MXPA01003605A - Reversal of viral-induced systemic shock and respiratory distress by blockade of the lymphotoxin beta pathway - Google Patents

Abstract

This invention provides methods of inducing an antiviral response in an individual comprising administering to the individual an effective amount of a LT-B blocking agent and a pharmaceutically acceptable carrier. In particular this invention provides methods for treating viral-induced systemic shock and respiratory distress.

Description

REVERSION OF SYSTEMIC SHOCK INDUCED BY VIRUSES AND RESPIRATORY INSUFFICIENCY BY BLOCKING THE ROUTE OF THE BETA LINFOTOXIN RELATED APPLICATIONS This is a continuation in part of the earlier United States Provisional Application No. 60 / 103,662 filed on October 9, 1998. The teachings of the previously filed provisional patent application are incorporated herein by reference.

FIELD OF THE INVENTION This invention relates in general to methods for inducing an antiviral response in an individual. In particular, this invention provides methods for treating systemic shock induced by virus and respiratory failure in an individual. The methods involve the administration of certain "lymphotoxin-beta blocking agents".

BACKGROUND OF THE INVENTION Various viruses including: Without Name (SNV), Ebola, Marburg, Lassa and Dengue, cause acute diseases with many of the following symptoms: rapid onset, fever, systemic shock and pulmonary insufficiency (Lacy et al. (1997) Adv. Ped. Inf. Dis. 12 :twenty-one). Another common feature among these infections is the systemic distribution of viral infection, which is directed to endothelial cells and to macrophages 5 (Lacy et al. (1997) Adv. Ped. Inf. Dis 12:21). Most of these emerging viruses, with the exception of SNV, were initially identified decades ago. Over the course of the years since they were discovered, these pathogens have re-emerged in outbreaks around the world. 10 In June 1998, 183 cases of SNV, the causative agent of hantavirus pulmonary shock syndrome, in the southwestern United States, had been confirmed due to an increase in kangaroo mouse populations. Only 55% of these cases survived the infection (Centers for Disease Control and Prevention, MMWR, 47, 449 (1998)). Currently little is known about the pathogenesis of these viruses, as well as the way to effectively treat the thousands of patients globally infected every year, who suffer systemic shock induced by viruses and respiratory failure. Thus, there is a need to identify novel methods for treating systemic shock induced by virus and respiratory failure in an individual.

SUMMARY OF THE INVENTION The present invention solves the problem _ ^ previously referred to in providing pharmaceutical compositions and methods for treating systemic shock induced by virus and respiratory failure in an individual. The methods and compositions of this invention partially exploit the discovery that certain agents, defined herein as blocking agents 10 of 1-beta-toxin (hereinafter also referred to as: LT-B) can be used in the treatment of systemic shock. induced by virus and respiratory failure in an individual. In one embodiment, the blocking agents of LT-B are a blocking agent of the lymphotoxin-beta receptor (also LT-B-R). In a preferred embodiment, LT-B-R is an antibody against a lymphotoxin-B receptor or a soluble lymphotoxin B receptor. In a most preferred embodiment, the blocking agent of LT-B-R is a protein recombinant LT-B-R fusion that has an extracellular ligand binding domain of LT-B-R fused to an immunoglobulin constant heavy chain domain. These and other objects, features, aspects and advantages of the present invention, as well as the The same invention will be more fully understood from the following description of the preferred embodiments. ^ BRIEF DESCRIPTION OF THE DRAWINGS 5 Figure 1 shows that the infection of mice NZB with clone 13 LCMV results in mortality. The mortality curve of NZB mice infected with LCMV-13 (n = 14) and viral titers in various tissues of mice infected with LCMV-13 (n = 7) six days after the infection. Figure 2 shows the histological profile of the infection with LCMV-13 in NZB mice. (A) normal lung a (100X, H + E), (B) interstitial pneumonitis with mononuclear cell infiltrates and thickening of the wall alveolar of the lung, 5 days after infection (100X, H + E) (C) lymphoid depletion, cell necrosis and obliteration of the follicular architecture of the spleen (25X, ^ H + E) (D) greater amplification showing cell necrosis and spleen caiorrectal debris (158, H + AND) (E) macrophages (white arrows) and endothelial cells positive to LCMV-13 (arrows) in the lung (100X, IHC), (F) endothelial cells positive for LCMV-13 (arrows) and mesothelial cells (arrowheads) and macrophages (white arrows) in the spleen (50X, IHC,) (G) cells positive endothelial cells to the LCMV-13 in heart (100X, IHC), (H) Kupffer cells positive to LCMV-13 and sinusoidal lining cells in the liver (100X, IHC). f ^ Figure 3 shows that the blocking signaling pathways of the LTßR significantly improve the survival rates between NZB mice infected with clone 13. We present here the mortality curves of NZB mice infected with clone 13 treated as described. NZB mice were given 2.5 x 106 pfu (plaque forming units) Cl 13 i.v. followed by ^ 10 two injections i.p. containing 250μg of the antibody TN3-19.12 in PBS without endotoxins (see reference S) on days 1 and 4 after infection. The control mice were injected with the same volume of PBS lacking antibodies on the same days. The mice were treated as described in reference R. To the triple treatment group, TNFR55-Ig and LTßR-Ig proteins were supplied on day 0 and day 3 after infection, i.p. in C amounts of 200 μg. Control mice were given human antibodies used in the synthesis of these fusion proteins (AY1943-29) on the same days and in identical amounts. Mice that received only LTßR-Ig were treated identically, except that injections of TNFR55-Ig were omitted. The data was compiled from various experiments with anti-TNF (TN3-19.12) alone, n = 16 for LTßR-Ig alone, n = 10 for the triple treatment group, (n = 10 for the triple treatment group, n = 22 for LTßR-Ig alone , n = 10 for the group of LTßR-Ig + TNFR55-Ig, n = 5 for the group treated with anti-TNF and TNFR55-Ig, n = 6 for the anti-TNF (TN3-19.12) alone and n = 25 for the control) . Figure 4 shows that blockade of the LTßR route results in a decrease in CD8 T cell function. The splenocytes of the mice in the different treatment groups were collected on the 6th day after infection and stained with tetramer that contained an NP118 peptide of 9mers, as previously described. The values provided were adjusted for a non-specific background dyeing. To monitor the production of gamma interferon in response to the same peptide, the cells were incubated for 5 hours at 37 ° C in the presence of NP118 at a final concentration of 0.1 μg / ml and IL-2. The values provided here were adjusted to background levels in the absence of the peptide. Splenocytes from three mice treated with control human Ig were mixed as were those from two LTßR-Ig mice (LT beta # 2/3). The other results come from individual mice. Figure 5 shows that depletion of CD8 + T cells, not CD4 + T cells, reverses the lethal effects of infection with LCMV-13 in mice in NZB. Mice were treated as described for depletion of cell populations in vivo. A mortality curve is presented for each of the treated groups (n = 4).

