304-7310, 2005) and enumeration of YopE-specific CD8 T cells by K^bYopE69_77 tetramer staining (Lin et ah, J. Immunol. 187:897-904, 2011) was described previously. In brief, cells isolated from lung tissue digested with collagenase and DNAse were incubated with Fc Block (clone 2.4G2) for 15 minutes at 4°C, washed, and incubated with tetramer for 1 hour at room temperature. The allophycocyanin-conjugated K^bYopE69_77 tetramer was supplied by the NIH Tetramer Facility. After washing again, cells were stained with anti-CD8-peridinin chlorophyll protein (clone 53- 6.7) for 30 minutes at 4°C. For enumeration of CD4 cells, NK cells and neutrophils, lung samples were stained on ice with anti-CD4 peridinin chlorophyll protein (clone RM4-5) and anti- NKl . l-phycoerythrin (clone PK136), anti-CD 1 lb-allophycocyanin (clone Ml/70) and anti- Ly6G-fluorescein isothiocyanate (clone 1 A8). Data were gated for forward scatter/side scatter and collected on a Beckton Dickinson FACSCanto II and analyzed using Flow Jo software.

Histology and immunofluorescent staining: Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Representative photomicrographs are presented at a magnification of 400x. The number of focal lesions per tissue section were quantified and assigned scores of: 1 when characterized by mixed inflammatory cells with little to no evidence of tissue damage, 2 when marked by contiguous areas of anoxic hepatocytes and/or mild hepatocellular necrosis with little to no evidence of bleeding, 3 when consisting of broad areas of hepatocellular necrosis interspersed with notable evidence of bleeding comprising 5-25% of the total lesion area, and 4 when presenting as broad areas of contiguous hemorrhage comprising greater than 25% of the total lesion area. For immunofluorescent staining, 7 μιη frozen sections of OCT-embedded liver tissues were air dried for 10 minutes and fixed in a mixture of acetone and ethanol (75:25) for 10 minutes at room temperature. Tissues were rinsed with PBS, blocked with 5% normal mouse serum for 30 minutes, and then incubated with primary antibodies diluted in PBS with 5% normal mouse serum for 1 hour at room temperature. The anti-Fl antibody was conjugated to Dylight 488 using the Dylight 488 microscale antibody labeling kit from (Thermo Fisher) and used at a 1 :200 dilution. Ly6G-phycoerythrin (BD Pharmingen) and F4/80-AF647 (AbD Serotech) were used at 1 :200 and 1 :75 dilutions, respectively. After rinsing, the tissues were counterstained with Hoechst nuclear dye. Slides were imaged using a Leica SP5 confocal microscope with 405, 488, 543, and 633 laser lines. Emission spectra were collected using the appropriate bandwidth settings for each fluorophore.

Measurements of hepatic fibrin deposition: Fibrin levels within tissue samples were quantified essentially as described previously (Johnson, et ah, J. Exp. Med. 197:801-806, 2003) and scored positive when above the limit of detection (10 ng/mg tissue). In brief, insoluble fibrin was extracted from homogenized tissue and quantified by Western blot using biotinylated fibrin- specific mAb 350 (American Diagnostica) followed by rabbit anti-biotin (Bethyl Labs), anti- rabbit horseradish peroxidase polymer (DakoCytomation), and chemiluminescent detection (Bio- Rad). Standard curves were generated by treating purified mouse fibrinogen (Sigma- Aldrich) with human thrombin (Enzyme Research Laboratories).

Real-time PCR: Tissue levels of mRNA encoding TNFa, IFNy, IL-6, IL-10, CXCL-1, lipocalin-2, TF, FXI, PAI-1, and TAFI were measured by real-time PCR (PerkinElmer), normalized to levels of mRNA encoding GAPDH, and expressed as fold change relative to levels in uninfected wild type mice (Mullarky et ah, J. Thromb. Haemost. 4: 1580-1587, 2006).

Statistics: Statistical analyses were performed using the program Prism 5.0 (GraphPad Software, Inc.). Survival data were analyzed by log rank tests. All other data were analyzed by Mann- Whitney tests, unless indicated otherwise in figure legends. When CFU or fibrin samples fell below the detection limit of our assays they were assigned log 10 values of 1.0 or 0.5, respectively.

Example 1: Fibrinogen contributes to innate defense against Y. pestis

Y. pestis strain KIM D27 retains much of its virulence when administered intranasally (Parent et al, Infect. Immun. 73:7304-7310, 2005). Figure 1A demonstrates that wild type C57BL/6 mice and RAG-deficient C57BL/6 mice succumbed with similar kinetics after intranasal challenge with 2xl0⁵ CFU KIM D27, which is approximately 20 times the median lethal dose for wild type mice (Parent et al, Infect. Immun. 73:7304-7310, 2005). Since RAG- deficient mice lack B and T cells, the mediators of adaptive immunity, these data indicate that adaptive immunity has little impact on the time to morbidity in naive mice challenged with KIM D27. Analyses of bacterial burden in lung and liver tissue at day 4 after challenge likewise failed to reveal a significant role for adaptive immunity during host defense against lethal challenge with KIM D27 in na^'ive mice.

Fibrinogen-deficient mice succumbed more rapidly to plague than littermate control fibrinogen-heterozygous mice when challenged intranasally with KIM D27 (Figure IB). The bacterial burden measured at day 4 after challenge revealed a modest but significant increase in numbers of CFU in the lung and liver tissues of fibrinogen-deficient mice, as compared with controls (Figure 1C). Thus, even though Y. pestis rapidly overwhelms innate host defense in wild type mice (Figure 1 A), fibrinogen nevertheless plays a significant protective role in that setting.

To formally demonstrate that fibrinogen contributes to innate defense, fibrinogen- deficient mice and RAG-deficient mice were intercrossed to generate RAG-deficient mice that were fibrinogen-deficient or fibrinogen-heterozygous. When these RAG-deficient mice were challenged with KIM D27, the modest but significant impacts of fibrinogen-deficiency on mortality and bacterial burden were still evident (Figures ID and IE). These data provide additional evidence of fibrinogen's role in innate defense against Y. pestis.

