MXPA97000710A - Identification of a region of the human citomegalovirus gene involved in the decreasing regulation of the expression of the heavy chain of the main complex of histocompatibility clas - Google Patents

Abstract

Infection of human fibroblast cells with human cytomegalovirus (HCMV) causes the down regulation of cell surface expression of the major histocompatibility class I complex. The present invention is directed to a mutant with a deletion of 9 kb in the S component of the HCMV genome (which includes the open reading markers IRS1-US9 and US11) which does not cause a down-regulation of the class I heavy chains. When examining the phenotypes of mutants with smaller supre-sions with this portion of the HCMV genome, it is found that a 7 kb region containing at least 9 open reading frames contains the genes required for the reduction of heavy chain expression. In addition, it has been determined that two subregions (A and B) of the 7 kb region each contain genes which are sufficient to cause the decreasing regulation of the heavy chain. In sub-region B, the product of the US11 gene is involved. It codes for an endoglycosidase glycoprotein sensitive to H which is intracytoplasmic, similar to the glycoprotein E3-19K of adenovirus type 2 which inhibits the expression on the surface of heavy chains class

Description

IDENTIFICATION OF A REGION OF THE HUMAN CITOMEGALOVIRÜS GENE INVOLVED IN REGULATION DECREASING THE EXPRESSION OF THE HEAVY CHAIN OF THE MAIN COMPLEX OF HISTOCOMPATIBILITY CLASS I FIELD OF THE INVENTION The present invention relates to a recombinant mutant human cytomegalovirus (HCMV) which does not express a decreasing regulation (regulation that decreases the activity) of the heavy chains class I of the major cellular histocompatibility complex (MHC) when it causes infection.

AMTg? SPgNTBS Pg THE INVENTION Human cytomegalovirus (HCMV) is a beta herpesvirus which causes a clinically serious disease in immunocompromised and immunosuppressed adults, as well as in some infants infected in utero or perinatally (Alford and Britt, 1990). The genome sequence of the 230 kb double-stranded DNA of HCMV has been determined (Chee et al., 1990) and has at least 200 open reading frames (ORF). For the purposes of this application, an open reading frame is defined as the REF: 23923 portion of a gene which codes for a sequence of amio acids and can therefore encode a protein. The function of some HCMV proteins are known or predicted due to their homology with other viral proteins (especially herpes simplex virus and cellular proteins). However, for most of the HCMV ORFs, the functions of the proteins they code are unknown. In order to study the function of the HCMV gene, mutants can be constructed by suppression of HCMV in order to determine their growth properties in vi tro (Jones et al., 1991, Jones and Muzithras, 1992). For purposes of this application, deletion mutants are defined as a human cytomegalovirus mutant which lack regions of the wild-type viral genome. This strategy involves the substitution site mutagenesis of a gene or HCMV genes selected by a prokaryotic reporter gene, usually / 3-glucuronidase, although guanosine phosphoribosyltransferase can also be used. In this manner, the recombinant virus can be isolated only if the replaced viral gene or genes are not essential. Several investigators have shown that infection by HCMV results in a decreasing regulation (regulation that decreases the activity) of heavy chains of MHC class I cellular (Bro ne et al., 1990, Beersma et al., 1993, Yamashita et al. , 1993). For purposes of this application, down-regulation is defined as a reduction in the synthesis, stability or surface expression of MHC class I heavy chains. Such phenomenon has been reported for some other DNA viruses, including adenovirus, mouse cytomegalovirus and herpes simplex virus (Anderson et al., 1985, Burget and Kvist, 1985, del Val et al., 1989, Campbell et al., 1992, Campbell and Slater, 1994, York et al., 1994). In the adenovirus and herpes simplex virus systems, the product of a viral gene which is available for in vitro replication is sufficient to cause the down regulation of MHC class I heavy chains (Anderson et al., 1985; Burget and Kvist, 1985). The genes involved in the down-regulation of the class I heavy chain by mouse cytomegalovirus have still been identified.

