MXPA97006483A - Methods and compositions for administrating ge therapy vectors - Google Patents

Abstract

A method of reducing the immune response during gene therapy is provided which includes the co-administration of the viral vector carrying a therapeutic transgene and a selected immunomodulator, capable of inhibiting the formation of neutralizing antibodies and / or the elimination of LTC from the vectors by administration. repeated

Description

METHODS AND COMPOSITIONS TO MANAGE GENES THERAPY VECTORS This invention was supported by the National Institutes of Health Grant Nos. KD 47757-02 and 01 34412-02. The government of the U.S. You have certain rights in this invention.

FIELD OF THE INVENTION The present invention relates, in general terms, to gene therapy, and more specifically, to methods of administering viral vectors used in gene therapy.

BACKGROUND OF THE INVENTION Recombinant adenoviruses have emerged as attractive vehicles for the transfer of genes in vivo to a wide variety of cell types. The first generation vectors, which became devoid of replication by suppression of the nearby primitive genes Ela and Elb, are capable of a highly efficient transfer of genes in vivo into target cells that do not divide CM. Kay and others, Proc. Nati Acad. Sci. USO, 91: 2353-2357 (1994); S. Ishibashi et al., 3. Clin. Invest., 92: 883-893 (1993); B. Quantin et al., Proc. Nati Ocad Sci. USO, 89_: 2581-2584 (1992); M. Rosenfeld et al., Cell, 6: 143 (1992); R. Simón and others Hu. Grena Thera. , 4: 771 (1993); Rosenfeld et al., Sience, 252: 431-434 (1991); Stratford-Perricaudet and others, Hum. Gene Ther. , 1_: 241-256 (1990) 1. The immune responses of the receptor to the viral vector, the transgene carried by the vector, and the cells infected with virus, have arisen as recurrent problems in the initial application of this technology to animals and humans (Yang et al. 3. Virol., 59. : 2004 -2015 (1995) (Yang I).] Virtually in all models, including lung-directed and liver-directed gene therapy, transgene expression is transient and is associated with the development of on-site pathology. The transient nature of transgenic expression from recombinant adenoviruses is due in part to the development of antigen-specific cellular immune responses against cells infected with the virus and to their subsequent elimination by the host. First generation vectors, although they are deleted in the Ela region of the vector, express viral proteins in addition to those of the transgene. Viruses activate cytotoxic T lymphocytes (LTC) EY. Dai et al., Proc. Nati Ocad Sci. USO, 92: 1401-1405 (1995); Y. Yang et al., Proc. Nati Ocad Sci. USO, 91_: 4407-4411 (1994) (Yang II); and Y. Yang et al., Immunity, 1: 433-442 (1994) (Yang III)]. The collaboration of LTCs directed against newly synthesized viral proteins and virus-specific helper T cells (Zabnr and O + ros, Cel, 75: 207-216 (1993); Crys + al and others, Nat Genet., 8: 42-51 (1994) 1 leads to the destruction of cells infected by the virus.Another target for the clarification of immunoregulated virally infected cells can be the transgene product when that transgene expresses a protein that is foreign to the virus. In this way, LTCs are an important effect in the destruction of target cells, with activation occurring in some cases in the context of the transgene product, or viralmen + e synthesized proteins, both of which are represented by MHC class I molecules (Yang T; and Zsengeller et al., Hu. Gene Thera., 6 457-467 (1995) .1 It has also been observed that these immune responses cause the occurrence of associated hepatitis that develops in the receptors. of therapy genes in vivo directed to the liver, in the course of 2 to 3 weeks after the initial treatment. Another limitation of recombinant adenoviruses for gene therapy has been the difficulty in obtaining detectable gene transfer after a second virus administration. This limitation is particularly problematic in the treatment of disorders or chronic diseases inherited from a single gene, such as cystic fibrosis (FO), which requires repeated therapies to obtain genetic reconstitution throughout life. Reduced gene transfer after a second therapy has been demonstrated in a wide variety of animal models after intravenous or intratracheal release of CT virus. Sinith and others, Gene Thera. , 5_: 397 (1993): S. Yei et al., Gene Thera. , 1 .: 192-200 (1994); K. Kozarsky and others 3. Biol.Chern. , _ 2_6_9: 13695 (1994)]. In each case, resistance to repeated gene therapy was associated with the development of the neutralizing antibody to adonovirus, which prevented the successful transfer of genes after a second virus administration. Potential solutions for these problems have been directed towards the development of second generation CY recombinant viruses. Yang et al., Nat. Genet., 7_: 362-369 (1994) (Yang IV); and 3. Engelhardt and others, Hurn. Gene Thera. , 5: 1217 (1994) 1, designed to decrease the expression of viral proteins synthesized again, and the use of non-immunogenic transgenes to prevent the activation of LTC. Thus, there remains a need in the art for a method and composition to improve the efficiency of gene transfer during repeated administrations of viral gene therapy.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method of gene therapy and compositions for use in the field, which results in a reduced immune response to the recombinant viral vector used to effect the therapy. The method includes coad ingesting with the gene therapy viral vector a selected mmunomodulator, which substantially reduces the occurrence of neutralizing antibody responses directed against the antigens encoded by the vector and / or the removal of cytolytic T cells from the cells. that contain the viral protein. This method is particularly useful when the recomrninistration of the recombinant virus is desired. In accordance with this method, the immunomodulator can be administered before, or concurrently with, the recombinant viral vector carrying the transgene to be released. Other aspects and advantages of the present invention are also described in the following detailed description of the preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS.

Figure 10 is a graph summarizing the neutralizing antibody titer present in samples of bronchoalveolar lavage fluid (LBO) of C57BL-6 mice infected with adenovirus on day 0 and subjected to necropsy on day 28, as described in the example 2. The control represents normal mice ("control"); CD4 rnOB represents mice deleted from CD4 + cells; IL-12 represents mice treated with IL-12 on days 0 and +1 and TFN-t represents mice treated with IFN-t on days 0 and +1. The data are presented as the mean ± 1 standard deviation for three independent experiments. Figure IB is a graph that summarizes the relative amounts (OD405) of TgG present in LBO samples. The symbols are as described in Figure 10. Figure 1C is a graph summarizing the relative amounts (OD405) of IgO present in LBO samples. The symbols are as described in Figure lfl. Figure 2 is a graph summarizing the neutralizing antibody titer, expressed as the reciprocal dilution of serum samples for the animals of example 4. The symbols representing the mice are described as follows: C57BL-6 mice infected with H5. OlOCMVlacZ on day 0 and with H5.010CBOLP on day 28 ("B6 mice"); C57BL-6 mice infected with HS.OlOCMVlacZ, inactivated with UV, on day 0 and with H5.010CBOLP on day 28 ("B6-UV mice"); MHC class II deficient mice - infected with HS.OlOCMVlacZ on day 0 and with H5.010CBOLP on day 28 ("class II-" mice); mice deficient in β2-rnicroglobulin infected with HS.OlOCMVlacZ on day 0 and with H5.010CBOLP on day 28 ("mice | 32pr"); and C57BL / 6 mice treated with Ob TK1.5 (anti-CD4) and infected with HS.OlOCMVlacZ on day 0 and with H5.010CBOLP on day 28 ("CD40b mice"). Figure 30 shows a graph summarizing the neutralizing antibody section, expressed as a reciprocal dilution of serum samples, to C57B / 6 mice mown in the tail vein on day 0 with H5.010CMVLDLR and on day 21 with H5.010CMVlacZ, and day 42 with H5.010CBOLP, and saline was administered on days -3, 0 and 3. The title is reported as a function of days after infection. Figure 3B shows a graph similar to that of Figure 30 for C57BL / 6 mice infused in the vein of the tail on day 0 with H5.010CMVLDLR and day 21 with HS.OlOCMVlacZ, and on the day 42 with H5.010CBOLP, and administered with rnOb GK1.5 ant? -CD4, on days -3, 0 and 3. Figure 3C is a graph similar to that in Figure 30 for C57BL / 6 mice infused into the vein of the tail the day 0 with H5.010CMVLDLR and day 21 with H5.010CMVLacZ, and day 42 H5.O10CBALP, and administered with mOb GK1.5 ant? -CD4 on days - 3, 0, 3, 18, 21 and 24. Figure 3D is a graph similar to that of Figure 30 for C57BL / 6 mice infused in the vein of the tail on day 21 with HS.OlOCMVLacZ, and on day 42 with H5.010CBOLP, and administered with mflb GK1.5 anti-CD4 on days -3, 0, and 3. Figure 40 is a graph that summarizes the relative amounts (OD * os) of IgGl present in the serum samples as a function of the dilutions of the sample, as described in example 6.