Roles for fibrin during innate defense against plague were more dramatic when fibrinogen-deficient mice were challenged with D27-pLpxL, a highly attenuated derivative of KIM D27. Intranasally administered D27-pLpxL is avirulent in wild type mice owing to its engineered, constitutive expression of hexa-acylated forms of LPS, which potently stimulate innate immunity by triggering TLR4 (Montminy et al, Nat. Immunol 7: 1066-1073, 2006). All wild type and most RAG-deficient mice survived intranasal challenge with 2xl0⁶ CFU D27- pLpxL (Figure IF), confirming a prominent role for innate immunity in defense against attenuated strains that constitutively express hexa-acylated LPS (Montminy et al., Nat. Immunol. 7: 1066-1073, 2006). In contrast, nearly all fibrinogen-deficient mice succumbed to challenge with D27-pLpxL (Figure 1G). Notably, some fibrinogen-heterozygous mice, which possess approximately 70% the level of fibrinogen as wild type mice (Suh et al, Genes Dev. 9:2020- 2033, 1995), also succumbed to D27-pLpxL challenge (Figure 1G). The day 4 bacterial burden in lung and liver were significantly elevated in fibrinogen-deficient mice challenged with D27- pLpxL, as compared with fibrinogen-heterozygous mice (Figure 1H). In fact, the burden of D27-pLpxL achieved levels approaching that of KIM D27 in fibrinogen-deficient mice (compare Figures 1C and 1H). RAG/fibrinogen-deficient mice also displayed high susceptibility to D27- pLpxL challenge, as measured by survival and bacterial burden (Figures II and 1 J), thus formally demonstrating that fibrinogen contributes substantially to the innate defense

mechanisms that mediate the attenuation of Y. pestis strain D27-pLpxL.

The attenuation of D27-pLpxL is thought to result from its capacity to evoke a strong immune response (Szaba et al., Infect. Immun. 77:4295-4304, 2009). The attenuation of KIM D28, another derivative of KIM D27, results from its inability to resist innate defense due to the loss of the pCDl plasmid-encoded T3SS. In contrast to fibrinogen-deficient mice challenged with D27-pLpxL (Figure 1G), fibrinogen-deficient mice readily survived intranasal challenge with KIM D28 (2xl0⁶ CFU). These data indicate that, depending upon the means of attenuation for a given Y. pestis strain, fibrinogen either can be critical or dispensable for innate immune defense. Fibrin may be dispensable with high titers of effective antibodies, but our data suggests it is critical for defense mediated by T cells or low doses of antibody.

Example 2: Fibrinogen can be dispensable for antibody-mediated defense against Y. pestis

The fibrinogen-deficient mice that survived challenge with KIM D28 subsequently withstood intranasal challenge with 2xl0⁵ CFU KIM D27. This observation suggested that fibrinogen can be dispensable for defense against KIM D27 in mice that have been immunized such that they possess Y. pestis-specific acquired immunity.

To definitively assess requirements for fibrinogen during acquired immunity, mice were challenged with Y. pestis and then supplied with antibody-mediated defense. Specifically, mice were infected intranasally with 2xl0⁵ CFU KIM D27 and then treated one day later with 10 μg LcrV-specific mAb 7.3. LcrV is a critical component of the T3SS (Viboud and Bliska, Annu. Rev. Microbiol. 59:69-89, 2005) and prior studies established that therapeutic administration of 10 μ mAb 7.3 protects wild type mice from intranasal challenge with KIM D27 (Kummer et al., Vaccine 26:6901-6907, 2008). This anti-LcrV immunotherapy protocol fully protected fibrinogen-deficient mice (Figure 2A). Analyses of day 4 bacterial burdens in lung and liver tissues likewise failed to discern differences between control and fibrinogen-deficient mice treated with 10 μg LcrV-specific mAb 7.3 (Figures 2B and 2C). Analogous studies using a mAb specific for Fl, the Y. pestis capsular protein, likewise demonstrated that antibodies could protect fibrinogen-deficient mice against lethal intranasal challenge with KIM D27 (Figure 2D).

Together, these data indicate that fibrinogen is not required for antibody-mediated defense against virulent Y. pestis.

Prior studies revealed distinct mechanisms of antibody-mediated protection depending upon the dose of immunotherapy (Kummer et al., Vaccine 26:6901-6907, 2008). When fibrinogen-deficient mice were infected intranasally with KIM D27 and then treated

therapeutically with a ten- fold lower, suboptimal dose of LcrV-specific mAb {i.e. 1 μg), most wild type and fibrinogen-heterozygous mice survived, whereas significantly fewer fibrinogen- deficient mice withstood the challenge (Figure 2E). Analyses of day 4 bacterial burdens revealed significantly increased bacterial CFU in lung and liver tissue from fibrinogen-deficient mice (Figures 2F and 2G). Similar results were observed when mice received a ten- fold lower dose of Fl -specific mAb (Figure 2H). Thus, fibrinogen is dispensable for immunotherapeutic protection against KIM D27 when optimal doses of protective mAb are used (Figures 2A-2D) but plays a significant role during suboptimal immunotherapy (Figures 2E-2H).

Example 3: Fibrinogen is essential for T cell-mediated defense against Y. pestis and neutrophils contribute to T cell-mediated defense

The cytokines TNFa and IFNy contribute to protection against plague in the suboptimal immunotherapy model, suggesting a role for type 1 T cells (Kummer et al, Vaccine 26:6901- 6907, 2008; Lin et al, Vaccine 29:357-362, 2010). To assess requirements for fibrinogen during T cell-mediated defense against Y. pestis, mice were supplied with Y. /?estz^'s-specific T cells and then challenged intranasally with 2xl0⁵ CFU KIM D27. In a first set of experiments, cellular immunity was supplied by intravenously injecting a polyclonal line of T cells derived from splenocytes of D27-pLpxL-immunized B cell-deficient μΜΤ mice that had been expanded in vitro by culture with heat-killed KIM D27 (Parent et ah, Infect. Immun. 73:7304-7310, 2005). One day following the transfer of 5xl0⁶ T cells, fibrinogen-deficient and control mice were challenged intranasally with KIM D27. All wild type mice that received culture medium alone succumbed to Y. pestis challenge, whereas all wild type mice that received T cells survived (Figure 3A). Most fibrino gen-heterozygous mice that received T cells also survived, whereas the fibrinogen-deficient mice all succumbed (Figure 3 A). Analyses of day 4 bacterial burdens confirmed that T cell transfer significantly reduced the number of Y. pestis CFU in lung and liver tissue of wild type mice (Figures 3B and 3C). The bacterial burden was significantly higher in fibrinogen-deficient mice, as compared with littermate controls, and resembled that of control wild type mice that did not receive T cells (Figures 3B and 3C).

