MX2007015638A - Methods and compositions for the treatment of persistent infections and cancer by inhibiting the programmed cell death 1 (pd-1) pathway - Google Patents

Abstract

The present invention provides methods and compositions for the treatment, prevention, or reduction of persistent infections, such as chronic infections, latent infections, and slow infections and cancer. The methods and compositions of the invention are also useful for the alleviation of one or more symptoms associated with such infections and cancer.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF PERSISTENT INFECTIONS AND CANCER BY INHIBITION OF THE CELLULAR DEATH PATH SCHEDULED (PD-1) RESEARCH CARRIED OUT WITH FEDERAL SPONSORSHIP This invention was made with the support of the government of the United States through grants AI39671 and CA84500 of the National Institute of Health (NHI or Na tional Heal th Insti tute). The government has certain rights over the invention.

FIELD OF THE INVENTION In general, the present invention relates to methods and compositions for the treatment of persistent infections and cancer.

BACKGROUND OF THE INVENTION Although the development of preventive vaccines has markedly decreased the mortality rate from viral infections, the use of these vaccines against viruses that cause persistent infections (for example, the hepatitis C virus) has had limited success. In contrast to the viruses that cause acute infections and spontaneous resolution, the immune response that is generated against the microbes causing infection persistent is often transient and insufficient to relieve the infection. Consequently, the infectious microbe remains inside the infected individual for prolonged periods of time, without necessarily causing constant damage to the host. One of the major impediments in the eradication of the microbes that cause persistent infection is the ability of these microbes to evade the immune system of the host organism. For example, certain viruses and parasites decrease the expression of host molecules necessary for the efficient recognition of infected cells by T cells. Persistent infections also cause functional deficiency of antigen-specific CD8 + T cells, vital for control and eradication of viral infections. Although the combination of therapeutic vaccines with auxiliary cytokines has been promoted, the resulting immune responses have not successfully eradicated the pathogen. Therefore, better methods are required to treat, prevent or alleviate persistent infections.

SUMMARY OF THE INVENTION The present invention provides methods and compositions for the treatment, prevention or reduction of a persistent infection or cancer or as an alternative the reduction of one or more of its symptoms. The invention is based on the discovery that the antigen-specific CD8 + T cells become functionally tolerant ("depleted") to the infectious agent after the induction of the programmed cell death polypeptide 1 (PD-1). Consequently, by reducing the expression or activity of PD-1, PD-L1 or PD-L2, the proliferation of functionally tolerant CD8 + T cells, the production of cytokines and the clearance rate of an infectious agent (eg, viral) increases. , bacterial, fungal, parasitic or cancer) so that the specific immune response to the infectious agent is intensified. Accordingly, the invention provides a method for alleviating or preventing a symptom of a persistent infection (eg, a viral infection, a bacterial infection, a fungal infection, a mycoplasmal infection and a parasitic infection) or cancer, by administering to a an individual in need thereof (e.g., a human being) a compound that reduces the activity or expression of a member of the analogue family to the CD-28 type (e.g., PD-1, CTLA-4, BTLA and a functional fragment or a variant thereof) or ligands of the family of analogs of the type CD-28 (for example, PD-L1 or PD-L2). Alternatively, the individual is administered an antigen-specific T cell or B cell that has been in contact with a compound that reduces the expression or activity of a PD-1 polypeptide in the cell. For example, the antigen-specific T cell or cell B is specific for a viral antigen. The T cell or the B cell is derived from an autologous source or is derived from another individual of the same or from a different species than the individual being treated. On the other hand, the invention presents a method for increasing the cytotoxic activity of a T cell (e.g., an anergic T cell or a T cell that has increased its tolerance to antigens) by contacting the T cell with a compound that reduces the activity or expression of a PD-1 polypeptide. In the aspects of the invention mentioned in the above, persistent viral infections are derived from infectious agents such as hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotrophic virus (HTLV), herpes virus, Epstein-virus. Barr or human papilloma virus. Persistent viral infections can also include infections caused by a latent virus. Types of cancer include lymphoproliferative disorders such as lymphoma angioimmunoblastic and nodular lymphocyte Hodgkin's lymphoma. Preferably, the compound of the invention increases a specific antigen immune response by increasing the cytotoxic activity of the T cell (for example, an increase in the production of cytotoxic cytokines such as IFNα, TNFα or IL-2, an increase in proliferation of T cell or an increase in viral clearance) in the individual being treated. For example, the compound reduces the expression or activity of a PD 1 ligand (PD-Ll) or a PD 2 ligand (PD-L2) or reduces the interaction between PD-1 and PD-Ll or the interaction between PD- 1 and PD-L2. Illustrative compounds include antibodies (eg, an anti-PD-1 antibody, an anti-PD-Ll antibody and an anti-PD-L2 antibody), RNAi molecules (eg, anti-PD-1 RNAi molecules, anti- PD-Ll RNAi and anti-PD-L2 RNAi), antisense molecules (eg, anti-PD-1 antisense RNA, anti-PD-Ll antisense RNA and anti-PD-L2 antisense RNA), dominant negative proteins (e.g. , dominant negative PD-1 protein, dominant negative PD-Ll protein and dominant negative PD-L2 protein) and small molecule inhibitors. The antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies and Ig fusion proteins. An anti-PD-Ll antibody includes clone EH12.

In addition to the compound that reduces the expression or activity of PD-1, the individual being treated may also be administered a vaccine which may or may not include an assistant or a primer booster. As an option, the individual who is administered a second compound, for example, an antiviral compound (eg, vidarabine, acyclovir, ganciclovir, valganclovir, nucleoside reverse transcriptase inhibitor (nucleoside-analog reverse transcriptase or NRTI) as AXT ( zidovudine), ddl (didanosine), ddC (zalcitabine), d4T (stavudine) or 3TC (lamivudine), non-nucleoside reverse transcriptase inhibitors (non-nucleoside reverse transcriptase inhibitor or NNRTI) such as nevirapine or delavirdine, protease inhibitor such as saquinavir , ritonavir, indinavir or nelfinavir, rivabirine and interferon), an antibacterial compound, an antifungal compound and an antiparasitic compound. The second compound can also be a compound that reduces the expression or activity of cytotoxic T lymphocyte 4 (CTLA-4) or attenuator of B and T lymphocytes (BTLA). Other exemplary compounds that can be administered to the individual are anti-CTLA-4 antibodies, anti-BTLA antibodies, anti-CD-28 antibodies, anti-ICOS antibodies, anti-ICOS-L antibodies, anti-B7-l antibodies, anti-B7-2 antibodies, antibodies anti-B7-H3 and anti-B7-H4 antibodies. The present invention also provides a method for identifying a candidate compound that modulates the activity or expression of a PD-1 polypeptide, the method consists of the following steps: (a) contacting a cell that expresses a PD-1 gene (by example, the PD-1 fusion gene) with a candidate compound; (b) measuring the expression or activity of PD-1 in the cell (for example, by measuring the expression of the mRNA or protein of PD-1); and (c) comparing the expression or activity of PD-1 in the cell with the corresponding expression or activity in a control cell that has not come into contact with the compound. An increase or decrease in the expression or activity of PD-1 indicates that the candidate compound is useful for modulating the expression or activity of a PD-1 polypeptide. As an alternative, the screening method can include the following steps: (a) contacting a T cell overexpressing a PD-1 gene with a candidate compound, (b) determining the cytotoxic activity of the T cell, (c) comparing the activity cytotoxicity of the T cell with the corresponding activity in a control cell that has not been in contact with the compound. An increase or decrease in this activity identifies the candidate compound as useful for modulating the expression or activity of a PD-1 polypeptide. The cytotoxic activity includes the production of cytokines, the proliferation of T cells and viral clearance. The invention also provides a screening method comprising the following steps: (a) contacting a PD-1 polypeptide with a candidate compound; (b) determining whether the candidate compound interacts with the PD-1 polypeptide; and (c) identifying a candidate compound as a compound useful for modulating the expression or activity of PD-1. Preferably, the identified candidate compound interacts with the PD-1 polypeptide and reduces its activity. The candidate compound identified with these screening methods reduces the interaction between PD-1 and PD-Ll or between PD-1 and PD-L2. Cells employed in any of the methods described herein include mammalian cells such as rodent cells or human cells. The cell is an immune cell, for example, a T cell. Preferably, the PD-1 polypeptide used in these screening methods is a human PD-1 polypeptide. We also present here a method to diagnose an individual who suffers from or is at risk of persistent infection or cancer, the method consists of the following steps: (a) obtain from a individual a sample containing immune cells (e.g., T or B cells), and (b) measuring the expression or activity of PD-1 in the sample. An increase in the expression or activity of PD-1 compared to this expression or activity in a control sample indicates that the individual suffers from or is at risk of suffering a persistent infection or cancer of choice, the passage (b) involves identifying antigen-specific immune cells, for example, a viral, bacterial, parasitic or fungal antigen. A method for selecting a treatment for an individual suffering from or at risk of suffering a persistent infection or cancer is also described. This method consists of the following steps: (a) obtaining from an individual a sample containing immune cells (e.g., T cells or B cells); and (b) measuring the expression or activity of PD-1 in immune cells, such that an increase in the expression or activity of PD-1 compared to this expression or activity in a control sample indicates that the individual suffers from or is at risk of persistent infection or cancer, and (c) select a treatment for the individual who is diagnosed as suffering from or at risk of persistent infection or cancer, so that the treatment includes a compound that reduces expression or activity of PD-1. Preferably, step (b) involves identifying antigen-specific immune cells, for example viral, bacterial, parasitic or fungal antigen. Samples derived from individuals include blood samples, tissue biopsies and bone marrow samples. On the other hand, the control cells can be obtained from an individual who does not suffer or is at risk of suffering a persistent infection. The invention also provides a composition containing: (a) a compound that reduces the level or activity of PD-1; and (b) a second compound, for example, an antibacterial compound, an antifungal compound, an anti-parasitic compound, an anti-inflammatory compound, an analgesic, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-CD- antibody. 28, an anti-ICOS antibody, an anti-ICOS-L antibody, an anti-B7-l antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody or an anti-B7-H antibody. The invention also provides a kit containing: (a) a compound that reduces the level or activity of PD-1; and (b) instructions for supplying the compound to an individual. Alternatively, the kit contains: (a) a first compound that reduces the level or activity of PD-1; (b) one second compound, for example, an antibacterial compound, an antifungal compound, an antiparasitic compound, an antiinflammatory compound, an analgesic, an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-CD-28 antibody, an anti-inflammatory antibody, ICOS, an anti-ICOS-L antibody, an anti-B7-l antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody or an anti-B7-H4 antibody; and (c) instructions for supplying the first and second compounds to an individual. The present invention offers important advantages with respect to common therapies for treatment, prevention and reduction or as an alternative relief of one or more symptoms of persistent infections. The administration of a therapeutic agent that reduces the level or activity of PD-1 increases the cytotoxicity of CD8 + cells which in turn increase the immune response to the infectious agent that has the ability to establish a persistent infection. On the other hand, the methods for selecting the candidate compound presented in this invention allow the identification of novel therapeutics that modify the deterioration process and not only mitigate the symptoms. Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as commonly the person skilled in the art to which the invention belongs knows. Even when methods and materials similar to those described herein can be used to carry out the invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are considered, in their entirety, part of the present by reference. In case of conflict, this specification, including definitions, will serve as control. On the other hand, the materials, methods and examples are illustrative only and have no limiting purpose. From the following detailed description and from the claims, other features and advantages of the invention will become apparent.