Figure 4B is a graph summarizing the relative amounts (OD-405) of TgG2a present in the serum samples as a function of the dilutions of the sample, as described in Example 6. Figure 5 is a graph that illustrates the percentage of specific lysis on pseudo-infected C57SV ("pseudo") cells and infected with H5.010CBOLP ("PLO"), as a function of the effector to target ratios for a 5 * Cr release test of Example 6B . Splenocytes of C57BL / 6 mice ("B6") and mice treated with IL-L2 ("B6 + IL12") were re-tested in vitro 10 days after administration of H5.010CBOLP, with H5. OlOCMVlacZ for 5 days. Figure 60 is a histogram that provides the neutralizing antibodies for OdS obtained in the LBA (lung experiment) of example 7. The results in column 1 are from C57BL / 6 mice (control), the results of column 2 are from knockout mice deficient in CD40L (CD40L-KO), and the results in column 3 are from C57BL / 6 mice treated with CD40L antibody (Ob CD40L). The data are presented as the mean neutralizing antibody titer of three samples +/- 1 D.E. Figure 6B is a histogram as described in Figure 60, with the data obtained from the serum for the liver experiment of example 7. Figure 70 is a line graph comparing the percentage of specific lysis in cultured lymphocytes of C57BL mice. 6 control (blank circles) and C57BL / 6 mice treated with antibody to CD40L (black circles). Seven days after administration of the virus, the lmfocytes were restimulated in v tro for 5 days and tested for cell-specific lysis of the pseudo-infected C57SV in a 6 hour s * Cr release test. The percentage of specific lysis is expressed as a function of different effector to target ratios (6: 1, 12: 1, 25: 1, and 50: 1). Splenocytes were used for this pulmon experiment. Figure 7B is a line graph as described in Figure 70, which uses C57SV cells infected with viruses. Figure 7C is a line graph as described in Figure 70 that uses pseudoinfected cells. Liver-mediated lymph node (NLM) cells were used for these liver experiments. Figure 7D is a line graph as described in Figure 70, using cells infected with viruses. NLM cells were used for these liver experiments.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides methods and compositions for improving an animal or human capacity to tolerate the administration of viral vectors for gene therapy. The invention provides methods to transiently prevent the activation of CD4 + T cells that are involved in both cellular and humoral immunological barriers to gene therapy. The methods include administration to an individual receiving a gene therapy vector of an adequate amount of an immunomodulator, preferably of short action. The immunomodulator is preferably administered concurrently with the administration of the vector for gene therapy, that is, a recombinant virus used to release a suitable therapeutic transgene for gene therapy. The immunomodulator can also be administered before or after administration of the vector. The method of this invention, which prevents the development of adverse cellular and humoral immune responses to viral gene therapy vectors, is based on immunosuppression to block the activation of helper T cells, specifically the CD4 function, and B cells. Or, difference from the prior art, which included chronic immunosuppression by continuous administration of drugs in non-specific suppressants, the present invention uses a transient approach to immunosuppression. Without wishing to be bound by theory, the inventors of the present believe that the main stimulus for immune activation are the viral capsid proteins of the recombinant vector. Chronic immunosuppression is not necessary in this case. Specifically, the transient suppression of the CD4 function at, or near, the time of administration of the recombinant virus, in accordance with this invention, prevents the formation of neutralizing antibody, thereby allowing for efficient gene transfer afterwards. minus two subsequent administrations of viral vectors for gene therapy. As illustrated below, the administration of immunomodulators according to the method of this invention is preferably carried out only at the time of administration of the virus. However, longer inrnunornodulation may be required which is necessary in the following examples of gene transfer to the liver and / or mouse lung, depending on the manner in which the antigens are present in different gene therapy protocols. In this manner, the transient immunomodulation method of the invention can, in certain circumstances, be combined with long-term immunosuppression and other immunomodulatory therapies.

I. Immunomodulators The selected immunomodulator is defined herein as an agent capable of inhibiting the formation, by means of activated B cells, of neutralizing antibodies directed against the recombinant viral vector and / or capable of inhibiting the elimination of cytolytic T lymphocyte ( LTC) of the vector. The immunomodulator can be selected to interfere with the interactions between the helper T subpopulations (THI or TH2) and the B cells to inhibit the formation of neutral antibody. Alternatively, the immunomodulator can be selected to inhibit the interaction of TH1 cells and LTCs to reduce the occurrence of CTL removal from the vector. More specifically, the immunomodulator desirably interferes with, or blocks, the function of CD4 T cells. Inunnomodulators for use in the inhibition of neutralizing antibody formation according to this invention can be selected based on the determination of the immunoglobume subtype of any neutralizing antibody produced in response to the viral vector. The neutralizing antibody that develops in response to the administration of a viral gene therapy vector, does so frequently based on the identity of the virus, the identity of the transgene, which vehicle is used to release the vector and / or the location of the virus. white or type of tissue for viral vector release. For example, TH2 cells are generally responsible for interference with the efficient transfer of genes administered during gene therapy. This is particularly true when the viral vector is adenovirus. More particularly, the inventors of the present have determined that the neutralization of antibodies of the subtypes, IgGi and Igfl, which depend on the interaction between TH2 cells and B cells, seems to be the main cause of most of the neutralizing antibodies against adenoviral vectors. .

The identity of the neutralizing antibody induced by the administration of a recombinant viral vector for specific gene therapy is easily determined in animal tests. See, eg, Example 6. For example, the administration of adenoviral vectors through the lungs generally induces the production of neutralizing antibody IgO, while the administration of viral vectors through the blood generally induces TyG_ neutralizing antibody. In these cases, an immune response dependent on TH2 interferes with the transfer of the adenovirus viral vector bearing a therapeutic transgene. Where the neutralizing antibody induced by viral administration is a TH2 mediated antibody, such as IgO or IgGi, in immunomodulator selected for use in this method desirably suppresses or prevents the interaction of TH2 cells with B cells. Alternatively, if it is found that the induced neutralizing antibody is an antibody mediated by THI, such as Ig (-2A, the immunomodulator desirably suppresses or prevents the interaction of THI cells with B cells. Where the reduction of the elimination of LTC from viral vectors is desired, as well As the blocking of neutralizing antibody formation, the immunomodulator is selected for its ability to suppress or block THI CD4 + cells to allow prolonged in vitro residence of the viral vector Inrnunomodulars may comprise soluble or naturally occurring proteins, including cytokines and monoclonal antibodies. The immunomodulators may comprise other pharmaceutical agents lithic. Odernás, the inrnunomod? lators in accordance with the invention may be used alone or in combination with another. For example, ida cyclophosphamide and the more specific immunomodulator, anti-CD4 monoclonal antibody, can be coadministered. In such a case, cyclophosphamide serves as an agent to block THI activation and stabilize transgenic expression beyond the period of transient immune block induced by anti-CD4 MOb treatment. An adequate amount or dose of the selected immunomodulator will depend primarily on the identity of the modulator, the amount of the recombinant vector carrying the transgene that is initially administered to the patient, and the method and / or site of the vector release. These factors can be evaluated empirically by a person skilled in the art using the methods described herein. Other secondary factors such as the condition to be treated, the age, weight, general health and immune status of the patient, can also be considered by a physician in determining the dosage of the immunomodulator that is to be released in the patient in set with a gene therapy vector in accordance with this invention. Generally, for example, a therapeutically effective human dose of a protein immunomodulator, eg, IL-12 or IFN-t, is administered on the scale of about 0.5 μg to about 5 mg for approximately 1x 07 pfu / Virus vector rnl. One skilled in the art can determine different dosages to balance the therapeutic benefit against any adverse side effects. 0. Monoclonal Antibodies and Soluble Proteins Preferred, the method of inhibition of. An adverse immune response to the gene therapy vector includes non-specific inactivation of CD4 + cells. One such method involves the administration of an appropriate monoclonal antibody. Preferably, such blocking antibodies are "humanized" to prevent the recipient from developing an immune response to the blocking antibody. A "humanized antibody" refers to an antibody having its regions determining the complomentarity (RDCs) and / or other portions of its light or heavy regions of variable domain basic structure derived from a non-human donor immunoglobulin, and the remaining portions of the molecule derived from immunoglobulin are derived from one or more human immunoglobulins. Said antibodies may also include antibodies characterized by a humanized heavy chain associated with an unmodified light chain of donor or acceptor or a chimeric light chain, or vice versa. Said "humanization" can be effected by methods known in the art. See, for example, G.E. Mark and E.O. Padlan, Cap. 4. "H? Manization of Monoclonal Ontibodies", The Handbook of Experimental Pharmacology, vol. 113, Spp gergerlag, New York (1994), pp. 105-133, which is incorporated herein by reference. Other suitable antibodies include those that specifically inhibit or deplete CD4 + cells, such as an antibody directed against the cell surface of CD4. The inventors of the present invention have shown that depletion of CD4 + cells inhibits the elimination of LTC from the viral vector. Such modulating agents include, but are not limited to, anti-T cell antibodies, such as or ant? -0KT3 + Cver, e.g., U.S. Patent No. 4,658,019; European Patent Application No. 501,233, published September 2, 19921. See example 2 below, which employs the commercially available antibody GK1.5 ATCC Accession No. TIB2071 to deplete CD4 + cells. Alternatively, any agent that interferes with, or blocks, the interactions necessary for the activation of B cells by TH cells, and thus the production of neutralizing antibodies, is useful as an immunomodulator in accordance with the methods of this invention. For example, activation of B cells by T cells requires the occurrence of certain C-H interactions. Dure and others, Inrnunol. Today, 15 (9): 406-410 (1994) 1, such as the binding of CD40 ligand on the T helper cell to the CD40 antigen on the B cell, and the binding of the CD28 and / or CTL04 ligands on the T cell to the B7 antigen on the B cell. Without both interactions, the B cell can not be activated to induce the production of the neutralizing antibody. The ligand interaction CD40 (LCD40) -CD40 is a desirable point to block the immune response to gene therapy vectors due to its broad activity in both the activation and the function of T helper cells, as well as in the absence of redundancy on your signaling route. A presently preferred method of the present invention thus includes transiently blocking the interaction of LCD40 with CD40 at the time of adduction of the adenoviral vector. This can be achieved by treatment with an agent that blocks the CD40 ligand on the TH cell and interferes with the normal binding of CD40 ligand on the helper T cell with the CD40 antigen on the B cell. Blocking the LCD40-CD40 interaction prevents Activation of T helper cells contributes to problems with transgenic stability and readministration. In this manner, an antibody for the CD40 ligand (anti-LCD40), available from Bristol-Myers Squibb Co; see, e.g., European Patent Application 555,880, published on August 18, 19931 or a soluble CD40 molecule may be one of the immunornodulators selected in the method of this invention. Alternatively, an agent that blocks the CD28 and / or CTL04 ligands present on the T helper cells interferes with the normal binding of those ligands with the B7 antigen on the B cell. Thus, a soluble form of B7 or an antibody to CD2B or CTLA4, v.gr .., CTL.04-Ig available from Bristol-Myers Squibb Co., see v. gr. , European Patent Application 606,217, published July 20, 19941, may be the immunornodulator selected in the method of this invention. This method has greater advantages than the previously described administration of cytokine to prevent activation of TH2, since it handles both cellular and humoral immune responses to foreign antigens.