SUMMARY OF THE INVENTION The present invention provides recombinant mutant human cytomegalovirus which does not down regulate the expression of the heavy chains of cellular MHC class I. The deletions of the sequence of the gene are carried out in the region of the recombinant cytomegalovirus (HCMV) genome containing open reading frames IRS-1-US11. Two such mutants, RV 798 and RV 799, both deleted from the open reading frames US2-US11, lack the ability to down regulate the MHC class I heavy chains. The present invention also provides a method for controlling the down-regulation of the expression of the major histocompatibility complex (MHC) class I in a cell infected with cytomegalovirus., method which uses recombinant mutant human cytomegalovirus. The present invention also provides a vaccine which uses the recombinant mutant human cytomegalovirus, as well as a method for preventing or reducing the susceptibility of an acute cytomegalovirus infection in an individual by administering an immunogenic amount of the recombinant mutant human cytomegalovirus. A live attenuated HCMV vaccine lacking gene sequences in the recombinant cytomegalovirus (HCMV) genome region containing open reading frames IRS-I - US11 will induce a better immune response compared to one containing this region of gene, based on the lack of the decreasing regulation of class I by the first. Therefore, a vira that lacks the region is a superior immunogen. The present invention also provides gene therapy vectors in which the HCMV gene involved in the decreasing region of the MHC class I heavy chain can be incorporated into adenovirus or similar virus vectors based on gene therapy vectors to minimize the immune response. This will allow the use of recombinant adenoviruses or similar viruses based on the therapy vectors that will be used in gene therapy. The invention can be understood more fully with reference to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the detection of the MHC class I cell surface by immunofluorescence-flow cytometry in cells infected with HCMV. Human foreskin fibroblast (HFF) cells are infected with the indicated virus at a multiplicity of infection of 5 PFU / cell for 72 hours. At this time, cells are fixed to 1% paraformaldehyde and stained with primary antibody specific for HLA-A, B, C (W6 / 32) or mouse IgG as control (paired in isotype) followed by goat IgG, against conjugated mouse with secondary FITC. The percent of positive cells (5 x 103 total) and the mean fluorescent intensity (MFI) based on forward light scattering versus integrated light scattering logarithm at 90 ° are calculated using the I muno Program Coulter MDADS I. Figure 2 shows the expression of the MHC class I heavy chains in infected cells of HCMV wild type AD169. Figure 2A is a Western blot analysis. HFF cells were uninfected (U) or infected with a multiplicity of infection of 5 PFU / cell. At 24, 48 and 72 hours post-infection, the total cellular proteins are harvested, subjected to electrophoresis through 15% SDS-polyacrylamide gel, electrotreated to nitrocellulose and probed with mouse monoclonal antibody TP25. .99 (specific for a non-conformational epitope, of MHC class I heavy chains) by using a chemiluminescent ECL detection kit (Amersham). Figures 2B and C are immunoprecipitation analyzes. HFF cells are uninfected or infected (as in the above), either in the absence or presence (+ PFA) of phosphonoformate and are radiolabeled either at 4 hours or in subsequent post-infection times (69-73 hours) ( Figure 2B) or for 2 hours at the indicated post-infection time (Figure 2C). The proteins are harvested immediately after radiolabelling and heavy class I chains are immunoprecipitated using the mouse monoclonal antibody TP25.99. Figure 3 shows the organization of recombinant virus genomes. Figure 3A, the first line, is a schematic diagram of the total organization of the wild-type HCMV genome. Single region sequences are shown by a line, while repeated region sequences are indicated by dark colored rectangles. The relevant HindIII fragments, within the L and S components, are indicated by their letter