MXPA99002217A - Method for tolerizing a mammalian patient to administration of gene therapy virus vectors - Google Patents

Abstract

A method for tolerizing a mammalian subject to administration of a live virus carrying a gene for delivery to a cell of the subject is disclosed. The method entails administering to the subject a suitable amount of an inactivated virus prior to administration of the live virus. The prior administration of the inactivated virus suppresses anti-virus cytotoxic T cells, permitting longer transgene persistence once the live virus is administered, and permitting effective readministration of live virus.

Description

METHOD TO INDUCE TOLERANCE TO THE ADMINISTRATION OF VIRAL VECTORS FOR GEN THERAPY IN A MAMMARY This invention was supported by the National Institute of Health, grant No. DK47757. The government of the United States has rights in this invention.

FIELD OF THE INVENTION The present invention relates to the field of somatic gene therapy and useful methods thereof.

BACKGROUND OF THE INVENTION Immune responses of the recipient to the viral vectors used in somatic gene therapy, ie to the viral proteins of the vector, and / or to the transgene carried by the vector, and / or the cells infected with the virus, have arisen as problems recurrent in the initial application of gene therapy technology for animals and humans [Yang et al., J. Virol. , 69: 2004-2015 (1995) (Yang I)]. Virtually in all models, including lung-directed gene therapy and liver-directed gene therapy, transgene expression is transient and associated with the development of pathology at the gene transfer site.

It has been found that, for example, the transient nature of transgene expression from recombinant adenovirus is due, in part, to the development of antigen-specific cellular immune responses to virus-infected cells and their subsequent elimination by the host. The collaboration of LTC (cytotoxic T lymphocytes) directed against newly synthesized viral proteins and virus-specific helper T cells [Zabner et al., Cell, 75: 207-216 (1993); Crystal and others, Nat. Genet , 8: 42-51 (1994)] leads to the destruction of cells infected by the virus. It has also been noted that these immune responses cause the appearance of associated hepatitis that develops in recipients of liver-directed gene therapy in vivo in the first two or three weeks of initial treatment and myositis in targeted gene therapy receptors. to muscle in vivo. Another antigenic target for the immune-mediated elimination of cells infected with virus is the product of the transgene. CTL are an important effector in the destruction of target cells with activation that occurs in some cases in the context of the transgenic product, as well as the viral proteins synthesized, both presented by MHC class 1 molecules [Yang I; and Zsengeller and others, Hum. Gene Thera. , 6: 457-467 (1995)]. Another limitation of the use of recombinant viral vectors for gene therapy has been the difficulty in obtaining a detectable gene transfer after a second administration of the virus. This limitation is particularly problematic in the treatment of inherited disorders of a single gene or in chronic diseases, such as cystic fibrosis (CF), which will require repeated therapies to obtain genetic reconstitution throughout life. It has been shown that there is a decreased gene transfer subsequent to a second therapy in a wide variety of animal models subsequent to intratracheal or intravenous delivery of adenovirus vectors [T. Smith and others, Gene Thera. , 5: 397 (1993); S. Yei and others, Gene Thera. , 1: 192-200 (1994); K. Kozarsky et al., J. Biol. Chem. , 269: 13695 (1994)].

Similar difficulties have been noted when the viral vector is different from the adenovirus ie retroviruses, vaccinia and the like. In each case, resistance to repeated gene therapy was associated with the development of neutralizing antiviral antibodies, which frustrated the successful transfer of the subsequent gene to a second administration of the virus. The proposed solutions to these antiviral immune responses have involved, to date, new designs of viral vectors which employ fewer viral genes as well as the pre- or co-administration of immuno-modulators, such as anti-CD40 ligands and others. modulators identified in the art [See, p. ex. , Yang et al., J. Virol. , 70 (9) (Sept., 1996); International Patent Application no.