B. Cytokines Other immunomodulators that inhibit the function of TH cells in the methods of this invention may also be employed. Thus, in one embodiment, it can be administered, at the time of primary administration of the viral vector, to an immunomodulator for use in this method that selectively inhibits the function of the THI subpopulation of CD4 + helper T cells. One of said immunomodulators is interleukin-4 (IL ~ 4). IL-4 increases the specific antigenic activity of TH2 cells at the expense of the function of THI Cver cells, e.g., Yokota et al., Proc. Nati Ocad Sci .. USA, 83_: 5894-5898 (19961; U.S. Patent No. 5,017,6911) It is contemplated that other immunomodulators that can inhibit THI cell function will also be useful in the methods of this invention. The immunomodulator can be a cytochrome that prevents the activation of the TH2 subpopulation of helper T cells.The success of this method depends on the relative contribution that the TH2 isotypes of Ig play in the neutralization of the virus, the profile to which they can be affected by the strain, the animal species as well as the mode of release of the virus and the target organ.A desirable immunomodulator for use in this method that selectively inhibits the function of the T-H2 subpopulation of CD4 + T cells at the time of administration. primary administration of the viral vector, includes interleukin-12 (IL-12) .Ill-12 increases the antigen-specific activity of THI cells at the expense of the function of the TH2 cell ~ l e.g., European Patent Application No. 441,900; P. Scott, Science, 260: 496-497 (1993); R. Manetti et al., 3. Exp. Med., 177: 1199 (1993); 0. D'flndrea and others, 3.Exp. Med., 176: 1387 (1992) 1. The IL-12 for use in this method is preferably in the protein form. Human TL-12 can be produced recombinantly using known techniques or can be obtained commercially. Alternatively, it can be engineered with a viral vector (which may optionally be the same one used to express the transgene) and expressed in a white cell in vivo or ex vivo. The specific suppression of TH2 with IL-12 is particularly effective in gene therapies directed to the lungs where Tgfl is the main source of neutralizing antibody. In gene therapy directed to the liver, both THI cells and TH2 cells contribute to the production of virus-specific antibodies. However, the total amount of neutralizing antibody can be decreased with IL-12. Another selected immunomodulator that performs a similar function is interferon gamma (IFN-t) CS.C. Morris et al., 3 Im unol., 152: 1047-1056 (1994); F.P. Heinzel and others, 3. Exp. Med., 177: 1505 (1993) 1. It is believed that TFN-t mediates many of the biological effects of IL-12 by secretion of activated macrophages and helper T cells. IFN-t also partially inhibits TH2 activation stimulated by IL-4. The TFN-t can also be obtained from a variety of commercial sources. Alternatively, it can be engineered into a viral vector and expressed in a living or ex vivo white cell using known genetic engineering techniques. Preferably, said cytokine immunomodulators are in the form of human recombinant proteins. These proteins can be produced by methods existing in the art. When the neutralizing antibodies are mediated by TH2, peptides, fragments, sibanities or active analogues of the known immunomodulators described herein, such as IL-12 or gamma interferon, which share the inhibitory function will also be useful in this method. of TH2 from these proteins. As illustrated in the examples below, the cytokines IL-12 (which activate THI cells to secrete IFN-t) and IFN-t have been shown to suppress only humoral immunity (ie, inhibit TH2 differentiation). The coadministering of any cytokine at the time of virus instillation prevented the formation of TgO and allowed efficient readinst ration of virus. To allow a second effective administration of viruses in gene therapy directed to the liver, the method may preferably comprise the administration of more than one cytokine, specific dosage regimens and / or the coadministration of an additional immunomodulator, such as one or more of the antibodies discussed above. The use of cytokines is advantageous since cytokines are natural products, and thus probably do not generate any adverse immune response in the patient to whom they are administered.

C. Other pharmaceutical agents In the methods of the invention other immunomodulators or agents that inhibit immune function can also be used nonspecifically, that is, cyclosporin 0 or cyclophosphamide. For example, it has been shown that a short course of cyclophosphamide successfully interrupts the activation of both CD4 and CD8 helper T cells for the adenovirus capsid protein at the time of virus release to the liver. As a result, the transgenic expression was prolonged and, at higher doses, the formation of newantial antibody was prevented, allowing the successful readministration of the vector. In the lung, cyclophosphamide prevented the formation of neutralizing antibodies at all doses and stabilized transgenic expression at high doses.

II. Viral Vectors Suitable viral vectors useful in gene therapy are well known, including retroviruses, vaccinia virus, poxivirus, adenovirus and adeno-associated viruses, among others. It is expected that the method of this invention will be useful with any virus that forms the basis of a gene therapy vector. However, exemplary viral vectors for use in the methods of the invention are adenovirus vectors see, e.g., M. S. Horwitz et al., "Odenoviri ae and Cheir Replication", Virolog, second edition, p. 1712, ed. B. N. Fields et al., Raven Press Ltd., New York (1990); M. Rosenfeld et al., Cell, 68: 143-155 (1992); 3. F. Engelhardt et al., Human Genet. Ther .. 4: 759-769 (1993); Yang IV; 3. L Jilson, Nature, 365: 591-692 (Oct. 1993); B. 3. C rter, in "Handbook of Parvoviruses", ed. P. Tijsser, CRC Press, pp. 155-168 (1990). Particularly convenient are human type C (Ad) adenoviruses, including serotypes d2 and Od5, which have become replication-free for gene therapy by suppressing the primitive genetic loci encoding Ela and Elb.

Much has been published about the use of adenoviruses deleted from El in gene therapy. See, K.F. Kozarsky and 3. M. Uilson, Curr. Qpm Genet Dev., 3: 499-503 (1993). ODN sequences of various types of adenovir? S are available from Genbank, including type Od5 CGenbank Occession No. M732601. Adenovirus sequences can be obtained from any type of known adenovirus, including the 41 human types currently identified by Chorwitz et al., Virology, 2nd ed., B. N. Fields, Raven Press, Ltd., New York (1990) 1. A variety of adenovirus strains are available from the Ornerican Type Culture Collection, Rockville, Maryland, or available upon request from a variety of commercial and institutional sources. In the following modality, adenovirus type 5 (0d5) is used for convenience. The selection of the virus useful for genetic engineering of recombinant vectors, including viral type, eg, adenovirus, and strain, is not expected to limit the following invention. Similarly, the selection of the transgene contained within the viral vector is not a limitation of this invention. It is expected that this method will be useful with any transgene. Suitable transgenes for releasing a patient in a viral vector for gene therapies can be selected by those skilled in the art. These therapeutic nucleic acid sequences typically encode products for administration and expression in an in vivo or ex vivo patient to replace or correct an inherited or non-inherited genetic defect or treat an epigenetic disorder or disease. Such therapeutic genes that are suitable for the development of gene therapy include, without limitation, a very low density lipoprotein receptor (R-LMBD) gene for the treatment of familial hypercholesterolemia or familial combined hyperlipidemia, the transmembrane regulatory gene of Cystic fibrosis (CFTR) for the treatment of cystic fibrosis, the DMD allele Becl > -er for the treatment of Duchenne muscular dystrophy, and a number of other genes that can be easily selected by a person skilled in the art to treat a particular disorder or disease. In this way, the selection of transgene is not considered a limitation of this invention, since said selection is within the knowledge of those skilled in the art. The viral vector carrying a therapeutic gene can be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. A suitable vehicle includes sterile saline. For this purpose, other non-aqueous sterile isotonic injectable solutions and sterile aqueous and non-aqueous suspensions known as pharmaceutically acceptable carriers well known to those skilled in the art may be employed. The viral vector is administered in amounts sufficient to transfect the desired cells and provide sufficient levels of translation and expression of the selected transgene to provide a therapeutic benefit without undue adverse effects or medically acceptable physiological effects, which can be determined by those skilled in the art. medical techniques. Conventional and pharmaceutically acceptable administration routes include direct delivery to the target organ, tissue or site intranasally, intravenously, intramuscularly, subcutaneously, in-radically, orally and other parenteral routes of administration. If desired, administration routes can be combined. The dosages of the viral vector will depend mainly on factors such as the condition to be treated, the selected gene, the age, the weight and the health of the patient, and thus may vary between patients. For example, a therapeutically effective human dosage of the viral vectors is generally in the range of about 20 to about 50 nl of saline containing concentrations of about 1 x 10 ~ 7 to 1 x irj-io pfu / rnl of virus. A preferred dosage for adult human is about 20 rnl in saline at the above concentrations. The dosage will be adjusted to balance the therapeutic effect against any side effects. The expression levels of the selected gene can be monitored to determine the selection, adjustment or frequency of dosage administration.