DETAILED DESCRIPTION OF THE INVENTION 5 Definitions To more clearly and concisely indicate the subject matter of the claimed invention, the following definitions of specific terms used in the following written description and in the appended claims are provided. The lymphotoxin-beta (LT-beta) is a member of the TNF family of ligands, which also includes the ligands of the receptors Fas, CD27, CD30, CD40, OX-40 and 4-IBB (Smith et al., Cell, 76, pp. 959-62 (1994)). The Signaling by various members of the TNF family, including TNF, LT-alpha, LT-beta and Fas, can induce tumor cell death by means of necrosis or apoptosis (programmed cell death). In cells that are not tumorigenic, TNF and many of the interactions of The ligand of the TNF-receptor family influences the development of the immune system and the responses to various agents that attack the immune system. The lymphotoxin-beta (also called p33), has been identified on the surface of T lymphocytes, lines of T cells, B cell lines and cytolytic cells activated by lymphokine (LAK). The LT-beta is the subject of the co-pending applications (international of the applicants with number PCT / US91 / 04588, published January 9, 1992 as WO 5 92/00329; and PCT / US93 / 11669, published June 23, 1994 as WO 94/13808, which are incorporated herein by reference. The LT-beta receptor, a member of the TNF receptor family, binds specifically to 10 surface LT ligands. LT-beta-R binds complexes Heteromeric LT (predominantly LT-alpha 1 / beta 2 and LT-alpha 2 / beta 1) although it does not bind to TNF or LT-alpha (Crowe et al., Science, 264, pp. 707-10 (1994)). Signaling by LT-beta-R may play a role in peripheral lymphoid organic development and in humoral immune responses. The mRNAs of LT-beta-R are found in the spleen, Vime and in other main human organs. The expression patterns of LT-beta-R are similar to those reported for p55-TNF-R except that LT-beta-R does not appear in peripheral blood T cells and in T cell lines. The term " LT-beta-blocker agent "refers to an agent that can decrease the binding of ligand with LT-beta, the LT-beta grouping on the cell surface or P125S LT-beta signaling or can influence how the LT-beta signal is interpreted within cells. (^ Examples of LT-beta-blocking agents include anti-LT-beta, LT-beta-R-Fc molecules, soluble and anti-LT-alpha, anti-LT-alpha / beta and anti-LT-beta- R Abs Preferably, the antibodies do not cross-react with the secreted form of LT-alpha The term "LT-beta-receptor blocking agent" refers to an agent that can decrease ligand binding to LT-beta-R, the LT-beta-R grouping on the cell surface or LT-beta-R signaling or that may influence how the LT-beta-R signal is interpreted within the cell. Examples of LT-beta blocking agents include soluble LT-15 beta-R-Fc molecules and anti-LT-beta-R Abs Preferably, the antibodies do not cross-react with the secreted form of LT-alpha. V The term "anti-LT-beta receptor antibody" refers to any antibody that specifically binds to at least one epitope of the LT-beta receptor. "anti-LT body" refers to any antibody that specifically binds to at least one epitope of LT-alpha, LT-beta or to an LT-25 alpha / beta complex.

P1255 The term "LT ligand" refers to a heteromeric LT complex or a derivative thereof that can bind specifically to the LT-beta receptor. The term "LT-beta-R signaling" refers to molecular reactions associated with the LT-beta-R pathway and the subsequent molecular reactions resulting therefrom. The term "ligand binding domain of LT-beta-R11 refers to the portion or portions of LT-beta-R 10 that are involved in the specific recognition of an LT ligand and in the interaction therewith." Heteromeric complex LT-alpha / beta "and" Heteromeric complex LT "refer to a stable association between at least one LT-alpha and one or more subunits LT-beta, which include the soluble, mutant, altered and chimeric forms of one or more of the subunits. Subunits can be associated through interactions, electrostatic, van der Waals or covalent. Preferably, the LT-alpha 162 heteromeric complex has the minus two adjacent LT-beta subunits and lacks the adjacent LT-alpha subunits. When the LT-alpha / beta heteromeric complex serves as an activating agent of LT-beta-R in a cell development test, the complex preferably is soluble and has the stoichiometry LT-alpha 1 / beta 2.

P1255 Soluble LT-alpha / 62 heteromeric complexes lack the transmembrane domain and can be secreted by an appropriate host cell that has been engineered to express LT-alpha and / or LT-beta 5 subunits (Crowe et al., J. Immunol. Methods, 168, pp. 79-89 (1994)). The terms "surface LT-alpha / 62 complex" and "surface LT complex" refers to a complex comprising LT-alpha and LT-beta membrane-bound subunits, which include the mutant, altered and mutant forms. chimeric of one or more of the subunits, which is displayed on the surface of the cell. "Surface Ligand LT" refers to a surface LT complex or a derivative thereof that can specifically bind to the LT-beta receptor. 15 An "effective amount" is an amount sufficient to obtain the beneficial or desired clinical results.The effective amount can be administered in a \, or more administrations. For the purposes of this invention, the effective amount of an agent that blocks the binding of The lymphotoxin-B with its receptor is an amount of the agent that is sufficient to improve, stabilize or retard the development of a viral response. In particular, an agent that is sufficient to improve, stabilize or retard the development of systemic shock induced by viruses and respiratory failure. The detection and P1255 measurement of these efficacy indicators are known to those experienced in the art. (V "^ By an" individual "reference is made to vertebrates, particularly to members of mammalian species 5 and includes, but is not limited to, domestic animals, animals for sporting activities and primates, including humans. An amino acid residue is: (i) an amino acid that has properties ^ 10 reactive similar to the amino acid residue that was replaced by the functional equivalent; (ii) an amino acid of an antagonist of the invention, the amino acid having properties similar to the amino acid residue that was replaced by the functional equivalent; (iii) a non-amino acid molecule that has similar properties to the amino acid residue that was replaced by the functional equivalent. A first polynucleotide encoding a protein antagonist of the invention is "functionally "Equivalent" in comparison to a second polynucleotide encoding the antagonist protein if it satisfies at least one of the following conditions: (a) the "functional equivalent" is a first polynucleotide that hybridizes to the second polynucleotide in standard hybridization conditions and / or is degenerate for the first polynucleotide sequence. More preferably, it encodes a mutant protein having the "activity of an integrin antagonist protein." (B) the "functional equivalent" is a first polynucleotide that encodes an expression of an amino acid sequence encoded by the second. polynucleotide The "functional equivalent" of an amino acid residue is: (i) an amino acid that has properties (^ 10 reactive similar to the amino acid residue that was replaced by the functional equivalent, (ii) an amino acid of an antagonist of the invention, the amino acid that has similar properties to the amino acid residue that was replaced by the functional equivalent, (iii) a non-amino acid molecule that has similar properties to the amino acid residue that was replaced by the functional equivalent. C, A first polynucleotide encoding a proteoceous antagonist of the invention is "functionally "Equivalent" in comparison to a second polynucleotide encoding the antagonist protein if it satisfies at least one of the following conditions: (a) the "functional equivalent" is a first polynucleotide that hybridizes to the second polynucleotide in standard hybridization conditions and / or is degenerate for the first polynucleotide sequence. More preferably, it encodes a mutant protein having the "activity of an integrin antagonist protein." And (b) the "functional equivalent" is a first polynucleotide that encodes an expression of an encoded amino acid sequence. by the second polynucleotide The LT-B blocking agents used in the invention include, but are not limited to, the agents listed herein, as well as their functional equivalents, as used herein, the term "equivalent". functionally refers therefore to a LT-B blocking agent or to a polynucleotide which encodes the LT-B blocking agent having the same beneficial effect or an improved one on the recipient or recipient as LT blocking agent. -B of what appears to be a functional equivalent.As you will appreciate, anyone of ordinary skill in the art, a functionally equivalent protein can be produced 20 me by recombinant techniques, for example, by expressing a "functionally equivalent DNA". In accordance with the foregoing, the present invention encompasses the LT-B blocking agent encoded by natural DNA, as well as by unnatural DNAs, which code for the same protein that encoded the wild-type DNA. Due to the degeneracy of the nucleotide coding sequences, other polynucleotides can be used to code for LT-B blocking agents. These include all the above sequences or portions thereof that are altered by the substitution of different codons encoding the same amino acid residue within the sequence, thus producing an imperceptible change. It is considered that the altered sequences are equivalent to these sequences. For example, Phe (F) is encoded by two TTC or TTT codons, Tyr (Y) is coded by TAC or TAT and His (H) is encoded by CAC or CAT. On the other hand, the Trp (W) is encoded by a single codon, the TGG. In accordance with the above, it will be appreciated that for a specific DNA sequence that codes for a In particular, there will be many degenerate DNA sequences that will encode it. It is considered that these degenerate DNA sequences are within the scope of this invention. The term "fusion" or "fusion protein" is refers to a covalent co-linear linkage of two or more proteins or fragments thereof, via their individual peptide backbones, most preferably, through the genetic expression of a polynucleotide molecule encoding those proteins. It is preferred that proteins or fragments of P125S are from different sources, so that this type of fusion protein is called a "chimeric" molecule. Thus, the preferred fusion proteins are chimeric proteins that include an LT-B blocking agent or a fragment covalently linked to a second entity that is not a LT-B blocking agent. Preferred fusion proteins of the invention may include portions of intact antibodies that retain the antigen-binding specificity, (1), for example, Fab fragments, Fab1 fragments, F (ab ') 2 fragments, F (v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of a heavy chain and a light and the like, the most preferred fusion proteins are and comprise a blocker agent entity of the LT-B fused or bound in some way to all or part of the hinge and constant regions of a v, immunoglobulin light chain, a heavy or both. Thus, this invention presents a molecule that includes: (1) a LT-B (2) blocking agent entity a second peptide, for example, one that increases the solubility or lifetime of the blocking agent entity of the LT-B, for example, a member of the immunoglobulin super family or a fragment or a portion thereof, eg, a portion or a P125S fragment of the IgG, for example, the heavy chain constant region of human IgGl, eg, CH2, CH3, and, the hinge regions. Specifically, a "fusion of LT-B or LT-B-R / Ig" is a protein comprising a blocking agent of the biologically active LT-B of the invention (for example, a soluble LT-BR or a biologically active fragment thereof, linked to an N-terminus of an immunoglobulin chain, wherein a portion of N-terminal immunoglobulin is replaced by the blocking agent of LT-B. A species of the LT-B or LT-BR / Ig fusion is a "LT-BR / Fc fusion" which is a protein comprising an LT-BR of the invention linked to at least a portion of the constant domain of an immunoglobulin. A preferred Fe fusion comprises a The LT-B blocking agent of the invention is linked to a fragment of an antibody containing the C terminal domain of the immunoglobulin heavy chains. , The "standard hybridization conditions", salt and temperature conditions practically equivalents to 0.5 X SSC at approximately 5 X SSC and 65 ° C for both hybridization and washing. The term "standard hybridization conditions" is therefore, as used herein, an operative definition and encompasses a range of hybridization conditions.