O96 / 12406, published May 2, 1996; and the International Patent Application no. PCT / US96 / 03035, all incorporated herein for reference. However, the successful induction of immuno-specific tolerance to the new gene products (both viral products and transgenic products) of the recombinant vectors of gene therapy remain one of the most formidable challenges in gene therapy. Failure to establish immune tolerance to viral gene therapy vectors can lead to immune rejection of the cells expressing the transgene and therefore to the loss of the transgenes. There is a need in the art for methods and compositions which enable the induction of specific immunological tolerance to the capsid proteins of the viral vector and to the products of both the viral and transgenic gene which are introduced and expressed in mammalian cells by the general methods of gene therapy.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a solution to the aforementioned needs in the art by providing a method for removing the immune barrier that prevents long-term gene therapy by selectively creating tolerance in a mammalian subject to multiple administrations of a live recombinant virus carrying a gene to be supplied to a cell of said subject. This method involves administering to the subject an appropriate amount of an inactivated virus before administration of the first live recombinant virus. The inactivated virus can be a whole virus, a replication-defective virus, a previously inactivated copy of the live recombinant virus carrying a transgene, or a recombinant virus previously inactivated without a transgene. Preferably an inactivated virus is administered via an oral or intravenous route. Essentially the administration of the inactivated virus deactivates the T cells specific for viral antigens, before the recombinant live virus carrying the transgene is administered. Other aspects and advantages of the present invention are described below in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure IA is a graphical representation of the data obtained from the proliferation test described in example 2. The graph plots the viral proliferation in mouse splenocytes measured using tritiated thymidine radioactivity in CPM X 10 units as a function of the number of adenovirus _ recombinate of serotype 5 carrying a beta-galactosidase gene (rAd5-LacZ) / ml x 10-8. Mice of type C57BL / 6 were fed with (1) PBS (Saline Solution Regulated with Phosphates) (open square), (2) 1012 rAd5-LacZ viral particles inactivated with heat and irradiated with UV, (rAd5-LacZ "dead") (open circle) or with cells from • r 1 mice injected intravenously (iv) with 10 particles of dead rAd5-LacZ virus (full circle), and subsequently live rAd5-LacZ virus was applied. Splenocytes were collected for analysis ten days after administration of live rAd5-LacZ virus. Figure IB is a graphical representation of the data obtained from the cytokine (IL-2) tests described in example 2. The plot plots IL-2 in pg / ml as a function of the rAd5-LacZ virus number / ml x 10 for the same mice as reported in Figure IA. Figure 1C is a graphical representation of the data obtained from the cytokine (IFN-gamma) tests described in example two. The graph plots IFN-gamma in pg / ml as a function of the virus number rAd5.LacZ / ml X 10"for the same mice as reported in figure 1A.The figure ID is a graphical representation of the data obtained from the cytokine test (IL-10) described in example 2. The plot plots IL-10 in pg / ml as a function of the number of rAd5-LacZ / ml X 10 ~ 8 viruses for the same mice as and as reported in figure A. Figure 2 is a graphic representation of the data obtained from the cytotoxicity test described in example 3. The symbols report to the treated mice as described in example 1. The square open, fed with PBS; the open circle fed with 10 dead virus particles rAd5-LacZ; the full circle, injected with 1012 particles of the dead rAd5-LacZ virus intravenously. The graph plots the percentage of splenocyte-specific death as a function of the effector-to-target ratio. Figure 3A is a histogram of mouse lung tissue for mice fed PBS and subsequently injected with the live rAd5-LacZ virus on day 10 after application. The expression of the LacZ gene is observed. Figure 3B is a histogram of mouse lung tissue for mice fed rAd5-LacZ killed virus and subsequently injected with rAd5-LacZ live virus on day 10 after application. LacZ gene expression is observed. Figure 3C is a histogram of mouse lung tissue for mice fed PBS and subsequently injected with rAd5-LacZ live virus on day 17 after application. Expression of the LacZ gene is observed. Figure 3D is a histogram of mouse lung tissue for mice fed rAd5-LacZ killed virus and subsequently injected with rAd5-LacZ live virus on day 17 after application. The expression of the LacZ gene is observed.

Figure 3E is a histogram of mouse lung tissue for mice fed PBS and subsequently injected with rAd5-LacZ live virus on day 28 after application. No expression of the LacZ gene is observed. Figure 3F is a histogram of mouse lung tissue for mice fed rAd5-LacZ killed virus and subsequently injected with rAd5-LacZ live virus on day 28 post-application. LacZ gene expression is observed.