BRIEF DESCRIPTION OF THE FIGURES Figure IA is a bar graph showing the levels of the mRNA of PD-1 in T cells specific for DbGP33-41 and / or DbGP276-286 of transgenic mice not previously subjected to experimentation, immune infected mice (FIG. approximately 30 days after infection) to the Armstrong virus of lymphocytic choriomeningitis (LCMV or lymphocytic tic choriomeningitis virus) or mice infected with LCMV-C1- 13 (approximately 30 days after infection) with CD4 deficiency, as determined by gene matrix analysis. Figure IB is a series of images from a flow cytometry experiment showing surface expression of PD-1 in CD8 + T-tetramer + cells in infected mice immune to LCMV Armstrong and mice infected with LCMV-C1-13 with CD4 deficiency . Anergic CD8 + T cells express high levels of PD-1 polypeptide on the cell surface approximately 60 days after chronic infection with LCMV-Cl-13 virus (labeled "chronic"), but virus-specific CD8 + T cells do not express the PD-1 polypeptide after clearance of an acute LCMV Armstrong infection (marked as "immune"). Figure IC is a series of images from a flow cytometry experiment demonstrating the presence of PD-Ll in splenocytes from chronically infected mice and uninfected mice. It is demonstrated that the expression of PD-Ll is the highest in the splenocytes that are infected by the virus. Figure 2A is a series of dispersive graphs showing that when mice infected with Cl-13 are treated from day 23 to 37 after infection there was an increase in approximately 3 times compared to the number of CD8 + T cells specific for DbNP396-404 and DbGP33-41 compared to the untreated controls. In order to determine any change in function, the production of IFN? and TNFa in response to 8 different LCMV epitopes. Figure 2B is a scatter plot showing that when all the known specificities of the CD8 + T cells are measured, there is an increase equivalent to 2.3 times in the total number of LC8-specific CD8 T cells. Figure 2C is a series of flow cytometry graphs showing the production of IFN? and TNFa in response to eight different LCMV epitopes. Figure 2D is a scatter plot showing that in the treated mice more CD8 T cells specific for the virus have the ability to produce TNFa. Figure 2E is a series of bar graphs showing that blockade of PD-Ll also results in an increase in viral control in spleen, liver, lung and serum. Figure 3A is a graph showing the increase in specific CD8 + T cells of DbGP33-41 and DbGP276-286 (labeled "GP33" and "GP276") in mice infected with Cl-13 who have CD4 deficiency, treated with anti-PD-Ll (marked as "aPD-Ll") from day 46 to day 60 after infection, compared to control (marked as "unta") "), which demonstrates that mice treated with anti-PD-Ll contained approximately 7 times more splenic CD8 + T cells specific for DbGP276-286 and approximately 4 times more splenic CD8 + T cells specific for DbGP33-41 than untreated mice. Figure 3B is a series of images demonstrating the increase in the frequency of CD8 + T cells specific for DbGP33-41 and DbGP276-286 in the spleen of mice infected with CD4 deficient Cl-13 treated with anti-PD-Ll (labeled as "aPD-Ll Tx") from day 46 to day 60 after infection compared to control (marked as "untx"). Figure 3C is a series of images demonstrating an increase in the proliferation of CD8 + T cells specific for DbGP276-286 in mice treated with PD-Ll, determined by BrdU incorporation and Ki67 expression. Figure 3D is a graph showing that mice that have high levels of expansion of CD8 + T cells manifest an appreciable response in peripheral blood mononuclear cells (PBMC or blood mononuclear cells), as shown when comparing CD8 + cells specific for DbGP276-286 in PBMCs and CD8 + T cells specific for DbGP276-286 in the spleen. Figure 4A is a series of graphs demonstrating the increase of CD8 + T cells specific for DbGP33-41 and DbGP276-286 producing IFN ?, in mice treated with anti-PD-Ll, compared to controls. High frequencies of CD8 + T cells specific for DbNP396-404, KbNP205-212, DbNP166-175 and DbGP92-101 were also detected in mice treated with anti-PD-Ll. Figure 4B is a graph showing that in mice treated with anti-PD-Ll, 50% of CD8 + T cells specific for DbGP276-286 produce IFNα. compared to 20% of CD8 + T cells specific for DbGP276-286 in the control mice. Figure 4C is a series of images demonstrating that chronically infected mice treated with anti-PD-Ll produce higher levels of TNFa than chronically infected untreated mice, but produce even lower levels of TNFa than immune mice infected with the LCMV Armstrong virus. Figure 4D is a graph showing that anti-PD-Ll treatment of infected mice with LCMV-C1-13, renews the ex vivo lytic activity of virus-specific T cells, compared to untreated infected mice, as determined by a 51Cr release assay. Figure 4E is a series of graphs demonstrating the reduction of viral values in various organs after treatment with a-PD-Ll of mice infected with LCMV-C1-13. Viral values decreased approximately 3 times in the spleen, 4 times in the liver, 2 times in the lung and 2 times in the serum after 2 weeks of a treatment with anti-PD-Ll, compared to the untreated mice. Figure 5A is a series of images from a flow cytometry experiment showing the surface expression of PD-1 and using 10 HIV-specific tetramers for dominant epitopes directed at chronic HIV C-clade infection. The percentages indicate the percentage of tetramer + cells that are PD-1 +. Figure 5B is a series of graphs demonstrating that the percentage and MFI of PD-1 has a significant regulated increase in HIV-specific CD8 T cells compared to the total CD8 T cell population (p <0.0001) in individuals who had not received previous treatment with antiretroviral therapy and PD-1 increases in the total population of CD8 T cells in HIV-infected individuals compared to controls seronegative to HIV (p = 0.0033 and p <0.0001, respectively). Staining of 120 HIV tetramers from 65 HIV-infected individuals and 11 HIV-seronegative controls were included in the analysis. Figure 5C is a series of graphs showing the percentage median and the MFI of PD-1 expression in tetramer + cells by epitope specificity. Figure 5D is a graph representing the variation in the percentage of PD-1 + cells in different specific populations of epitope within individuals with multiple detectable responses. The horizontal bars indicate the median percentage of HIV + PD-1 + tetramer cells in each individual. Figure 6A is a series of graphs showing that there is no correlation between the number of HIV-specific CD8 + T cells, determined by tetramer staining and plasma viral load, while there is a positive correlation between the percentage and MFI of PD -1 in tetramer + cells and plasma viral load (p = 0.0013 and p <0.0001, respectively). Figure 6B is a series of graphs that show that there is no correlation between the number of HIV tetramer cells and the CD4 count, while there is an inverse correlation between the percentage and the MFI of PD-1 in tetramer + HIV cells and the CD4 figure (p = 0.0046 and p = 0.0150, respectively). Figure 6C is a series of graphs showing that the percentage and MFI of PD-1 in the total CD8 T cell population correlates positively with plasma viral load (p = 0.0021 and p <; 0.0001, respectively). Figure 6D is a series of graphs that represent that the percentage and mean fluorescence intensity (MFI or mean fl uorescence in tensi) of the expression of PD-1 in the total CD8 T cell population correlates inversely with the CD4 figures (p = 0.0049 and p = 0.0006, respectively). Figure 7A is a series of images of a flow cytometry experiment showing the representative phenotypic staining of CD8 + T cells specific for B * 4201 TL9 of an individual SK222 in which 98% of the CD8 + T cells specific for B 201 TL9 they are PD-1 +. Figure 7B is a graph illustrating a summary of phenotypic data of people in whom > 95% of HIV-specific CD8 + T cells are PD-1 + were analyzed from 7 to 19 samples for each of the phenotypic markers indicated. The horizontal bar indicates the median percentage of tetramer + PD-1 + cells that were positive for the indicated marker. Figure 8A is a series of images of a flow cytometry experiment showing the representative data of the proliferation assay of an individual positive to B * 4201. After 6 days of stimulation with the peptide, the percentage of CD8 T cells specific for B 201 TL9 increased from 5.7% to 12.4% in the presence of an anti-PD-Ll blocking antibody. Figure 8B is a line plot representing the summary data of the proliferation assay, which indicates a significant increase in the proliferation of HIV-specific CD8 T cells in the presence of an anti-PD-Ll blocking antibody (n = 28, p = 0.0006, test t for paired data). Figure 8C is a bar graph showing the differential effects of blocking PD-1 / PD-L1 on the proliferation of HIV-specific CD8 T cells by individual patient. The white bars indicate the increase of tetramer + cells in the presence of peptide alone, the black bars indicate the increase of tetramer + cells in the presence of peptide plus anti-PD-Ll blocking antibody. Individuals in whom CFSE trials were performed for more than one epitope are indicated with the symbols of asterisk, square or triangle.

DETAILED DESCRIPTION OF THE INVENTION In recent decades the use of antibiotics has notably reduced the mortality rate caused by microbial infections. The success of antimicrobial treatment modalities has been limited by the ability of certain infectious agents to evade the immune system of the host organism and in turn establish a persistent infection. For example, the immune response that is generated against viruses such as hepatitis and HIV is not enough to purge the infectious agent, which remains in the infected individual. In these infections, the antigen-specific CD8 + T cells become functionally tolerant to the infectious agent and enter a state known as "anergy" or "exhaustion". The anergic T cells lose their cytotoxic activity, that is, their ability to produce cytokines, proliferate and purify the infectious agent. The present invention is based on the surprising discovery that the anergy of the T cells is concurrent with an induction of the expression of PD-1 and that the expression of PD-1 correlates with certain types of disorders lymphoproliferatives Accordingly, the invention features methods for increasing the cytotoxicity of T cells by contacting a T cell with an agent that reduces the expression or activity of PD-1, PD-1 ligand (PD-Ll) or ligand 2 of PD-1 (PD-L2). More specifically, the invention provides methods of treating or preventing persistent infection or lymphoproliferative disorders (eg, cancer such as angioimmunoblastic lymphoma and predominant nodular lymphocyte lymphoma) by administering to an individual an agent that reduces expression or activity of PD-1. The reduction of the expression or activity of PD-1, PD-Ll or PD-L2 results in an increase in the activity of the cytotoxic T cells, increasing the specific immune response towards the infectious agent. The results presented here show that the administration of anti-ligand-1 blocking antibodies programmed death (PD-Ll) in mice with persistent infection, increased the cytotoxic activity of the anergic T cells. Specifically, alteration of PD-1 signaling induced the expression of CD8 + T cells, enhanced cytokine production and increased viral clearance. On the other hand, CD8 + T cells generated during persistent infections in deficient mice in CD4 they proliferated and recovered much of their function with the anti-PD-Ll treatment. For T cells to respond to exogenous proteins, the antigen presenting cells (APCs) have to emit two signals to resting T lymphocytes. The first signal, which confers specificity to the immune response, is transduced through the T cell receptor (TCR) after recognition of the exogenous antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, called co-stimulation, induces the proliferation of T cells and makes them functional. The co-stimulation is neither antigen-specific nor is it restricted by the MHC and is derived from one or more distinct cell surface polypeptides expressed by the APCs. If the T cells are stimulated only through the T cell receptor, without receiving an additional costimulatory signal, they become insensitive, anergic or die, which results in a decrease in the immune response. The CD80 (B7-1) and CD86 (B7-2) proteins, expressed in the APC are critical co-stimulatory polypeptides. Even though B7-2 has a predominant function during the primary immune responses, B7-1 increases later in the course of an immune response and prolongs the responses of primary T cells or co-stimulate the responses of secondary T cells. B7 polypeptides are capable of providing costimulatory or inhibitory signals to immune cells to promote or inhibit immune cell responses. For example, when bound to a co-stimulator receptor, PD-Ll (B7-4) induces costimulation of immune cells or inhibits the co-stimulation of immune cells when present in soluble form. When they bind to an inhibitory receptor, B7-4 molecules can transmit an inhibitory signal to an immune cell. Exemplary members of the B7 family include B7-1, B7-2, B7-3 (recognized by antibody BB-1) B7h (PD-Ll) and B7-4 and soluble fragments or derivatives thereof. The members of the B7 family bind to one or more receptors in an immune cell, for example, CTLA-4, CD-28, ICOS, PD-1 and / or other receptors and depending on the receptor, have the ability to transmit a inhibitory signal or a costimulatory signal to an immune cell. CD-28 is a receptor that is constitutively expressed in resting T cells. After signaling through the T cell receptor, the ligation of CD-28 and the transduction of a costimulatory signal causes the T cells to proliferate and secrete IL-2. CTLA-4 (CD152), a homologous receptor of CD28 is absent in resting T cells but its expression is induced after the activation of T cells. CTLA-4 has a role in the down-regulation of T cell responses. ICOS, a related polypeptide with CD-28 and CTLA-4, participates in the production of IL-10. PD-1, the receptor to which PD-Ll and PD-L2 bind, is also rapidly induced on the surface of T cells. PD-1 is also expressed on the surface of B cells (as a response to an anti-IgM) and in a subset of thymocytes and myeloid cells. The addition of PD-1 (for example, by cross-linking or by aggregation) results in the transmission of an inhibitory signal in an immune cell, causing a reduction in immune responses concomitant with an increase in the anergy of the immune cell. Members of the PD-1 family bind to one or more receptors, for example, PD-Ll and PD-L2 on the antigen-presenting cells. PD-Ll and PD-L2, which are PD-1 ligand polypeptides, are members of the B7 polypeptide family. Each PD-1 ligand contains a signal sequence, an IgV domain, an IgC domain, a transmembrane domain and a short cytoplasmic tail. These ligands are expressed in placenta, spleen, ganglia lymphatic, thymus and heart. PD-L2 is also expressed in pancreas, lung and liver, while PD-L1 is expressed in fetal liver, activated T cells and endothelial cells. The two PD-1 ligands are increased in activated monocytes and dendritic cells.