Conditions of higher stringency may include, P1255 for example, hybridization with a buffer for sieving on plate (0.2% polyvinylpyrrolidone, Ficoll 400 (0.2%) 0.2% coil serum albumin, 50 mM Tris-HCl (pH 7.5), 1 M NaCl, 0.1% sodium pyrophosphate, 5% SDS); 10% dextran sulfate and 100 μg / ml salmon sperm DNA subjected to sonication and denatured at 65 ° C for 12 to 20 hours and washed with NaCl / 75 mM / 7.5 mM sodium citrate (0.5 x SSC) / SDS at 1% at 65 ° C. Lower stringency conditions may include, for example, hybridizing with buffer for plaque screening, 10% dextran sulfate and 110 μg / ml salmon sperm DNA subjected to sonication and denatured at 55 ° C for 12 to 20 minutes. hours and washing with 300 mM NaCl / 30 mM sodium citrate (2.0 X SSC) / 1% SDS at 55 ° C. See also Current 15 Protocols ln Molecular Biology, John Wiley & Sons, Inc. New York, Sections 6.3.1-6.3.6, (1989). As used herein, a ^ - ^ "therapeutic composition" is defined as comprising the proteins of the invention and other ingredients biologically compatible. The therapeutic composition may contain excipients such as water, minerals and carriers such as protein.

II. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention depends in part on the P1255 discovery that blocking agents of LT-B can induce an antiviral response in an individual. It was found that treatment of an individual infected with a virus can greatly increase the survival rate of the individual. Specifically, the treatment of NZB mice infected with LCMV-13 with an LT-B blocking agent, such as the LTßR-Ig fusion protein, was shown to increase its survival rate to 73%. The current treatment for Ebola, Dengue, SNV ^ 10 and other viruses mentioned herein is preventive by means of education about the transmission of the disease. There are no vaccines for these highly pathogenic viruses. Ribavirin, an analogue of guanidine, has been used as a generic antiviral drug for some of those infections with a reproducible success documented only in the treatment of the Fever of the Lassa when used at the beginning of the disease (M.D., Lacy and R.A. Smego, Adv. Ped. Inf. Dis., 12, 21 (1997) .Our data indicate that some of the pathologies associated with these viruses can be mediated in immune form. Blocking the LT system could greatly increase the chances of survival by transiently reducing the numbers of the virus-specific CD8 T cells and their functionality. Tests clinics that use various means to block the route P1255 of TNFa are already in process for the treatment of various conditions (HI Pass, D. Mew, HA Pass, et al., Chest Surg. Clin. N. Amer. 5, 73 (1995). Treatment with LTßR-Ig should be considered for 5 additional tests in animal models for eventual use in human trials involving patients with rapidly progressive acute viral infections involving shock and / or pulmonary insufficiency.

Blossing agents of LT-B In one embodiment of this invention, the LT-beta blocking agent comprises an antibody (Ab) directed against LT-beta that inhibits LT-beta signaling. Preferably, anti-LT-beta Ab is an antibody monoclonar (mAb). Ab inhibitors of anti-LT-beta and other LT-beta blocking agents can be identified using screening method that detects the ability of one or more agents to bind to an LT ligand or to inhibit the effects of LT-signaling. beta about the cells. In another embodiment of this invention, the LT-beta blocking agent comprises a LT-beta receptor blocking agent (LT-B-R). In a preferred embodiment, the blocking agent of LT-B-R is an antibody (Ab) directed against LT-beta-R that inhibits P125S signaling of LT-beta-R. Preferably, the anti-LT-beta-R Ab is a monoclonal antibody (mAb). One of X ^ these MAb inhibitors of anti-LT-beta-R is the BDA8 mAb. Ab inhibitors of anti-LT-beta-R and other LT-beta-R blocking agents can be identified using screening methods that detect the ability of one or more agents to either bind to LT-beta-R or to LT ligand or to inhibit the effects of LT-beta-R signaling on cells. (10 A screening method uses the cytotoxic effects of LT-beta-R signaling on tumor cells carrying LT-beta-R.) Tumor cells are exposed to one or more LT-beta-R activating agents to induce LT-beta-R signaling LT-beta-R activating agents include LT-alpha / 62 heteromeric complexes (preferably soluble LT-alpha l / beta 2) in the presence of IFN-gamma or one that activates to Ab - ^ ^ anti-LT-beta-R (see below, also described in the co-pending application of the United States with the series 08 / 378,968), of the applicants. Antibodies and other agents that can block the signaling of LT-beta-R are selected based on their ability to inhibit the cytotoxic effect of LT-beta-R signaling on tumor cells in the next essay: P1255 1) Tumor cells, such as HT29 cells are cultured for three or four days in a series of tissue culture cavities containing a medium and at least one activating agent of LT-beta-R in presence or absence of serial dilutions of the agent to be tested; 2) A vital staining dye that measures mitochondrial function, such as MTT and is reacted for several hours is added to the tumor cell mixture; 10 3) The optical density of the mixture of each cavity is quantified with a light of 550 nm wavelength (OD 550). The OD 550 is proportional to the number of tumor cells remaining in the presence of the activating agent of LT-beta-R and the blocking agent of LT-beta-15 R in each cavity. An agent or combination of agents that can reduce the cytotoxicity of the tumor cell activated by LT-beta-R by at least 20% in this assay is a blocking agent of LT-beta-R within the scope of this invention. In the above assay, any agent or combination of agents that activate LT-beta-R signaling can be used to identify LT-beta-R blocking agents. LT-beta-R activating agents that induce LT-beta-R signaling (such as activation of 25 mAb of anti-LT-beta-R) can be selected on the basis of P1255 of its ability, alone or combined with other agents, to enhance the cytotoxicity of the tumor cell using ("^ the tumor cell assay described above. \ And Another method to select a blocking agent 5 of the LT-beta-R is to monitor the ability of the putative agent to directly interfere with the LT-receptor ligand binding.An agent or combination of agents that can block the ligand-receptor binding by at least 20% is a blocking agent of the LT-beta-R within the scope of the Invention Any number of assays that measure the resistance of the ligand-receptor binding can be used to conduct competition assays with the putative LT-beta-R blocking agents.