DETAILED DESCRIPTION OF THE INVENTION The method of the present invention provides for the induction of selective tolerance in a mammalian subject to multiple administrations of a recombinant virus in gene therapy which carries a therapeutic gene (transgene) for delivery to a cell, in which the transgenic product is expressed in vivo According to the present invention, it was unexpectedly observed that administering to a package insert for gene therapy treatment, an appropriately high dose of an inactivated virus prior to the administration of a live recombinant virus carrying a transgene suppresses the antiviral immune responses shown by the patient and allows prolonged expression of the transgene in vivo. As detailed below in Example 1, oral administration of a heat-inactivated and UV-irradiated rAd5-LacZ virus induced immune suppression of anti-adenoviral antigenic immunity in mice that were subsequently injected with rAd5-LacZ live virus. Immune tolerance was measured by determining the persistence of the transgene, LacZ, in mouse tissue several days after application. The mice that received the live rAd5-LacZ virus did not reject the live recombinant virus carrying the transgene. In contrast, mice that received similar oral doses of rAd5-LacZ live virus or splenocytes infected with rAd5-LacZ live virus showed strong anti-adenoviral antigenic immunity by administering the live virus. This result indicated that the previous administration of the killed or inactivated virus induced immuno-tolerance in the mice-to the administration of the live recombinant virus carrying the transgene. The additional experiments described below in detail in Example 2 were conducted to determine whether immune tolerance to subsequent administrations of the live recombinant virus resulting from the oral administration of the inactivated virus could be similarly induced by intravenous administration of the inactivated virus. Following the same protocol as in Example 1, the mice were administered phosphate buffered saline (PBS) as an inactivated rAd5-LacZ control or virus orally or the same dose of inactivated rAd-LacZ i.v. virus. Subsequently, rAd5-Lacz was delivered to the three groups of mice. The splenocytes of the mice were subjected to proliferation tests to measure the immune tolerance based on a comparison of the number of splenocytes that proliferated as a function of the live virus number rAd5-LacZ / ml. As illustrated in Figure 1A, the splenocytes decreased in number in the mice to which the inactivated virus was administered, both orally and intravenously in comparison to the control. Cytokine tests were conducted on the same mice to evaluate the mechanism of tolerance. As shown in Figure IB the IL-12 production was suppressed only with the inactivated virus administered orally. However, both IFN-gamma (FIG. 1C) and IL-10 (FIG. ID) were deleted in response to the administration of inactivated virus administered orally or i.v. in comparison to the control. The oral administration suppressed the last two cytokines to a much greater degree than that of the control and that of the inactivated virus administered iv. Because immune rejection of the transgene expressing cells is mediated primarily by cytotoxic T cells, anti-adenoviral cytotoxicity was tested for oral or intravenous administration of killed viral antigens using the thymidine H-release test (JAM test). as described in example 3. As shown in figure 2, both oral and intravenous administration of rAd5-LacZ killed virus suppressed the generation of adenovirus-specific cytotoxic T cells, although under these conditions, the oral route appeared to be more effective than the intravenous route. It should be noted that this is the first demonstration that oral antigen administration activates antiviral cytotoxic T cells. Resistance to readministration of recombinant virus can be partially mediated by neutralizing antibodies specific for viral antigens. Therefore, to determine if oral administration of rAd5-LacZ virus suppresses humoral immunity, antiadenovirus antibody responses were examined in mice fed rAd5-LacZ by conventional methods as described in Example 4 above. 40-60% for IgM and IgA antiadenovirus responses. These data strongly suggest that specific immunological tolerance can be induced by oral or intravenous administration of inactivated viruses, but that the oral route may be more effective than the intravenous route. To test if oral tolerance helps to prolong the expression of the transgene in vivo, the expression of the LacZ gene was examined by X-gal histochemistry according to the normal procedures as described in example 5 for mice that were administered inactivated virus rAd5-LacZ or PBS orally and subsequently live recombinant virus as in example 2. As indicated in figures 3A to 3F, expression of the LacZ gene lasted approximately 17 days in the lung tissue of non-tolerized mice (fed with PBS). Expression of the transgene was prolonged to at least 28 days in mice fed 10 rAd5-LacZ killed viruses before being given live virus according to this invention. Therefore, the pre-administration of inactivated virus has a "tolerizing" effect after subsequent administration of the live recombinant virus. This allows a prolonged persistence of transgene expression from a single administration of recombinant virus and allows multiple effective administrations of a recombinant virus carrying the transgene to the gene therapy candidate, i.e. to a patient suffering from a disorder due to to the mutation, deletion or functional deletion of a normal gene or to a patient with an acquired disease. The method of the present invention is useful for any recombinant virus carrying a useful transgene and gene therapy, such as adenovirus, adeno-associated virus retrovirus and others. Many appropriate recombinant viral vectors carrying a multitude of desired genes are currently known in the art. The design of a recombinant virus for gene therapy is not a limitation on this method, nor is the selection of the transgene and the regulatory sequence required to express the transgenic product in vivo. The selection of the virus is also within the field of gene therapy technique. Similarly, it is anticipated that the method of this invention will operate to suppress undesired antiviral immune responses and possibly, unwanted antitransgenic immune responses without considering the disease or disorder being treated. Therefore, this method is expected to have broad applicability in the field of gene therapy. In one embodiment that invention, the inactivated virus and the live recombinant virus carrying the transgene have substantially, and completely preferable, viral capsid proteins and identical viral genes. For example, as described in the following examples, the "dead" virus is simply the same recombinant virus, except that it has been killed or inactivated. Subsequent administration of live virus uses the same virus, which has not been inactivated. Alternatively, the inactivated virus may be the same virus but does not carry a transgene, i.e. a "void" recombinant virus. Still in another modality, the inactivated virus and the live virus can have viral proteins that are equally related. • "antigenically related" means that each virus activates an immune response capable of killing the other viruses, because each virus expresses a gene product with at least one antigen present on the other gene product.