Definitions As used herein, the term "persistent infection" refers to an infection in which the infectious agent (e.g., virus, bacteria, parasite, mycoplasma or fungi) is not cleared or removed from the infected host , even after the induction of an immune response. Persistent infections can be chronic infections, latent infections or slow infections. Even though acute infections are relatively brief (lasting from a few days to a few weeks) and are overcome by the body through the immune system, persistent infections can last for months, years or even a lifetime. These infections can also recur frequently over a long period of time with asymptomatic stages and productive infection without cytolytic activity or even without causing excessive damage in the host cells. The infectious causative agents can also be detected in the host (for example, within specific cells or infected individuals), even after the immune response has disappeared, using standard techniques. Persistent infection is diagnosed in mammals according to any standard method known in the art and described, for example, in U.S. Patent Nos. 6, 368, 832, 6, 579, 854 and 6, 808, 710 and the publications of patent application Nos. 20040137577, 20030232323, 20030166531, 20030064380, 20030044768, 20030039653, 20020164600, 20020160000, 20020110836, 20020107363 and 20020106730, both of which are considered part of the present, by reference. For example, an individual can be diagnosed with a persistent chlamydial infection after detecting chlamydial species in a biological sample of this individual through PCR (polymerase chain reaction) analysis. It is not necessary that mammals have been diagnosed with a persistent infection for treatment according to this invention. Microbial agents capable of establishing a persistent infection include viruses (e.g., papilloma virus, hepatitis virus, human immunodeficiency virus and herpes virus), bacteria (e.g., Escherichia coli and Clamydia spp.), Parasites (e.g. , Plasmodimum, Leishmania spp., Schistosoma spp., Trypanosoma spp., Toxoplasma spp.) And fungi. In the sense that the term "alleviating a symptom of a persistent infection" is used herein, it refers to improving any of the conditions or symptoms associated with the persistent infection before or after it has occurred. Alternatively, alleviating a symptom of a persistent infection may be the decrease in the infectious microbial load (eg, viral, bacterial, fungal, mycoplasmic or parasitic) in the individual compared to the corresponding load in an untreated control. In comparison with an equivalent untreated control, this reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% depending on determined by any standard technique. Preferably, the persistent infection is completely eliminated when detected by any standard method known in the art, in which case the persistent infection is considered to have been treated. A patient who is treated for a persistent infection is one to whom a doctor has diagnosed this condition. Diagnosis and monitoring or surveillance may include, for example, detecting the level of microbial load in a biological sample (for example, a tissue biopsy, a test in blood or urine), detect the level of surrogate marker of the microbial infection in a biological sample, detect symptoms associated with persistent infections or detect immune cells that participate in the immune response typical of persistent infections (for example, detection of antigen-specific T cells that are anergic). A patient who undergoes the prevention of a persistent infection may or may not have received the corresponding diagnosis. A person related to the technique will understand that these patients may have undergone the same standard tests described above or may have been identified, without any examination, as someone at high risk due to the presence of one or more risk factors (for example, family history or exposure to the infectious agent). As used herein, the term "PD-1" refers to a polypeptide that complexes with PD-Ll or PD-L2 proteins and therefore participates in immune responses, for example, the T-cell co-stimulation. The PD-1 proteins of the invention are virtually identical to naturally occurring PD-1's (see, for example, Ishida et al., EMBO J. 11: 3887-3895, 1992, Shinohara et al., Genomics 23: 704-706, 1994; and Patent of the States United No. 5, 698,520, which is considered part of this, as a reference). PD-1 signaling can reduce, for example, the cytotoxicity of CD8 + T cells by reducing T-cell proliferation, cytokine production or viral clearance. According to this invention, PD-1 polypeptide reduces the cytotoxic activity of CD8 + T cells by at least 5%, 10%, 20%, 40%, 50%, 60%, 70%, 80%, 90% or more than 100 % below control levels, determined by any standard method. The term "PD-1 gene" refers to a nucleic acid encoding a PD-1 protein. The term "PD-1 fusion gene" refers to a PD-1 promoter and / or to a total or partial PD-1 coding region operably linked to a second heterologous nucleic acid sequence. In preferred embodiments, the second heterologous nucleic acid sequence is a reporter gene, i.e., a gene whose expression can be analyzed; reporter genes include, among others, those encoding glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), alkaline phosphatase and beta-galactosidase. The expression "reduce the expression or activity of PD-1" refers to reducing the level of biological activity of PD-1 in relation to the level of activity of PD-1 in an untreated control. According to this invention, this level or activity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or even more than 100%, with respect to a control without treating. For example, the biological activity of PD-1 is reduced if the binding of PD-1 to PD-Ll and / or PD-L2 decreases, so there is a reduction in PD-1 signaling and therefore there is an increase in the cytotoxicity of CD8 + T cells. In the sense that is used herein, the term "activity" with respect to a PD-1 polypeptide includes any activity that is inherent to the PD-1 protein in the wild, for example, the ability to modulate an inhibitory signal in an activated immune cell, for example, by associating a natural ligand in an antigen presenting cell. This modulation of the inhibitory signal in an immune cell results in the modulation of the proliferation of cytokine secretion in an immune cell. PD-1 can also modulate a costimulatory signal by competing with a costimulatory receptor by binding to a B7 molecule. Thus, the term "PD-1 activity" includes the ability of a PD-1 polypeptide to bind to its natural ligand, the ability to modulate inhibitory or co-stimulatory signals and the ability to modulate the response immune. Accordingly, the reduction of PD-1 activity includes reducing the interaction of PD-1 with PD-Ll or PD-L2. This can be done, for example, by blocking PD-Ll or PD-L2. The term "immune cell" refers to a cell of hematopoietic origin and which has a function in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes). The term "T cell" refers to a CD4 + T cell or a CD8 + T cell. The term T cell includes both TH1 cells and TH2 cells. The term "T-cell cytotoxicity" includes any immune response mediated by the activation of CD8 + T cells. Exemplary immune responses include cytokine production, proliferation of CD8 + T cells, production of granzyme or perforin and clearance of the infectious agent. The term "insensitivity" includes the refractivity or lack of response of immune cells to stimulation, for example, to stimulation via activating receptor or a cytokine. Insensitivity can be presented, for example, by the exposure to immunosuppressants or exposure to high doses of antigen. In the sense that is used in the present, the term "anergy" or "tolerance" includes refractivity to stimulation mediated by an activation receptor. This refractivity is usually antigen-specific and persists after the exposure to the tolerizing antigen has ended. For example, anergy in T cells (in contrast to insensitivity) is characterized by the absence of cytokine production, for example, IL-2. Anergy of the T cell occurs when the T cells are exposed to an antigen and receive a first signal (a signal mediated by a T cell receptor or by CD-3) in the absence of a second signal (a costimulatory signal). Under these conditions, the re-exposure of the cells to the same antigen (even if the re-exposure occurs in the presence of a co-stimulatory molecule) results in the inability to produce cytokines and therefore to proliferate. However, the anergic T cells generate responses to unrelated antigens and can proliferate if cultured with cytokines (eg, IL-2). For example, the anergy of the T cells can also be observed by the lack of IL-2 production in the T lymphocytes as determined by ELISA or by a proliferation assay using a cell line Indicator Alternatively, a reporter gene construct can be used. For example, anergic T cells can not initiate gene transcription of IL-2 induced by a heterologous promoter under the control of the 5'-gene enhancer IL-2 or by a multimer of the API sequence that can be found within the enhancer (Kang. et al., Science 257: 1134, 1992). Antigen-specific antigenic T cells can have a minimum reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 100% in cytotoxic activity with respect to the corresponding antigen-specific control T cell. The term "purified antibody" refers to an antibody that at least 60% by weight is free of proteins and organic molecules of natural origin with which it is normally associated. Preferably, the preparation is at least 75%, more preferably 90% and most preferably 99%, by weight, an antibody, for example, an antibody specific for PD-1, PD-Ll or PD-L2. A purified antibody can be obtained, for example, by affinity chromatography using a recombinantly produced protein or conserved peptide motifs and standard techniques. The term "specifically binds" refers to to an antibody that recognizes and binds to an antigen such as the PD-1, PD-Ll or PD-L2 polypeptides but that practically does not recognize or bind to other non-antigenic molecules in a sample, for example, a biological sample, which naturally includes the protein. A preferred antibody that binds to the PD-1, PD-Ll or PD-L2 polypeptides is set forth in U.S. Patent Nos. 6, 808,710 and the publications of patent application Nos. 20040137577, 20030232323, 20030166531, 20030064380, 20030044768, 20030039653, 20020164600, 20020160000, 20020110836, 20020107363 and 20020106730, both of which are considered part of the present, by reference. The term "neutralizing antibodies" refers to antibodies that interfere with any of the biological activities of a PD-1 polypeptide, in particular, the ability of a PD-1 polypeptide to reduce an immune response such as the cytotoxicity of T cells. neutralizing antibody can reduce the ability of a PD-1 polypeptide to reduce the immune response, preferably by 50%, more preferably by 70% and most preferably by 90% or more. Any standard test can be used to measure immune responses, including those described here, to potentially evaluate antibodies neutralizing. The term "virtually identical" with reference to a protein or polypeptide, refers to a protein or polypeptide exhibiting at least 75%, but preferably 85%, more preferably 90% and most preferably 95% or even 99 % identity with a reference amino acid sequence. For proteins or polypeptides, the length of the comparison sequences will generally be 20 amino acids, preferably at least 30 amino acids, more preferably at least 40 amino acids and most preferably 50 amino acids or the total length of the amino acid. protein or polypeptide. The nucleic acids encoding these "virtually identical" proteins or polypeptides constitute an example of "virtually identical" nucleic acids; it is known that by the degeneracy of the genetic code, the nucleic acids include some sequence that encodes those proteins or polypeptides. On the other hand, a "virtually identical" nucleic acid sequence also includes a polynucleotide that hybridizes with a reference nucleic acid molecule under very stringent conditions. The term "very stringent conditions" refers to any group of conditions that are characterized by high temperature and low ionic strength and allow hybridization comparable to that resulting from the use of a DNA probe with a minimum length of 40 nucleotides, in a buffer containing 0.5 M NaHP0, pH 7.2, SDS (sodium dodecyl sulfate) 7%, 1 mM EDTA and BSA 1 % (fraction V), at a temperature of 65 ° C or a buffer containing 48% formamide, 4.8XSSC, 0.2 M Tris-Cl, pH 7.6, Denhardt IX solution, 10% dextran sulfate and 0.1% SDS, a temperature of 42 ° C. Other conditions of stringent hybridization, eg, PCR, Northern blotting, Southern blotting or hybridization in itself, DNA sequencing, etc., are well known to those skilled in the art of molecular biology. See, for example, F. Ausubel et al. , Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1998, which is considered part of this, as a reference. The term "substantially pure" refers to a nucleic acid, polypeptide or other molecule that has been separated from the components with which it is found in the natural state. In general, the polypeptide is practically pure when it is at least 60%, 70%, 80%, 90%, 95% or even 99% by weight, free of the proteins and organic molecules with which it is associated in natural state. For example, a practically pure polypeptide can be obtained by extraction to from a natural source, by extraction of a recombinant nucleic acid in a cell that does not normally express that protein or by chemical synthesis. The term "isolated DNA" refers to the fact that DNA is free of genes that flank DNA in the natural genome of the organism from which this DNA is derived. Thus, the term "isolated DNA" encompasses, for example, cDNA, cloned genomic DNA and synthetic DNA. The term "an effective amount" refers to the amount of a compound, alone or in combination, required to reduce or prevent hypertension or to treat or prevent a chronic infection in a mammal. The effective amount of the active compound (s) varies depending on the route of administration, age, body weight and the general health of the individual. Finally, the doctor or veterinarian who takes care of the case will decide the appropriate amount and the dosage scheme. The term "candidate compound" refers to a chemical substance of natural or artificial origin. The candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, organic molecules of natural origin, nucleic acid molecules, peptide nucleic acid molecules and components derived therefrom. By example, a candidate compound useful according to the present invention reduces the binding of PD-1 to PD-Ll, PD-L2 or both. The term "pharmaceutical composition" refers to any composition that contains at least one therapeutically active or biologically active agent and that is suitable for administration to the patient. Any of these formulations can be prepared by methods accepted and well known in the art. See, for example, Remington: The Science and Practice of Pharmacy, 20 th. ed. (Ed. A.R. Gennaro), Mack Publishing Co. , Easton, Pa., 2000.

Methods of treatment The cytotoxicity of T cells is increased by contacting a T cell with a compound that reduces the expression or activity of PD-1. The T cell is a naive T cell, a memory T cell or an activated T cell. Alternatively, the T cell is an antigen-specific T cell. Antigen-specific T cells are either anergic or tolerant to the infectious agent. The cytotoxicity of the T cell is characterized by an increase in cell proliferation and the release of cytokines. The methods are useful to alleviate symptoms of a variety of infections and types of cancer. An infection or cancer is treated, prevented, or a symptom is alleviated by administering an inhibitor of PD-1 to an individual. The individual is a mammal, for example, a person, a primate, a mouse, a rat, a dog, a cat, a cow, a horse and a pig. The individual suffers or is at risk of developing an infection. An individual who suffers or is at risk of developing an infection is according to standard methods, susceptible to a particular infection. The infection, for example, bacterial, viral, fungal, mycoplasmic or parasitic, is a persistent infection. Persistent infections unlike acute infections are not effectively eliminated by the induction of an immune response. The infectious agent and the immune response arrive at a balance, in such a way that the infected individual remains that way for a long period of time without necessarily manifesting symptoms. Persistent infections include, for example, latent, chronic and slow infections. In a chronic infection, the infectious agent can be detected in the body at all times. However, the signs and symptoms of the disease may be present or absent for a period of time dragged on. Examples of chronic infection include hepatitis B (caused by HBV) and hepatitis C (caused by HCV), adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpes virus 6 , varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, BK polyomavirus, JC polyoma virus, measles virus, rubella virus, human immunodeficiency virus (HIV), leukemia virus I of human T cell and human T cell leukemia virus II. Persistent parasitic infections can arise as a result of infection with Leishmania, Toxoplasma, Trypanosoma, Plasmodium, Schistosoma and Encephalitozoon. In a latent infection, the infectious agent (for example, a virus), apparently, is inactive and latent so that the individual does not always show signs or symptoms. In a latent viral infection, the virus remains in equilibrium with the host for a long period of time before the symptoms appear again, however, the real viruses can not be detected until the reactivation of the disease occurs. Examples of latent infections include infections caused by VSH-1 (cold sores), VSH (genital herpes) and VZV (varicella-zoster).

In a slow infection, infectious agents gradually increase in number over a long period of time in which no significant signs or symptoms are observed. Examples of slow infections include AIDS (caused by HIV-1 and HIV-2), lentiviruses that cause tumors in animals and prions. On the other hand, persistent infections often occur as later complications of acute infections. For example, sclerosing panencephalitis (SSPE) may develop after an acute measles infection or regressive encephalitis secondary to a rubella infection may also occur. Types of cancer include, for example, angioimmunoblastic lymphoma or nodular lymphocyte predominant Hodgkin lymphoma. Angioimmunoblastic lymphoma (AIL or angioimmunoblastic lymphoma) is an aggressive (fast-moving) type of non-Hodgkin's T-cell lymphoma characterized by enlarged lymph nodes and hypergammaglobulinemia (increased antibody in the blood). Other symptoms may include rash, fever, weight loss, positive Coombs test or night sweats. This malignant tumor usually occurs in adults. The age of the patients is usually 40 to 90 years (the median around 65) and more often adults. As the LAI progresses, hepatosplenomegaly, hemolytic anemia, and polyclonal hypergamaglubulinemia may develop. Approximately in 40 to 50% of the patients the skin is involved. Nodular lymphocyte-predominant Hodgkin lymphoma is a B cell neoplasm that appears to be derived from germinative center B cells with non-functional immunoglobulin mutant genes. Like angioimmunoblastic lymphoma, neoplastic cells are associated with a network of follicular dendritic cells. The expression of PD-1 is observed in T cells that are closely related to the lymphocytic CD20 + neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma, < J in a pattern similar to that observed for CD57 + T cells. CD57 + has been identified as another marker of T cells associated with associated T-cell germinal center together with CXCR5, findings that support the conclusion that neoplastic cells in nodular lymphocyte-predominant Hodgkin lymphoma are closely related to germinal centers of cells T associated. A PD-1 inhibitor is any agent that has the ability to reduce the expression or activity of PD-1, PD-Ll or PD-2 in a cell. The expression or activity of PD-1 is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the corresponding expression or activity in a control cell. The control cell is a cell that has not been treated with PD-1 inhibitor. The expression or activity of PD-1 is determined by any standard method known in the art, including those described herein. As an option, the PD-1 inhibitor inhibits or reduces the binding of PD-1 to PD-Ll, PD-L2 or both. PD-1 inhibitors include polypeptides, polynucleotides, small molecule antagonists or siRNA. A PD-1 inhibitor polypeptide includes, for example, an antibody or fragment thereof that reduces the expression or signaling of PD-1. Illustrative antibodies include anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-BTLA antibodies, anti-CD-28 antibodies, anti-ICOS antibodies, antibodies anti-ICOS-L, an anti-B7-l antibody, anti-B7-2 antibody, anti-B7-H3 antibodies or anti-B7-H antibodies. Alternatively, the PD-1 inhibitor is a dominant negative protein or a nucleic acid that encodes a dominant negative protein that interferes with the biological activity of PD-1 (ie, the binding of PD-1 to PD-Ll, PD-L2 or both). A dominant negative protein is an amino acid molecule having a sequence that is at least 50%, 70%, 80%, 90%, 95% or even 99% sequence identity with at least 10, 20, 30, 35 , 50, 100 or more than 150 amino acids of the wild-type protein to which the dominant negative protein corresponds. For example, a dominant PD-1 negative has a mutation that does not allow it to bind to the PD-Ll. The dominant negative protein can be administered as an expression vector. The expression vector can be a viral vector or a non-viral vector (e.g., retrovirus, recombinant associated adenovirus or a recombinant adenoviral vector). Alternatively, the dominant negative protein can be administered directly systemically as a recombinant protein or to the infected area using microinjection techniques. Small molecules include, among others, peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic and inorganic compounds (including hetero-organic compounds and organometallic) with a molecular weight less than about 5000 grams per mole, organic or inorganic compounds with a molecular weight less than about 2000 grams per mole, organic or inorganic compounds with a molecular weight of less than about 1000 grams per mole, organic or inorganic compounds with a molecular weight less than about 500 grams per mole, and salts, esters and other pharmaceutically acceptable forms of these compounds. The PD-1 inhibitor is an antisense molecule, a silencing RNA molecule (siRNA) or a small molecule antagonist that targets the expression or activity of PD-1. The term "siRNA" refers to a double-stranded RNA molecule that prevents the translation of a particular RNA. Standard techniques are used to introduce the siRNA into the cell, including those in which DNA is a template from which an siRNA is transcribed. The siRNA includes a nucleic acid sequence of PD-1, PD-Ll or PD-L2, an antisense nucleic acid sequence of PD-1, PD-Ll or PD-L2 or both. As an option, the siRNA is constructed in such a way that a single transcript has the complementary sense and antisense sequences of the target gene, eg, a hairpin. The union of SiRNA to a transcript of PD-1, PD-Ll or PD-L2 in the target cell results in a reduction in the production of PD-1, PD-Ll or PD-L2 in the cell. The length of the oligonucleotide is at least 10 nucleotides and can be as long as the transcript of PD-1, PD-Ll or natural PD-L2. Preferably, the oligonucleotide has a length of 19 to 25 nucleotides. Most preferably, the oligonucleotide is less than 75, 50 or 25 nucleotides in length. Other suitable PD-1 inhibitors are described, for example, in U.S. Patent No. 6,808,710 and Patent Application Publication Nos. 20040137577, 20030232323, 20030166531, 20030064380, 20030044768, 20030039653, 20020164600, 20020160000, 20020110836, 20020107363 and 20020106730, both of which are considered part of the present, by reference. The preferred dose of the PD-1 inhibitor is a biologically active dose. A biologically active dose is a dose that induces an increase in the cytotoxic activity of the CD8 + T cell that increases the specific immune response to the infectious agent. Preferably, the PD-1 inhibitor has the ability to reduce the expression or activity of PD-1 in antigen-specific immune cells (e.g., T cells such as CD8 + T cells) in a minimum proportion of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 100% below the levels corresponding to the untreated control. The levels or activity of PD-1 in immune cells is determined by any of the methods known in the art, including, for example, Western blot analysis, immunochemistry, ELISA, Northern blot analysis. Alternatively, the biological activity of PD-1 is determined by evaluating the binding of PD-1 to PD-Ll, PD-L2 or both. The biological activity of PD-1 is determined according to its ability to increase the cytotoxicity of CD8 + T cells, including, for example, cytokine production, clearance of the infectious agent and proliferation of antigen-specific CD8 + T cells. Preferably, the agent that reduces the expression or activity of PD-1 increases the specific immune response to the infectious agent in a minimum proportion of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 90% or more than 100% above the levels corresponding to the untreated control. Therefore, the agent of the present invention is any agent having one or more of these activities. Although the agent of the invention is preferably expressed on CD8 + T cells, it should be understood that any cell that can influence the immune response to infections Persistent antibodies are also suitable for the methods of the invention and include, for example, B cells. Optionally, the individual is administered one or more additional therapeutic agents. Additional therapeutic agents include, for example, antiviral compounds (eg, vidarabine, acyclovir, ganciclovir, valganciclovir, nucleoside reverse transcriptase inhibitor analogue (NRTI or nucleoside-analog reverse transcriptase inhibitor) (eg, AZT (zidovudine), ddl (didanosine), ddC (zalcitabine), d4T (stavudine) or 3TC (lamivudine), non-nucleoside reverse transcriptase inhibitor (NNRTI or non nucleoside reverse transcriptase inhibitor) (eg, (nevirapine or delavirdine), protease inhibitor ( saquinavir, ritonavir, indinavir or nelfinavir), ribavirin or interferon), antibacterial compounds, antifungal compounds, antiparasitic compounds, anti-inflammatory compounds, antineoplastic or analgesic agents.The additional therapeutic agent is administered before, at the same time or after administration of the inhibitor. PD-1 For example, the PD-1 inhibitor and the additional agent are administered in formulations eparadas in a range of 1, 2, 4, 6, 10, 12, 18 or more than 24 hours. Optionally, the additional agent is formula together with the PD-1 inhibitor. When the other agent is presented in a different composition, different administration routes can be used. The agent is administered in known effective doses to treat, reduce or prevent an infection. The concentrations of the PD-1 inhibitor and the additional agent depend on different factors, including the form of administration, the point at which they are directed, the physiological state of the mammal and some other medication that is administered. In this way, the treatment dosage should be adjusted to optimize safety and efficacy and this is left to the ability of the experienced technician. The determination of the appropriate posology and the administration scheme for a particular situation are within the skill in the art. Optionally, the individual is also administered a vaccine that produces a protective immune response against the infectious agent causing the persistent infection. For example, the individual receives a vaccine that generates an immune response against the human immunodeficiency virus (HIV), tuberculosis, influenza or hepatitis C. Illustrative vaccines are described, for example, in the publication of Berzofsky et al. (J. Clin Invest. 114: 456-462, 2004). If desired, the vaccine is administered with a booster or with auxiliaries. The PD-1 inhibitors are administered in an amount sufficient to increase the cytotoxicity of T cells, for example, CD8 + T cells. An increase in the cytotoxicity of T cells results in an increase in the immune response and a reduction in persistent infection. An immune response is measured, for example, by an increase in the proliferation of immune cells, for example, T cells or B cells, an increase in the production of cytokines and an increase in clearance of the infectious agent. This reduction includes relief of one or more of the symptoms associated with the persistent infection. Administration of the PD-1 inhibitor reduces persistent infection or alleviates one or more of the symptoms associated with persistent infection in a minimum proportion of 10%, 20%, 30%, 40%, 50%, 60%, 70% 80%, 90% or 100% compared to an untreated individual. The treatment is effective if it results in a clinical benefit, for example, a decrease in the burden of the infectious agent in the individual. When the treatment is applied for prophylactic purposes, the term "effective" means that the treatment delays or prevents the infection. Efficacy can be determined by any known method for diagnosis or treatment of the particular infection.