The strength of the binding between a receptor and a ligand can be measured using an enzyme linked immunosorbent assay (ELISA) or a radio immunoassay (RIA). The Union (_ "Specific can also be measured by tagging fluorescent antibody-antigen complexes and performing analysis of fluorescence activated cell sorting (FACS) or performing other of these immunodetection methods, all of which are well-known techniques in the area. The interaction of the ligand-receptor binding can also be measured with the TM instrument (Pharmacia P1255 Biosensor) that takes advantage of plasmon resonance detection (Zhou et al., Biochemistry, 32, pp. 8193-98 (1993); (^ Faegerstram and O 'Shannessy), "Surface plasmon resonance detection in affinity technologies", in Handbook of 5 Affinity Chromatography, pp. 229-52, Marcel Dekker, Inc., New York (1993)). The BIAcore TM technology allows the receptor to attach to a gold surface and make the ligand flow over it. Resonance detection Plasmon 10 provides direct quantification of the amount of mass bound to the surface in real time. This technique produces on and off rate constants, and, thus, a ligand-receptor dissociation constant and an affinity constant can be determined directly in the presence and absence of the putative LT-beta-R blocking agent. With any of these or other techniques for measuring receptor-ligand interactions, we can evaluate the ability of a LT-beta-R blocking agent, alone or in combination with other agents, to inhibit the binding of surface LT ligands. or soluble to surface or soluble LT-beta-R molecules. These assays can also be used to test LT-beta-R blocking agents or derivatives of said agents (eg, 25 fusions, chimeras, mutants and chemically forms).

P1255 altered), alone or in combination, to optimize the ability of that altered agent to block the activation of LT-beta-R. Blocking agents of LT-beta-R, in a modality of this invention, comprise soluble LT-beta receptor molecules. The sequence of the extracellular portion of human LT-beta-R, which codes for the ligand-binding domain, is shown in Figure 1 of U.S. Patent No. 5,925,351, (10 incorporated herein by reference). Using the sequence information in Figure 1 of U.S. Patent No. 5,925,351 and the recombinant DNA techniques well known in the art, the functional fragments encoding the ligand binding domain of LT-beta -R can be cloned into a vector and expressed in an appropriate host to produce a soluble LT-beta-R molecule.The soluble LT-beta-R molecules that can compete with the native LT-beta receptors for binding to Ligand LT, in accordance with the assays described herein, are selected as LT-beta-R blocking agents.A soluble LT-beta receptor comprising amino acid sequences selected from those shown in FIG. 1 of the United States Patent No. 5,925,351, may be linked to one or more heterologous protein domains ("fusion domain") to increase the in vivo stability of the fusion protein of the ^ receptor or to modulate its activity or biological location. Preferably, the plasma stable proteins, which normally have a half-life greater than 20 hours in circulation, are those which are used to construct the fusion proteins with the receptor. These plasma proteins include, but are not limited to: immunoglobulins, serum albumin, lipoproteins, apolipoproteins and transferrin. Sequences that can direct the soluble LT-beta-R molecule to a particular cell or tissue type can also bind to the ligand-binding domain of LT-beta-R to create a soluble LT-beta-R fusion protein. specifically located. The entire extracellular region of LT-beta-R or a functional portion thereof (Figure 1 of U.S. Patent No. 5,925.35), which comprises the domain of The ligand binding of LT-beta-R can be fused to an immunoglobulin constant region such as the Fe domain of a human IgGl heavy chain (Browning et al., J. Immunol., 154, pp. 33-46 ( 1995).) Soluble receptor-IgG fusion proteins are common immunological reagents and methods for their construction are known in the art (see for example, U.S. Patent No. 5,225,538) A functional ligand-binding domain of LT-beta-R can be fused to an immunoglobulin (Ig) Fe domain derived from a class or subclass of immunoglobulin other than IgGl The Fe domains of antibodies belonging to different classes or subclasses of Ig can activate various functions of secondary effector Activation occurs when the Fe domain is linked to a cognate Fe receptor The functions of the secondary effector include the ability to activate the complement system, to cross the placenta, and to bind 10 different proteins. nas microbial. The properties of the different classes and subclasses of immunoglobulins are described in Roitt et al., Immunology, p. 4.8 (Mosby-Year Book Europe Ltd., 3d ed., 1993). The complement enzyme cascade can be activated by the Fe domains of IgGl, IgG3 and IgM antibodies linked to antigen. The Fe domain of IgG2 appears to be less effective and the Fe domains of IgG4, IgA, IgD and IgE are not effective in activating complement. In this way, a Fe domain can be selected on the basis of whether the functions of its associated secondary effector are desirable for the particular immune response or disease that will be treated with the LT-beta-R-Fe fusion protein if it were advantageous. In order to damage or annihilate the target cell carrying the LT ligand, a particularly active Fe domain (IgG1) could be selected to prepare the LT-beta-R-Fc fusion protein.

Alternatively, if it were desirable to direct the LT-beta-R-Fe fusion to a cell without triggering the complement system, an inactive Fe IgG4 domain could be selected. Mutations in the Fe domains that reduce or eliminate Fe receptor binding and complement activation have been described (S. Morrison, Annu, Rev. Immunol., 10, pp. 239-65 (1992)). These or other mutations can be used, alone or in combination, to optimize the (^ 10 activity of the Fe domain used to construct the LT-beta-R-Fc fusion protein.) The production of a soluble human LT-beta-R fusion protein comprising ligand binding sequences fused to a Fe domain of human immunoglobulin (mLT-beta-R-Rc) is described in Example 1 of U.S. Patent No. 5,925,351, incorporated by reference herein.A CHO line prepared from y- ^ conforming to Example 1 which secretes hLT-beta-R-Fc is called "hLT beta; R-hGl CHO # 14." A sample of that line was deposited on July 21, 1995 in the American Type Culture Collection (ATCC) (Rockville, Md.) In accordance with the provisions of the Budapest treaty and assigned ATCC accession number CRL 11965. The production of a soluble LT-25 beta-R fusion molecule murine (mLT-beta-R-Fc) is described in P1255 Example 2 of U.S. Patent No. 5,925,351, incorporated by reference herein.