In another embodiment the inactivated virus can be an inactivated whole virus, whose genome provides the viral sequences of the recombinant virus carrying the transgene, or a virus with defective replication comprising at least a portion of the genome from which the recombinant virus carrier was made of the transgen. The replication defective virus may or may not carry the transgene. Another embodiment of this invention allows the induction of tolerance of a patient to the open reading frame of transgenic therapeutics. According to this method the inactivated virus is a different virus or strain different from the live viral vector, but carries and expresses the same transgene as the living viral vector does. The inactivated virus can be prepared by conventional means. Preferably by heat inactivation the live virus is subjected to a temperature of at least 56 ° C for about 30 minutes. Similarly, the UV irradiation necessary to inactivate a virus can range from exposures of about 100 to 4000 rads for an exposure time of about several seconds to several hours. Another means of inactivation, such as inactivation, chemical with organic solvents, toxic bleaches, imines, formalin and the like can be used and are well known in the art. Combinations of these conventional mechanisms of viral inactivation can also be used to produce an inactivated virus of this invention.

In the practice of this invention, it is preferred that a high dose of the inactivated virus be administered orally, as demonstrated in the following examples and figures referred to herein. However, another appropriate route of inactivated virus administration is intravenous (iv). An appropriately high dose of inactivated virus to induce tolerance is greater than 10 viral particles / kg. of the patient's body weight for any of the administration routes. More preferably, where the route is oral, a preferred amount of the inactivated virus is greater than 1013 particles / kg. and can be close to, or greater than, 1016 particles / kg. Where the route is i.v., a preferred amount of the inactivated virus is greater than 10 particles / kg. , and can be close to or greater than 10 particles / kg. The amount of a particular inactivated virus necessary for a particular patient according to the methods of this invention can be determined on an empirical basis by one skilled in the art. An amount as such may also be impacted by the specific virus used (i.e., the degree to which said virus expresses the viral antigens), and can be easily determined without incurring undue experimentation. The inactivated virus is generally administered between about 6 hours to 28 days prior to the first administration of live recombinant virus carrying the transgene to allow immune suppression or deactivation of the T cells to occur prior to administration of the live recombinant virus. The pre-administration of inactivated virus creates a climate of immune tolerance, or at least reduces the immunological rejection, of the live recombinant virus. Although not anticipated to be necessary, administration of the inactivated virus can be repeated before a second administration or subsequent administration of the live virus, under conditions such as cystic fibrosis, in which it is likely that repeated transgenic administration is necessary for the treatment is effective. However, it is anticipated that only a single administration of the inactivated virus before the first administration of the live recombinant virus carrying the transgene will be necessary to induce long-term enduring tolerance. Immune tolerance can be mediated by many different mechanisms which include deletion, anergy and deviation of specific lymphocytes. It is anticipated that several mechanisms of immune tolerance are involved in this method of gene therapy. While you do not want to be united by theory, the mechanisms by which the present invention operates can be similar to "clonal anergy", that is, the lack of response of the T cells, which causes the deactivation of the immune attack of the T cells specific to a particular antigen, resulting in a reduction of the immune response to this antigen. See for. example, the description in application of International Patent No. W095 / 27500, published on