The polyclonal T cell lines contained, on average, 38% CD4- and 62% CD8-positive T cells. A large fraction of Y. /?estz^'s-specific CD8 T cells in C57BL/6 mice recognize YopE₆9-77, a peptide fragment of the Y. pestis T3SS YopE protein (Lin et ah, J. Immunol. 187:897-904, 2011). To specifically assess roles for fibrinogen during CD8 T cell-mediated defense against virulent Y. pestis, fibrinogen-deficient mice were immunized with YopE₆9-77 and then challenged intranasally with KIM D27. Consistent with prior studies (Lin et al., J. Immunol. 187:897-904, 2011), YopE₆9-77-immunized wild type mice showed significantly improved survival as compared with wild type mice immunized with a control ovalbumin peptide (Figure 3D).

YopE₆9-77-immunized fibrinogen-deficient mice displayed significantly reduced survival as compared with fibrmogen-heterozygous controls (Figure 3D). Analyses of day 4 bacterial burdens revealed that YopE₆9_77 immunization reduced bacterial CFU to a significantly greater extent in the lung and liver tissue of fibrinogen-heterozygous mice, as compared with fibrinogen- deficient mice (Figures 3E and 3F).

The failure of YopE₆9_77 immunization to protect fibrinogen-deficient mice from plague (Figure 3D-3F) could have resulted from a failure to prime and expand T cells. To assess this possibility, flow cytometric studies were performed using a K^bYopE₆9-77 tetramer that specifically binds YopE₆9_7₇-specific CD8 T cells (Lin et al., J. Immunol. 187:897-904, 2011). In comparison with unimmunized mice, the number of pulmonary YopE69_77-specific CD8 T cells increased approximately 100-fold in YopE69_77-immunized mice (Figure 4A). The magnitude of this priming was similar in fibrinogen-deficient and littermate control fibrinogen-heterozygous mice. Four days after challenge with KIM D27, fibrinogen-deficient and heterozygous mice still harbored similar numbers of pulmonary YopE₆₉_77-specific CD8 T cells (Figure 4A). The fibrinogen-deficient and heterozygous mice also harbored similar numbers of total pulmonary CD8 cells, CD4 cells, and NK cells. Interestingly, the number of Ly6G-expressing neutrophils was similar in the unchallenged mice but decreased significantly in the fibrinogen-deficient mice, as compared with control heterozygous mice, at day 4 after challenge with KIM D27 (Figure 4B).

To assess whether a decrease in neutrophil numbers compromises T cell-mediated defense against plague, wild type mice were immunized with YopE₆9-77 and then neutrophils were depleted at the time of Y. pestis challenge. Specifically, mice were treated with Ly6G- specific mAb or isotype-matched control mAb on days -1, 1, 3, and 5 relative to challenge with KIM D27. YopE₆₉_77-immunized mice treated with Ly6G-specific mAb displayed significantly reduced survival in comparison with YopE₆₉_77-immunized mice treated with control mAb (Figure 4C).

Example 4: Fibrinogen restrains hemorrhagic pathology during innate and T cell- mediated defense against Y. pestis

Fibrin(ogen) can function protectively during infection by restraining bacterial growth/dissemination and/or by suppressing hemorrhagic pathology (Luo et al. , J. Immunol. 187: 1866-1876, 2011; Flick et al, J. Clin. Invest. 113: 1596-1606, 2004; Mullarky et al, Infect. Immun. 73:3888-3895, 2005; Johnson, et al. , J. Exp. Med. 197:801-806, 2003; Ahrenholz and Simmons, Surgery 88:41-47, 1980; Echtenacher et al , Infect. Immun. 69:3550-3555, 2001; Massberg et al , Nat. Med. 16:887-896, 2010; Sun et al , Science 305:1283-1286, 2004; Sun et al, Blood 113: 1358-1364, 2009). The data presented in Figures 1-3 indicate that fibrinogen restrains the bacterial burden during Y. pestis infection. To investigate whether fibrin(ogen) also suppresses hemorrhage in this setting, tissues were subjected to histological analysis (Figure 5). On day 4 after challenge with KIM D27, fibrinogen-heterozygous mice displayed evidence of hepatic necrosis with lesions showing many pyknotic nuclei (Figures 5A and 5M). Fibrinogen- deficient mice displayed similar pathology but fewer lesions contained identifiable nuclei and some lesions showed areas of hemorrhage (Figures 5D and 5M). Infection with highly attenuated D27-pLpxL induced a robust influx of inflammatory cells in both fibrinogen- heterozygous and fibrinogen-deficient mice, but there was a greater propensity for hemorrhage in the fibrinogen-deficient mice (Figures 5E and 5M). Likewise, there was a greater level of hemorrhagic pathology in YopE-immunized fibrinogen-deficient mice challenged with KIM D27, as compared with littermate controls (Figures 5F and 5M). Quantitative scoring revealed that a significantly greater percentage of the lesions in the fibrinogen-deficient mice showed evidence of hemorrhage (Figure 5N). Together, these observations indicate that innate and T cell-mediated defense against Y. pestis is associated with hemorrhagic pathology in fibrinogen- deficient mice.

Example 5: Fibrinogen supports phagocyte viability during innate and T cell- mediated defense against Y. pestis

To investigate the nature of the cellular response during innate and T cell-mediated defense against Y. pestis, samples of hepatic tissue were subjected to immuno fluorescent staining. Many of the infiltrating cells observed in the na^'ive fibrinogen-heterozygous mice infected with D27-pLpxL (Figure 5H) and in the YopE-immunized fibrinogen-heterozygous mice infected with KIM D27 (Figure 51) stained specifically with mAb that recognize macrophages (F4/80; blue color) or neutrophils (anti-Ly6G; red color). The leukocyte staining in these samples co-localized with dense clusters of nuclei, as revealed by co-staining with the DNA intercalating Hoescht dye (white color). In striking contrast, the lesions observed in all the fibrinogen-deficient mice (Figures 5 J, 5K, 5L), as well as the na^'ive fibrinogen-heterozygous mice infected with KIM D27 (Figure 5G), generally lacked evidence of Hoescht-staining nuclei even though they still stained specifically with macrophage and neutrophil markers.