^ A CHO line prepared according to Example 2 of U.S. Patent No. 5,925,351 which secretes 5 mLT-beta-R-Fc is called "mLT beta; R-hGl CH0 # 1.3.BB". A sample of this line was deposited on July 21, 1995 in the American Type Culture Collection (ATCC) (Rockville, Md.) In accordance with the provisions of the Budapest Treaty and assigned the access number ATCC CRL 11964 C 10 Different amino acid residues that form the binding site of the receptor-Ig fusion protein can alter the structure, stability and ultimate biological activity of the soluble LT-beta receptor fusion protein at the C-terminus. One or more amino acids 15 of the selected LT-beta-R fragment is added to modify the point of attachment with the selected fusion domain. The N-terminus of the LT-beta-R fusion protein can also be varied by changing the position at which the LT-beta-R DNA fragment is cleaved at its 5 'end for insertion into the expression vector recombinant. The stability and activity of each LT-beta-R fusion protein can be tested and optimized using routine experimentation and assays to select the LT-beta-R blocking agents described herein. 25 Using the sequences of the binding domain P1255 with LT-beta-R ligand within the extracellular domain shown in Figure 1, can also be constructed / N variants to the amino acid sequence to modify the affinity of the soluble LT-beta receptor or the fusion protein of the LT ligand. The soluble LT-beta-R molecules of this invention can compete for binding with the surface LT ligand with the endogenous cell surface LT-beta receptors. It is contemplated that any soluble molecule comprising a ligand-binding domain with LT-beta-R ligand that can compete with the cell surface LT-beta receptors for binding with the LT ligand is an LT-blocking agent. beta-R which falls within the scope of the invention. In another embodiment of this invention, the antibodies directed against the human LT-beta receptor (anti-LT-beta-R Abs) function as blocking agents of LT-beta-R for use in the treatment of conditions that place individuals, including human being, at risk of systemic shock induced by virus and respiratory failure. The anti-LT-beta-R Ab of this invention can be polyclonal or monoclonal (m? Bs) and can be modified to optimize their ability to block LT-beta-R signaling, their bioavailability in vivo, stability or other desired traits.

P1255 Sera of the polyclonal antibody directed against the LT-beta receptor are prepared using Conventional techniques, by injecting animals such as goats, rabbits, rats, hamsters or mice, in subcutaneously, with a human fusion protein LT-beta receptor-Fe (Example 1 of the Patent of the States No. 5,925,351) in complete Freund's adjuvant, followed by intraperitoneal booster injection or subcutaneous injection in incomplete Freund's adjuvant. The polyclonal antiserum containing the desired antibodies directed against the receptor of the LT-beta are screened by conventional immunological procedures. Mouse monoclonal antibodies (m? Bs), directed against a human LT-beta receptor-Fe fusion protein were prepared as described in U.S. Patent No. 5,925,351, example 5. A hybridoma cell line ( BD.A8.AB9) which produces the BDA8 of mouse human LT-beta-R mAb was deposited on January 12, 1995, at the American Type Culture Collection (ATCC) (10801 University Boulevard, Manassas, Va. 20110- 2209), in accordance with the provisions of the Budapest Treaty and assigned accession number ATCC HB 11798. Various forms of anti-LT-beta-R 25 antibodies can also be prepared using standard anti-TB techniques.

P1255 Recombinant DNA (Winter and Milstein, Nature, 349, pp. 293-99 (1991)). For example, "chimeric" antibodies may be constructed in which the antigen binding domain of an animal antibody is linked to a human constant domain, (e.g., Cabilly et al., U.S. Patent No. 4,816,567; Morrison et al., Proc. Nati, Acad. Sci. USA, 8 1, pp. 6851-55 (1984)). Chimeric antibodies reduce the observed immunogenic responses, produced by animal antibodies when used in human clinical treatments. In addition, recombinant "humanized antibodies" that recognize LT-beta-R can be synthesized. The humanized antibodies are chimeras comprising mainly human IgG sequences in which regions responsible for the binding with specific antigen have been inserted (for example WO 94/04679). The animals are immunized with the desired antigen, the antibodies are isolated and "> (corresponding) and the portion of the variable region sequences responsible for antigen binding is removed. specific. The antigen-binding regions derived from animals are then cloned into the appropriate position of human antibody genes in which the antigen-binding regions have been deleted. Humanized antibodies minimize the use of sequences heterologous (between species) in human antibodies and P1255 are less likely to produce immune responses in the treated subject. c The construction of different classes of recombinant anti-LT-beta-R antibodies can also be can be achieved by preparing chimeric or humanized antibodies comprising the variable domains of anti-LT-beta-R and human constant domains (CH1, CH2, CH3) isolated from different classes of immunoglobulins. For example, anti-LT-beta-R IgM antibodies with an increase in the 10-valent antigen-binding site may be produced recombinantly by cloning the antigen binding site into vectors carrying the human mu-chain constant regions ( Arulanandam et al., J. Exp. Med., 177, pp. 1439-50 (1993); Lañe et al., Eur. J. Immunol., 22, pp. 2573-78 (1993); Traunecker et al., Nature, 339, pp. 68-70 (1989)). In addition, standard recombinant DNA techniques can be used to alter the binding affinities and ^ ^ _ of recombinant antibodies with their antigens, altering the amino acid residues in the vicinity of the sites of binding with antigen. The binding affinity with antigen of a humanized antibody can be increased by mutagenesis based on molecular modeling (Queen et al., Proc. Nati Acad. Sci. U.S.A., 86, pp. 10029-33 (1989); WO 94/04679). 25 It may be desirable to increase or decrease the P125S affinity of the anti-LT-beta-R Ab for LT-beta-R, depending on the type of target tissue or the target ^ particular treatment contemplated. For example, it can be It is advantageous to treat a patient with constant levels of Ab 5 anti-LT-beta-R with a reduced ability to send signals through the LT-beta pathway for semi-prophylactic treatments. Similarly, anti-LT-beta-R inhibitors Ab with an increased affinity for LT-beta-R may be advantageous for short term treatments. In testing other antibodies directed against the human LT-beta receptor, it is expected that additional anti-LT-beta-R antibodies that function as blocking agents of LT-beta-R in humans can be identified to treat conditions that place individuals, including humans, in systemic shock induced by viruses and in respiratory failure or in and - ^ risk thereof, using routine experimentation and the assays described herein. Another preferred embodiment of this invention includes compositions and methods comprising antibodies directed against the LT ligand that function as blocking agents for LT-beta-R. As described above for the anti-LT-beta-R Ab, the anti-LT ligand antibodies that function as agents P1255 LT-beta-R blockers can be polyclonal or monoclonal and can be modified in accordance with? ^ routine procedures to modulate their antigen bindproperties and their immunogenicity. The anti-LT antibodies of this invention can oppose either one of the two LT subunits individually, includthe soluble, mutant, altered and chimeric form of the LT subunit. If LT subunits are used as an antigen, they are preferably subunits of LT-beta. If LT-alpha subunits are used, it is preferred that the resultanti-LT-alpha antibodies bind to surface LT ligand and not cross-react with secreted LT-alpha or modulate TNF-R activity (from in accordance with the assays described in Example 3 of U.S. Patent No. 5,925,351). Alternatively, directed antibodies and ^ V ^, against a homomeric (LT-beta) or a heteromeric (LT-alpha / 62) complex comprisone or more subunits of the LT can be developed and screened for their activity as LT-beta-R blockagents. Preferably, LT-alpha l / beta 2 complexes are used as the antigen. As mentioned above, it is preferred that the resultanti-LT-alpha l / beta 2 antibodies bind to the surface LT ligand without bindto secreted LT-alpha P1255 and without affectthe activity of TNF-R. The production of anti-human antibodies LT-alpha? Polyclonal is described in the co-pendapplication (WO 94/13808) of the applicants. They have also described the anti-LT-alpha and anti-LT-beta monoclonal antibodies (Brownet al., J. Immunol., 54, pp. 33-46 (1995)). Mouse anti-human LT-beta mAbs were prepared as described in Example 6 of the US Patent.