designation (Oram et al., 1982). The second line is an expansion of the wild-type HindlII-Q, -X and -V regions of the S component. Significant open reading frames and their orientation are shown with clear rectangles (Chee et al., 1990). The position of the repeated IRS sequences is indicated by a dark colored rectangle. The positions of the restriction endonuclease sites HindIII (H) and Xhol (X) are shown. Figures 3B-I show the genomic organization of the indicated HCMV mutant. In each case, the first line is the organization of the wild type AD169 genome, the second line represents the organization of relevant sequences of the linearized plasmid used to produce the recombinant virus. The inclined lines indicate the limits of the viral flanking sequences which may be involved in the homologous recombination to generate the desired mutation. The suppressed region is indicated by a dark rectangle below the first line. Figure 3J shows the derivation and organization of RV799. The first two lines are the same representations as Figure 3B-I, and the third line represents the organization of the relevant sequences the linearized plasmid used to produce RV799 from original RV134 (second line). Figures 4A-C show the analysis of heavy chain expression in cells infected with HCMV mutants. HFF cells were uninfected (U) or infected with the indicated virus (multiplicity of infection of 5 PFU / cells) and radiolabeled for 4 hours in later times, after infection (69-73 hours). The proteins were collected immediately after radiolabeling. Figure 4A is an X-ray of heavy class 1 chains which were immunoprecipitated using mouse monoclonal antibody TP25.99. Figure 4B is an x-ray of the total radiolabeled proteins to verify the equivalent radiolabel efficiency approximately. Figure 4C is an x-ray that verifies the same progression through the viral replicative cycle. UL80 proteins were immunoprecipitated by using polyclonal rabbit antiserum against the assembly protein. Figures 5A-C show immunoprecipitation of class I heavy chains from cells infected with RV798-, RV799-, RV134- or wild-type AD169. The HFF cells were uninfected (U) or infected with the indicated virus (multiplicity of infection of 5 PFU / cell) and radiolabeled for 2 hours in late post-infection times (71-73 hours). The proteins were harvested immediately after radiolabeling. Figure 5A is an X-ray of the class I heavy chains which were immunoprecipitated using the mouse monoclonal antibody TP25.99. The equivalent radio-labeling efficiency was verified (Figure 5B) and progression through the viral replicative cycle (Figure 5C) as described for Figure 4B and C. Figure 6 is an X-ray showing the sensitivity of endoglycosidase H of heavy class I chains synthesized in infected cells with RV798. HFF cells were infected with RV798 (multiplicity of infection of 5 PFU / cells) and radiolabeled for 2 hours at early times (6-8 hours) or late times (80-82 hours) post-infection. For comparison purposes, uninfected cells were radiolabelled for 2 hours. The proteins were harvested either immediately after radiolabeling (pulse) or after 2 hours of saturation (saturation) in complete unlabelled medium. Class I heavy chains were immunoprecipitated using the mouse monoclonal antibody TP25.99. The immunoprecipitated protein is incubated for 6 hours either in the presence (+) or absence (-) of 1.5 mU of endoglycosidase H, before electrophoresis in SDS-polyacrylamide gel and fluorography. Figure 7A-C shows immunoprecipitation of the class I heavy chains from cells infected with RV798-, RV7181-, RV7177-, or wild-type AD169. The HFF cells were uninfected (U) or infected with the indicated virus (multiplicity of infection of 5 PFU / cell) and radiolabeled for 2 hours at later times post-infection (65-67 hours). The proteins were collected immediately after radiolabeling. Figure 7A is an X-ray of heavy class I chains which were immunoprecipitated using mouse monoclonal antibody TP25.99. The equivalent radiolabelling efficiency (Figure 7B) and the progression through the viral replicative cycle (Figure 7C) were verified as described for Figures 4B-C.