Figures 5G-5L also show staining of Y. pestis bacteria using a mAb that recognizes the capsular Fl protein (green color). Specifically, anti-Fl staining readily detected bacteria within many of the lesions of na^'ive mice infected with KIM D27 (Figures 5G and 5J). Bacteria were rarely observed in the lightly colonized na^'ive fibrinogen-heterozygous mice infected with D27- pLpxL (Figure 5H) and YopE-immunized fibrinogen-heterozygous mice infected with KIM D27 (Figure 51) but, consistent with the CFU data (Figures 1H and 3F), bacteria were often visualized in the lesions of the corresponding fibrinogen-deficient mice (Figures 5K and 5L). Altogether, the data suggest that phagocytes were recruited to sites of Y. pestis infection in fibrinogen- deficient mice but failed to survive their encounters with Y. pestis bacteria. Example 6: Unimpaired inflammatory responses in fibrinogen-deficient mice infected with Y. pestis

The increased bacterial burden in liver tissue collected from fibrinogen-deficient mice infected with Y. pestis could have resulted from impaired induction of a hepatic immune response. However, real-time PCR measurements failed to discern any impairment in the upregulation of mediators of inflammation and immunity (Figure 6). Rather, the fibrinogen- deficient and fibrinogen-heterozygous mice showed similar inductions of the cytokines TNFa and IFNy, the neutrophil chemoattractant CXCL1, and the antibacterial peptide lipocalin-2.

Levels of IL-6 and IL-10 mRNA also did not differ significantly between fibrinogen-deficient and fibrinogen-heterozygous mice. These data are consistent with prior studies demonstrating unimpaired induction of hepatic inflammatory proteins in fibrinogen-deficient mice infected with T. gondii, L. monocytogenes, and Y. enterocolitica (Luo et al, J. Immunol. 187: 1866-1876, 2011; Mullarky et al, Infect. Immun. 73:3888-3895, 2005; Johnson, et al, J. Exp. Med. 197:801-806, 2003).

Example 7: Fibrin contributes to innate and T cell-mediated defense against Y. pestis

The data presented thus far indicate a role for fibrinogen during innate and T cell- mediated defense against plague. Accordingly, we next assessed whether fibrin itself contributes to defense against plague. Kinetic analyses in wild type mice revealed that intranasal inoculation of either strain KIM D27 or strain D27-pLpxL resulted in similar levels of hepatic CFU on day 2 post infection (Figure 7A), at which time quantitative immunoblotting of liver tissue first revealed detectable levels of hepatic fibrin (Figure 7B). By day 4, liver tissue from mice infected with KIM D27 contained significantly higher bacterial loads (Figure 7A) and fibrin (Figure 7B), as compared with mice infected with D27-pLpxL. Likewise, by day 4 the expression of genes whose products might contribute to fibrin formation had increased to a significantly greater extent in mice infected with KIM D27 as compared with D27-pLpxL (Figures 7C-F). While levels of mRNA encoding the procoagulant TF did not change significantly during the course of either KIM D27 or D27-pLpxL infection (Figure 7C), levels of the procoagulant FXI and the anti-fibrinolytic TAFI increased modestly and achieved significantly higher levels in mice infected with KIM D27 as compared with D27-pLpxL (Figures 7D and 7E). Levels of another anti-fibrino lytic, PAI-1, increased markedly in mice infected with either KIM D27 or D27- pLpxL, but achieved significantly higher levels in the KIM D27-infected mice (Figure 7F).

To assess functional roles for fibrin, studies were performed with mice possessing reduced capacities to generate fibrin. First, mice with genetic impairments in coagulation pathways leading to the production of thrombin were infected with Y. pestis. One set of mice was deficient for expression of TF, the key initiator of thrombin-generating coagulation pathways (Tilley and Mackman, Semin. Thromb. Hemost. 32:5-10, 2006; Pawlinski and Mackman, Thromb. Res. 125 Suppl l :S70-73, 2010). A second set of mice was deficient for expression of FXI, a key component of the intrinsic coagulation pathway, which is not critical for thrombin production but amplifies and sustains thrombin levels in certain settings (Gailani and Broze, Jr., Science 253:909-912, 1991; Naito and Fujikawa, J. Biol. Chem. 266:7353-7358, 1991; von dem Borne et ah, J. Thromb. Haemost. 5:1106-1112, 2007; 54. Gailani and Renne, J. Thromb. Haemost. 5: 1106-1112, 2007). As shown in Figure 8A, all FXI deficient mice survived challenge with D27-pLpxL, whereas mice engineered to express very low levels of TF displayed significantly reduced survival as compared with littermate control mice. YopE- immunized mice expressing very low levels of TF also displayed significantly decreased protection as compared with littermate control mice (Figure 8B). These observations suggest that TF-dependent production of thrombin, which leads to the formation of fibrin, contributes to innate and T cell-mediated defense against Y. pestis.

To confirm a role for thrombin, wild type mice were treated with Coumadin, a pharmacologic anticoagulant that reduces production of thrombin. Treatment with Coumadin significantly decreased survival of wild type mice infected with attenuated D27-pLpxL

(Figure 8C). Likewise, YopE-immunized wild type mice showed significantly decreased protection when treated with Coumadin at the time of challenge with KIM D27 (Figure 8D). Given that Coumadin reduces thrombin-mediated fibrin formation without impacting fibrinogen levels, these findings suggest essential roles for fibrin during innate and T cell-mediated defense against Y. pestis.

Mice deficient in fibrin due to increased fibrinolysis, rather than decreased coagulation, were used to further assess roles for fibrin in innate and T cell-mediated defense against plague. PAI-1 and TAFI are two important regulators of fibrinolysis (Rijken and Lijnen, J. Thromb. Haemost. 7:4-13, 2009). PAI-1 suppresses the activation of plasmin, the primary mediator of fibrinolysis (Lijnen, J. Thromb. Haemost. 3:35-45, 2005), whereas TAFI cleaves lysine residues from fibrin, thereby decreasing fibrinolysis by removing binding sites for plasmin (Bajzar, Arterioscler. Thromb. Vase. Biol. 20:2511-2518, 2000; Morser et al., J. Thromb. Haemost.

8:868-876, 2010). Prior studies established that genetic depletion of both PAI-1 and TAFI decreases fibrin levels more than depletion of either one alone (Luo et al., J. Immunol. 187: 1866- 1876, 2011; Vercauteren et al., J. Thromb. Haemost., 10(12):2555-2562, 2012). Control mice largely survived infection with D27-pLpxL, whereas significantly fewer PAI-1 /TAFI-deficient mice survived (Figure 8E). Likewise, YopE-immunized PAI-1 /TAFI-deficient mice showed significantly reduced survival after challenge with KIM D27 (Figure 8F).

Discussion: The studies described below reveal dramatic impairments in both innate and T cell-mediated defense in (i) mice lacking fibrinogen, (ii) mice with very low levels of TF procoagulant activity, (iii) mice with elevated levels of fibrinolytic activity, and (iv) mice treated with the pharmaceutical anticoagulant Coumadin (Figures 1, 3, and 8). The similar impairments displayed by the mice used in this study strongly suggest that fibrin is an important contributor to both innate and T cell-mediated defense in mouse models of pneumonic plague.