United No. 5,925,351. The hybridoma cell line "^ 10 (B9.C9.1) which produces the B9 mAbs of the mouse anti-human LT-beta-R was deposited on July 21, 1995 in the American Type Culture Collection (ATCC) (10801 University Boulevard, Manassas, Va. 20110-2209), in accordance with the provisions of the Budapest Treaty and assigned the number of access ATCC 11962. The monoclonal hamster anti-mouse LT-alpha / 62 antibodies were prepared as described in example r V and 7 of U.S. Patent No. 5,925,351. A hybridoma cell line (BB.F6.1) that produces mAbs BB.F6 LT-alpha / 62 anti-mouse hamster was deposited on July 21, 1995 in the American Type Culture Collection (ATCC) (10801 University Boulevard, Manassas, Va. 20110-2209) in accordance with the provisions of the Treaty of Budapest and was assigned the access number ATCC MB 11963.

A cell sortassay was developed Fluorescence-activated P1255 (FACS) to screen targeted antibodies with LT subunits and LT complexes XN can act with LT-beta-R blockagents, as described in Examples 6 and 7 of the United States Patent No. 5,925,351. In this assay, the soluble LT-beta-R-Fc fusion protein was added to PMA-activated 11-23 cells, which express to the surface LT complexes (Brownet al., J. Immunol., 154, p. 33-46 (1995)) in the presence of increasamounts of (N 10 test antibody: An antibody that can inhibit the interaction of LT-beta receptor-ligand by at least 20% is selected as a blockagent for LT-beta-R.) Usan LT-alpha / beta complex instead of a subunit of LT as an antigen to immunize an animal may lead to a more efficient immunization or may result in antibodies that have higher affinities for the surface LT ligand. It is conceivable that, when immunizwith the LT-alpha / 62 complex, antibodies recognizthe amino acid residues in the Two subunits, LT-alpha and LT-beta (for example, residues that can form an LT-alpha / 62 cleft) when testantibodies directed against heteromeric LT-alpha / 62 human complexes, it is expected that anti-HIV antibodies can be identified. -LT additional that work as agents LT-beta-R blockers using experimentation P1255 routine and the tests described here. f ~ Administration The compositions described herein shall be administered in effective doses in methods to treat systemic shock induced by viruses and respiratory failure in an individual. The determination of a preferred pharmaceutical formation and a therapeutically efficient dosage regimen for an application The determination is very well within the capabilities of the technique, taking into consideration, for example, the condition and weight of the patient, the extent of the desired treatment and the patient's tolerance to the treatment. It is expected that doses of approximately 1 mg / kg of a soluble LT-beta-R are suitable starting points to optimize treatment doses. The determination of a therapeutically effective dose can also be evaluated by performing in vi tro experiments that measure the concentration of the LT-beta-R blocking agent required to coat the target cells (LT-beta-R or LT ligand-positive cells, depending on the blocking agent) from one to 14 days. The receptor-ligand binding assays described herein can be used to monitor the coating reaction of the cells. Cells positive for LT-beta-R or LT P1255 ligand can be separated from the population of activated lymphocytes using FACS. Based on the results of these in vitro binding assays, a range of concentrations of the LT-beta-R 5 blocking agent suitable for testing in animals can be selected, in accordance with the assays described herein. The administration of the soluble LT-beta-R molecules, the anti-LT ligand and the anti-LT-beta-R Ab of this invention, alone or in combination, including the isolated and purified forms of the antibodies or complexes, their salts or pharmaceutically acceptable derivatives, can be carried out using any of the administration forms of conventionally accepted agents, which show immunosuppressive activity. The pharmaceutical compositions used in these therapies can also be in a variety of forms. These include, for example, dosage forms, solid, semi-solid and liquid, such as tablets, pills, powders, solutions or suspensions. liquids, suppositories and injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. Administration forms may include oral, parenteral, subcutaneous, intravenous, intralesional or topical administration. The molecules of LT-beta-R Soluble P1255, the anti-LT ligand and the anti-LT-beta-R Ab of this invention can be placed, for example, in sterile, isotonic formulations with or without cofactors that stimulate absorption or stability. The formulation is preferably liquid or can be a lyophilized powder. For example, the soluble LT-beta-R molecules, the anti-LT ligand and the anti-LT-beta-R Ab of this invention can be diluted with a buffer formulation comprising 5.0 mg / ml of citric acid. monohydrate, 2.7 mg / ml of trisodium citrate, 41 mg / ml of mannitol, 1 mg / ml of glycine and 1 mg / ml of polysorbate 20. This solution can be lyophilized, stored in refrigeration and reconstituted before administration with sterilized water for injection (USP). The compositions will also preferably include conventional pharmaceutically acceptable carriers, well known in the art (see, for example, V, example, Remington's Pharmaceutical Sciences, 16th Edition, 1980, Mac Publishing Company). These carriers Pharmaceutically acceptable agents may include other medicinal agents, carriers, genetic carriers, adjuvants, excipients, etc., such as preparations of human serum albumin or plasma. The compositions are preferably in the form of a unit dose and will usually be administered one or more times a day.

P1255 The pharmaceutical compositions of this invention can also be administered using microspheres, liposomes, other microparticulate delivery systems or prolonged release formulations, placed in, near or in some way in communication with the affected tissues or the bloodstream. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shaped articles, such as suppositories or microcapsules. The 10 implantable or microcapsule extended release matrices include polylactics (U.S. Patent No. 3,773,319; European Patent 58,881), L-glutamic acid and ethyl-L-glutamate copolymers (Sidman et al., Biopolymers, 22, pp. 547-56 (1985)); poly (2- 15 hydroxyethyl methacrylate) or ethylene vinyl acetate (Langer et al., J. Biomed, Mater. Res., 15, pp. 167-277 (1981); Langer, Chem. Tech., 12, pp. 98-105 (1982)). .. Liposomes containing soluble LT-beta-R molecules, anti-LT ligand and anti-LT-beta-R Abs of this The invention, alone or in combination, can be prepared by well known methods (see, for example, DE 3,218,121; Pstein et al., Proc. Nati, Acad. Sci. USA, 82, pp. 3688-92 (1985); Hwang; et al., Proc. Nati, Acad. Sci. USA, 77, pp. 4030-34 (1980); Patents of the States United States Nos. 4,485,045 and 4,544,545). In an ordinary way.