Therapeutic Administration The invention includes administering to an individual a composition containing a compound that reduces the expression or activity of PD-1 (referred to herein as "PD-1 inhibitor" or "therapeutic compound"). An effective amount of a therapeutic compound is preferably from about 0.1 mg / kg to 150 mg / kg. Effective doses vary, as will be recognized by one skilled in the art, depending on the route of administration, the use and type of excipients, and co-administration with other therapeutic treatments that include the use of other anti-infective agents or therapeutic agents to treat, prevent or alleviate a symptom of a particular infection or cancer. A therapeutic scheme is carried out, using standard methods, by identifying a mammal, for example, a human patient suffering from (or at risk of developing) an infection or cancer, using standard methods. The pharmaceutical compound is administered to this individual by methods known in the art. Preferably, the compound is administered orally, rectal, nasal, topical or parenteral, for example, subcutaneously, intraperitoneally, intramuscularly and intravenously. The compound is administered prophylactically or after the detection of an infection. Optionally, the compound is formulated as a component of a mixture of therapeutic agents intended to treat an infection. Examples of formulations suitable for parenteral administration include aqueous solutions of the active ingredient in an isotonic saline solution, a 5% glucose solution or other common pharmaceutically acceptable excipient. Common solubilizing agents such as PVP (polyvinylpyrrolidone) or cyclodextrins are also used as pharmaceutical excipients for the delivery of therapeutic compounds. The therapeutic compounds described herein are formulated in compositions intended for other routes of administration, by conventional methods. For example, for oral administration, the PD-1 inhibitor is formulated in a capsule or tablet. The capsules may contain any common pharmaceutically acceptable material such as gelatin or cellulose. Tablets can be formulated according to conventional procedures by compressing the mixture of a therapeutic compound with a solid carrier and a lubricant. Examples of solid carriers include starch and bentonite modified with sugar. The compound is administered in the form of a hard shell tablet or a capsule containing a binder, for example, lactose or mannitol, a conventional filler and a tableting agent. Other formulations include Ointments, suppositories, pastes, sprays, patches, creams, gels, resorbable sponges or foams. These formulations are produced by methods known in the art. If the therapeutic compound is a nucleic acid encoding a protein, the therapeutic nucleic acid is administered in vivo to promote the expression of its encoded protein, constructing it as part of a nucleic acid expression vector and administering it to be made intracellular (by example, by the use of a retroviral vector, by direct injection, by the use of microparticle bombardment, by coating with lipids or cell surface receptors or transfection agents, or by administering it bound to a homeobox-like peptide that is known to enter the nucleus (see, Joliot et al., 1991. Proc. Na ti. Acad. Sci. USA 88: 1864-1868), and the like As an alternative, a therapeutic nucleic acid is introduced intracellularly and incorporated into the cell's DNA. host for expression, for Homologous recombination or remains in episomal form. For local administration of DNA, standard vectors are used for gene therapy. These vectors comprise viral vectors, including those derived from deficient replication hepatitis viruses (eg, HBV and HCV), retroviruses (see, for example, WO 89/07136; Rosenberg et al., 1990 , N. Eng. J. Med. 323 (9): 570-578), adenovirus (see, for example, Morsey et al., 1993, J. Cell. Biochem., Supp. 17E) adeno-associated virus (Kotin et al. 1990, Proc. Na ti, Acad. Sci. USA 87: 2211-2215), replication-defective herpes simplex virus (HSV, Lu et al., 1992 Abstract, page 66, Abstracts of the Meeting on Gene Therapy , Sept. 22-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) and any modified versions of these vectors. The invention can use any other delivery system that performs the in vivo transfer of nucleic acids into eukaryotic cells. For example, nucleic acids can be packaged in liposomes, for example, cationic liposomes (Lipofectin), receptor-mediated delivery systems, non-viral nucleic acid vectors, phantom erythrocytes or microspheres (e.g., microparticles; see, e.g., U.S. Patent Nos. 4,789,734, 4,925,673, 3,625,214; Greogoriadis, 1979, Drug Carriers in Biology and Medicine, pgs. 287-341 (Academic Press), pure DNA can also be administered. The DNA in gene therapy can be administered to the patient parenterally, for example, intravenously, subcutaneously, intramuscularly and intraperitoneally. The DNA or an inducing agent is administered in a pharmaceutically acceptable carrier, ie, a biologically compatible carrier that is suitable for administration to an animal, for example, a physiological saline solution. A therapeutically effective amount is an amount that is capable of producing a desirable medical result, for example, the decrease of a gene product of PD-1 in a treated animal. This amount can be determined by a technician with ordinary experience. As is known in the field of medical technology, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, particular compound being administered, sex, time and the route of administration, general health and the concurrent administration of other medications. The dosage may vary, although a preferred dose for intravenous administration of DNA is from about 10 6 to 10 22 copies of the DNA molecule.

Typically, the plasmids are administered to a mammal in an amount of about 1 nanogram 5000 micrograms of DNA. Preferably, the compositions contain about 5 nanograms to 1000 micrograms of DNA, 10 nanograms to 800 micrograms of DNA, 0.1 micrograms to 500 micrograms of DNA, 1 microgram to 350 micrograms of DNA, 25 micrograms to 250 micrograms of DNA or 100 micrograms to 200 micrograms of DNA. Alternatively, the administration of recombinant adenoviral vectors encoding the PD-1 inhibitor in a mammal can be done at a minimum concentration of 105, 106, 107, 108, 109, 1010 or 1011 plaque forming units (pfu). The gene products of PD-1 are administered to the patient intravenously in a pharmaceutically acceptable carrier, for example, a physiological saline solution. Standard methods for intracellular delivery of peptides, for example, packaged in liposomes, can be used. These methods are well known to those of ordinary skill in the art. It is expected that an approximate dose of 1 to 100 moles of polypeptide of the invention per kg of body weight per day will be administered. The compositions of the invention are useful for parenteral administration, for example, administration intravenous, subcutaneous, intramuscular and intraperitoneal. The PD-1 inhibitors are effective at direct contact of the compound with the affected tissue. Therefore, the compound can be administered topically. Alternatively, the PD-1 inhibitors can be administered systemically. On the other hand, the compounds can be administered by implantation (directly onto an organ (for example, the intestine or the liver) or subcutaneously) of a solid or a resorbable matrix that slowly releases the compound into the surrounding tissues or adjacent to the individual. For example, for the treatment of a gastrointestinal infection, the compound can be administered systemically (for example, intravenously, rectally or orally) or locally (for example, directly in the gastric tissue). Alternatively, a wafer or a resorbable sponge impregnated with PD-1 inhibitor is placed in direct contact with gastric tissue. The PD-1 inhibitor is released in vivo slowly by diffusion of the active principle from the wafer and the erosion of the polymer matrix. As another example, liver infection (i.e., hepatitis) is treated by infusing a solution containing the PD-1 inhibitor into the vasculature of the liver. For the treatment of infections In neurological studies, the PD-1 inhibitor can be administered intravenously or intrathecally (ie, by direct infusion into the cerebrospinal fluid). For local administration, a wafer or a resorbable sponge impregnated with PD-1 inhibitor is placed in direct contact with CNS tissue. The compound or mixture of compounds are released slowly in vivo by diffusion of the active principle from the wafer and the erosion of the polymer matrix. Alternatively, the compound is infused into the cerebrospinal fluid or cerebrospinal fluid using standard methods. For example, a surgical drilling ring of the skull with an integrated catheter that is used as an injection port is placed to fix the skull in the surgical perforation. what was done to him The access to the reservoir of fluid connected to the catheter is made by medinating a needle or stylet inserted through the septum placed in the upper part of the piercing ring. The catheter unit (described, for example, in U.S. Patent No. 5, 954,687) allows a fluid flow path suitable for transferring fluids between a particular location, near or within the brain that makes the administration of the medication for a period of time.

For cardiac infections, the compound can be delivered, for example, into cardiac tissue (i.e., myocardium, pericardium or endocardium), by direct intracoronary injection through the chest wall or using methods using a percutaneous catheter as a base standard and fluoroscopic guidance. In this way, the inhibitor can be injected directly into the tissue or can be infused from a mesh or stent or a catheter inserted into a body lumen. A variety of perfusion or coronary catheters can be used to administer the compound. Alternatively, the compound is coated or impregnated into a stent that is placed in a coronary vessel. Pulmonary infections can be treated, for example, by administering the compound by inhalation. The compounds are supplied in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide or a nebulizer. A person skilled in the art will understand that patients treated according to the invention may have undergone the same tests to diagnose a persistent infection or they may have been identified without examination as someone at high risk due to the presence of one or more risk factors (for example, exposure to an infectious agent, genetic predisposition or having a pathological condition that predisposes to secondary infections). The reduction of symptoms or damage caused by the persistent infection may also include, among other effects, the relief of symptoms, the decrease in the degree of disease, stabilization of the disease (ie, without worsening), delay or delay of the advance of the disease and improving or mitigating the state of the disease. The treatment can be applied at home with the close supervision of the responsible physician or in the medical assistance facilities.

Methods for measuring the immune response Methods for measuring the immune response after treatment according to the present invention are well known in the art. The activity of the T cells can be evaluated, for example, by assays that detect the production of cytokines, assays that measure the proliferation of T cells, assays that measure the clearance of the microbial agent and assays that measure the cytotoxicity of CD8 + T cells . These tests are described, for example, in the Patent of the States U.S. No. 6,808,710 and the publications of patent application Nos. 20040137577, 20030232323, 20030166531, 20030064380, 20030044768, 20030039653, 20020164600, 20020160000, 20020110836, 20020107363 and 20020106730, both of which are considered part of the present, as reference . Optionally, the ability of a PD-1 inhibitor to increase the cytotoxicity of CD8 + T cells is evaluated by assays that measure the proliferation of CD8 + T cells (eg, thymidine incorporation, BrdU assays and staining with markers). of the cell cycle (eg, Ki67 and CFSE), described, for example, by Dong et al. (Nature 5: 1365-1369, 1999) In one example, the proliferation of T cells is monitored by culturing the purified T cells expressing PD-1 with a PD-1 inhibitor, a primary activation signal as described above and 3H-thymidine The level of T cell proliferation is determined by measurement of thymidine incorporation. Cytotoxicity of CD8 + T cells is also assessed by lysis assays (eg, 51Cr release assays or assays that detect the release of perforin or granzyme), assays that detect caspase activation or assays that measure n the release of the microbial agent of the infected individual. For example, the viral load in a biological sample of the infected individual (eg, serum, spleen, liver, lung or tissue to which the virus is tropic) can be measured before or after treatment. The production of cytokines such as IFN ?, TNFa and IL-2 can also be measured. For example, purified T cells are cultured in the presence of the PD-1 inhibitor protein and a primary activation signal. The level of several cytokines in the supernatant can be determined by sandwich enzyme-linked immunosorbent assays or other conventional assays described, for example, in the Dong et al. (Nature 5: 1365-1369, 1999). If desired, the efficacy of the PD-1 inhibitor is assessed through its ability to induce T cell co-stimulation. For example, a method for in vitro co-stimulation comprises providing purified T cells expressing PD-1. with a first or primary activation signal by an anti-CD3 monoclonal antibody or a phorbol ester or by an antigen in combination with an MHC class II. The ability of a candidate compound to reduce the expression or activity of PD-1 and therefore to generate the costimulatory or secondary signal needed to modulating immune function, for these T cells, can be evaluated by any of the conventional assays well known in the art. A B-cell response is assessed by a specific ELISA antigen assay (eg, LCMV, HIV, tuberculosis or malaria), plasma cell ELISPOT, B-cell memory assay, B-cell phenotyping and analysis of germinal centers by immunohistochemistry .