III. The method of the invention The method of this invention includes the co-administration of the selected immunomodulator with the selected recombinant viral vector. Coadministration occurs so that the immunomodulator and the vector are administered within a period of time u and close to one resp > ecto of the other. It is currently preferred to administer the modulator concurrently with, or no more than, one to three days before the administration of the vector. The immunomodulator can be administered separately from the recombinant vector, or if desired, can be administered in admixture with the recombinant vector. As illustrated by the examples below, the in unomod? Lator, either? Anti-CD40L antibody, anti-CD4 antibody or cytokine, is conveniently administered at a time close to the administration of the viral vector used for gene therapy. . Particularly, the administration of IL-12 or IFN-T causes reduction in the levels of TH2 cells for approximately 2 to 3 days. Therefore, IL-12 and / or IFN-t are conveniently administered within a day after administration of the viral vector carrying the gene to be released. However, preferably IL-12 and / or IFN-t are administered essentially simultaneously with the viral vector. The immunomodulator can be administered in a pharmaceutically acceptable carrier or diluent, such as saline. For example, when formulated separately from the viral vector, the nanometer is conveniently suspended in saline. Said solution may contain conventional components, for example pH adjusters, preservatives, and the like. Said components are known and can be easily selected by a person skilled in the art. Alternatively, the immunomodulator can be administered alone as ODN, either separately from the vector- or mixed with the recombinant vector carrying the transgene. There are methods in the art for the pharmaceutical preparation of the modulator as a protein or as ODN CVer, e.g., 3. Cohen, Science, 259: 1691-1692 (1993) with respect to ODNI vaccines. Conveniently, the immunomodulator is administered by the same route as the recombinant vector. The immunomodulator can be formulated directly in the composition containing the viral vector administered to the patient. Alternatively, the immunomodulator can be administered separately, preferably shortly before or after administration of the viral vector. In another alternative, a composition containing an immuno odometer, such as IL-12, can be administered separately from a composition containing a second immunomodulator, such as an anti-CD40L antibody, and so on, depending on the number of immunomodulators administered. These administrations can be, independently, before, simultaneously with, or after, the administration of the vector v i ra1. The administration of the selected immunomodulator can be repeated during the treatment with the recornbinating viral vector carrying the transgene during the period that the transgene is expressed (monitored by tests that detect the expression of the transgene or its intended effect), or with each recombinant vector recombinant. . Alternatively, each reinjection of the same viral vector can employ a differential injector. An advantage of the method of this invention is that it represents a transient manipulation, necessary only at the time of administration of the gene therapy vector. This strategy is expected to be safer than strategies based on the induction of tolerance, which can permanently impair the recipient's ability to respond to viral infections. Furthermore, it is expected that the preferred use of immunomodulators such as the aforementioned cytokines or antibodies is safer than the use of agents such as cyclosporin or cyclophosphamide (which cause immunosuppression) because the transient immunomodulation is selective ( this is, CTL-mediated responses are retained as are the humoral responses dependent on the THI function). In an example of efficient gene transfer in accordance with the methods of this invention, the selected immunoreactors are TL-12, which causes the selective induction of THI cells, and / or TFN-t, which suppresses cell induction. TH2 - Another preferred immunomodulator is anti-CD4 + antibody, GK1.5, which suppresses THI V cells reduces the elimination of LTC from the vector. Another preferred immunomodulator is the monoclonal anti-CD40 ligand antibody, MRl, available from the American Type Culturo Collection, Rockville, Maryland. As exemplified below, the use of the immunomodulators identified above allowed efficient genetic transfer as well as repeated use of the same viral vector. In conjunction with gene therapy we used an adenovir? S vector that contains either a transgene of alkaline phosphatase ("ALP"), a transgene of beta-galactosidase ("lacZ"), or a transgene of the lipoprotein receptor. low density. The following examples illustrate the preferred methods for preparing suitable viral vectors useful in the gene therapy methods of the invention. These examples are illustrative only and do not limit the scope of the invention.

E1EMPLO 1 CONSTRUCTION AND PURIFICATION OF ADENOVIRUS VECTORS RECOMBINANT COPIES The recombinant adenovirus H5.010CMVlacZ was constructed as follows. The plasmid pfld.CMVlac [described in Kozarsky et al., 3. Biol. Chem., 269 (18): 13695-13702 (1994)], which contains 0-1 units of the adenovirus map, was used, followed by a enhancer / promoter of cytomegalovirus TBoshart et al., Cell, 41: 521-530 (1985) 1, an E. coli beta-galactosidase gene (lacZ), a polyadenylation signal (pfl), units 9.2-16 (Od 9.2 -16) of the adenovirus 5 rnapa and generic plasmid sequences that include an origin of replication and resistance gene of arnpicillin. pOd.CMVlacZ was linearized with Nhel and cotransfected in 293 cells (OTCC CRL1573) with ODN s? b360 (derived from adenovir? s type 5) that had been digested with Xbal and Clal, as previously described CK. F. Kozarsky, Somatic Cell Mol. Genet , 19: 449-458 (1993) and Kozarsky (1994), cited above !. The resulting recombinant viruses, HS.OlOCMVlacZ, contain the 0-1 units of the adenovirus map, followed by a better dor / CMV promoter, a lacZ gene, a polyadenylation signal (pA), units 9.2-100 of the adenovirus map. , with a small deletion in the E3 gene in 78.5 to 84.3 mu of the basic structure of the sub360 of Ad 5. The recombinant adenovirus H5.010CBOLP contains the 0-1 units of the adenovirus map, followed by a promoter of (.- chicken cytoplasmic actin CMV increased CT 0. Kost et al, Nuci, Oci ds Res., 1JL (23): 8287 (1983) 1, an OLP gene from human placenta, a polyadenylation signal (pO), and units 9 -100 of the map of adenovirus type 5, with a small suppression in the E3 gene in 78.5 to 84.3 rnu of the basic structure of Od3 sub360. This adenovirue recornbinante was built substantially in a similar way to the adenovir? S Hd.OlOCMVlacZ described above, see, also, Kozarsky (1994), cited earlier, these recombinant adenoviruses s, Hd.OlOCMVlacZ and H5.010CBALP, were isolated after transfection CGraham, Virol. , 52_: 456-467 (1974) 1, and were subjected to two rounds of plaque purification. Lysed products were purified on two gradients of sequential density of cesium chloride as previously described CEnglehardt et al., Proc. Nati Ocad Sci. USO, 88: 11192-11196 (1991) 1. Cesium chloride was removed by paeing the virus on BioRad DG10 gel filtration columns, using pH regulated saline with phosphate (PB?). For mouse experiments, the virus was used, either fresh or after column purification, glycerol was added to a final concentration of 10% (v / v), and the virus was stored at -70 ° C until used.

E3EMPL0 2 ME30RAMIENTO OF THE TRANSFERENCE OF GENES MEDIATED BY ADENOVIRUS BY SECOND ADMINISTRATION WITH IL-12 AND IFN-t IN THE MOUSE PULMON In this example, the recolbinant adenoviruses Hd.OlOCMVlacZ and H5.010CBOLP were used. Each virus expresses a different transgene reporter whose expression can be discriminated from the first transgene reporter. Female C57BL / 6 mice (from 6 to R weeks of age) were infected with suspensions of H5.010CBOLP (1 x 109 pfu in 50 μl of PBS) via the trachea on day 0 and then with HS.OlOCMVlacZ on the day 28. A group of said mice was used as a control. Another group of mice were acutely suppressed from CD4 + cells by i.p. of antibody for CD4 + cells (GK1.5, OTCC No. TIB207, dilution of aecitis 1:10) at the time of initial gene therapy (days -3, 0, and +3). A third group of mice was injected with IL-12 (injections of 1 μg intratracheal or 2 μg, μ.p.) at the time of the first administration of virus (days 0 and +1). A fourth group of mice was injected with interferon gamma (1 μg mtratracheal injections or 2 μg, i.p.) at the time of the first administration of virus (days 0 and +1). When the mice were subsequently subjected to euthanasia and necropsy on days 3, 28 or 31, lung tissues were prepared for cryoses, while bronchoalveolar lavage (LBO) and lymph nodes were used for immunological tests. 0. Cryosections The expression of OLP in lung tissues was evaluated on day 3 and day 28 by histochemical staining following the procedures of Yang T, cited above. The expression of (3-galactosidase on day 31 was determined by histochemical staining of X-gal.) The results described below were obtained from histological stains of alkaline phosphatase (amplification per 100) or X-gal stains of β -galactosidase (amplification per 100) The instillation of ALP virus (109 pfu) into the airways of all groups of C57BL / 6 mice resulted in a high level of transgene expression in most respiratory conduction pathways which decreased to undetectable levels on day 28. It was observed that the loss of transgenic expression is due to the CTL-mediated elimination of the genetically modified hepatocytes (Yang T, cited above.) In the control mice, gene expression was not detected recombinant 3 days after the second administration of the virus, this is day 31. The administration of virus to the suppressed animals of CD4 + - was associated with expression n recombmante high-level transgene was stable for a month. The expression of the second virus was detectable on day 31. In this way, the suppression of CD4 + cells effectively allows the readministration of the vector without immediate elimination of LTC. The high level of initial gene transfer decreased after approximately one month in mice treated with IL-12. However, unlike control, high-level gene transfer to airway epithelial cells was achieved when the virus was administered to animals treated with IL-12 on day 28, as observed in the results of day 31 The animals treated with interferon gamma were virtually indistinguishable from • the animals treated with IL-12 since efficient gene transfer was achieved by a second virus administration. In this way, the use of these cytokines as immunomodulators allowed the repeated administration of the vector without its immediate elimination by neutralizing antibody. In other experiments, TH2 cells were not inhibited at the expense of increased THI activation. In mice treated with the ALP virus parenterally and IL-12 i.p., IL-12 does not increase the activity of adenovirus-specific CTL as shown by the chromium release tests. More importantly, the treatment of animals with IL-12 at the time of intratracheal instillation of virus does not stimulate inflammation or decrease transgenic persistence after a second virus administration.

B. Immunological Tests - NLM NLM lymphocytes from the control group and the group treated with IL-12 of ratonee C57BL / 6, 28 days after the administration of H5.010CBOLP, were cultured and challenged in vitro with H5. .OlOCMVlacZ inactivated with UV at 10 particles / cell for 24 hours. Cell-free supernatants were tested for the presence of IL-2 or IL-4 on HT-2 cells (a cell line dependent on IL-2 or IL-4) CYang I, cited above !. The presence of IFN-μ in the same culture supernatant of lymphocytes was measured on L.929 cells as described by CYang I, cited above !. The stimulation index (SI) was calculated by dividing cpm of 3H-thymidine incorporated in the cultured HT-2 cells in supernatants of restimulated lymphocytes with virus by cpn of 3H-tirnidine, incorporated in HT-2 cells grown in supernatants of lymphocytes incubated in antigen-free medium. The results are shown in Table I below.