Figures 8A-D are photographs which show the location of the US11 gene product (gpUSll) in cells infected by immunofluorescence. HFF cells were uninfected or infected with wild type AD169 or RV699 (deleted from the US11 gene) at a multiplicity of infection of 5 PFU / cells. After 8 hours, uninfected or infected cells were fixed with 4% paraformaldehyde. Then some cells were perbilized with 0.2% Triton X-100. The main antibody was rabbit polyclonal antiserum raised against the fusion protein US11 (Jones and Muzithras, 1991). The fluorescence was visualized through a Zeiss microscope. Figure 9A-D shows the analysis of heavy chain expression in cells infected with HCMV mutants in early post-infection times. The HFF cells were uninfected (U) or infected with the indicated virus (multiplicity of infection of 5 PFU / cells) and radiolabeled for 4 hours from 6-10 hours post-infection. The proteins were collected immediately after radiolabeling. Figure 9A is an X-ray of heavy class I chains which were immunoprecipitated using mouse monoclonal antibody TP25.99. Figure 9B is an x-ray in which, to verify approximately equal infection, an immediate-early protein of 72 kDa IE1 is immunoprecipitated, using the mouse monoclonal antibody 9221. Figure 9C is an X-ray of the immunoprecipitation of the transferrin receptor. cell with Ber-T9 mouse monoclonal antibody to approximately verify an equal expression of this glycoprotein. Figure 9D is an x-ray of the total radiolabeled proteins to approximately verify the equivalent radiolabel efficiency. Figure 10 provides a summary of the MHC class I heavy chain expression data from HFF cells infected with wild-type and mutant HCMV. The first line is the total organization of the wild-type HCMV genome, and the second line is an expansion of the wild-type H ± ndlII-Q and -X regions of the S component. ORFs are indicated by a clear rectangle; Unlabeled ORFs that overlap US4 and US5 are US4.5. Deletions within the various HCMV mutants are indicated by the darkened rectangle. RV670 is deleted from IRS2-US9 and USll; RV35 is deleted from US6-US11; RV67 is deleted from US10-US11; RV80 is deleted from US-8-US9; RV725 is deleted from US7; RV69 is deleted from US6; RV47 is deleted from US2-US3; RV5122 is deleted US1; RV46 is deleted from IRS1; RV798 is deleted from US2-US11; RV7181 is deleted from IRS1-US9; RV7177 is deleted from IRS1-US6; and RV7186 is deleted from IRS1-US11. The results of down-regulation of the MHC class I heavy chain are obtained from immunoprecipitation experiments (using the heavy chain conformation of an independent monoclonal antibody TP25.99) in which the HFF cells infected with HCMV were radiolabeled in later times post-infection The last line shows the location of the two subregions which contain the genes which are sufficient for the down regulation of the MHC class I heavy chain. Subregion A contains ORF US2-US5 (bases 193119-195607) and subregion B contains the ORF US10 and USll (bases 199083-200360). Figure 11A-B are results of Spotting Weestern cell lines that express the USll gene of HCMV. Uninfected human U373-MG astrocytoma cells are stably transformed with a USll expression plasmid and analyzed by Western blot analysis for the MHC class I heavy chain expression (HA figure) and for USll expression ( Figure 11B) using the monoclonal antibody TP25.99 and the polyclonal antisera USll, respectively.