Screening Assays The present invention provides screening methods for identifying compounds that can inhibit the expression or activity of PD-1. Useful compounds include any agent that inhibits biological activity or reduces the cellular level of PD-1. For example, candidate compounds can reduce the binding of PD-1 to PD-Ll or PD-L2 or both. Using these agents as breakthrough compounds, for example, these screening methods also allow the identification of other new PD-1 specific inhibitors that function to treat, reduce or prevent persistent infections or alternatively, that alleviate one or more symptoms associated with these infections. The screening method can include high-tech techniques performance . The term "candidate compound" refers to a chemical substance that may be of natural origin or an artificial derivative. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, organic molecules of natural origin, nucleic acid molecules, peptide nucleic acid molecules, and components derived therefrom. For example, a candidate compound useful according to the present invention reduces the binding of PD-1 to PD-Ll or to PD-L2 or both. There are several methods to carry out these screening tests. According to one approach, the candidate compounds are added in varying concentrations to the culture medium of cells expressing PD-1. The term "PD-1 gene" refers to a nucleic acid encoding a PD-1 protein. The term "PD-1 fusion gene" refers to a PD-1 promoter and / or all or part of a PD-1 coding region operably linked to a second heterologous nucleic acid sequence. In preferred embodiments, the second heterologous nucleic acid sequence is a reporter gene, i.e., a gene whose expression can be evaluated; reporter genes include, among others, those that encode glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), green fluorescent protein (GFP), alkaline phosphatase and beta-galactosidase. The genetic expression of PD-1 is measured, for example, by Northern blot analysis (Ausubel et al., see above), using a fragment prepared from the PD-1 nucleic acid molecule as a hybridization probe or by real-time PCR with appropriate primers. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. If desired, the effect of the candidate compound, alternatively, can be measured by the level of PD-1 polypeptide using the same general approach and standard immunological techniques, eg, Western blot or immunoprecipitation with an antibody specific for PD-1. For example, immunoassays can be used to detect or monitor the level of PD-1. Monoclonal or polyclonal antibodies that can bind to PD-1 can be used in any standard immunoassay format (e.g., ELISA or RIA) to measure PD-1 levels. PD-1 can also be measured by mass spectrometry, high performance liquid chromatography, spectrophotometric or fluorometric techniques, or combinations thereof.

Alternatively, the screening methods of the invention can be used to identify candidate compounds that decrease the biological activity of PD-1 by reducing the binding of PD-1 to PD-Ll or PD-L2 or both, in a proportion minimum of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% with respect to an untreated control. For example, a candidate compound can be analyzed for its ability to decrease the activity of PD-1 in cells that naturally express PD-1, after transfection with cDNA for PD-1 or in solutions containing PD-1 lacking cells, as described below. The effect of a candidate compound on the binding or activation of PD-1 can be assessed by radioactive and non-radioactive binding assays, competition assays and receptor signaling assays. As a specific example, mammalian cells (e.g., rodent cells) are cultured that express a nucleic acid encoding PD-1, in the presence of a candidate compound (e.g., a peptide, polypeptide, a synthetic organic molecule, a organic molecule of natural origin, a nucleic acid molecule, or components thereof). Cells can be those that express PD-1 endogenously or manipulate genetically, by some technique standard known in the art, (eg, transfection and viral infection) to overexpress PD-1. The level of expression of PD-1 is measured in these cells by Western blot analysis and then compared to the level of expression of the same protein in control cells that have not been in contact with the candidate compound. A compound that promotes a decrease in the activity level of PD-1 as a result of the decrease in its synthesis or its biological activity is considered useful in the invention. In a particular example, a compound that interferes with the binding of PD-1 to PD-Ll or PD-L2 or both (thereby reducing the biological activity of PD-1), resulting in an increase in the immune response , it is useful according to the present invention. Given its ability to decrease the biological activity of PD-1, this molecule can be used, for example, as a therapeutic agent to treat, reduce or prevent a persistent infection or alternatively to alleviate one or more symptoms associated with these infections. As a specific example, a candidate compound can be contacted with two proteins, the first protein is a polypeptide virtually identical to PD-1 and the second protein is PD-Ll or PD-L2 (i.e., a protein that binds to the PD-1 polypeptide under conditions that allow binding and which result in a reduced immune response). According to this particular screening method, the interaction between these two proteins is measured after the addition of the candidate compound. A decrease in the binding of PD-1 to the second polypeptide after the addition of the candidate compound (with respect to this binding in the absence of the compound), identifies the candidate compound as a compound having the ability to inhibit the interaction between the two proteins . Finally, the screening assay of the invention can be carried out, for example, in a cell-free system or using a hybrid system of two yeasts. If desired, one of the proteins or candidate compound can be immobilized on a support, as described above, or it can have a detectable group. Alternatively or in addition, the candidate compounds can be selected according to the binding specificity to PD-1 and therefore inhibit it. The efficacy of this candidate compound depends on its ability to interact with PD-1. This interaction can be easily analyzed using any of the standard binding techniques and functional assays (for example, those described by Ausbel et al., See above). For example, a candidate compound can be analyzed in vi tro in terms of its interaction and binding with PD-1 and its ability to modulate immune responses can be assessed by any of the standard assays (eg, those described herein). For example, a candidate compound that binds PD-1 can be identified by a chromatographic technique. For example, a recombinant PD-1 can be purified by standard techniques from cells genetically engineered to express PD-1 (for example, those described above) and can be immobilized on a column. Alternatively, PD-1 of natural origin can be immobilized in a column. A candidate compound solution is then passed through a column and a specific compound for PD-1 is identified based on its ability to bind PD-1 and is immobilized on the column. To isolate the compound, the column is washed and the molecules that did not bind specifically are removed, then the compound of interest is released from the column and harvested. The compounds isolated by this method (or any other suitable method), if desired, can be further purified (for example, by high performance liquid chromatography). The screening of new inhibitors and the optimization of forward compounds can be tested, for example, evaluating their ability to modulate the cytotoxic activity of T cells or the immune response, by standard techniques. On the other hand, these candidate compounds can be analyzed for their ability to function as antimicrobial agents (e.g., as described herein). The compounds isolated by this approach can also be used, for example, as therapeutic agents to treat, reduce or prevent persistent infections or alternatively to alleviate one or more symptoms associated with these infections. Compounds that are identified as compounds that bind to PD-1 with an affinity constant less than or equal to 10 mM are considered especially useful in the invention. Potential therapeutic agents include organic molecules, peptides, pseudopeptides, polypeptides and antibodies that bind to a nucleic acid or polypeptide sequence encoding PD-1 and thereby inhibit or extinguish its activity. Potential antimicrobial agents also include small molecules that bind to the point of attachment of this polypeptide and thereby prevent binding to cell binding molecules, such that normal biological activity is impeded. Other potential antimicrobial agents include antisense molecules.

Diagnostic and Prognostic Methods Cancer, for example, angioimmunoblastic T cell lymphoma or nodular lymphocyte predominant Hodgkin lymphoma, is detected by determining the amount of a PD-1 polypeptide in a test sample (ie, a sample taken from a patient) . A change in the level of PD-1 polypeptide with respect to a control sample is indicative of cancer in the individual. The change may be an increase or decrease in the PD-1 polypeptide relative to a control sample. The control sample is prepared (i.e., fractionated) in a manner similar to the test sample. A sample is, for example, blood, serum, ascites, urine or other body fluids. Preferably, the sample is a T cell or a B cell. The amount of PD-1 is determined in the test sample and compared to the expression of the normal control level. By "normal control level" is meant the level of expression of a PD-1 polypeptide that is normally found in an individual not suffering from cancer. An increase in the level of PD-1 in the sample from the patient indicates that the individual suffers from or is at risk of developing cancer. By contrast, when the methods are applied prophylactically, a similar level or a decrease in the level of the PD-1 polypeptide in the sample drawn from the patient, indicates that the individual does not suffer is not at risk of developing cancer. An increase in the level of PD-1 polypeptide in the sample derived from the patient, indicates that the individual suffers or is at risk of developing cancer. The alteration in the amount of the PD-1 polypeptide is statistically significant. The term "statistically significant" means that the alteration is greater than what could be expected by the change alone. The statistical significance is determined by the method known in the art. For example, statistical significance is determined by the p-value. The p-value is a measure of the probability of a difference between groups occurring during an experiment. (P (z = checked)). For example, a "p" value of 0.01 means that there is a probability in 100 of the result. The smaller the "p" value, the more likely the difference between the groups was caused by the treatment. An alteration is statistically significant if the "p" value is at least 0.05. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less. The "diagnostic accuracy" of a test, The test or method in relation to the ability of the test, assay or method to distinguish between patients who have cancer or are at risk of suffering from it is based on the "clinically significant presence" of a PD-1 polypeptide in a patient. The term "clinically significant presence" means that the presence of the PD-1 polypeptide in the patient (usually in a sample drawn from the patient) is greater or less than the threshold (or limit value) for that PD-1 polypeptide and by thus it indicates that the patient has cancer for which the sufficiently high presence of that protein is a marker. The terms "high degree of diagnostic accuracy" and "very high degree of diagnostic accuracy" refer to the test or assay for the PD-1 polypeptide with a certain threshold that correctly (accurately) indicates the presence or absence of cancer. A perfect test would have perfect accuracy. Thus, for individuals who have diabetes, the test would indicate only positive test results and would not report any of the individuals that were "negative" (there would be no "false negatives"). In other words, the "sensitivity" of the test (the proportion of true positives) would be 100%. On the other hand, for individuals who did not have diabetes, the test would indicate only the Negative test results and would not report any of the individuals as "positive" (there would be no "false positives"). In other words, the "specificity" (the proportion of true negatives) would be 100%. See, for example, O'Marcaigh AS, Jacobson RM, "Estimating The Predictive Value of a Diagnostic Test, How to Prevent Misleading or Confusing Results," Clin. Ped. 1993, 32 (8): 485-491, which analyzes the specificity, sensitivity and negative and positive prognostic values of a test, for example, a clinical diagnostic test. Changing the threshold or limit value of a test (or test) usually changes the sensitivity and specificity but in a qualitatively inverse relationship. For example, if the threshold is lowered, more individuals in the evaluated population will generally have test results above the threshold or limit value. Because individuals who have the results above the threshold or limit value, are reported as individuals who have the disease, condition or syndrome for which the test is performed, lowering the threshold or limit value will cause more individuals to report with positive results (that is, they have cancer). In this way, the test will indicate that cancer has a greater proportion of those who have it. However, at the same time, there will be more fakes positive because the test will indicate more people who do not have the disease, condition or syndrome (ie, people who are really "negative"), PD-1 polypeptide values above the threshold and therefore will be reported as positive (ie , who has the disease, condition or syndrome) instead of the test reporting as negative. Therefore, the specificity (actual negative proportion) of the test will decrease. In the same way, the elevation of the threshold or limit value will tend to decrease the sensitivity and increase the specificity. Therefore, to assess the accuracy and usefulness of the medical test, trial or proposed methods to assess the patient's condition, sensitivity and specificity should always be taken into account and be careful to consider the limit value at which they are reported. sensitivity and specificity because these can vary significantly in the range of limit values. However, there is an indicator that allows the representation of the sensitivity and specificity of a test, test or method across the entire range of limit values with only one value. This indicator is derived from the curve "receiver operating characteristics" ("ROC") for the test, test or method in question. See, for example, Shultz, "Clinical Interpretation of Laboratory Procedures," chapter 14 of Teitz, Fundamentals of Clinical Chemistry, Burtis and Ashwood (eds.), 4th ed. 1996, W.B. Saunders Company, pages 192-199; and Zweig et al. , "ROC Curve Analysis: An Example Showing The Relationships Among Lipid And Apolipoprotein Serum Concentrations In Identifying Patients With Coronary Artery Disease", Clin. Chem., 1992, 38 (8): 1425-1428. An ROC curve is an xy graph of sensitivity on the "y" axis, on a scale of zero to one (that is, 100%), against a value equal to one minus specificity on the "x" axis, on a scale of zero to one (that is, 100%). In other words, it is a graph of the proportion of true positives versus the proportion of false positives for that test, trial or method. To construct the ROC curve of the test, test or method in question, patients are evaluated by a perfectly accurate method or "gold standard" that is independent of the test, test or method in question, to determine if patients are negative or true positives for the disease, condition or syndrome (for example, coronary angiography is a gold standard test to detect the presence of coronary atherosclerosis). The patients also they examine using the test, test or method in question and for variable limit values patients are reported as positive or negative according to the test, trial or method. The sensitivity (proportion of true positives) and the value equal to one minus the specificity (whose value is equivalent to the proportion of false positives), are determined for each threshold or limit value and each pair of values x and are plotted as a single point in the diagram xy. The "curve" that connects these points is the ROC curve. The area under the curve ("ABC") is the indicator that allows the representation with a single value of the sensitivity and specificity of a test, test or method across the entire range of threshold or limit values. The maximum ABC is one (in a perfect test) and the minimum area is half. The closer the ABC is to one, the better the accuracy of the test. The term "high degree of diagnostic accuracy" refers to a test or assay (such as the test of the invention to determine the clinically significant presence of PD-1 polypeptide, by which the presence of diabetes is indicated) in which the ABC (area under the ROC curve for the test or test) is at least 0.70, preferably at least 0.75, more preferably at least 0.80, preference of at least 0.85, more preferably at least 0.90, and most preferably at least 0.95. The term "very high degree of diagnostic accuracy" refers to a test or assay in which the AUC (area under the ROC curve for the test or test) is at least 0.875, preferably at least 0.90, with greater preference of at least 0.925, preferably at least 0.95, more preferably at least 0.975, and most preferably at least 0.98. Optionally, the expression of other known biomarkers for a particular cancer is also determined as another indication of whether or not the individual is a carrier of cancer. For example, CD10, bcl-6, CD20, CD57 or CXCR5 are detected. The PD-1 polypeptide and additional biomarkers are detected in any way that is suitable, but are usually detected by placing a patient-derived sample in contact with an antibody that binds PD-1 or biomarker and then detecting the presence or absence of a reaction product. The antibody can be monoclonal, polyclonal, chimeric or a fragment of the foregoing, as discussed in detail above; the detection stage of the reaction product can be carried out by any suitable method of immunoassay. The sample of the individual is usually a biological fluid as described above and may be the same sample of biological fluid that is used for the method described above. The expression of a PD-1 polypeptide also allows to monitor the course of cancer treatment. In this method, a biological sample is obtained from an individual subject to treatment, for example, surgical, chemotherapeutic or hormonal treatment, for cancer. If desired, biological samples are obtained from the individual at various times, before, during or after treatment. The expression of PD-1 is determined and compared against a reference, for example, a control of which the cancer state is known. The reference sample has been exposed to the treatment. Alternatively, the reference sample has not been exposed to the treatment. Optionally, this monitoring is carried out in a preliminary manner with surgical surveillance procedures and subsequent surgical surveillance procedures. For example, samples may be collected from individuals who have received initial surgical treatment for cancer and subsequent treatment with antineoplastic agents for cancer. same and monitor the progress of the treatment. If the reference sample is from an individual who does not have cancer, the similarity or a decrease of 1 amount of the PD-1 polypeptide in the test sample and the reference sample, indicates that the treatment is effective. However, an increase in the amount of PD-1 polypeptide in the test sample and the reference sample indicates a less favorable clinical outcome or prognosis. The term "effective" means that the treatment causes a decrease in the amount of PD-1 polypeptide or a decrease in the size, prevalence or metastatic potential of a tumor in an individual. Cancer evaluation is done using standard clinical protocols. The efficacy is determined together with any known method for diagnosis or treatment of the particular tumor. The expression of a PD-1 polypeptide also allows the identification of patients who are sensitive to systemic treatment, for example, chemotherapeutic, hormonal or radiation therapy. In this method, the biological sample of an individual is obtained before it undergoes surgical treatment for cancer. The expression of a PD-1 polypeptide is then determined and compared to a biological sample obtained from a patient after the Surgical removal of cancer. Probably, the patient will be sensitive to systemic treatment if the amount of PD-1 polypeptide decreases after the surgical removal of the cancer. On the contrary, a patient will not be sensitive to systemic treatment if the amount of polypeptide remains constant or increases after the surgical removal of the cancer. The expression of PD-1 polypeptide or other cancer biomarkers is determined at the protein or nucleic acid level by any of the methods known in the art. For example, Northern blot analysis using probes that specifically recognize one or more of these sequences can be applied to determine gene expression. Alternatively, expression is measured using reverse transcription PCR assays, for example, by means of specific primers for the sequence of differentiated expression genes. The expression is also determined at the protein level, that is, by measuring the levels of peptides encoded by the gene products described herein or the activities thereof. These methods are well known in the art and include, for example, immunoassays based on antibodies of proteins encoded by the genes. Any biological material can be used for detection and / or quantification of the protein or its activity. As an alternative, a suitable method can be selected to determine the activity of proteins encoded by the marker genes according to the activity of each protein analyzed. Preferably, the individual is a mammal. The mammal is, for example, a person, a non-human primate, mouse, rat, dog, cat, horse or cow. In general, individuals are men or women. The individual has previously been diagnosed as having cancer and may have already undergone treatment. As an alternative, the individual has not been previously diagnosed as a carrier of cancer. The present invention is useful with all patients who are at risk of developing cancer. Although each type of cancer has its own set of factors, the risk of developing cancer increases with age, gender, race and personal and family medical history. Other risk factors are closely related to lifestyle, at the same time certain infections, occupational exposure and some environmental factors may also be related to the development of cancer. The diagnosis of cancer is usually made through the identification of a mass when doing an examination, although it can also be done by other means such as radiological diagnosis or ultrasound. The treatment usually involves cytoreductive surgery followed by treatment with antineoplastic agents such as docetaxel, vinorelbine, gemcitabine, capecitabine or combinations of cyclophosphamide, methotrexate and fluorouracil; cyclophosphamide, doxorubicin and fluorouracil; doxorubicin and cyclophosphamide; doxorubicin and cyclophosphamide with paclitaxel; doxorubicin followed by CMF; or cyclophosphamide, epirubicin and fluorouracil. On the other hand, many patients will require radiation therapy. The immunoassays performed in accordance with the present invention can be homogeneous or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves the specific antibody (e.g., PD-1 polypeptide), a labeled analyte and the sample of interest. The signal that comes from the tag or tag is modified directly or indirectly by binding the antibody to the labeled analyte. Both the immunological reaction and the detection of the intensity thereof are carried out in a homogeneous solution. The immunochemical labels that may be used include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages or coenzymes. In a heterogeneous trial approach, the Reagents, in general, are the sample, the antibody and the means to produce a detectable signal. Samples such as those described above can be used. The antibody, usually, it is immobilized on a support, for example, a bead, a plate or a slide and is brought into contact with the specimen that supposedly contains the antigen in a liquid phase. Then, the support is separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means to produce said signal. The signal is related to the presence of the analyte in the sample. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels or enzymatic labels. For example, if the antigen to be detected has a second point of attachment, an antibody that binds to that point can be conjugated to a detectable group and added to the reaction solution in liquid phase before the separation step. The presence of the detectable group in the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are radioimmunoassays, immunofluorescence methods or immunoassays linked to enzymes. For those skilled in the art they will be family the specific immunoassay formats and their variations, which may be useful to carry out the methods set forth herein. See, in general, E. Maggio, Enzyme-Immunoassay (1980) (CRC Press Inc., Boca Raton, Fia.); see also U.S. Patent No. 4, 727,022 to Skold et al. entitled "Methods for Modulating Ligand-Receptor Interactions and their Application", U.S. Patent No. 4, 659,678 to Forrest et al. entitled "Immunoassay of Antigens", U.S. Patent No. 4, 376,110 by David et al. , entitled "Immunometric Assays Using Monoclonal Antibodies", U.S. Patent No. 4, 275,149 to Litman et al. entitled "Macromolecular Environment Control in Specific Receptor Assays", U.S. Patent No. 4, 233,402 to Maggio et al. , entitled "Reagents and Methods Employing Channeling" and U.S. Patent No. 4, 230,767 of Boguslaski et al. entitled "Heterogeneus Specific Binding Assay Employing a Coenzyme as Label". The antibodies are conjugated to a solid support suitable for a diagnostic assay (for example, beads, plates, slides or wells formed with materials such as latex or polystyrene) according to known techniques, such as precipitation. The antibodies, as described herein, can also be conjugated with detectable groups such as radiolabels (eg, 35 S, 125 I, 131 I), enzymatic labels (e.g., horseradish peroxidase, alkaline phosphatases) and fluorescent labels (e.g., fluorescein) in accordance with known techniques. The diagnostic kits for carrying out the methods described herein, are produced in different ways. In one embodiment, the diagnostic kit comprises (a) an antibody (e.g., PD-1 polypeptide) conjugated to a solid support and (b) a second antibody of the invention conjugated to a detectable group. The reagents may also include auxiliary agents such as buffers and protein stabilizers, for example, polysaccharides and the like. The diagnostic kit may also include, if necessary, other members of the signal producing system of which the detectable group is an element (eg, enzyme substrates), agents to reduce background interference in a test, control reagents , devices to perform the test, and the like. Alternatively, a kit contains (a) an antibody and (b) a specific binding element for the antibody conjugated to the detectable group. The auxiliary agents described in the foregoing may also be included. This case is packed in any way adequate, usually, with all the elements in a single container along with a printed instruction sheet to perform the test. This invention, in part, is based on the experiments described in the following examples. These examples are intended to illustrate the invention and should not be construed as limiting thereof.