A prior study by Lathem and colleagues indirectly suggested functional roles for fibrin during innate defense in mice inoculated intranasally with Y. pestis (Lathem et al, Science

315:509-513, 2007). Our findings support that possibility by directly demonstrating increased Y. pestis CFU within lung tissue of mice lacking the capacity to produce fibrin (Figures 1-3). Multiple forms of fibrin-mediated immunity have been proposed. None are mutually exclusive and their relative importance may be context dependent. Fibrin-mediated "hemostatic immunity" appears to play critical roles during Listeria and Toxoplasma infections. Challenging fibrin-deficient mice with low doses of these pathogens causes hepatic bleeding, severe anemia, and increased mortality (Mullarky et al., Infect. Immun. 73:3888-3895, 2005; Johnson, et al., J. Exp. Med. 197:801-806, 2003). The time at which fibrin-deficient mice succumb to Listeria and Toxoplasma infections coincides with the time of peak anemia in wild type mice, suggesting that fibrin prevents infection-induced hemorrhage in these models (Mullarky et al., Infect.

Immun. 73:3888-3895, 2005; Johnson, et al. , J. Exp. Med. 197:801-806, 2003). Blood-tinged sputum is a diagnostic symptom of fulminant pneumonic plague in humans (Lien-Teh, A

Treatise on Pneumonic Plague. Leaugue of Nations Health Organisation, Geneva, 1926;

Inglesby et al., JAMA 283:2281-2290, 2000), suggesting a failure of hemostatic responses in that setting. Although, Y. pestis infection did not cause significant anemia in the models of septic pneumonic plague described in this report (data not shown), both innate and T cell-mediated defense against plague clearly benefit from some degree of hemostatic immunity since the hepatic tissue of Y. pestis-infected fibrinogen-deficient mice, but not littermate controls, was characterized by lesions full of red blood cells (Figure 5).

Proteolytic fragments of fibrinogen may also contribute to fibrin-mediated defense against plague. When it is cleaved by thrombin to create fibrin, fibrinogen releases a set of peptides that are chemoattractant for monocytes, macrophages and neutrophils (Jennewein et al, Mol. Med. 17:568-573, 2011). However, these fibrinopeptides seem unlikely to play major roles during fibrin-mediated defense against plague since PAI-l/TAFI-deficient mice should produce wild type levels of fibrinopeptides yet they appear phenotypically similar to fibrinogen-deficient mice during innate and T cell-mediated defense against plague (Figure 8). Fibrin degradation products (FDPs) that result from fibrinolysis also can regulate leukocyte functions (Jennewein et al, Mol. Med. 17:568-573, 2011). FDP levels should be normal or elevated in PAI-l/TAFI- deficient mice, reduced in lowTF mice, and absent in fibrinogen-deficient mice. Given that all those mice display similar phenotypes during innate and T cell-mediated immune defense against plague (Figure 8), FDPs seem unlikely to play a major role in the models of fibrin-mediated immunity described herein.

Fibrin could contribute to innate and T cell-mediated immune defense against plague by physically trapping Y. pestis bacteria, thereby limiting their growth and dissemination (Titball and Oyston, Nat. Med. 13:253-254, 2007; Esmon et al., J. Thromb. Haemost. 9 Suppl 1: 182- 188, 2011). Trapping has long been considered a logical means of fibrin-mediated innate defense against bacteria, and there is evidence for this mechanism during E. coli infections (Ahrenholz and Simmons, Surgery 88:41-47, 1980; Echtenacher et al, Infect. Immun. 69:3550- 3555, 2001; Massberg et al, Nat. Med. 16:887-896, 2010). Physical restraint of Y. pestis bacteria by fibrin is one possible explanation for the decreased dissemination of Pla-deficient strains from peripheral sites of infection (Sebbane et al, Proc. Natl. Acad. Sci. USA 103:5526- 5530, 2006; Sodeinde et al, Science 258: 1004-1007, 1992; Welkos et al., Microb. Pathog.

23:211-223, 1997). However, Lathem and colleagues provided evidence that Pla is less important for Y. pestis dissemination from lung tissue (Lathem et al, Science 315:509-513, 2007). While the data presented here do not rule out a role for trapping, the liver histology provides direct evidence that successful innate and T cell-mediated defense against plague corresponds with fibrinogen-dependent accumulation of cellular infiltrates at sites of Y. pestis dissemination (Figure 5). Fibrinogen is a ligand for a number of cell surface receptors that facilitate leukocyte adhesion and activation, including CD 1 lb/CD 18, CD1 lc/CD18, CD44, and TLR4 (Wright et al, Proc. Natl. Acad. Sci. USA 85:7734-7738, 1988; Loike et al, Proc. Natl. Acad. Sci. USA 88: 1044-1048, 1991; Altieri et al, J. Biol. Chem. 268: 1847-1853, 1993; Weber and Springer, J. Clin. Invest. 100:2085-2093, 1997; Ugarova et al, J. Biol. Chem. 273:22519- 22527, 1998; Sitrin et al, J. Immunol. 161: 1462-1470, 1998; Forsyth et al., J. Exp. Med.

193: 1123-1133, 2001; Rubel et al, J. Immunol. 166:2002-2010, 2001; Rubel et al, Eur. J.

Immunol. 33: 1429-1438, 2003; Flick et al, Exp Biol Med (Maywood) 229: 1105-1110, 2004; Alves et al, J. Biol. Chem. 284: 1177-1189, 2009; Szaba and Smiley, Blood 99:1053-1059, 2002; Smiley et al., J. Immunol. 167:2887-2894, 2001; Barrera et al, Blood 117:5674-5682, 2011). Since most phagocytes express the fibrin(ogen)-binding integrins CD1 lb/CD18 and/or CD1 lc/CD18, it is easily conceivable that fibrin acts as an inducible matrix supporting the accumulation of phagocytes at sites of infection. In addition to providing an adhesive substrate, ligation of fibrin by fibrin(ogen)-binding receptors can also activate phagocyte functions, including the secretion of chemokines that recruit additional leukocytes (Sitrin et al, J. Immunol. 161: 1462-1470, 1998; Forsyth et al, J. Exp. Med. 193: 1123-1133, 2001; Rubel et al, J.