DESCRIPTION OF THE PREFERRED MODALITIES A recombinant HCMV mutant, named RV670, has been constructed which expresses a marker gene (3-glucuronidase) in place of a group of viral genes. By infecting human fibroblast cells with this mutant, the expression of the heavy chains class I of the major histocompatibility complex (MHC) is not reduced in comparison when these cells are infected with wild-type HCMV. In contrast to wild type HCMV, the virus of the present invention does not produce a down regulation of the expression of the cellular MHC class I heavy chain proteins. A 7 kb region of the HCMV genome, which contains the genes which are required for the down regulation of heavy chain expression, are used in the invention. One skilled in the art will appreciate that efficient processing and presentation of the antigen is required to activate and expand cytotoxic T lymphocyte precursors for an efficient cellular mediated immune response. The presentation of efficient viral antigen requires the continuous expression of MHC class I proteins during infection. Infection of cells with RV670 results in a continuous expression in class I heavy chains. One skilled in the art will appreciate that the virus (RV670) or other human cytomegalovirus with a deletion of similar genes can be used to produce an effective live or inactivated vaccine because the class I heavy chains are still expressed in cells infected by RV670, as well as in uninfected cells, and therefore the presentation of the viral antigen is carried out for the purpose of initiating cytotoxic T cell responses. In the present invention, experiments by flow cytometry and immunofluorescence confirmed that cell surface expression of heavy class I chains is greatly reduced in later post-infection times in HFF cells infected with HCMF wild-type strain 8169. The radiolabelling-immunoprecipitation experiments indicate that the down-regulation of the newly synthesized MHC class I heavy chains is carried out throughout the course of the infection, and that they are initiated at very early (3 hours) post-infection times (FIG. 2 C) . This reduction has been reported to be at the post-translational level: heavy class I chains have a higher turnover rate in cells infected with HCMV compared to uninfected cells (Beersma et al., 1993). Such instability of the class I heavy chains results in a reduced cell-mediated immune response to HCMV infection since the viral peptides will be presented inefficiently. Therefore, the reduction in the expression of the heavy chain of class I is important in terms of evasion of the host immune system and in the establishment of persistent or latent infections by HCMV (Gooding, 1992). The bank of HCMV mutants, which represent 180 ORF which can be supplied for viral replication in tissue culture, was examined for its ability to cause down-regulation of the MHC class I heavy chains. It was shown that a 7 kb region of the S component of the HCMV genome containing the ORF US2-US11 (bases 193119-200360), contains genes which are required for this phenotype (the data are summarized in Figure 10). Within this region, there are two subregions, each of which contains enough genes for the down regulation of the heavy chain. Sub region A contains the ORF US2-US5 (bases 193119-195607). It has been proposed that US2 and US3 encode membrane glycoproteins (Chee et al., 1990). US3 is a differentially divided gene which is expressed through the viral replicative cycle and encodes a protein with transcriptional transactivating function (Tenney and Colberg-Poley, 1991).; Colberg-Poley et al., 1992; Tenney et al., 1993; Weston, 1988). Other smaller ORFs are also present in this sub-region (between ORFs US3 and US5), but their expression characteristics or functions have not been reported. Gretch and Stinski (1990) reported that there is an early mRNA of 1.0 kb transcribed from this region of the HCMV genome, but a fine map determination has not been made. It is not yet known which of these genes is involved in the decreasing regulation of the heavy chain. Sub-region B, which is also sufficient for the MHC class I heavy chain reduction, contains the US10 and USll genes (figure 10), bases 199083-200360. However, based on the data using HCMV mutant RV670 which expresses wild-type levels of the US10 gene product, the expression of US10 is not sufficient for a down-regulation of heavy chain expression (Figure 2B). The genetic data implies that the USll gene product is necessary. It has been shown that the expression of USll is sufficient to cause a down-regulation of the MHC class I heavy chain in uninfected cells, stably transformed, in the absence of other MCNV proteins (FIG. 11). RNA and protein expression from these ORF start early and progress through the course of infection (Jones and Muzithras, 1991), - US10 and USll encode glycoproteins of 22 kDa, (gpUSlO) and 32 kDa ( gpUSll) respectively; both glycoproteins have sugar residues bound to N which are completely sensitive to endoglycosidase H. These glycoproteins are retained in the endoplasmic reticulum or in the golgi apparatus. Consistent with this conclusion are the immunofluorescence data in which gpUSll is not detected on the cell surface, but is detected in the cytoplasm of cells infected with HCMV (figure 8). The characteristics of HCMV gpUSll (as well as of gpUSlO) are similar to that of a 25 kDa glycoprotein (E3-19K) encoded from the E3 region of adenovirus type 2. Ad E3-19K is not essential for viral replication. It has been shown to contain N-linked sugar residues sensitive to endoglycosidase H, which are retained in the endoplasmic reticulum and bind to the MHC class I heavy chains, thus preventing their transport to the cell surface 9 (Anderson et al. al., 1985; Burgert and Kvist, 1985). In contrast to Ad E3-19K, no direct association was detected between gpUSll (or gpUSlO) and class I heavy chains (ie, by coinmunoprecipitation) (data not shown).