Example 1: Inhibition of the path of PD-1 in chronically infected mice, by means of anti-PD-Ll antibodies. Mice infected with several strains of lymphocytic choriomeningitis virus (LCMV) were used to study the effect of chronic viral infection on the function of CD8 + T cells. The Armstrong LCMV strain causes an acute infection that is eliminated in 8 days, leaving behind a long-standing population of resting high-functioning CD8 + T cells. In contrast, strain LCMV Cl-13 establishes a persistent infection in the host characterized by a viremia that lasts up to 3 months. The virus remains in some tissues indefinitely and the antigen-specific CD8 + T cells become functionally deficient. The CD8 + T cells DbNP396-404 are physically removed, while the CD8 + T cells DbGP33-41 and DbGP276-286 persist but lose their ability to proliferate or secrete antiviral cytokines, like IFN? and TNFa. C57BL / 6 mice were purchased from the National Cancer Institute (Na tional Cancer Ins ti tute) (Fredecrick, MD). The mice were infected intravenously with 2 x 106 pfu of LCMV-C1-13. CD4 depletion was performed by injecting 500 μg of GK 1.5 in PBS on the day of infection and the next day. Mice immune to LCMV were generated by infecting them intraperitoneally with 2 x 105 pfu of Armstrong LCMV. Genomic matrix analysis was performed on naive DbGP33-41 P14 transgenic specific CD8 + T cells purified by FACS, DbGP33-41 specific CD8 + T cells derived from Armstrong LCMV immune mice and DbGP33-41 or DbGP236 specific CD8 + T cells. -286 derived from mice infected with LCMV Cl-13 deficient in CD4 +. RNA isolation and gene array analysis were done as described in the publication by Kaech et al. , (Cell 111: 837-51, 2002). He PD-1 mRNA was strongly expressed in depleted CD8 + T cells compared to memory CD8 + T cells (Figure IA). On the other hand, PD-1 was expressed on the surface of CD8 + T cells in mice infected with LCMV Cl-13, but was not present in the surface of the CD8 + T cells after the removal of the Armstrong LCMV (Figure IB). Mice with chronic infection also expressed high levels of the PD-1 ligands, PD-Ll, in the majority of the lymphocytes and APC compared to the uninfected mice. In this way, viral antigenic persistence and depletion of CD8 + T cells are concurrent with the induction of PD-1 expression. To test the hypothesis that blocking the PD-1 / PD-L1 route can restore T cell function and increase viral control during chronic LCMV infection, the PD-l / PD-Ll coinhibitory path was interrupted during chronic LCMV infection by blocking antibodies aPD-Ll. Intraperitoneally and every third day, a monoclonal blocking antibody against PD-Ll was administered to mice infected with LCMV Cl-13 (200 μg of mouse anti-PD-Ll rat IgG2b monoclonal antibodies (clone 10F.5C5 or 10F.9G2 )) from day 23 to day 37 post-infection. On day 37, there were approximately 2.5 times more CD8 + T cells specific for DbNP396-404 and 3 times more CD8 + T cells specific for DbGP33-41 in treated mice compared to the untreated controls (Figure 2A). The induction of proliferation was specific for CD8 + T cells since the number of CD4 + T cells in the spleen was approximately the same in both the treated and untreated mice (~6 x 10 4 IAbGP61-80 CD4 + T cells per spleen). In addition to an increase in the proliferation of CD8 + T cells, the inhibition of PD-1 signaling results in an increase in antiviral cytokine production in virus-specific CD8 + T cells. The production of IFN? and TNFa in CD8 + T cells for eight different epitopes. The combined response was 2.3 times higher in the treated mice compared to the untreated mice (Figures 2B and 2C). After treatment, a double increase in the frequency of TNFa producing cells was also observed (Figure 2D). Viral clearance also accelerated as the virus was removed from the serum, spleen and liver of the treated mice. Reduced viral values (~ 10-fold) were observed at day 37 post-infection (14 days after the start of treatment) in the lung and kidney of the treated mice. However, untreated mice showed significant levels of virus in all these tissues (Figure 2E). Viral values in serum and tissue homogenates were determined by Vero cells, as described in the publication by Ahmed et al. (J. Virol. 51: 34-41, 1984).

The results show that a PD-1 inhibitor increases the proliferation of CD8 + T cells and viral clearance and this indicates that the inhibition of PD-1 signaling re-establishes the function of CD8 + T cells. On the other hand, the inhibition of PD-1 signaling also enhances B-cell responses since the number of LCMV-specific antibody-secreting cells also increased (> 10-fold) after treatment. CD4 + T cells play a key role in the generation and maintenance of CD8 + T cell responses. In this regard, sensitized CD8 + T cells in the absence of CD4 + T cells (termed "useless" CD8 + T cells) are unable to generate normal immune responses. On the other hand, chronic LCMV infection is more severe in the absence of CD4 + T cells. Consequently, the useless T cells generated during infection by LCMV Cl-13 show an even more intense functional deficiency than the T cells generated in the presence of CD T cells. The CD8 + T cells specific for DbNP396-404 are reduced to undetectable levels and the CD8 + T cells DbGP33-41 and DbGP276-286 completely lose the ability to secrete IFNα. and TNFa. The CD4 + T cells were eliminated at the time infection with LCMV Cl-13 and mice were treated with anti-PD-Ll antibody treatment from day 46 to day 60 post-infection. LCMV specific CD4 + T cells were not detectable by IFNα staining. intracellular before or after treatment. After the treatment, the treated mice had approximately 7 times more CD8 + T cells DGP276-286 and 4 times more CD8 + T cells DbGP33-41 in the spleen than the untreated control mice (Figure 3A). The number of virus-specific CD8 + T cells in the spleen also increased (Figure 3B). This increase in virus-specific CD8 + T cells in the treated mice was attributed to an increase in proliferation, as detected by incorporation of BrdU. 43% of CD8 + T cells DGP276-286 incorporated intermediate levels of BrdU and 2% incorporated high levels of BrdU in untreated mice, while in treated mice 50% of CD8 + T cells DbGP276-286 incorporated intermediate levels of BrdU and 37% incorporated high levels of BrdU. The BrdU analysis was performed by adding 1 mg / ml of BrdU in the drinking water during the treatment and the staining was done according to the manufacturer's protocol (BD Biosciences, San Diego, CA). On the other hand, the treated mice had a higher percentage of CD8 + T cells that expressed the KI67 protein associated with the cell cycle (60% versus 19% in untreated mice, Figure 3C). Response to treatment in CD8 + T cells in PBMC was restricted to mice that had high levels of CD8 + T cell expansion. The inhibition of PD-1 also increased the production of antiviral cytokine in diseased and useless CD8 + t-cells of the virus. After treatment, the number of CD8 + T cells DbGP33-41 and DbGP276-286 that produce IFN? it was markedly increased (Figure 4A), although higher numbers of CD8 + T cells specific for DbNP396-404, KbNP205-212, DbNP166-175 and DbGP92-101 were also detected in treated mice (Figure 4A). 50% of CD8 + T cells specific for DbGP276-286 of treated mice can produce IFN? compared to 20% of CD8 + T cells specific for DbGP276-286 in untreated control mice (Figure 4B). However, the levels of IFN? and TNF [alpha] produced by T-cell-specific contents of DbGP276-286 of treated mice, were lower than functional memory cells specific for DbGP276-286 (Figure 4C). The inhibition of PD-1 also increased the lytic activity of the CD8 + T cells specific to the virus, exhausted and useless. Ex vivo lytic activity of virus-specific CD8 + T cells was detected after treatment by a release assay of 51 Cr (Wherry et al., 2003. J. Virol. 77: 4911-27). Viral values were reduced after 2 weeks of treatment, approximately 3 times in the spleen, 4 times in the liver, 2 times in the lung and 2 times in the serum with respect to the untreated mice (Figure 4E). Therefore, these results demonstrate that blocking the PD-1 pathway breaks down the peripheral tolerance of cytotoxic T cells (CTL) to a chronic viral infection and that depleted CD8 + T cells lacking the support of CD4 + T cells do not they are inactivated irreversibly.

Example 2: Administration of an antiviral vaccine and a PD-1 inhibitor. An approach to reinforce T cell responses during a persistent infection is therapeutic vaccination. The rationale for this approach is that endogenous antigens may not present in an oal or immunogenic manner during chronic viral infection and that supplying an antigen in the form of a vaccine may provide a more effective stimulus to the virus-specific B and T cells. Using the chronic LCMV model, mice were administered a variolovacunal virus expressing the epitope LCMV GP33 as the therapeutic vaccine (VVGP33), which resulted in a modest increase in CD8 + T cell responses in some chronically infected mice. Four of the nine mice with chronic infection that received the therapeutic vaccine showed a positive response while none of the control mice had a significant increase in the immune response against GP33. When this therapeutic vaccination was combined with an inhibitor of PD-Ll, LCMV specific T cell responses were reinforced to a greater degree compared to treatment alone and the effect of the combined treatment was more than additive.

Example 3: Inhibition of the PD-1 pathway in chronically infected mice, by means of PD-1 RNAi. The interfering RNA (RNAi) is capable of silencing the gene expression in mammalian cells. Long-stranded double-stranded RNA (dsRNA) is introduced into the cells and then processed and smaller silencing RNA molecules (siRNAs) are formed that target specific mRNA molecules or a small group of mRNA. This technology is very useful in situations in which antibodies are not functional. For example, RNAi can be used in a situation in which unique splice variants produce soluble forms of PD-1 and CTLA-4. The silencing RNA of PD-1 is inserted into a commercial siRNA expression vector, for example, pSilencer * expression vectors or adenoviral vectors (Ambion, Austin, TX). These vectors are contacted with depleted white T cells, ex vivo or in vivo (See, Example 4).

Example 4: Ex vivo rejuvenation of depleted T cells Depleted CD8 + T cells specific for the virus are isolated from mice chronically infected with LCMV Cl-13, using magnetic beads or density centrifugation. The transfected CD8 + T cells are contacted with a monoclonal antibody that is directed to PD-Ll, PD-L2 or PD-1. As described in Example 1, inhibition of the PD-1 pathway results in the rejuvenation of CD8 + T cells. Accordingly, there is an increase in the proliferation of CD8 + T cells and the production of cytokines, for example. These rejuvenated CD8 + T cells are reintroduced into the infected mice and the viral load is measured as described in Example 1.