October 19, 1995 and incorporated herein by reference. It is anticipated that oral tolerance to the recombinant virus can be increased by manipulation of the antigenic doses as described above. Other factors may also increase oral tolerance, such as the resistance of the co-stimulation and the cytokine medium. For example, oral tolerance can be increased by administering the inactivated virus with a selected immuno-modulator. An optional step in the method of this invention involves co-administration to the patient, either concurrently with, or before or after administration of the inactivated virus, an appropriate amount of a short-acting immuno-modulator. The selected immuno-modulator is defined herein as an agent capable of inhibiting the formation of neutralizing antibodies directed against the recombinant virus of this invention or capable of inhibiting the elimination of the virus by the cytolytic T lymphocytes (CTL). The immuno-modulator can interfere with the interactions between the subgroups T (JJI or jj2) and the B cells to inhibit the formation of neutralizing antibodies. Alternatively, the immuno-modulator can inhibit the interaction between TJJI cells and CTLs to reduce the occurrence of deletion of the recombinant virus by CTLs.

A variety of useful immuno-modulators and doses of use thereof are described, for example, in Yang et al., J. Virol. , 70. (9) (Sept., 1996); International Patent application no. 096/12406, published May 2, 1996; and the International Patent Application no. PCT / US96 / 03035, all incorporated herein for reference. The following examples and analysis of the method of this invention utilize a gene therapy model for cystic fibrosis (CF), using a recombinant human replication defective adenovirus type 5 (rAd5) as a vector and Escherichia coli lacZ as a transgene. However, it should be clear to the person skilled in the art that the methods of this invention have broad application to other disorders, other recombinant and inactivated viruses and other transgenes. Therefore the field of this invention is not limited to the components of the following examples used to illustrate the invention.

EXAMPLE 1 Antifungal selection based on the ability to induce oral tolerance As it is known that the form of an antigen plays a major role in the development of oral immunity versus tolerance, 3 types of viral antigens were examined for their ability to induce oral tolerance or immunity.

These include a live recombinant adenovirus, serotype 5 (Ad5) carrying a gene coding for beta-galactosidase (LacZ). This recombinant virus is described in Y. Yang et al., J. Virol. , 69 (4): 2004-2015 (1995) as H5. OlOCMVLacZ. This virus is based on a sub360 base structure Ad5 deleted from Ela and Elb (1.0 to 9.6 map units) as well as 400 bp from the E3 region. The LacZ gene is expressed from a cytomegalovirus promoter. For ease of reference, this recombinant virus is referred to hereafter as "live rAd5-LacZ virus". A second viral antigen is rAd5-LacZ virus which is inactivated with heat and irradiated with ultraviolet (UV) light. The recombinant virus was heated for 0.5 hours at 56 ° C, followed by exposure to ultraviolet radiation of about 1000 rads for several minutes. The third antigen is syngeneic splenocytes infected with live virus rAd5-LacZ. To determine whether these antigenic preparations induce oral tolerance, mice of the C57BL / 6 type were first fed with 10 viral particles from one of the live or inactivated viral vectors described above, or with 10 splenocytes infected with 1012 live viral particles. Two days later, the mice were given 5 x 1010 particles of live rAd5-LacZ virus intratracheally. The mice were sacrificed 10 days after the application and their spleens were collected. The splenocytes were tested for their anti-adenoviral immune responses in a conventional proliferation test as described in Yang et al., J. Virol. , 6_9: 2004 (1995), incorporated herein for reference. Surprisingly, oral administration of heat-inactivated and UV-irradiated rAd5-LacZ viruses induced profound immune suppression (tolerance) of antiadenovirus immunity. In contrast, oral administration of live virus rAd5-LacZ or syngeneic splenocytes infected with live virus rAd5-LacZ did not induce tolerance.