Immunol. 166:2002-2010, 2001; Rubel et al, Eur. J. Immunol. 33:1429-1438, 2003; Flick et al, Exp Biol Med (Maywood) 229: 1105-1110, 2004; Szaba and Smiley, Blood 99: 1053-1059, 2002; Smiley et al, J. Immunol. 167:2887-2894, 2001; Walzog et al, FASEB J. 13: 1855-1865, 1999). In this manner, extravascular fibrin(ogen) can be viewed as a danger-associated molecular pattern triggering leukocyte migration, accumulation, and activation (Flick et al, Exp. Biol. Med. (Maywood) 229: 1105-1110, 2004; Smiley et al, J. Immunol. 167:2887-2894, 2001). A particularly elegant in vivo demonstration of the phagocyte-activating capacity of fibrin(ogen) was provided by Flick and colleagues who engineered mice to only express fibrinogen molecules that lack a key binding site for CD1 lb and CD1 lc integrins (Flick et al, J. Clin. Invest.

113: 1596-1606, 2004; Flick et al, Exp Biol Med (Maywood) 229: 1105-1110, 2004). These mice displayed elevated bacterial burden in a model of acute peritonitis and their peritoneal neutrophils appeared unable to kill phagocytosed S. aureus bacteria (Flick et al. , J. Clin. Invest. 113: 1596-1606, 2004; Flick et al, Exp Biol Med (Maywood) 229: 1105-1110, 2004). A failure to control bacterial replication is likewise observed in fibrinogen-deficient mice infected with S. aureus, group A Streptococcus, L. monocytogenes and Y. enterocolitica (Luo et al.,

J. Immunol. 187: 1866-1876, 2011; Mullarky et al, Infect. Immun. 73:3888-3895, 2005; Sun et al, Science 305: 1283-1286, 2004; Sun et al, Blood 113:1358-1364, 2009).

Although a specific role for the CD1 lb/c-binding site on fibrinogen has yet to be demonstrated in all these settings, it seems likely that fibrin formation at sites of bacterial infection supports innate host defense by providing a non-diffusible cue for the accumulation and activation of inflammatory cells that express fibrin-binding integrins (Degen et al. , J. Thromb. Haemost. 5 Suppl 1:24-31, 2007).

The studies reported here suggest that fibrin also affects leukocyte survival at sites of Y. pestis infection. Fibrinogen can suppress neutrophil apoptosis in vitro (Rubel et al,

J. Immunol. 166:2002-2010, 2001; Sakamoto et al, Infect. Immun. 78: 1004-1011, 2010), likely via interactions with CD 1 lb/CD 18 (Walzog et al. , FASEB J. 11 : 1177- 1186, 1997; Whitlock et al., J. Cell Biol. 151:1305-1320, 2000). Fibrinogen-deficient mice implanted with

inflammatory microspheres develop suppurative lesions, whereas wild type mice develop cellular infiltrates containing viable neutrophils (Sakamoto et al, Infect. Immun. 78:1004-1011, 2010). In na^'ive fibrin(ogen)-sufficient mice infected with Y. pestis, the hepatic lesions that form contain pyknotic nuclei (Figure 5A) and the presence of residual leukocyte membranes within these lesions suggests that Y. pestis infection leads to the death of recruited leukocytes

(Figure 5G). In the presence of effective innate or T cell-mediated immunity, the hepatic tissue of fibrin(ogen)-sufficient mice contains clusters of viable neutrophils and macrophages

(Figures 5B, 5C, 5H, 51), whereas fibrinogen-deficient mice develop lesions characterized by residual leukocyte membranes (Figures 5K, 5L). These findings extend the conclusions of Degen et al. (J. Thromb. Haemost. 5 Suppl 1 :24-31 , 2007) by suggesting that fibrin formation at sites of Y. pestis infection provides a non-diffusible cue for the accumulation, activation, and enhanced survival of inflammatory cells.

While prior studies have demonstrated that fibrin(ogen) contributes to innate defense against bacterial infection, this is the first study to demonstrate that fibrin can be essential for T cell-mediated defense. To our knowledge, there are no prior reports of defective T cell responses in fibrin(ogen)-deficient mice. To the contrary, T cell-mediated control of pathogen burden appears unimpeded in fibrin(ogen)-deficient mice challenged with T. gondii ((Mullarky et al, Infect. Immun. 73:3888-3895, 2005; Johnson, et al., J. Exp. Med. 197:801-806, 2003) or M. tuberculosis (Sakamoto et al, Infect. Immun. 78: 1004-1011, 2010), and here we showed unimpeded expansion of CD8 T cells in response to vaccination with YopE₆9-77, an antigenic Y. pestis peptide (Figure 5 A). Nevertheless, those YopE-immunized mice displayed a significantly impaired capacity to restrain Y. pestis growth and prevent plague (Figure 3 and 4A).

The failure of T cell-mediated defense in fibrin(ogen)-deficient mice infected with Y. pestis appears to reflect a failure of neutrophils to survive encounters with bacteria in the absence of fibrin(ogen)-dependent signals. The T cells primed in this Y. pestis model produce TNFa and IFNy (Lin et al., J. Immunol. 187:897-904, 2011). These cytokines can stimulate neutrophils (McCall et al, Eur. J. Immunol. 21:2523-2527, 1991; Evans et al, Proc. Natl. Acad. Sci. USA 93:9553-9558, 1996; Scapini et al, Immunol. Rev. 177: 195-203, 2000), which can reduce Y. pestis growth in vivo and in vitro (Laws et al, Microbes Infect. 12:331-335, 2010). CD8 T cells producing TNFa and IFNy may help to amplify neutrophil functions, perhaps augmenting their oxidative mechanisms (McCall et al, Eur. J. Immunol. 21:2523-2527, 1991; Evans et al, Proc. Natl. Acad. Sci. USA 93:9553-9558, 1996) and/or their production of inflammatory cytokines and chemokines (Scapini et al, Immunol. Rev. 177:195-203, 2000). Exposure to TNFa and IFNy also can render macrophages non-permissive for intracellular Y. pestis replication (Lukaszewski et al, Infect. Immun. 73:7142-7150, 2005). Thus, T cells may combat plague by producing cytokines that help macrophages restrict intracellular Y. pestis replication while enabling neutrophils to survive Y. pestis encounters and kill extracellular bacteria in a fibrin(ogen)-dependent manner. Regardless of the precise mechanism, the findings presented here demonstrate that one previously unappreciated function of fibrin is to support neutrophil-dependent T cell-mediated defense against bacteria.