TABLE I Incorporation of 3H-Ti? N? d? na (cpm + DE) Title of IFN-T Medio Hd.OlOCMVlacZ S.I. ÜJI / inl? * C57BL / 6 175 +. 40 2084 + 66 11.91 80 ant? -IL2 (1: 5000) 523 + 81 2.98 ant -ll_4 (1: 5000) 1545 +, 33 8.83 C57BL / 6 + TL12 247 + 34 5203 j_ 28 21.07 160 ant? -iL2 ( 1: 5000) 776 + 50 3.14 ant? -iL4 (1: 5000) 4608 + 52 18.66 The stimulation of lymphocytes from regional lymph nodes with recombinant adenoviruses led to the secretion of specific cytokines for the activation of both subpopulations of T helper cells, THI (ie, IL-2 and IFN-y) and TH2 (ie , TL-4) (Table I). The analysis of lymphocytes from animals treated with IL-12 stimulated in vitro with virus revealed an increased secretion of IL-2 and IFN-t in relation to the production of TL-4, when compared with animals that did not receive IL-12 ( that is, the ratio of IL-2 / IL-4) increased from 3 to 6 when IL-12 was used; Table I). c) Immunological tests-LBA LBO samples obtained from animals were evaluated 28 days after the first exposure to recirculating virus to determine the neutralizing antibodies for adenovirus and neutralizing anti-adenovirus antibody isotypes, as follows. The same four groups of C57BL / 6 mice, this is control, suppressed from CD ^, treated with IL-12 and treated with IFN-t, were infected with Hd.0.10CBOLP. Neutralizing antibody was measured in serially diluted I.BO samples (100 μl) which were mixed with Hd.OlOCBlacZ (1 x 106 pU n 20 μl), incubated for 1 hour at 37 ° C, and applied to HELA cells confluent 80% in 95-well plates (2 x 10 * cells per well). After 60 minutes of incubation at 37 ° C, 100 μl of DMEM containing 20% FBS was added to each well. The cells were fixed and stained for [.galactosidase] expression the next day. All cells were lacZ positive in the absence of anti-adonoviral antibodies. The adenovirus-specific antibody type was determined in BAL using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 100 μl of PBS containing 5 x 109 particles of HS.OlOCBlacZ for 18 hours at 4 ° C. The wells were washed 5 times with PBS. After blocking with 200 μl of 2% BSA in PBS, the plates were rinsed once with PBS and incubated with BAL samples diluted 1:10 for 90 minutes at 4 ° C.

The wells were then washed extensively and filled with 100 μl of I G or TgO against mouse, conjugated with OLP, dLluid at 1: 1000 (Sigma). The plates were incubated, subsequently washed 5 times, and 100 μl of the substrate solution (? -n? T rofelin phosphate, PNPP) was added to each well. The substrate conversion was stopped by the addition of 50 μl of 0.1 M EDTA, and the reactions were measured at 405 nrn. The results are shown graphically in Figures 10 to 1C, which summarize the neutralizing antibody titer, and the relative amounts (0D405) of TgG and IgO present in BAL samples. The neutralizing antibody titer for each sample was reported as the highest dilution at which 50% of the cells stained blue. As demonstrated by the first bar of Figures 10 to IC, the cytokines identified in Table I above were associated in the control mice with the appearance of antibodies to adenovir? S proteins in LBO of both isotypes IgG and IgA, which were capable of neutralizing the human recombinant vector AD5 in an in vitro test outside a 1: 800 dilution. As shown in the second bar of the graphs of FIGS. A to 1C, the transient suppression of CD4 + cells inhibited the formation of neutralizing antibody (FIG. IA) and virus-specific IgA antibody (FIG. 1C) by 80-fold, thereby allowing efficient gene transfer occurred after a second virus administration. Figure IB also shows a slight inhibition of TgG. As shown in the third bar of the three graphs, IL-12 selectively blocked the secretion of antigen-specific Igfl (figure 1C), without significantly impacting the formation of IgG (figure IB). This was concurrent with a 20-fold reduction of the viral specific neutralizing antibody (Figure 10). The animals treated with methylferon ga (fourth bar of FIGS. 10 and IB) were virtually indistinguishable from the animals treated with IL-12., since the virus-specific Igfl (figure 1C) and neutralizing antibody (figure 10) decreased compared to the control animals not treated with cytokine, but not to the extent obtained with those treated with IL-12. These studies show that the administration of selected immunomodulators to recombinant viral vector receptors for gene therapy at or near the time of first exposure to the vector can prevent the formation of blocking antibodies and / or the elimination of LTC from the vector both initially co or at the time of repeated exposure to the viral vector. The concordant reduction of neutralizing antibody with antiviral IgO suggests that immunoglobulin of the IgO subtype is mainly responsible for the blocking of gene transfer.

E3EMPL0 3 IMPROVEMENT OF MEDIUM TRANSFERENCE BY ADENOVIRUS BY SECOND ADMINISTRATION WITH IL-12 AND IFN-t IN MOUSE LIVER Experiments substantially identical to those described in Example 2 above were conducted in which viral vectors were administered in the blood for introduction of the transgene into the liver (instead of intratracheal delivery into the lung). In this example, the reclosing adenoviruses H5.010CMVlacZ and H5.010CBALP were used. Female C57BL / 6 mice (6 to 8 weeks old) were injected with suspensions of H5.010CBOLP (1 x 109 pfu in 50 μl PBS)? .p., On day 0 and similarly with Hd.OlOCMVlacZ on day 28. A group of these mice was used as control. Another group of mice was acutely suppressed from CD4 + cells by i.p. of antibody for cells CD4 + (GK1.5; OTCC No. TIB207, dilution of aßcitis 1:10) at the time of initial gene therapy (days -3, o and 3). A third group of mice was injected with IL-12 (injections of 2 μg, i.p.) at the time of the first administration of virus (days 0 and +1). A fourth group of mice was injected with interferon gamma (injections of 2 μg, i.p.) at the time of the first administration of virus (day 0 and +1). When the mice were subsequently submitted to euthanasia and necropsy on days 3, 28, or 31, liver tissues were prepared for cryoscopy *. in accordance with the procedures previously used for lung tissues in Example 2. The results of the disposition were substantially similar for gene therapy directed to the liver, in accordance with this method, as well as for the lung-directed therapy of Example 2 previous. The results described below were obtained from histoquinine injections of alkaline phosphatase (X100 amplification) or X-gal staining of β-galactosidase (amplification XI 00). Administration of OLP virus (109 pf?) In the veins of all groups of C57BL / 6 mice resulted in high-level transgene expression in liver tissue that decreased to undetectable levels on day 28. Loss of expression of transgenes appeared to be due to the LTC-mediated removal of genetically modified hepatocytes (see also Yang I, cited above). In the control mice, no recombinant gene expression was detected 3 days after the second virus administration, this is day 31. The administration of virus to the suppressed CD4 + animals was associated with substantially lower neutralizing antibodies and high level of expression of recombinant transgenes that was stable for one month. The expression of the second virus was detectable on day 31.

The initial transfer of genes at high level decreased after approximately one month in the mice treated with IL-12; however, unlike the control, some transfer of genes to the liver was achieved through the blood when the virus was readinrninistered to animals treated with IL-12 on day 28 and the level of neutralizing antibody was reduced. The animals treated with inter-rum rum were virtually indistinguishable from the animals treated with TL-12 since efficient gene transfer was achieved by a second administration of virus . In this way, the use of these cytokines and anti-CD4 + antibodies as immunomodulators allowed the repeated administration directed to the liver of the vector without its immediate elimination by neutralizing antibodies.