Example 5: In vitro screening of new CD8 + T cell rejuvenating compounds Compounds that modulate the PD-1 pathway can be identified by in vivo and ex vivo assays based on their ability to reverse the depletion of CD8 + T cells derived from a chronic viral infection. Exhausted CD8 + T cells are extracted from mice chronically infected with LCMV Cl-13 and then contacted with a test compound. The amount of antiviral cytokines (e.g., IFNα or TNFα) released from the T cells that were contacted is measured, for example, by ELISA or other quantitative method and compared with the amount, if any, of antiviral cytokines released from depleted T cells that did not come into contact with the test compound. An increase in the amount of antiviral cytokine released by the treated cells compared to the amount released in the untreated cells identifies the compound as an inhibitor of PD-1 which is useful for modulating the activity of T cells.

Example 6: In vivo screening of new CD8 + T-cell rejuvenating compounds Exhausted CD8 + T cells are extracted from mice chronically infected with LCMV Cl-13. Intravenously, a test compound is administered to the infected mice. The number of cytokines is measured antivirals (e.g., IFNα or TNFα) that are released in the serum of treated and untreated mice, for example, by ELISA or other quantitative method and the comparison is made. An increase in the amount of antiviral cytokines found in the serum of treated mice, relative to the amount in the untreated mice, identifies the test compound as an inhibitor of PD-1. Alternatively, the viral value (e.g., serum viral value) can be determined before and after treatment with the test compound.

Example 7: Chimpanzees as a model for immunotherapy of persistent HCV infection Chimpanzees provide a model for the persistence of HCV in humans. The deficient immunity of the T cells results in the persistence of a long-lived virus that includes both a deficit in HCV-specific CD4 T helper cells and a deficient or altered activity of the effector CD8 + T cells. Persistently infected chimpanzees are treated with antibodies against CTLA-4, PD-1 or a combination of the two. The efficacy of blocking inhibitory pathways is determined, combined with vaccination using structural and non-structural HCV proteins, and It is also determined if these strategies can increase the frequency and longevity of the T cells of virus-specific memory. The immunity defect of T cells is exclusively specific for HCV in humans and chimpanzees with persistent infection. The blood and liver of infected chimpanzees are examined to evaluate the expression of CTLA-4, PD-1, BTLA and its ligands and to detect the presence of Treg cells. The antiviral activity can then be reestablished by supplying chimpanzees with humanized monoclonal antibodies that block signaling through these molecules. Chimpanzees with persistent infection are treated with humanized aCTLA-4 antibodies (MDX-010, Medarex) or aPD-1 antibodies. The initial dose of MDX-010 is 0.3 mg / kg, at 2 weeks 1.0 mg / kg and then 3, 10, and 30 mg / kg at three-week intervals. After treatment with antibodies to coinhibitory molecules, the cellular and humoral immune responses as well as the HCV RNA load are determined. Samples are collected at weeks 1, 2, 3, 5 and 8 and then at monthly intervals. Samples include: 1) serum for the analysis of transaminases, autoantibodies, neutralizing antibodies for HCV and cytokine responses, 2) plasma for viral load and genomic evolution, 3) PBMC for measurements of immunity in vi tro, 4) natural liver (unprepared) for the isolation of intrahepatic lymphocytes and RNA, and 5) prepared liver (incorporated in formalin / paraffin) for histological and immunohistochemical analysis. Regional lymph nodes are also collected at 2 or 3 time points, to evaluate the expression of coinhibitory molecules and variants of excision by immunohistochemical and molecular techniques. Trials to evaluate the efficacy and safety of these therapies will be performed as described herein. To determine if vaccination with HCV antigens reinforces the therapeutic effect of antibodies against PD-1, chimpanzees were treated as follows: 1) intramuscular immunization with recombinant envelope glycoproteins El and E2 (in auxiliary MF59) and other proteins (core plus NS 3, 4 and 5 formulated with ISCOMS) at weeks 0, 4 and 24; 2) intramuscular immunization with the vaccine used in 1) but co-administered with aCTLA-4 antibodies (30 mg per kg of body weight, intravenously at weeks 0, 4 and 24 when the vaccine was applied); 3) as well as 2) with the exception that aPD-1 (or BTLA) antibodies are substituted with CTLA-4 antibodies; 4) as well as groups 2) and 3) with the exception that in addition to the vaccine, a combination of CTLA-4 antibodies and PD-1 (or BTLA). The responses of HCV-specific T and B cells are monitored at monthly intervals after immunization for a period of 1 year. The markers examined in the HCV + tetramer and in the total T cells in this analysis include markers of differentiation (e.g., CD45RA / RO, CD62L, CCR7 and CD27), activation (for example, CD25, CD69, CD38 and HLA-DR), of survival / proliferation (for example, bcl-2 and Ki67), of cytotoxic potential (for example, granzymes and perforin) and cytokine receptors (CD122 and CD127). There is an interesting correlation between the levels prior to the chemokine IP-10 therapy and the response to PEG IFNα / ribavirin. The IP-10 levels are determined to investigate a possible correlation between negative regulatory pathways or HCV-specific T cell responses and IP-10 levels. The expression of inhibitory receptors and ligands in PBMC is carried out by flow cytometry.

Example 8: Immunostaining of PD-1 in reactive lymphoid tissue Ma terials The materials were obtained at Bringham & Women's Hospital, Boston, MA, in accordance with institutional policies. All diagnostics were based on the histological and immunophenotypic features described in the lymphoma classification system of the World Health Organization (Jaffe ES, et al., 2001) and in all cases the diagnostic material was reviewed by a hematopathologist.

Immunostaining Immunostaining of PD-1 was performed on formalin-fixed and paraffin-embedded tissue sections after antigen retrieval by microwave in 10 mM citrate buffer, pH 6.0 with a previously described human anti-PD-1 monoclonal antibody ( 2H7; 5), using a standard indirect method with avidin-biotin proxidase and color development with diaminobenzidine, as described previously (Jones D., et al., 1999, Dorfman DM, et al., 2003). The cases were considered immunoreactive for PD-1 if at least 25% of neoplastic cells showed positive staining. The staining of PD-1 was compared with that of the control antibody isotype IgG of mouse diluted to an identical concentration of protein in all the cases studied, to confirm the specificity of the staining. The monoclonal antibody 2H7 for PD-1 was used for staining specimens, fixed in formalin and embedded in paraffin, of reactive lymphoid tissue, thymus and a group of cases of lymphoproliferative disorders of T cells and B cells. In amygdala specimens showing reactive changes, including follicular hyperplasia, a subset of predominantly small lymphocytes in the germinal centers exhibited cytoplasmic staining for PD-1, and PD-1 positive cells were observed infrequently in the interfollicular zones of the T cells. The staining pattern of PD-1 in the germinal centers was almost identical that is observed with an antibody against CD3, a marker of pan-T cells, while an antibody against CD20, a marker of pan-B cells, stained the vast majority of germ-center B cells. Similar results were observed in the histological sections of reactive lymph nodes and spleen. No staining of PD-1 was observed in adult thymus.

Example 9: Immunostaining of PD-1 in tissue sections, embedded in paraffin, of B-cell and T-cell lymphoproliferative disorders A group of B-cell and T-cell lymphoproliferative disorders were studied for the expression of PD-1; the results are summarized in the Table 1. Forty-two cases of B cell lymphoproliferative disorders were studied for DP-1 expression, including representative cases of lymphoblastic lymphoma / B-cell precursor lymphoblastic leukemia, as well as a group of B-cell lymphoproliferative disorders. mature B cells, including several B-cell non-Hodgkin lymphomas of follicular origin, including 6 cases of follicular lymphoma and 7 cases of Burkitt's lymphoma. None of the B cell lymphoproliferative disorders showed staining for PD-1. In some cases, non-neoplastic reactive lymphoid tissue was present and showed a staining pattern of PD-1 as seen in amygdala and other reactive lymphoid tissues indicated above. Similarly, in 25 cases of Hodgkin's lymphoma, including 11 cases of classic Hodgkin's lymphoma and 14 cases of lymphocyte-predominant Hodgkin's lymphoma, the neoplastic cells did not show staining for PD-1. Interestingly, in the 14 cases of lymphocyte-predominant Hodgkin lymphoma, the T cells surrounding the neoplastic CD20 + L positive H cells were immunoreactive for PD-1, similar to the staining pattern observed for CD57 + T cells in the lymphoma of Hodgkin with lymphocytic predominance. These positive cells to PD-1 were a subset of the total population of CD3 + T cells present. A group of T cell lymphoproliferative disorders were studied for the expression of PD-1; the results are summarized in Table 1. Representative cases of lymphoblastic lymphoma / T-cell precursor lymphoblastic leukemia, an immature T-cell neoplasm, were negative for PD-1 as were T-cell neoplasms subsequent to thymus, including cases of T-cell prolymphocytic leukemia, peripheral T-cell lymphoma, large anaplastic T-cell lymphoma, non-specific and adult T-cell leukemia / lymphoma. In contrast, the 19 cases of angioimmunoblastic lymphoma had foci of PD-1 positive cells that were immunoreactive for markers of pan-T cells such as CD3. PD-1 positive cells were consistently found in foci of expanded CD21 + follicular dendritic cell networks, a characteristic feature of angioimmunoblastic lymphoma.

TABLE 1. Immunostaining of PD-1 in lymphoproliferative disorders Abbreviations: B-LL / LL - lymphoblastic lymphoma / lymphoblastic leukemia B cell precursor; CLL - chronic lymphocytic leukemia; MCL - cerebral cortex cell lymphoma; FL - follicular lymphoma; MZL - marginal zone lymphoma; HCL - hair cell leukemia; DLBCL diffuse large cell lymphoma; BL - Burkitt's lymphoma; LPL - lymphoplasmacytic lymphoma; MM - multiple myeloma; T-LL / L - lymphoblastic lymphoma / T-cell precursor lymphoblastic leukemia; T-PLL - T-cell prolymphocytic leukemia; AIL - angioimmunoblastic lymphoma; PTCL - peripheral T cell lymphoma, nonspecific; ALCL anaplastic large cell lymphoma; ATLL leukemia / adult T cell lymphoma.

* Number of immunoreactive cases / total number of cases * * Cells positive to PD-1 that form rosettes around neoplastic L & H cells in 14/14 cases.

Example 10: General Methods for Studying PD-1 Expression in HIV-specific human CD8 T cells The following methods were used to perform the detailed experiments in Examples 11-14.

Study subjects Study participants with chronic HIV-1 clade C infection were recruited from outpatient clinics at McCord hospital, Durban South Africa and St hospital. Mary's, Mariannhill, South Africa. Peripheral blood was obtained from 65 individuals in this cohort, all of whom were patients who had not received previous treatment of antiretroviral therapy at the time of analysis. To be included in the study, individuals were selected based on their alleles of human leukocyte antigen (HLA or human leukocyte antigen) expressed and compatible with the ten class I tetramers that were constructed (see below). The median viral load of the cohort was 42,800 copies of HIV-1 RNA / ml plasma (range 163-750000) and the median absolute CD4 count was 362 (range 129-1179). Information relative to the duration of the infection was not available. All the individuals presented written informed consent for the study, which was approved by the institutional review committees. Construction of PD-1 and PD-Ll antibodies Monoclonal antibodies to human PD-Ll (29E.2A3, mouse IgG2b) and PD-1 (EH12, mouse IgGl) were prepared as previously described and it has been shown that block the PD-1 interaction: PD-Ll.

MHC class I tetramers For this study ten MHC class I MHC tetramers were used, synthesized as previously described (Altman JD, et al., 1966): A * 0205 GL9 (p24, GAFDLSFFL; SEQ ID NO: 1), A * 3002 KIY9 (Integrase, KIQNFRVYY; SEQ ID NO: 2), B * 0801 DI8 (p24, DIYKRWII; SEQ ID NO: 3), B * 0801 FL8 (Nef, FLKEKGGL; SEQ ID NO: 4), B * 4201 RM9 (Nef, RPQVPLRPM; SEQ ID NO: 5), B * 4201 TL9 (p24, TPQDLNTML; SEQ ID NO: 6), B * 4201 TL10 (Nef, TPGPGVRYPL; SEQ ID NO: 7), B * 4201 YL9 (RT , YPGIKVKQL; SEQ ID NO: 8), B * 8101 TL9 (p24, TPQDLNTML; SEQ ID NO: 9) and Cw0304 YL9 (p24, YVDRFFKTL; SEQ ID NO: 10). Constructs of plasmids expressing A * 0205, A * 3002 and Cw * 0304 were kindly provided by Drs. Eugene Ravkov and John Altman, NIH Tetramer Core facility, Atlanta Georgia.

Staining and phenotypic analysis of the HLA class I tetramer Peripheral blood mononuclear cells (PBMC, 0.5 million) were stained with tetramer for 20 minutes at 37 ° C. The cells were washed once with phosphate-buffered saline (PBS or phosphate buffered saline), pelleted and stained directly with fluorescein isothiocyanate-conjugated anti-CD8 (FITC) (Becton Dickinson), conjugated anti-PD-1. with phycoerythrin (clone EH12) and ViaProbe (Becton Dickinson). The cells were incubated for 20 minutes at room temperature, washed once in PBS and resuspended in 200 μl of PBS with 1% paraformaldehyde and purchased in a fluorescence activated cell sorter (FACSCalibur, Becton Dickinson). A minimum of 100,000 events were acquired in the FACSCalibur.

CFSE Proliferation Assays One million freshly isolated PBMCs were washed twice in PBS, pelleted and resuspended in 1 ml of carboxy-fluorescein diacetate, 0.5 μM succinimidyl ester (CFSE, Molecular Probes) for 7 minutes at 37 ° C . The cells were washed twice in PBS, resuspended in 1 ml of RIO medium (RPMI 1640 supplemented with glutathione, penicillin, streptomycin and 10% fetal bovine serum [FCS]) and deposited in a well of a 24-well plate. Initial studies revealed that a final concentration of 0.2 μg / ml of peptide gave the optimal proliferative responses, therefore, this was the final concentration of the peptide in the well used for each analysis. The negative control wells had PBMC in medium alone or PBMC in a medium with purified anti-PD-Ll (10 μg / ml) and the positive control wells were stimulated with 10 μg / ml of phytohemagglutinin (PHA). After day 6 of incubation in an incubator to 37 ° C, the cells were washed with 2 ml of PBS and stained with MHC class I tetramers conjugated with PE, ViaProbe (Becton Dickinson) and anti-CD8-APC antibodies. The cells were acquired in a FACSCalibur and analyzed by CellQuest® software (Becton Dickinson). The cells were housed in ViaProbe CD8 + lymphocytes. "The degree of increase in tetramer + cells was calculated by dividing the percentage of tetramer + CD8 + cells in the presence of peptide among the percentage of tetramer + CD8 + cells in the absence of stimulation with peptide.

Statistical analysis Analysis of the correlation of Spearman, the Mann-Whitney test and the t test for paired data, using the GraphPad Prism Version 4.0a software. All the tests were bilateral and the values p < 0.05 were considered significant.