This report also demonstrates that fibrin(ogen) is dispensable for immune defense against plague mediated by optimal levels of protective Y. pestis-specific antibody even though it contributes significantly to the partial protection conferred by suboptimal levels of antibody (Figure 2). These observations are reminiscent of prior work demonstrating that TNFa and IFNy are dispensable for defense against plague mediated by optimal levels of antibody but critical for defense mediated by suboptimal levels of antibody (Kummer et al, Vaccine 26:6901-6907, 2008; Lin et al, Vaccine 29:357-362, 2010). Given that T cells also contribute to antibody- mediated defense against plague (Levy et al., Vaccine 29:6866-6873, 2011), these observations suggest that T cells producing TNFa and IFNy may provide fibrin-dependent defense that is particularly critical when suboptimal levels of antibodies are present.

Further studies are required to establish why fibrin is dispensable for defense against plague mediated by high levels of protective antibody. Prior studies indicate that antibodies to either LcrV or Fl can interfere with the transport of effector proteins by the Y. pestis T3SS (Pettersson et αί, Μοί Microbiol. 32:961-976, 1999; Cowan et al.Jnfect. Immun. 73:6127-6137, 2005; Cornelius et al., Infect. Immun. 77: 1807-1816, 2009). That observation suggests that fibrin may be dispensable for protection in the presence of sufficient titers of antibody to effectively neutralize the T3SS, which functions to intoxicate phagocytes. Consistent with that possibility, fibrin is not required when mice are challenged with attenuated Y. pestis strain KIM D28 (data not shown), which lacks the T3SS. However, another intriguing possibility is that optimal levels of antibody may bypass requirements for fibrin-mediated enhancement of phagocyte survival by cross-linking phagocyte Fc receptors, thereby providing phagocytes with fibrin(ogen)-independent survival signals.

Studies over the past few decades have revealed remarkable interplay between inflammation, immunity, and coagulation (Esmon et al. , J. Thromb. Haemost. 9 Suppl 1 : 182- 188, 2011; Delvaeye and Conway, Blood 114:2367-2374, 2009; Levi and van der Poll, Crit. Care Med. 38:S26-34, 2010; Stearns-Kurosawa et al., Annu. Rev. Pathol. 6:19-48, 2011). This report extends those connections by demonstrating that coagulation leading to fibrin formation can critically influence both innate and adaptive immunity. The accumulating evidence for essential protective roles for fibrin during a variety of infections could help to explain why potent anticoagulant therapies have failed to extend survival in septic patients (Riedemann, J. Clin. Invest. 112:460-467, 2003; Dyson and Singer, Crit. Care Med. 37:S30-37, 2009), and suggests that treatments for sepsis should strive to mitigate coagulopathy without preventing the formation of protective fibrin.

Example 8:

Having demonstrated essential roles for fibrin during neutrophil-dependent T cell- mediated defense against Y. pestis, we next determined whether Pla functions to limit T cell responses. As shown in Figure 9, survival is dramatically higher following challenge with strains of Y. pestis in which Pla is inactivated. Wild type C57BL/6 mice were treated intranasally with cholera toxin (CT) adjuvant along with YopE or control ovalbumin (Ova) peptides on days -35, - 28, and -14 and then challenged intranasally on day 0 with 10 median lethal doses of Y. pestis strains C092, C092deltaPla, or C092 Pla-D206A. C092 is a wild type strain, whereas delta Pla and Pla-D206A lack the plasminogen activating activity of the Y. pestis gene encoding Pla, a potent plasminogen activator. Pla antagonism rendered Y. pestis susceptible to host defense mediated by T cells.

We anticipate that fibrinolytic mechanisms similarly circumvent T cell responses against more common invasive pathogens, such as S. aureus. If so, therapeutics targeting bacterial plasmin-generating factors should act synergistically with T cells, or vaccines that prime T cells, thereby providing a new treatment option for the growing threat of antibiotic-resistant infections by bacteria that have proven difficult to subdue with antibody-based vaccines.

Our data suggest that treating mice, and presumably people, with antagonists of Y. pestis Pla may improve T cell-mediated defense against plague. Since Pla activates mammalian plasminogen, creating the fibrinolytic enzyme plasmin, our data also suggest that antifibrinolytic drugs targeting plasmin or plasminogen may also enhance T cell-mediated defense. We hypothesize that antifibrinolytic drugs will improve T cell responses against Y. pestis and other bacterial pathogens that degrade fibrin. If so, then new or existing antifibrinolytics can be exploited as synergistic enhancers of antibacterial T cell responses. We imagine bandages, lotions, inhalers and sprays containing antifibrinolytics as treatments for a number of skin, tissue or respiratory diseases caused by or aggravated by bacterial infection, including dermatitis, fasciitis, sinusitis and pneumonia. It may or may not be necessary to prime specific T cell responses in such settings, depending on whether or not the patient already possesses T cells that recognize the causative bacteria. The studies described below aim to establish proof-of-concept for a new paradigm that may be broadly applicable to the treatment of many types of bacterial infections.

Future Experimental design: Exposed carboxyl-terminal lysine residues act as receptors for the binding of plasmin and plasminogen to fibrin and bacteria. Lysine analogs like tranexamic acid competitively interfere with those interactions, thereby suppressing fibrinolysis (Boyle and Lottenberg, Thromb. Haemost. 77: 1-10, 1997; Bergmann and Hammerschmidt, Thromb. Haemost. 98:512-520, 2007; Lucas et al., Ann. N. Y. Acad. Sci. 408:71-91, 1983; Lucas et al., J. Biol. Chem. 258:4249-4256, 1983). Tranexamic acid is approved for clinical use as an antifibrinolytic and is available "over the counter" in Europe. It reportedly blocks plasminogen activation by many bacteria in vitro (Boyle and Lottenberg, Thromb. Haemost. 77:1-10, 1997; Bergmann and Hammerschmidt, Thromb. Haemost. 98:512-520, 2007; Lucas et αΙ., Αηη. N. Y. Acad. Sci. 408:71-91, 1983; Lucas et al. , J. Biol. Chem. 258:4249-4256, 1983), and we have found that it suppresses plasminogen activation by Y. pestis in vitro. Thus, tranexamic acid should block Pla-mediated plasminogen activation in vivo, while also blocking feedback amplification of fibrinolysis by antagonizing plasminogen and tPA binding to exposed carboxyl- terminal lysine residues on partially degraded fibrin (Hoylaerts et al., J. Biol. Chem. 257:2912- 2919, 1982; Christensen, FEBS Lett. 182:43-46, 1985; Fleury and Angles-Cano, Biochemistry 30:7630-7638, 1991; Sakharov and Rijken, Circulation 92: 1883-1890, 1995).