P12SS Liposomes are of the small unilamellar type (approximately 200-800 Angstrom) in which the lipid content is greater than about 30% in mol of cholesterol. The cholesterol ratio is selected to control the optimal release rate of the soluble LT-beta-R molecule, the anti-LT ligand and the anti-LT-beta-R Ab. The soluble LT-beta-R molecules, the anti-LT ligand and the anti-LT-beta-R Ab of this invention can also be linked to liposomes containing other LT-beta-R blocking agents, immunosuppressive agents or cytokines to modulate the blocking activity of LT-beta-R. The binding of the soluble LT-beta-R molecules, the anti-LT ligand and the anti-LT-15 beta-R Ab to the liposomes can be effected by any known crosslinking agent, such as heterobifunctional crosslinking agents which are They have used ^ - Widely for attaching toxins or chemotherapeutic agents to antibodies for delivery directed. Conjugation to liposomes can also be effected using the crosslinking reagent directed to the carbohydrate 4- (4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et al., J. Cell. Biochem. Abst. Suppl. 16E 77 (1992) ). 25 The LT-beta-R blocking agents of the P1255 compositions and methods of this invention can be modified to obtain a desirable level of signaling - ~ ^, of LT-beta-R, depending on the condition, disorder or disease to be treated. It is contemplated that the The absolute level of LT-beta-R signaling can be finely tuned by manipulating the concentrations and affinities of the LT-beta-R blocking agents for their respective molecular targets. For example, in one embodiment of this invention, the subject is administered X-10 compositions comprising soluble LT-beta-R molecules. The soluble LT-beta receptor can compete effectively with the cell surface LT-beta receptors by binding to surface LT ligands. The ability to compete with LT ligands The surface area depends on the relative concentrations of the soluble LT-beta-R molecules, and on the cell surface and their relative affinities for binding to the ligand. Soluble LT-beta-R molecules harbor mutations that increase or decrease the binding affinity of that soluble LT-beta-R mutant with the LT surface ligand can be effected using standard recombinant DNA techniques well known to those skilled in the art. The ability of a large number of molecules with mutations directed to the site or P1255 randomized to act as blocking agents for LT-beta-R using routine experimentation and ("Techniques described herein.) Similarly, in another embodiment of this invention, antibodies directed either against the LT-beta receptor or one or more subunits of LT ligand function as LT-beta blocking agents. -R The ability of these antibodies to block the signaling of the LT-beta receptor can be modified by mutation, V-10 chemical modification or by other methods that can vary the concentration or effective activity of the antibody delivered to the subject.

Uses 15 As a general matter, the methods of the present invention can be used to induce an antiviral response in an individual, comprising C administering to the individual an effective amount of a LT-B blocking agent and a pharmaceutically carrier acceptable. The viral response that will be treated can be caused by any of several known viruses, which include, but are not limited to, the virus without a name (SNV), Ebola, Marburg, Lassa and Dengue.

Equivalents P1255 The invention can be incorporated into other specific forms without deviating from the spirit or essential characteristics thereof. Therefore, it is considered that the foregoing modalities are illustrative 5 in all respects, rather than limiting the invention disclosed herein. The scope of the invention is thus indicated by the appended claims rather than by the foregoing description and, all changes that may fall within the meaning and scope of equivalence of the claims are intended to be included in this. .

Example The tumor necrosis factor (TNF a) plays an important role in the facilitation of acute shock responses to viral infections and other immunogens (K.C.F. Sheehan, N.H. Ruddle, and R.D.C.: Schreiber., J. "Immupol., 142, 3884 (1989); G.W.H. Wong and D.V. Goeddel Nature 323, 819 (1986); B. Beutler, I.W.

Milsark, A. Cerami, Science 229, 869 (1985); F. Mackay, P.R. Bourdon, D.A. Griffiths, et al. J. Iwmunol. 159, 3299 (1997); P.S. Crowe, T.L. VanArsdale, B.N. Walter, et al. Science 264, 707 (1994)). During episodes of Dengue Fever that involve shock, TNFa levels in the serum of patients is elevated as are the levels P125S of soluble TNFR-75 (D. Hober, et al., J. Trop. Med. Hyg., 48, 324 (1993); D.B. Bethell, K. Flobbe, C.X.T. Phuong, et C ^ al., J. Infect. Dis. , 177, 778 (1998)). We measured the levels of TNFa in the serum of mice infected with a variant of lymphocytic choriomeningitis virus, LCMV, clone 13 (LCMV-13) (HH, II). It was found that the levels of TNFa in the serum of mice infected with LCMV-13 was just above the level of detection for the assay until 4 days after infection (the levels (Serum TNFa _ 10 were measured by the ELISA assay (Genzyme Corporation, catalog number 80-2802-00)). On the 5th and 6th days, when the disease is at its peak, the levels of soluble TNFa in the serum increased 3-6 times above normal (data not shown).

Therefore, we chose to block TNFa function using a monoclonal antibody, TN3-19.12, which is known to bind to both secreted TNFa, thereby causing its exhaustion in V the mouse, as verified by an ELISA (K.C.F. Sheehan, N.H. Ruddle, and R.D. Schreiber., J. I m nol., 142, 3884 (1989) G.W.H. Wong and D.V. Goeddel Nature 323, 819 (1986); B. Beutler, I.W. Milsark, A. Cerami, Science 229, 869 (1985); F. Mackay, P.R. Bourdon, D.A. Griffiths, et al. J. Immunol. 159, 3299 (1997); P.S. Crowe, T.L. VanArsdale, B.N. Walter, et al. Science 264, 707 (1994); D.

Hober, et al. , J. Trop. Med. Hyg. , 48, 324 (1993); D.B.

P1255 Bethell, K. Flobbe, C.X.T. Phuong, et al. , J. Infect. Dis. , 177, 778 (1998)). Serum TNFa levels were V-measured by the ELISA assay (Genzyme Corporation, catalog number 80-2802-00). The NZB mice were given 2.5 x 10 6 pfu Cl 13 i.v. followed by two injections i.p. containing 250μg of TN3- 19.12 antibody in PBS without endotoxins (see reference S) on the 1st and 4th days after infection. The control mice were injected in the same days with the same volume of PBS lacking the antibody. This treatment (anti-TNF) had little effect on the survival rate of these mice (figure 3). Alpha lymphotoxin (LTa), also known as TNFβ, although it shares identical receptors and many of its biological effects with TNFα, is not recognized by this antibody (F. Mackay, Bourdon PR, DA Griffiths, et al., J. Imrminol 159, 3299 (1997).) It may be necessary to target both TNFa and LTa r to increase survival rates. To test this hypothesis we used the previous mAb TN3-19.12 and a fusion protein with the receptor that fused the extracellular domain of the p55 TNF receptor with the CH2 and CH3 domains of the human IgG1 (TNFR55-Ig) (WR Forcé, BN Walter, C. Hession, et al, J. Immunol. , 155, 5280 (1995), GT Miller, PS Hochman, W. Meier, et al., JEM., 178, 211 (1993); J.L. Browning, I. Dougas, A. Ngam-ek, et al., J.