EXAMPLE 4 TRANSFER OF GENES MEDIATED BY ADENOVIRUS IN LIVER OF MOUSE Immune responses to the primary administration of recombinant viruses were characterized after using different strains of mice. Male recornbinant viruses (Hd.OlOCMVLacZ or H5.010CBALP) were inactivated with ultraviolet light in the presence of 8-methoxypsoralen. Briefly, the purified virus was resuspended in 0.33 mg / ml of 8-rnetox? Psoralen solution and exposed to a 365 nm UV light source on ice at k cm of the lamp filter for 30 minutes. After, the virus was passed over a Sephadex G-50 column equilibrated with PBS. Limit dilution translation tests of inactivated virus supply solutions demonstrated less than one functional virus per 105 particles of mactivated virus. The suspensions of the viruses (2xl09 pfu in 100 μl of PBS) were infused into the tail vein of female mice from 6 to 8 weeks of age, as detailed in the following experiments. Each experiment was carried out with a minimum of 3 mice in which transgeme expression was quantified in a section of each of 5 lobes. The minimum analysis was 15 sections per experimental condition. to. C57BL / 6 CH-2b mice were injected; Jackson Laboratories, Bar Harbor, MEl with suspensions of H5.010CMVlacZ on day 0 and similarly with H5.010CBALP on day 28 ("B6 mice"); b. C57BL / 6 mice were injected with UV inactivated Hd.OlOCMVlacZ on day 0 and H5.010CBALP on day 28 ("mice B6-UV ") c) Mice deficient MHC class II (II-) 'CGenPharm International, Mo? Ntain View, CAI, procreated in a background of C57BL / 6 (between 5 to 10 generations) and carrying the H haplotype -2b, they are incapable of expressing the I-At determinants and can not develop responses mediated by CD4 + T cells [Gr? Sby et al., Science, 53: 1417-1420 (1991) 1. These mice were infected with H5. 010CBOLP on day 0 and with H.SOlOCMVlacZ on day 28 ("class II- mice") d) Mice deficient in microglobulm (32 ((32rn-) [GenPharn International, Mointain View, COI, procreated on the background C57BL / 5 (between 5 to 10 generations) and carrying the haplotype H ~ 2b, are unable to develop MHC class I associated responses.These mice were infected with HS.OlOCMVlacZ on day 0 and with H5.010CBALP on day 28 ("ß2pr mice"): e C57BL / 6 mice were inoculated with 0.5 ml aliquots of mouse ascites fluid of a 1:10 dilution containing the GK1.5 (MOb anti-C. , OTCC TIB207), on days -3, 0 and +3, as described in example 2. This was equivalent to 100 μg of purified monoclonal antibody by injection. These mice depleted of CD4 + cells were infected with Hd.OlOCMVlacZ on day 0 and H5.010CBOLP on day 28 ("CD40b mice"); and f. C57BL / 6 mice, treated with IL-12 (2 μg in 200 μl PBS) i.p. on day 0 and day +1 as described in example 2, with HS.OlOCMVlacZ on day 0 and H5.010CBALP on day 28 ("IL-12 mice"). The mice of each group were subsequently subjected to euthanasia and the liver tissues were evaluated for LacZ expression by X-gal histochemistry (amplification x 100) on day 3 and day 28; and for ALP expression by histochemical stain on day 31 (amplification x 100). The development of neutralizing antibody for adenovirus in each group of mice was examined in serum samples obtained on day 28. Infusion of lacZ virus in C57BL / 6 mice was associated with high-level but transient expression of the reporter gene and the development eventual neutralizing antibody directed against adenoviral antigen (figure 2). No gene transfer was detected when the ALP virus was subsequently infused into these animals. In contrast, TI-class mice did not produce neutralizing antibody (Figure 2) and were receptive to high-level gene transfer from a second virus administration. Similarly, animals transiently depleted of CD4 partially stabilized lacZ expression and did not develop neutralizing antibody, allowing efficient virus re-administration. In the B6-UV mice, which received recombinant virus inactivated with UV, the inactivated virus generated a complete neutralizing antibody response (figure 2) that completely avoided the subsequent gene transfer. This result demonstrates that the incoming virus capsid proteins are sufficient to activate a blocking auxiliary T cell and a humoral immune response mediated by B cells. The experiment with mice / 32? Tr was carried out to evaluate the role of CD8 cells and MHC class T expression in the primary response to CZijlstra and others virus, Nature, 344: 742-746 (1990) 1. Transgenic expression was stable in these animals, consistent with previous reported data [Yang III, cited above !. However, gene transfer occurred at a significant level in the establishment of a virus strand readrnini. This was unexpected because this strain of mice must have all the components of the immune response necessary to produce a neutralizing antibody (ie, CD4 cells, MHC class IT and B cells). These animals did not develop a significant neutralizing antibody response to the adenoviral antigens. These results suggest dysregulation of the activation of T helper cells and / or B cells (Figure 2). Analysis of lymphocytes from ß2m animals demonstrated antigen-activated IFN-t secretion in excess of that measured in C57BL / 6 mice, possibly due to the persistence of cells infected with virus and the chronic activation of THI cells. The THI amplified response in ß2m- mice could lead to an inhibition of TH2 cells that cause the decreased production of antiviral antibodies. It was found that the expression of transgenes is stabilized in animals deficient in CD8 and MHC class I cells, by virtue of an interruption of the germinal line β2m-. The specific extirpation of perforin, the molecule on LTCs and natural killer cells (ON) that regulates cytolysis, prolongs the expression of the transgene synthetically (data not shown).E3EMPL0 5 EFFECT OF CD4 ANTIBODY ON TRANSFER OF GENES MEDIATED BY AD BY REPEATED ADMINISTRATIONS Suspensions of adenoví recornbinant.es were infused expressing different transgenes in the tail vein of Cd7BL / 6 (H-2 &) mice at 21-day intervals. Hd.OlOCMVLDLR is an adenovirus deleted from the Ela and Elb genes and has a deletion in the E3 gene at 78. gives 84.3 nu of the s? B360 from the basic structure of Od d, with the LDL receptor gene in place of deletion The [described by Kozarsky et al., Biol. Chem., 269: 1-8 (1994) 1. Hd.OlOCMNVLDLR was administered on day 0; H. OlOCMVlacZ on day 21; and H5.10CBOLP on day 42. To a control group of mice i.p. injections were administered. of saline on days -3, 0 and +3, with respect to each virus infusion. A second group of mice were given i.p. of s4-pressor antibody to block the activation of T helper cells (GKl.d mAb) in the same protocol. A third group of mice were given i.p. of mAb GK1.5 on days -3, 0, 3, 18, 21 and 24. A fourth group of mice, which did not receive the initial administration of Hd.OlOCMVLDLR, was treated with mflb GK1.5 on days -3, 0 and 3. The mice were subsequently euthanized, and the liver tissues were evaluated for the expression of LDLR by means of immunohistochemistry on day 3, for expression of lacZ by X-gal histochemistry on day 24, and for OLP expression by histochemical staining on day 4d. The tests were carried out as follows: 0. Inmununluorescent staining was performed for LDLR expression as follows: frozen sections (6 μm) were fixed in ethanol as described by Morris et al., Cited above. After blocking with 10% goat serum in PBS (GS / PBS), sections were incubated with a polyclonal antibody for LDLR (1: 200) for 60 minutes, and then with goat anti-rabbit IgG-FITC for 30 minutes . The sections were washed and mounted with Citiflour (Citifluor, UK). B. X-gal histochemistry was performed as follows: Sections of fresh frozen tissue (6 μ) were fixed in 0.5% glutaraldehyde for 10 minutes, rinsed twice for 10 minutes in PBS containing lMM MgCl 2, and were incubated in 1 mg / rnl of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), 5 mM of K3Fe (CN) β, 5 M of K «Fe (CN) β, and 1 mM MgCl2 in PBS for 3 hours. C. The OLP histochemistry was performed as follows: Frozen sections (6 μ) were fixed in 0.5% glutaraldehyde for 10 minutes, rinsed in PBS, incubated at 65 ° C for 30 minutes to inactivate the endogenous activity of ALP, were washed in 100 mM Tris (pH 9.5), 100 inM NaCl and 50 mM MgCl2, and stained in the same pH buffer containing 0.16d mg / ml 5-bromo-4-chloro-3 phosphate -indole (BCIP) and 0.33 rng / l of nitroazole tetrazolium (NBT) at 37 ° C for 30 minutes. The results discussed below were obtained from analysis of cytochemical injections of liver tissue 3 days after each virus infusion, including LDLR virus on day 0, lacZ virus on day 21 and OLP virus on day 42 (amplification by 150) . Serum samples were also collected on days 0, 3, 7, 14, 21, 28, 35 and 24 of each group of animals and analyzed for the neutralizing antibody titer. Serum samples were incubated at 56 ° C for 30 minutes and then diluted in DMEM in duplicate steps beginning at 1:20. Each serum dilution (100 μl) was mixed with Hd.OlOCMVlacZ (2 x 106 pfu in 20 μl), incubated for 1 hour at 37 ° C, and applied to 80% confluent Hela cells in 96 well plates ( 2 x 10 * cells per well). After 60 minutes of incubation at 37 ° C, 100 μl of DMEM containing 20% FBS was added to each well. The cells were fixed and stained for β-galactosidase expression the next day. All cells stained blue in the absence of serum samples. The neutralizing antibody titer for each sample was reported as the highest dilution with which less than 50% of the cells were stained blue. Figures 3A-3D report the antibody titer expressed as a function of days after infection. These experiments showed that in the group of mice that did not receive CD4 antibodies, efficient gene transfer occurred after the first virus. However, the development of neutralizing antibody blocked the transfer of genes with the two subsequent viruses. Neutralizing antibody appeared rapidly in the serum after the second virus if CD4 antibodies were not coadministered (Figure 3B). The third virus was not effective in these animals. In contrast, the administration of CD4 antibodies at the time of the first virus infusion prevented the formation of neutralizing antibodies (Figure 3B) and allowed high-level gene transfer with the second virus. The appearance of neutralizing antibody after the second virus was accelerated (Figure 3B) compared to the time course of a primary response in Figure 3A), suggesting activation of some level of cellular immunity even in the presence of CD4 antibodies. The administration of CD4 antibodies with the second virus again blocked the neutralizing antibody in the group of mice receiving CD4 antibodies and both viruses, allowing efficient transfer of genes with the third virue. These experiments demonstrate that transient immune blockade at the time of virus release, as opposed to chronic immunosuppression, is sufficient for efficient gene transfer. The fourth group of animals received CD4 antibodies on day -3 without administration of H5.010CMVLDLR, that is, 21 days before the primary administration of virus. The neutralizing antibody that developed after primary exposure to the virus blocked gene transfer 21 days later (Figure 3D) in a manner indistinguishable from that observed in intact animals not pretreated with CD4 antibodies (Figure 30). This result confirms the transient nature of CD4 suppression.

EXAMPLE 6 EFFECT OF IL-12 ON AD SPECIFIC ANTIBODY TYPES AND LTC RESPONSES fl. Serum samples obtained from the C57BL / 6 ("B6") and C57BL / 6 ("B6 + IL12") mice treated with IL-12 from example 5, 28 days after infection for IgGl antibody isotypes were tested. and adenovirus-specific IgG2a. An immunosorbent solid-phase enzyme-linked immunosorbent assay (ELISA) was performed using the purified Hd.OlOCMVlacZ virus as the antigen. Microtiter plates Immunolon-2-U (Fisher) were coated with 200 ng / well of viral antigen in 100 ml of PBS for 6 hours at 37 ° C, washed three times in PBS, and blocked in PBS / 1% BSA overnight at 4 ° C. The next day, serially diluted serum samples of multiples of 4 were added to the plates coated with antigens and incubated for k hours at 37 ° C. Plates were washed 3 times in PBS / 1% BSA and incubated with goat anti-mouse IgGl-biotm or goat-to-mouse IgG2a-b? Otna (COLT? G Laboratories, San Francieco, CA) at dilution 1 : 5000 for 2 hours at 37 ° C. The plates were washed as before and Avidm-ALP (Sigma) was added to each well at a dilution of 1: 5000 for 1 hour at 37 ° C. The wells were again washed as before and a PNPP substrate was added. The optical densities were read in a microscope reader model .SO from Biorad. Figures 4A and 4B summarize the relative amounts (OD405) of IgG1 and IgG2a, respectively, present in serum samples as a function of the dilutions of the sample. Serum ELISA tests revealed anti-v antibodies? of both IgG1 and IgG2a subtypes, consistent with the activation of both THI and TH subpopulations, respectively. Animals that received IL-12 produced anti-viral IgGl at the expense of increased production of IgG2a. B. Cultured splenocytes from Cd7BL / 6 mice ("B6") and C57BL / 6 mice treated with IL-12 ("B6 + IL12") from example 5 were restimulated in vitro 10 days after the administration of H5.010CBALP, with H5.010CMVlacZ for 5 days in DMEM supplemented with 5% FBS and 2 ~ 50 mM mercaptoethanol. These cells were tested for specific lysis on peeudo-infected ("pseudo") cells and C57SV cells infected with H5.010CBALP ("ALP") in a 6-hour sicr release test performed subsequently using the following effector ratios. white cells (C57SV, H-2b) in 200 μl of DMEM with 10% FBS in 96 well plates with V bottom (E: B = 50: 1, 25: 1, 12: 1, 6: 1, 5 : 1 and 3: 1). Before mixing with the effector cells, the target cells (l x 10 *) were labeled with 100 μCi siCr after a 24 hour infection with H5. OlOCMVlacZ at a rate of 50 and was used at 5 x 103 cells / well. After incubation for 6 hours, 100 μl aliquots of the supernatant were withdrawn for counting in a gamma counter. The percentage of specific release of sicr was calculated as: Sample cyclin - cpm spontaneous release) / (cp> max release - cpm spontaneous release)! x 100. The results, reported as percentage of specific lysis as a function of different relationship of effects to target (figure d), show that CTL activity against target cells infected with virus was not affected by treatment with IL-12 . The net result was a 3-fold reduction in neutralizing antibody, the magnitude of which was insufficient to allow efficient transfer of genes by virus re-administration. Differences in efficiency of virus readministration between ß2m- and loe Cd7BL / 6 mice treated with IL-12 could reflect inadequate repression mediated by cytokine after i.p. of TL-12 or different mechanisms of inhibition of TH2-d4 activation EXAMPLE 7 THE DEFICIENT MICE IN LCD40 ILLUSTRATES THE NECESSARY ROLE OF T-CELL ACTIVATION IN THE GUEST'S RESPONSES TO ADENOVIRAL VECTORS The role of T cell LCD40-mediated signaling in immune and cellular and humoral responses to vectors aderovir, was studied in genetically deficient mice in LCD40. Previous studies have demonstrated abnormalities in B-cell responses dependent on or in these mice (J. Xu et al., Immumty, .1: 423-431 (1994) and B. Renshaw et al., 3. Exp. Med., 180 : 1889-1900 (1994) - Deficient mice of LCD40 (CD40L KO) and their normal breeding partners in a C57BL / 5-129 chimeric background (3. Xu and others, cited above), were administered with lacZ containing suppressed adenovirus of El (HS.OlOCMVlacZ) on day 0 in the trachea (1 x 109 in 50 μl of PBS), to effect gene transfer to the lung, and in the peripheral circulation through the tail vein (2 x 109 in 100 μl in PBS), to effect gene transfer to the liver.The animals were repeatedly treated with H5.010CBALP, an adenoviral vector containing a different reporter gene (alkaline phosphatase, ALP), on day 28. The blood was analyzed before the second administration of vector for neutralizing antibodies, and tissues were cultured for analysis of the ression of the reporter gene 3 days later (this on day 31). Animals were sacrificed 3 and 28 days later to determine the efficiency and stability of transgene expression, respectively. The table summarizes orfomét rich analyzes of these tissues.