EXAMPLE 2 Induction of tolerance of T-helper cells specific for adenovirus A. Tolerance / administration experiment Three groups of C57BL / 6 mice were either fed with phosphate buffered solution (SRP) or 10-L "S particles of rAd5-LacZ virus inactivated with heat, irradiated with UV (virus "dead"). As a control, the third group of mice was injected intravenously with 10 particles of killed rAd5-LacZ virus to determine if tolerance could be induced via an intravenous route of administration. Two days later, the mice were administered with 5 x 10 10 particles of live rAd5-LacZ virus intratracheally. The mice were sacrificed 10 days after administration and their spleens were collected. Splenocytes were tested for antiadenovirus immune responses in a proliferation test and a cytokine test described below. The experiment was repeated in duplicate with similar results.

B. Proliferation test A proliferation test was conducted as described in Yang et al., J. Virol. , 6_9: 2004 (1995), incorporated herein for reference. Briefly described, the splenocytes, 1 x 106 cells / well were cultured in 0.2 ml. of serum-free medium containing several concentrations of killed rAd5-LacZ virus. One μCi of 3 H-thymidine was added to each culture 72 hours after the initiation of the culture and the cells were collected and their radioactivity was measured 16 hours later. The results of the proliferation test can be seen in Figure IA. Radioactivity is measured in counts per minute of the tritiated thymidine incorporated into the DNA. These data points are proportional to cell proliferation. Each point of the data represents an average for a minimum of 3 mice. The standard deviations of the CPM data are within 25% of the averages.

As illustrated in Figure 1A, both oral and intravenous administrations of killed rAd5-LacZ virus suppressed splenocyte proliferation when compared to SRP controls. The splenocytes decreased in number for the mice given the inactivated virus, both orally and intravenously in comparison to the control ones.

C. Cite-quina test To assess the mechanisms behind immune suppression and cell proliferation, cytokine tests were conducted as described in Chen et al., Science, 265: 1237 (1994), incorporated herein by reference. Briefly described, the culture supernatants were collected at 40 hours and the cytokine concentration was determined by ELISA. Each point of the data represents an average of a minimum of three mice and the standard deviations of the cytokine data are within 20% of the averages. The results of the cytokine tests can be seen for IL-2 in figure IB, for IFN-gamma in figure IC, and for IL-10 in figure ID. Oral, but not intravenous, pre-administration of inactivated rAd5-LacZ virus suppressed IL-2 production. However, both IFN-gamma. (Fig. IC) and IL-10 (Fig. ID) were suppressed in response to the administration of inactivated virus administered orally or i.v. in comparison to the control.

EXAMPLE 3 Induction of tolerance of specific cytotoxic T cells for viruses To test whether oral or intravenous administration of dead viral antigens affects the cytotoxic T cell responses, antiadenovirus-induced cytotoxicity was tested with the H-thymidine release test (JAM test) as described in P. Metzinger, J. Iimxtunol. Meth., 145: 185 (1991), incorporated herein by reference. Briefly described, the mice were treated as in example 2 above. Their splenocytes were cultured in vitro for 4 days with rAd5-LacZ and their specific cytotoxicity was tested using a line of syngeneic fibroblastic cells infected with rAd5-LacZ. The CTL will cause lysis of cells that have viral antigens exposed on their surfaces. The cells are loaded with a radio marker and the release of the label is proportional to cell lysis. The results are shown in Figure 2. Each point of the data represents an average of a minimum of 3 mice. Standard deviations are within 15% of the averages. The experiment was repeated in duplicate with similar results. Both oral and intravenous administration of killed rAd5-LacZ virus suppressed the generation of adenovirus-specific cytotoxic T cells, although under these conditions, the oral route appeared to be the most effective compared to the intravenous route.