Here we will assess whether tranexamic acid enhances T cell responses to plague (a prophetic example). We will immunize mice with YopE or control ovalbumin peptide and challenge with C092. Following published procedures (Busuttil et al., J. Throm. Haemost. 2: 1798-1805, 2004; Swaisgood et al., Am. J. Respir. Crit. Care Med. 176:333-342, 2007; Paul et al., J. Exp. Med. 204: 1999-2008, 2007; and Yamamoto et al., Biochem. Biophys. Res.

Commun. 430:999-1004, 2013), we will supplement drinking water with tranexamic acid beginning one day prior to bacterial challenge.

Interpretations: Data obtained to date suggest that Pla-mediated plasminogen activation circumvents T cell-mediated defense against C092 (Figure 9), so we anticipate that treatment with tranexamic acid will improve survival of YopE-immunized mice that are challenged with C092. With 10 mice per group, the experiment is designed using a log-rank test at a power of 0.8 to detect significant improvements (p<0.05) in the median survival time to greater than 20 days (i.e. greater than 50% survival to end of study).

Follow-up studies will repeat the experiment to establish reproducibility and increase statistical power. Once we obtain positive results, we will move from a prophylactic tranexamic acid treatment regimen to a therapeutic regimen and extend our studies to other bacteria, focusing initially on MRSA. If the tranexamic acid studies are unsuccessful, then we will pursue a number of alternative approaches. The serine protease inhibitor (serpin) alpha2-antiplasmin is a potent natural plasmin antagonist. Unfortunately, it is not ideal for our purposes since plasmin typically becomes resistant to alpha2-antiplasmin once bound to bacteria (Boyle and Lottenberg, Thromb. Haemost. 77: 1-10, 1997; Bergmann and Hammerschmidt, Thromb. Haemost. 98:512- 520, 2007). Another serpin, aprotinin, is a potent plasmin inhibitor that antagonizes bacterial fibrinolytic activities, so it could be tested in our model (Boyle and Lottenberg, Thromb.

Haemost. 77: 1-10, 1997; Hollands et al. , J. Biol. Chem. 287:40891-40897, 2012). Engineered serpins with improved specificity for plasmin have also been described (Markland et al, Biochemistry 35:8045-8057, 1996; Bajaj et al. , J. Biol. Chem. 286:4329-4340, 2011). We could acquire and test these serpins by injecting them into mice. However, given our success with HDI, we would first attempt to supply serpins by that method, hopefully achieving sustained levels without the need for purified proteins or repeated injections.

We are not aware of any highly selective, commercially available, small-molecule plasmin antagonists that are suitable for in vivo studies. However, there is significant research ongoing in this area (Swedberg and Harris, Chembiochem. 13:336-348, 2012; Saupe et al, J. Med. Chem. 56:820-831, 2013). DNA/RNA-based aptamer technology is also possible.

Potent and specific aptamers targeting thrombin, FIX, and other serine proteases are being developed for clinical use (Wang et al, Curr. Med. Chem. 18:4169-4174, 2011; Avino et al, Curr. Pharm. Des. 18:2036-2047, 2012). Another approach would be to demonstrate that gene- targeted mice with impaired fibrinolysis exhibit improved T cell defense against bacteria. Such studies would serve as proof-of-concept fostering the development of drugs for this purpose. Plasminogen-, uPA-, or tPA-deficient mice could be investigated, depending on the bacteria. For example, plasminogen bound to B. burgdorferi is activated by uPA, so uPA-deficient mice may be useful in that setting, whereas plasminogen bound to E. coli is activated by tPA (Boyle and Lottenberg, Thromb. Haemost. 77:1-10, 1997). A key distinction from prior studies of this type is that the mice in our studies will have primed T cells.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a patient who has been exposed, or who is at risk of exposure to, a bacterial pathogen, the method comprising administering to the patient:

(a) an antigen expressed by the bacterial pathogen or cells expressing the antigen; and

(b) an agent that inhibits fibrinolysis.

2. The method of claim 1, wherein the patient is a human.

3. The method of claim 1, wherein the bacterial pathogen is of the genus Yersinia, Haemophilus, Salmonella, Streptococcus, Staphylococcus, Mycobacterium, Escherichia, Helicobacter, Moraxella, Mycoplasma, Neisseria, Proteus, Pseudomonas, or Borrelia.

4. The method of claim 1, wherein the agent that inhibits fibrinolysis inhibits the production or activity of plasmin, tissue-type plasminogen activator (tPA), or urokinase-type plasminogen activator.

5. The method of claim 1, wherein the agent that inhibits fibrinolysis increases the expression or activity of a plasminogen activator inhibitor (PAI).

6. The method of claim 5, wherein the PAI is PAI-1.

7. The method of claim 1, wherein the agent that inhibits fibrinolysis increases the expression or activity of a thrombin-activatable fibrinolysis inhibitor (TAFI).

8. The method of claim 1, wherein the agent that inhibits fibrinolysis inhibits the expression or activity of a bacterial plasminogen-binding protein or plasminogen activator.

9. The method of claim 8, wherein the bacterial plasminogen activator is streptokinase, staphylokinase, Pla protein, or a bacterial protease in the omptin family.

10. The method of claim 1, wherein the agent that inhibits fibrinolysis is a nucleic acid construct, a polypeptide, or a small molecule.

11. The use of a therapeutically effective amount of:

(a) an antigen expressed by a pathogen or cells expressing the antigen; and

(b) an agent that inhibits fibrinolysis;

for treating a patient who has been exposed to, or who is at risk of exposure to, the pathogen.

12. The use of claim 11, wherein the pathogen is of the genus Yersinia, Haemophilus, Salmonella, Streptococcus, Staphylococcus, Mycobacterium, Escherichia, Helicobacter, Moraxella, Mycoplasma, Neisseria, Proteus, Pseudomonas, or Borrelia.

13. The use of claim 11, wherein the agent that inhibits fibrinolysis is a nucleic acid construct, a polypeptide, or a small molecule.

14. The use of claim 11, wherein the agent that inhibits fibrinolysis inhibits the expression or activity of a bacterial plasminogen-binding protein or plasminogen activator.

15. A kit comprising (a) an antigen expressed by a bacterial pathogen or cells expressing the antigen; (b) an agent that inhibits fibrinolysis; and (c)instructions for use.

16. A dressing for a bodily wound, the dressing comprising a substrate and, in contact with the substrate (a) an antigen expressed by a bacterial pathogen or cells expressing the antigen; and/or (b) an agent that inhibits fibrinolysis.