TABLE II QUANTITATIVE ANALYSIS OF LUNG AND LIVER FROM MOUSE FOR EFFICIENCY OF EXPRESSION OF TRANSGENES Day 3 Day 28 Day 31 i Lung (% of airway> 25% expression of transgenes) 15 Control 76 0 0 Ob CD40L 72 42 30 CD40L KO 75 30 45 2 Liver (% expression of transgenes) 20 Control 90. .5 ± 2, .6 0 0 flb CD40L 89, .3 +3. .1 46, .7 +4, .8 8. .2 + 4. .2 CD40L KO 92. .3 ± 4. .0 60, .4 +2. .8 8d. .9 + 3, .4 •? D i The data were quantified by examining a total of 100 respiratory tracts from 3 mice for the presence of respiratory epithelial cells containing transgene using the criterion of a positive airway when the transgene was greater than 2d%. 2 The data are presented as the mean ± D.E.

Normal partners of bait that were 3d administered with vectors showed high level of expression of transgenes on day 3 in the lung and liver that decreased to indetactablee levels on day 28 (similar to what is observed in mice without Cd7BL / 6 disease; II). Serum and alveolar bronchial lavage (LBO) were analyzed for neutralizing antibody for human Add as described in Y. Dai et al., Proc. Nati, acad. Sci. USA, 92: 1401 ~ 140d (199d). Substantial neutralizing antibody was developed for adenoviral capsid proteins on day 28 either in BAL fluid from animals that received vector int ratracheally or in blood from animals that received vector in the venous circulation (Figure 6). The readrnimstration of the vector on day 28 was not successful as was evident by the lack of transgene expression in the target organ 3 days later (similar to what is observed in mice 0 Cd7BL / 6, table II). Substantially different results were obtained in mice deficient in CD40L. Transgene expression was stable as little decrease during 28 days in both lung and liver (Table II). In addition, the neutralizing antibody was not developed (Figure 6), d resulting in highly efficient expression of transgenes after a second virus administration (Table I).

EXAMPLE 8 TRANSIENT TRANSFER BLOCKING OF CD40 WITH AN ANTIBODY THAT AVOID OR PRIMARY ACTIVATION OF T CELLS AND PROLONG EXPRESSION OF TRANSGENES The following example demonstrates that transient inhibition of CD40L with antibody blocked the activation of 5 CD * + T cells in the gene therapy lung model and effectively eliminated the effector responses of CD4 and B T cells. Persistent transgene expression and the efficiency of vector readmission in the lung was essentially identical in animals genetically deficient in CD40L (example 7 above) compared to those transiently inhibited with CD40L antibody (see below). The favorable results obtained in the CD40L-deficient mice, as shown in example 7 above, provided a basis for the development of a gene therapy adjunct with adenoviral vectors in the pharmacological inhibition of CD40L signaling. This therapy is based on the findings that the proteins of the incoming virus capsid are the main antigen source for the activation of CD4 + T cells, thereby restricting the costimulatory blocking time for a short interval when the vector is administered. Experiments in C57BL / 6 mice (6 weeks of age, females) not treated with antibodies or treated with isotype control antibody demonstrated high level expression of transgenes on day 3 in lung (Table II) and liver (Table II), which decreased to undetectable levels on day 28 (Table II). Gene transfer experiments were also performed on injected Cd7BL / 6 animals i.p. with 100 μg of mAb for CD40L (MRl, ATCC Hybridoma HB11048) i.p. days -3, 0, +3 and +6 in relation to the initial administration of vector or equivalent amounts of a control guinea pig monoclonal antibody. The studies in murine lung demonstrated stabilization of transgene expression in animals treated with CD40L mAb: the number of respiratory tracts that showed transgenes in more than 2d% of epithelial cellulae ee showed a minimum decrease of 72% on day 3 to 42% on the day 28 (table II). Transgene expression was also stabilized in the liver of animals treated with CD40L, in which hepatocytes expressing transgenes decreased slightly from 89% to 47% for a 28-day interval (Table II). The expression t ransgemca was stabilized in animals treated with CD40L antibody, for at least 6 weeks, which was the largest time point evaluated (data not shown). The recipient animals were analyzed for specific antigen activation of CD4 + and CD8 + T cells, which were tested both in vitro and in vivo. The effect of blocking CD40L on CD4 + T cells was studied in proliferation tests of lymphocytes stimulated with UV-activated adenovirus essentially as it is later deciphered (Table III). Briefly, nodule lymphocytes were restimulated with UV inactivated virus for 4 hours. mediatmal lymphatics (for the lung experiment) or splenocytes (for liver experiment) of mice, 10 days after virus administration. Supernatants were tested on HT-2 cells (ATCC, CRL 1841) for cytokine secretion, and proliferation was determined 72 hours after 2d measure the incorporation of 3H-thymidine. The activation of adenoviral specific T cells quantified by the stimulation index was initially documented on day 7 in animals treated without antibodies that were admixed by means of the vector in the lung or liver. The activation of adenoviral specific T cells increased progressively for the next 14 days. The stimulation index was calculated by dividing the 3H counts in the presence of antigen between those in the absence of antigen. Activation of T cells was substantially inhibited in both models by the coadministration of CD40L antibody. The greatest inhibition was observed in animals administered with vector in the lung.

TABLE III Responses of CD4 + T Cells and Neutralizing Antibody Index of Stimulation1 Neutralizing Antibody2 Day 7 Day 21 Day 28 Lung Control 11.2 ± 1.1 52 ± 2 267 + 92 CD40L Ab 1.2 ± 0.1 10 ± 1 20 ± 0 CD40L KO N.D N.D 20 + 0 Liver Control 20 ± 1 53 ± 2 533 ± 185 CD40L Ab 4.1 + 0.5 21 ± 1 66 t 23 CD40L KO N.D N.D 20 ± 0 i The data is presented as the average stimulation index of three determinations +1 D.E. N.D.- Not determined.

Data are presented as the mean neutralizing antibody titer (reciprocal dilution of three samples ± 1 D.E.

Activation of CD8 + T cells by virus infected cells was analyzed in chromium release tests using compatible MHC H-2 target cells infected with adenoviral vectors. As shown above, specific lysis was demonstrated with cultured lymphocytes of C57BL / 6 receptors on day 7, which were stimulated in vitro with cells that presented antigen, infected with adenovirus, and incubated with adenoviral infected targets (figure 7). No lysis was demonstrated for pseudo-infected targets. The lymphocytes cultured from animals treated with CD40L antibody also demonstrated CTL activity for adenovirus-infected cells. However, the extent of the lisie was consistently lower than that obtained from immunosuppressed animals. The need to extend CTL by in vitro stimulation before the cytolytic test may mask more significant differences in CTL activation that occurs after primary exposure in vivo. The main effect of CD4 + inhibition on the activation of adenoviral specific CD4 + T cells was also evaluated in vivo using in? Nocytochemistry techniques. These experiments were restricted to the model of gene transfer directed to the liver due to technical limitations of immunofluorescence in lung sections. Liver tissues were analyzed on day 14 for infiltration of CD4 + and C08 + T cells by double immunofluorescence. Animals not treated with antibody showed a typical mixed lymphocyte infiltrate that was dominated by CD4 + T cells and associated with substantial MHC class I eo-regulation on the basolateral surface of hepatocytes. Previous studies have suggested that the secretion of TFN-t from CD4 + T cells activated with THI, contributes to the increase in MHC class I that can sensitize hepatocytes to mediated elimination of LTC. Animals treated with antibody to CD40L still mobilized a mixed lymphocyte infiltrate. However, the proportion of CD4 + T cells is substantially lower and the increase in subcntial MHC class I is less effective. The specificity of the immunofluoroscence tests was demonstrated in pseudo-mfectadoe animals.