EXAMPLE 4 Effects on humoral immunity To test whether oral administration of inactivated rAd5-LacZ virus suppresses humoral immunity, antiadenovirus antibody responses in mice fed inactivated rAd5-LacZ virus were examined by conventional methods as described in Yang et al., J.

Virol. , 6_9: 2004 (1995), incorporated herein for reference.

Briefly described, the mice were treated as described in Example 2 and the isotype of antibodies specific for adenovirus was determined in alveolar bronchial lavage (BAL) using the enzyme-linked immunosorbent assay (ELISA). The resulting data showed that although mucosal IgA was not drastically suppressed by oral administration of. rAd5-LacZ inactivated virus, a suppression of between 40-60% was observed for the IgM and IgA antiadenovirus responses. These data strongly suggest that specific immunological tolerance can be induced by oral or intravenous administration of inactivated viruses and that the oral route may be more effective than the intravenous route.

EXAMPLE 5 Histochemistry of X-GAL Expression of the LacZ gene was examined by X-gal histochemistry according to normal procedures, for example, the procedures described in J. Price et al., Proc. Nati Acad. Sci., U.S.A., 84 .: 156-160 (1987). The mice were treated as described in Example 2 and their lung tissue was examined at several times after administration of the live virus. As indicated in Figures 3A to 3F, expression of the LacZ gene lasted approximately 17 days in the lung tissue of mice not induced to tolerance (fed with PBS). Expression of the transgene was prolonged for at least 28 days in mice fed with 10 particles of dead rAd5-LacZ virus, according to this invention. All references listed above are incorporated herein for reference. Numerous modifications and variations of the present invention are included in the specifications identified above and are expected to be obvious to those skilled in the art. It is believed that such modifications and alterations to the compositions and methods of the present invention, such as selections of different transgenes or dose selection of the immune modulators or vectors are within the scope of the appended claims.

Claims (14)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of an inactivated virus in the preparation of a medicament for administration to a mammalian subject, characterized in that said inactivated virus is administered in a high dose prior to the administration of a live recombinant virus carrying a transgene to be supplied or said subject, characterized in that said medicament creates tolerance to said mammalian subject towards said living recombinant virus.

2. The use according to claim 1, characterized in that said inactivated virus is administered orally.

3. The use according to claim 1, characterized in that said inactivated virus is administered intravenously.

4. The use according to any of claims 1 to 3 characterized in that said dose is greater than 1011 viral particles / kg. of the patient's body weight.

5. The use according to any of claims 1 to 4 characterized in that said inactivated virus is a whole virus.

6. - The use according to any of claims 1 to 4 characterized in that said inactivated virus is a virus with defective replication.

7. The use according to claim 6, characterized in that said inactivated virus is chosen from the group consisting of retroviruses with defective replication, vaccinia virus with defective replication and canaripox virus of replication defective.

8. The use according to any of claims 1 to 7 characterized in that said inactivated virus comprises said recombinant virus carrier of a transgene, previously subjected to inactivation.

9. The use according to any of claims 1 to 7 characterized in that said inactivated virus has a viral sequence identical to said recombinant virus carrying the transgene, but said inactivated virus does not carry said transgene.

10. The use according to any of claims 1 to 7 characterized in that said inactivated virus and said live virus have antigenically related capsid proteins or express antigenically related viral proteins.

11. The use according to any of claims 1 to 7 characterized in that said inactivated virus is administered between 6 hours and approximately 28 days before the administration of said live virus.

12. - The use according to any of claims 1 to 7 characterized in that said inactivated virus is readministered before a second administration and subsequent administrations of said live virus.

13. The use according to any of claims 1 to 7 characterized in that said inactive virus is prepared by a method selected from the group consisting of thermal inactivation, physical inactivation, chemical inactivation and combinations thereof.

14. The use according to any of claims 1 to 7 characterized in that said inactivated virus and said live virus have different viral genes but carry substantially identical transgenic sequences and express substantially identical transgenic products in vivo.