Example 11: Expression of PD-1 in HIV-specific CD8 T cells A panel of 10 MHC class I tetramers specific for CD8 T cell epitopes of the HIV clade C virus was synthesized, based on prevalent HLA alleles and frequent white epitopes in Gag, Nef, Integrasa and RT (Kiepiela P, et al., 2004), which allow the direct visualization of the surface PD-1 expression in these cells. High-resolution HLA typing was performed throughout the cohort, and a subgroup of 65 people who had not previously received antiretroviral therapy, based on the relevant HLA alleles, was selected for the study. A total of 120 individual epitopes were analyzed and staining of representative ex vivo PD-1 in the tetramer + cells is shown in Figure 5A. The expression of PD-1 was very evident in these tetramer + cells and was significantly higher than in the total CD8 T cell population of the same individuals (p < 0.0001); in turn, the expression of PD-1 in both CD8 T cells tetramer + as in the total population of CD8 T cells was significantly higher than in controls seronegative to HIV. For eight of the ten tetramers analyzed, at least one person was identified in whom the level of expression of antigen-specific CD8 cells was 100% (Figure 5C). PBMCs of 3 to 25 individuals were stained for each response of the HIV tetramer and a median expression level of PD-1 in the 68 to 94% range of tetramer + T cells was obtained (Figure 5C). These findings were also confirmed by analysis of the mean fluorescence intensity (MFI or mean fl uorescence intensi ty) of PD-1 in the tetramer + cells and in the total population of CD8 T cells (Figure 5B, C). We then determined whether there was evidence of epitope-specific differences in the levels of PD-1 expression in people with several detectable responses. Of the 65 people examined, 16 individuals each had between 3 and 5 positive responses to tetramer. The expression of PD-1 was almost identical and close to 100% for each response analyzed in three of the 16 individuals; however, the other 13 individuals showed different expression patterns of PD-1 depending on the epitope (Figure 5D). These data indicate that the expression of PD-1 can be expressed differentially in contemporary epitope-specific CD8 T cells from a single person, perhaps consistent with recent data indicating epitope-specific differences in antiviral efficacy (Tsomides TJ, et al., 1994; Yang 0, et al., 1996; Loffredo JT, et al., 2005).

Example 12: Relationship between the expression of PD-1 and the progression of HIV disease The relationship between the expression of PD-1 in HIV-specific CD8 T cells and plasma viral load and CD4 levels, both prognostic factors of the progression of HIV disease. Consistent with previous studies, the relationship between the number of positive tetramer cells and the viral load or the CD4 numbers showed no significant correlation (Figure 6A, B). On the contrary, there were significant positive correlations with the viral load and in the percentage and MFI of the expression of PD-1 in HIV positive tetramer cells (p = 0.0013 and p <0.0001, respectively, Figure 6A). There were also inverse correlations between the CD4 figure and the percentage and MFI of PD-1 in HIV positive tetramer cells (p = 0.0046 and p = 0.0150, respectively, Figure 6B). Since the tetramers analyzed probably represent only a fraction of The population of HIV-specific CD8 T cells in these individuals was also analyzed for the relationship between PD-1 expression in all CD8 cells and these parameters. There were significant positive correlations between the viral load and the percentage and MFI of PD-1 expression in the total CD8 T cell population (p = 0.0021 and p <0.0001, respectively, Figure 6C) and inverse correlations were also observed between the CD4 count and the percentage and MFI of PD-1 expression in the total CD8 T cell population (p = 0.0049 and p = 0.0006, respectively, Figure 6D). In this same group, the expression of PD-1 in CMV-specific CD8 + T cells of 5 individuals was analyzed and it was observed that PD-1 was expressed significantly less in these cells in comparison with HIV-specific CD8 T cells (median 23% tetramer + PD-1 + CMV, p = 0.0036, no data are presented) and there was no difference with respect to the total of CD8 T cells in the same individuals, which indicates that the high expression of PD-1 is not a uniform feature of all CD8 T cells specific to the virus. These data suggest that the increase in antigen amounts in chronic infection results in an increase in the expression of PD-1 in CD8 T cells and is consistent with murine data in chronic LCMV infection in which the expression of PD-1 is associated with functional depletion of CD8 T cells (Barber DL, et al., 2005). On the other hand, in a large study that includes the analysis of several epitopes, there is the first obvious association between HIV-specific CD8 T cells and the viral load or the CD4 count.

Example 13: Relationship between the expression of PD-1 and the memory status and function of CD8 T cells The expression of PD-1 was analyzed in the context of several additional phenotypic markers associated with memory status and function. CD8 + T cells that include CD27, CD-28, CD45RA, CD57, CD62, CD127, CCR7, perforin, granzyme B and Ki67 (Figure 7). Representative stains for these markers in tetramer * B * 4201 TL9 cells from one individual are presented in Figure 7A and the global data from 13 individuals are presented in Figure 7B. These studies were limited to the tetramer responses that gave more than 95% positive to PD-1, since multiparameter flow cytometry of more than 4 colors was not available in KwaZulu Natal. The tetramer + PD-1 + HIV cells express high levels of CD27 and granzime B, very low levels of CD-28, CCR7 and intracellular Ki67, low levels of CD45RA and perforin and intermediate levels of CD57 and CD62L (Figure 7B). These data indicate that HIV-specific PD-1 + T cells display an effector / effector memory phenotype and are consistent with previous reports of skewed maturation of HIV-specific CD8 T cells (Champagne P, et al 2001).; Appay V, et ai. 2002; Hess C, et al. 2004). On the other hand, the virus was sequenced to determine if these cells directed an immune escape (Brown JA, et al., 2003). Of 45 of these positive tetramer responses evaluated, in only 5 viral epitopes were different from the South African C clade conss sequence (no data presented), indicating that these cells exert little selection pressure in vivo. Previous experiments in mice using the LCMV model showed that in vivo blocking of the PD-1 / PD-L1 interaction by infusion of anti-PD-Ll blocking antibodies results in an increase in the functionality of the CD8-specific T cells. LCMV, as determined by the production of cytokines, the ability to kill, the proliferative capacity and more strictly, the reduction of viral load (Barber DL, et al., 2005).

Example 14: Effect of blocking the PD-1 / PD-L1 pathway on the proliferation of HIV-specific CD8 T cells Because HIV-specific CD8 T cells have a poor proliferative capacity (Migueles SA, et al 2002, Lichterfeld M, et al 2004), it was determined whether blocking PD-1 / PD-L1 could increase this function in vi tro. Representative data of an individual positive to B * 4201 are shown in Figure 8A. Incubation of freshly isolated and CFSE labeled PBMCs, in a medium alone or in a medium with an anti-PD-Ll antibody, resulted in the conservation of a specific CD8 T cell population of B * 4201-TL9 (1.2% of CD8 T cells) that remained CFSEhi after six days of culture. Stimulation of PBMC labeled with CFSE for 6 days with TL9 peptide alone, resulted in a 4.8-fold expansion of CFSE10 B * 4201-TL9 + tetramer cells, while stimulation of CFSE PBBS labeled with CFSE, with TL9 peptide in the presence of an anti-PD-Ll blocking antibody increased the proliferation of TL9-specific cells, which resulted in a 10.3-fold increase in tetramer + cells. The CFSE proliferation assays were performed on 28 samples in the presence and absence of purified anti-human PD-Ll blocking antibody. A significant increase in proliferation of HIV-specific CD8 + T cells was observed in the presence of peptide plus anti-PD-Ll blocking antibody compared to the amount of proliferation after stimulation with peptide alone (Figure 8B, p = 0.0006, t test with paired data). The degree of increase of the tetramer + cells in the presence of anti-PD-Ll blocking antibody varied by individual and by epitope in a given individual (Figure 8C), which again suggests epitope-specific differences in the degree of functional exhaustion of these responses .

OTHER MODALITIES Although the invention has been described in a detailed manner, this description has the purpose of illustrating and not limiting the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are also within the scope of the following claims.

Claims (50)

1. CLAIMS 1. A method for alleviating or preventing a symptom of persistent infection or cancer, which is to administer to an individual in need thereof a compound that reduces the expression or activity of a programmed cell death peptide (PD-1) in that individual . The method according to claim 1, wherein the persistent infection is a viral infection, a bacterial infection, a fungal infection, a mycoplasmal infection or a parasitic infection. The method according to claim 2, wherein the viral infection is an infection with hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus or a human papilloma virus. 4. The method according to claim 2, wherein the parasitic infection is malaria. 5. The method according to claim 2, wherein the bacterial infection is tuberculosis. The method according to claim 1, wherein the cancer is angioimmunoblastic lymphoma or Hodgkin's lymphoma with nodular lymphocyte predominance. 7. The method according to claim 1, wherein the compound reduces the expression or activity of a ligand 1 of PD-1 (PD-Ll) or a ligand 2 of PD-1 (PD-L2). The method according to claim 1, wherein the compound reduces the interaction between PD-1 and PD-Ll or the interaction between PD-1 and PD-L2. The method according to claim 1, wherein the compound increases the activity of the cytotoxic T cells in the individual. The method according to claim 9, wherein the activity of the cytotoxic T cells is the production of cytokines, the proliferation of T cells or the clearance of the infectious agent. The method according to claim 10, wherein the infectious agent is a virus, a bacterium, a fungus, a mycoplasma or a parasite. The method according to claim 9, wherein the T cell is a cancer-specific T cell. The method according to claim 10, wherein the cytokines are IFN ?, TNFa or IL-2. The method according to claim 1, wherein the compound is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, anti-PD-1 RNAi, anti-PD-Ll RNAi, anti-PD-L2 RNAi, anti-PD-1 antisense RNA, anti-PD-Ll antisense RNA, anti-PD-L2 antisense RNA, a dominant PD-1 negative protein, a dominant PD-Ll negative protein and a PD-L2 protein dominant negative. The method according to claim 12, wherein the antibody is a monoclonal antibody, a humanized antibody, a deimmunized antibody or an Ig fusion protein. 16. The method according to claim 1, wherein the compound is a small molecule. 17. The method according to claim 1, further comprising administering to the individual a vaccine. 18. The method according to claim 17, wherein the vaccine comprises an auxiliary or a booster. 19. The method according to claim 1, further comprising administering to the individual a second compound. The method according to claim 19, wherein the second compound is an antiviral compound, an antibacterial compound, an antifungal compound, an antiparasitic compound, an antiinflammatory compound, an antineoplastic compound or an analgesic. The method according to claim 19, wherein the second compound reduces the expression or activity of antigen 4 of the cytotoxic T lymphocyte (CTLA-4) or of the B and T lymphocyte attenuator (BTLA). 22. The method according to claim 19, wherein the second compound reduces the volume of the cell Of cancer. 23. The method according to claim 19, wherein the second compound is an anti-CTLA-4 antibody, an anti-BTLA antibody, an anti-CD-28 antibody, an anti-ICOS antibody, an anti-ICOS-L antibody. , an anti-B7-l antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody or an anti-B7-H4 antibody. 24. The method according to claim 1, wherein the individual is a person. 25. A method to alleviate or prevent a symptom of a persistent infection or cancer, which consists of administering to an individual in need thereof a compound that reduces the expression or activity of a family member type CD28, wherein the member of the family type CD-28 is selected from the group consisting of PD-1 , CTLA-4, BTLA and a functional fragment or variants thereof. 26. The method according to claim 25, wherein the persistent infection is a viral infection. The method according to claim 26, wherein the viral infection is an infection with hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus or a human papilloma virus. 28. The method according to claim 25, in where the compound increases the activity of cytotoxic T cells in the individual. 29. The method according to claim 28, wherein the activity of the cytotoxic T cells is the production of cytokines, the proliferation of T cells or the clearance of the infectious agent. 30. The method according to claim 25, wherein the compound is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-anti-PD-1 antibody. BTLA, an anti-CD-28 antibody, an anti-ICOS antibody, an anti-ICOS-L antibody, an anti-B7-l antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody or an antibody anti-B7-H4. 31. The method according to claim 25, further comprising administering to the individual a vaccine. 32. The method according to claim 25, further comprising administering to the individual a second compound. 33. A method for alleviating or preventing a symptom of a persistent infection or cancer in an individual, the method is to administer to the individual an antigen-specific T cell that has been in contact with a compound that reduces the expression or activity of an antigen. PD-1 polypeptide in that cell. 34. The method according to claim 33, wherein the individual is also administered an antigen-specific B cell that has been in contact with a compound that reduces the expression or activity of a PD-1 polypeptide in that cell. 35. The method according to claim 33, wherein the T cell comes from an autologous source. 36. The method according to claim 33, wherein the T cells are derived from an individual of the same species. 37. The method according to claim 33, wherein the T cells are derived from an individual of different species than the individual being treated. 38. The method according to claim 33, wherein the antigen-specific T cell is specific for a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen or a cancer antigen. 39. The method according to claim 38, wherein the viral antigen is an antigen of a hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus. or a human papilloma virus. 40. A method to increase the cytotoxic activity of a T cell, which consists of putting in contacting the T cell with a compound that reduces the expression or activity of a PD-1 polypeptide. 41. The method according to claim 40, wherein the biological activity is reduced by reducing the expression or activity of the PD-1 ligand (PD-Ll) or the ligand 2 of PD-1 (PD-2L). 42. The method according to claim 40, wherein the compound reduces the interaction between PD-1 and PD-Ll or the interaction between PD-1 and PD-L2. 43. The method according to claim 40, wherein the cytotoxic activity is the production of cytokines, the proliferation of T cells or the clearance of the infectious agent. 44. The method according to claim 40, wherein the compound is an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, anti-PD-1 RNAi, anti-PD-Ll. RNAi, anti-PD-L2 RNAi, anti-PD-1 antisense RNA, anti-PD-Ll antisense RNA, anti-PD-L2 antisense RNA, a PD-1 dominant negative protein, a dominant PD-Ll negative protein or a PD-L2 dominant negative protein. 45. The method according to claim 40, further comprising contacting the cell with an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody. , a anti-BTLA antibody, an anti-CD-28 antibody, an anti-ICOS antibody, an anti-ICOS-L antibody, an anti-B7-l antibody, an anti-B7-2 antibody, an anti-B7-H3 antibody or an anti-B7-H4 antibody. 46. A method for making a diagnosis in an individual who suffers from or is at risk for persistent infection or cancer, the method consists of: (a) obtaining a sample from the individual, the sample contains immune cells; and (b) measuring the expression or activity of PD-1 in the sample of the individual, wherein an increase in expression or activity of PD-1 as compared to the expression or activity in a control sample, identifies the individual as a individual who suffers or is at risk of suffering a persistent infection or cancer. 47. A method for selecting a treatment for an individual suffering from or at risk of suffering a persistent infection or cancer, the method consists of: (a) obtaining a sample of the individual containing immune cells; (b) measuring the expression or activity of PD-1 in immune cells, wherein an increase in the expression or activity of PD-1 compared to the expression or activity in a control sample, identifies the individual as an individual suffering from or at risk of suffering a persistent infection or cancer; (c) selecting a treatment for the individual diagnosed as suffering identifies the individual as an individual suffering from or at risk of suffering a persistent infection or cancer, wherein the treatment comprises a compound that reduces the expression or activity of the PD -1. 48. The method according to claims 46 or 47, wherein step (b) also involves identifying antigen-specific immune cells, wherein the antigen is a viral antigen, a bacterial antigen, a parasitic antigen or a fungal antigen. 49. The method according to claim 46 or 47, wherein the sample is a blood sample, a tissue biopsy or a sample of bone marrow. 50. The method according to claims 46 or 47, wherein the control cells are from an individual who is neither suffering from nor at risk of suffering a persistent infection or cancer.