EXAMPLE 9 THE CD40L ANTIBODY PREVENTS BLOCKING ANTIBODY FORMATION IN TRANSFER OF GENES DIRECTED TO THE LUNG In this experiment the impact of the transient blockade of CD40L signaling at the time of vector administration on the production of neutralizing antibody and the efficiency of repeated vector administration was evaluated. The animals that received vector on day 0 with or without mAb were again treated with an adenoviral vector. which contained a different reporter gene on day 28. Blood was analyzed before administration of the second vector to determine neutralizing antibodies, and 3 days later (that is, day 31) tissues were cultured for reporter gene expression analysis. The most impressive results were obtained in the gene therapy model directed to the lung. The development of neutralizing antibody in L.BO after gene transfer directed to the vector lung was 20-fold inhibited in animals supplemented with CD40L antibody (FIG. 7). The transfer of genes with the second vector was not successful in animals not treated with antibody or animals treated with an isotype of control mOb (data not shown), as is evident by the complete absence of transgene expression 3 days after readministration of the vector. This contrasts with animals treated during the first administration of vector with CD40L antibodies in which gene transfer was effected after a second vector administration. Transgene expression was detected in >25% of airway epithelial cells of 30% of airways after the second vector, which is only slightly lower than the number of airways expressed by the transgene in an intact animal treated with vector (ie, 75%; Table II). The antibody to CD40L partially blocked the production of neutralizing antibody in the serum after intravenous infusion of virus (Figure 7). This was sufficient to allow some gene transfer to the liver with the second vector (8% hepatocytes), which did not occur in the absence of antibody, but is substantially reduced from that achieved after primary administration of vector in intact animals ( 89%).

EXAMPLE 10 A SHORT COURSE OF CYCLOPHOSPHAMID AVOIDS IMMUNE RESPONSES DESTRUCTIVE IN LUNG AND LIVER OF MOUSE Cyclophosphamide was administered to C57BL / 6 mice at different dosing regimens while administering a lacZ virus deleted in FA in the blood, to study gene transfer directed to the liver, and in the trachea, to study gene transfer directed to the lung . A second virus deleted from El, which expresses the reporter gene of alkaline phosphatase, is readministered in the same organ that received the first vector. Lymphocytes were isolated from regional sites and evaluated m vitro to determine the activation of vector-specific T cells. The tissues were cultured at different times for analysis of inflammation and its consequences as well as the expression of reporter genes both lacZ and OLP.

A. Animal Studies Cd7BL / 6 female mice were injected with lx109 pfu of Hd. OlOCMVlacZ through the trachea (lung studies) or tail vein (liver studies) on day 10. The d injections of cyclophosphat ida were given i.v. as indicated (in 200 rnl of PBS). Hd was injected. 010CBOLP as described before on day 28. The animals were sacrificed on day 3, 28, 31 and dO for analysis of transgenic expression. When necropsy was performed, lung and liver tissues were prepared for dispositions, while the vessel, bronchoalveolar lavage (LBfl) and mediastinal lymph node (NLM) were cultured for immunological tests.

B. Morphological Analysis. 5 For immunocytochemical analysis, frozen liver tissue was sectioned, while the lungs were inflated with a 1: 1 mixture of PBS / OTC, frozen and cryosected in blocks. For X-Gal (5-bromo-4-chloro-3-? Ndol? L-bD-galactopyranoside) histochemistry, 6 mm or frozen tissue sections were fixed in 0.5% glutaraldehyde for 10 minutes, washed twice with PBS containing 1 mM MgCl 2, and incubated in 1 mg of X-Gal per ml, 5 mM of K3Fe (CN) β, 5 mM of K4Fe (CN) β, and 1 mM of MgCl 2 in PBS, for 4 hours. hours. For alkaline phosphatase staining, frozen d-sections were fixed in glutaraldehyde at 0.d% for 10 minutes and washed twice in PBS. The sections were incubated at 65 ° C for 30 minutes to activate the endogenous alkaline phosphatase, washed once in PBS and made in 100 nM Tris-HCl (pH 9.5), 100 mM NaCl, 50 mM MgCl 2 contained 0.165 rng of BCIP (5-bromo-4-oloro-3- ndo.lilo phosphate) and 0.33 rng nitroblue tetrazolium per ml at 37 ° C for 30 minutes.

C. Immunofluorescence Frozen sections were fixed in methanol at ~ 20 ° C for 10 minutes, air dried and rehydrated in 0 PBS twice and the non-specific binding in 10% goat serum / PBS was blocked for 30 minutes. The sections were incubated for 1 hour with any rat CD4 anti-mouse antibody (anti L3T4, GibcoBRL, 1: 100 dilution in 2% goat serum), followed by a 30 minute incubation with d rng of d in unoglob? Lina G (IgG) anti-rat fluorescein or rat CD8a anti-mouse-fluorescein isothiocyanate (anti- | _y-2, GibcoBRL, 1: 100 dilution in 2% goat serum). For MHC class I staining, sections were incubated with diluted mouse hybridoma supernatant l: d0 for H-2KbDb (20-8-4S), for 60 minutes, followed by a 30 minute incubation with 5 mg / ml of goat anti-mouse IgG-fluorescein conjugated isothiocyanate (FITC). The sections were washed twice and mounted with the Citifluor antidecubator (Canterbury Chemical Lab., Canterbury, United Kingdom). 5 D. CTL test For CTL tests, splenocytes from three mice or lymphocytes from 10 mice were concentrated. The cells are restimulated in vitro for 5 days with Hd.OlOCMVlacZ (MOT 0. 5) and tested on MHC-compatible white cells, which were previously infected with Hd. OlOCMVlacZ and loaded with 51Cr, using different effector / cel ratios the target. The specific release percentage of sicr as [(cpm sample - cpm spontaneous release) / (cpm maximum release - cpm spontaneous release)! x 100. Spontaneous release was determined by analyzing target cells without effector cells in the medium, while maximum release was estimated by adding d% SDS to the target cells during the 6 hours incubation time.

E. Cytokine Release Test 6 x 106 splenocytes were cultured with or without antigen (ie, H5, OlOCMVlacZ inactivated with UV at an MOI of 10) for 24 hours in a 24-well plate. 100 ml of cell-free supernatant was transferred onto 2 x 10 3 HT-2 cells (cell line dependent on IL-2 and IL-4) in 96 well round bottom plates. 10% rat concanavalin A medium and supernatant were used as negative controls and 2d positive. Proliferation was determined 48 hours later by means of a 6 hour pulse of [SHlti idina (0.35 rnCi / well).

F. Neutralizing Antibody Test Serum and LBñ were incubated for 30 minutes at 56 ° C to inactivate the complement. Serial dilutions of serum and LBO were incubated in DMEM without FBS (50 ml, starting at 1:20) with 1 x 106 pfu of H5. OlOCMVlacZ, for 60 minutes and was applied on 2 x 1.0 * of Hela cells (confluent 80%) in 96-well plates. After 60 min of incubation, 100 nmol of DMEM containing 20% FBS was added. The cells were fixed 16 to 18 hours later and stained for β-galactoeidase activity. All cells stained blue when medium was added instead of serum or LBO. The neutralizing antibody titer was determined by means of the highest dilution with which less than 50% of the cells were stained blue. All items identified herein are incorporated by reference. Numerous modifications and variations of the present invention are included in the previously identified specification and are expected to be obvious to the person skilled in the art. Such modifications and alterations including the specific immunomodulator selected, the manner of administration, the recombinant vector, the selected transgene, route of administration, etc., are considered to fall within the scope of the appended claims.

Claims (4)

NOVELTY OF THE INVENTION CLAIMS

1. The use of an immunomodulator for the manufacture of a medicament for inhibiting an immune response against a coadministered recombinant virus, said drug inhibits the function of neutralizing antibodies directed against said virus and reduces the elimination of LTC from virally infected cells.

2. The use according to claim 1, further characterized in that said immunomodulator comprises an agent selected from the group consisting of (a) a cytokine; (b) an agent that depletes or inhibits CD4 + cells; (c) an antibody against T cells; (d) an agent that blocks the interaction between CD40 ligand on a T cell and CD40 on a B cell; (e) an agent that blocks the interaction between the ligand of CD28 or CTLA4 on a T cell and B7 on a B cell; and (f) cyclophosphamide.

3. The use according to claim 2, further characterized in that the cytochrome is selected from the group consisting of? -terleucin-4,? -terleucin-12 and gamma-interferon.

4. The use according to claim 2, characterized in that said agent that depletes or inhibits CD4 + cells comprises an anti-CD4 antibody. d. The use according to claim 2, further characterized in that said agent that blocks the interaction between CD40 ligand on a T cell and CD40 on a B cell, inhibits the CD40 ligand on the T cell. 6. The Use according to claim d, further characterized in that said agent is selected from the group consisting of soluble CD40 molecule and anti- CD40 ligand antibody. 7. The use according to claim 2, further characterized in that said agent that blocks the interaction between the ligand of CD28 or CTL04 on a T cell and B7 on a B cell, binds to the ligand of CD28 or CTL04 on the cell T. ld 8. The use according to claim 7, further characterized in that said agent is selected from the group consisting of soluble CD28, soluble CTLA4, anti-CD28 antibody and anti-CTLA4 antibody. 9. The use according to any of claims 1 to 8, further characterized in that said immunomodulator comprises a DNA molecule. 10. The use according to any of claims 1 to 8, further characterized in that said immunomodulator comprises a protein. 2d. The use according to claim 1, further characterized in that said recombinant virus is an adenovirus. 12. The use according to any of claims 1 to 11, further characterized in that the medicament is administered simultaneously with said recom binant virus. 13. The use according to claim 11, further characterized in that the medicament comprises said recirculating blood. and. 14. The use according to any of embodiments 1 to 11, further characterized in that the drug is administered prior to the administration of said recombinant virus. Id. The use according to any of claims 1 to 11, further characterized in that the medicament is administered after the administration of said recombinant virus.