P1255 Ixrununol., 154: 33 (1995). The mice were treated as described in reference R. For the triple treatment group, the TNFR55-Ig and LTßR-Ig proteins were provided on day 0 and on day 3 after infection, 5 ip route, in quantities of 200μg. Control mice were given human antibodies used in the synthesis of these fusion proteins (AY1943-29) on the same days and in identical amounts. Mice that received only LTβR-Ig were treated identically,, r 10 except that injections of TNFR55-Ig were omitted). This treatment also did not significantly alter the survival rates in NZB mice infected with LCMV-13 (see the anti-TNF group and TNFR55-Ig). The membrane form of lymphotoxin, a heteromer of LTa and LTß, does not recognize TNFR-75 or TNFR-55, but rather binds to a third receptor called LTßR (15). We chose to use a fusion protein containing the extracellular domain LTßR also linked to the CH2 and CH3 domains of human IgGl (LTßR-Ig). The treatment of mice with anti-TNFa mAb, TNFR55-Ig and LTßR-Ig (triple treatment or TNFR55-Ig and LTßR-Ig) resulted in a marked increase in survival, up to 80% and 70%, respectively. In contrast, only 20% of the mice treated with anti-TNFa mAb and TNFR55-Ig, survived the infection. Recently it was identified P12S5 a second ligand for LTßR, LIGHT, (DN Mauri, R. Ebner, RI Montgomery, et al., Imity 8, 21 (1998), RI Montgomery, MS Warner, B. Lum, et al., Cell 87, 427 (nineteen ninety six) ) . It has also been shown that LIGHT binds to the mediator of entry to the herpes virus (HVEM), a transmembrane type I protein with significant homology to members of the TNFR family that is expressed on activated CD4 and CD8 T cells. (DN Mauri, R. Ebner, RI Montgomery, et al., Immunity 8, 21 (1998), RI 10 Montgomery, MS Warner, B. Lum, et al., Cell 87, 427 (1996)). Based on the results presented here, the prevention of LTßR signaling and, potentially, the signaling of HVEM through the binding of LTß2a_. and LIGHT by LTßR-Ig was probably responsible for the majority of the effect observed in the triple treatment group. We affirm this hypothesis by treating NZB mice infected with LCMV-13 just with the LTßR-Ig fusion protein. The survival rate of the mice in this group (73%) was almost as high as in the group with triple treatment (figure 3). Taken together, these data represent the first demonstration that the signaling pathway of the LTßR and / or HVEM is involved in the orchestration of an acute lethal disease involving systemic shock and respiratory failure. 25 In an effort to determine the mechanism of P12S5 survival behind treatment by blocking the LTß, both the CD8 tetramer co-stained specific T cells C NP118, the dominant CD8 epitope in the LD system of NZB and the intracellular dyeing for the production of gamma interferon by splenocytes stimulated with peptide NP118 were performed on samples of NZB mice infected with LCMV-13 that were treated with the control antibody, LTßR-Ig alone or with triple treatment. Figure 4 shows a reduction in the (10 number of CD8 T cells specific for NP118 with the highest effect observed in mice with triple treatment) In mice treated with the control antibody, only 10% of the tetramer positive cells actively produced INF. Anergic T, during the infection with LCMV-13 has been previously documented and probably due to the high levels of the viral antigen in the mouse (figure 1). In mice C treated with NP118 has not only declined the number of specific LTßR-Ig cells, but crue has also been reduced the percentage of cells that produce INF ?. This effect is even more pronounced in the triple treatment group. In this way, it is possible that the CD8 compartment could be the source of this lethal NZB response to infection with LCMV-13. He fact that it is known that activated CD8s show P1255 LTß2a ?, is consistent with this hypothesis (Y. Abe, A. Horiuchi, Y. Osuka, et al., Lymph, Ctyok, Res., 11, 115 (1992), CF Ware, PD Crowe, MH Grayson, et. al., J. Immunol., 149, 3881 (1992), JL Browning, A. Ngam-ek, P. 5 Lawton, et al., Cell, 72, 847 (1993)). To support this acceleration, we depleted infected CDN or CD4 positive T cells in infected NZB mice in vivo (the male NZB mice were given 2.5 x 106 pfu LCMV-13 i.v. followed by two i.p. injections of 500μl of the T cell antibody.

Lyt2.43 mAb was used to deplete the CD8 + T cells while the GK1.5 (MI) antibody was used for the depletion of CD4 + T cells. Both antibodies were prepared by precipitation with ammonium sulfate from supernatants. of hybridoma, followed by dialysis against PBS. FACS analysis was used to verify depletion in several of the mice). The depletion of CD4 T cells did not increase the c; survival. In contrast, the depletion of CD8 T cells resulted in 100% survival in absence of the symptoms of the disease, unlike the mice treated with LTßR-Ig (figure 5). Because the viral titers were higher in various tissues of mice with CD8 depleted than in the untreated ones, it is likely that death will result from a toxic immune response mediated by CD8 T cells, in P12S5 instead of tissue destruction due to viral infection. We have reported here that NZB mice when infected with a high intravenous dose of LCMV-13 developed an acute and rapidly progressive disease that shares several common features with Ebola, Marburg, Lassa, Dengue and Sin Nombre infections. The lethality of this disease was dependent on the presence of CD8 + T cells that are known to express TNFα, LTa and LTβ when they are activated. Although this is an encouraging finding, treatment of viral infection by depleting CD8 + T cells would not be advisable. This treatment could leave patients vulnerable to other opportunistic infections. In addition, since viral clearance or clearance is unlikely in the absence of CTLs, the risk of the patient tolerating the virus with the restoration of the CD8 + compartment is very real. We have shown that the block of the LTßR / HVEM pathways by administration of LTßR-Ig represents a powerful treatment that is transient in nature, with rapid recovery to homeostasis once the treatment is stopped (Mackay and Browning, unpublished) . The surviving mice treated in this way, eventually eliminated the virus from the tested tissues (data not shown) and no longer showed signs of the P12SS disease These data represent the first demonstration that LTßR signaling plays an important role in antiviral responses and function CD8 T cells. The lymphotoxin system is intimately linked to the organization of the lymphoid architecture, most likely by controlling the expression of several chemosins that direct the organization of T and B cells (.Chaplin et al. Curr Opin. I munol ^ "10 10, 289 (1998), J. Cyster, in press.) The mature functional state of follicular dendritic cells is maintained by constant B cell signaling and these cells disappear the next day after the arrest of the cell. LTßR signaling.These cells are reviews for antigen presentation to the compartments of B and T cells. Reasonable speculation is that some aspect of the presentation and antigen to CD8 cells or the proper placement of these cells in a cruimiosin gradient during the maturation, by interrupting the LTßR signaling. Previous studies of the LT function have focused mainly on the biology of the B cell and involvement in a T cell function was not foreseen. Either the LT has additional functions or this data reflect the role of the novel LIGHT ligand. Currently P12SS is not clear the role that HVEM and LIGHT can play in the progress of the disease documented here. P12S5

Claims (8)

1. CLAIMS: 1. A method for inducing an antiviral response in an individual, comprising administering to the individual an effective amount of an agent that blocks the 5 binding of lymphotoxin-β to its receptor and a pharmaceutically acceptable carrier.
2. 2. A method according to claim 1, wherein the agent is a blocking agent of LT-beta-R.
3. 3. The method according to claim 2, in which the agent is an antibody against the lymphotoxin-β receptor or against a soluble lymphotoxin-β receptor.
4. 4. The method according to claim 3, wherein the agent is a receptor fusion protein of the 15 lyfotoxin-ß / Ig.
5. The method according to claim 1, wherein the agent is a soluble lymphotoxin-β or a and anti-lymphotoxin-β.
6. 6. A method for inducing an antiviral response in an individual, comprising administering to the individual an effective amount of an agent that blocks the lymphotoxin-β receptor and / or the route of HVEM signaling. The method according to claims 1 to 6, 25 wherein the individual is infected with one of the following viruses: Nameless, Ebola, Marburg, Lassa or Dengue. c: The method according to claim 7, wherein the agent is a 1-phoxin-β-5 R / Ig fusion protein. C