The identification of the region of the US2-US11 gene as the region of the HCMV genome required for the down-regulation of the MHC class I heavy chains is significant in several aspects. As mentioned above, expression from this region of the genome during the course of infection acts to interfere with an effective cell-mediated immune response. The surface expression of MHC class I molecules is required for the presentation of antigen to activate and expand the precursor populations of cytotoxic T lymphocytes (CTL) (Scwartz, 1985). In addition, they are additionally required for objective recognition by activated CLT (Zinkernagel and Doherty, 1980). In MCMV, CTLs against primary immediate early protein protect against lethal infection by this virus (Jonjic et al., 1988). However, in individuals infected with HCMV, the frequency of CTL against the immediate early protein of HCMV analogous IE1, has been reported to be extremely rare (Gilbert et al., 1993). Recent studies have shown that IE peptides represent more efficiently by cells infected with HCMV treated with interferon? in comparison with untreated infected cells (Gilbert et al., 1993). Interferon μ causes increased surface expression of MHC class I proteins.

Therefore, increasing the expression of class I heavy chains in cells infected with HCMV may be important for the efficient generation of specific CTLs for IE or CTL against other important HCMV antigens. An HCMV mutant lacking the US2-US11 region could have this effect since the class I heavy chains are not subject to down-regulation when cells are infected with this mutant. Therefore, a deletion of this region from the viral genome is important in the development of an inactivated HCMV vaccine to induce an effective immune response against HCMV. Several years ago it was reported that the UL18 ORF of HCMV codes for a protein that resembles MHC class I heavy chains (Beck and Barrell, 1988). We hypothesized that the decreasing regulation of heavy chains of cells infected with HCMV was due to the competition of the product of the UL18 gene for β2 -microglobulin, which effectively prevented the normal association of class I heavy chains and 02-microglobulin (Browne et al., 1990). This hypothesis was essentially discarded when a HCMV mutant with UL18 suppression retained its ability to down regulate the heavy chain suppression (Browne et al., 1992). It remained possible that the UL18 gene product was only one of several HCMV genes whose expression is sufficient for this phenotype. However, the data of the present invention indicate that only the genes within the US2-US11 region are sufficient for the decreasing regulation of the heavy chain of class I. The existence of two independent mechanisms which result in the decreasing regulation of the MHC class I expression emphasizes the importance of this phenotype for successful infection and persistence in the host. One mechanism can serve as a support system for the other, but it is also possible that there is specificity of cell type for each system. In the case of the HFF cell system, both mechanisms are functional. However, in U373-MG cells, the decreasing regulation of heavy chain expression depends more on the presence of subregion A. In this case, there may be qualitative or quantitative differences in cellular proteins which interact with the products of the gene of sub-region B. A similar situation occurs in the herpes simplex virus system. It has recently been reported that a product of the US12 gene of 88 amino acids (ICP47) is sufficient to sequester the class I heavy chain in the endoplasmic reticulum (York et al., 1994). However, heavy chain expression is not affected by mouse cells infected with herpes simplex virus, although ICP47 is expressed in this cell and mouse heavy chains are not subject to down-regulation when expressed in a fibroblast system human infected with HSV (York et al., 1994). A pharmaceutical composition containing the recombinant HCMV mutant of the present invention can be prepared in which the genome lacks the gene sequence capable of down-regulating the expression of MHC class I in infected cells. A stabilizer or other suitable vehicle can be used in the pharmaceutical composition. As described above, the recombinant HCMV mutant of the present invention, which lacks the gene sequence capable of down-regulating the expression of MHC class I can be used in a vaccine for the prevention of cytomegalovirus infections. Optionally, an adjuvant can be added to the vaccine. A method for immunizing an individual against cytomegalovirus can be carried out by administering to the individual an immunogenic amount of the recombinant HCMV mutant of the present invention which lacks the gene sequence capable of down-regulating the expression of MHC class I A method for preventing or reducing the susceptibility in an individual to an acute infection by cytomegalovirus can be carried out by administering to the individual an immunogenic amount of the recombinant HCMV mutant of the present invention which lacks the genetic sequence capable of subjecting Decreasing regulation of MHC class I expression. The down regulation of the expression of MHC class I in a cytomegalovirus infected cell can be controlled by a method that presents the steps of identifying a gene sequence capable of down-regulating the major histocompatibility complex and suppressing the gene sequence identified from the cytomegalovirus genome. As described above, the sequence of the gene involved in the down-regulation of the MHC class I heavy chain can be incorporated into adenovirus or similar virus vectors based on the gene therapy vectors to minimize the immune response and allow the use of vectors in gene therapy. A virus based on the gene therapy vector comprises the gene sequence of the open reading frame of USll. Another virus based on the gene therapy vector comprises the gene sequences of subregions A and B (open reading frame US2-US5 and US10-US11, respectively).

EXAMPLE 1 Viruses and cells You get HCMV strain AD169 from the American Type Culture Collection and is propagated according to conventional or standard protocols known to those skilled in the art. Cells of human foreskin fibroblasts (HFF) are isolated in this laboratory and then used for twenty passages (Jones and Muzithras, 1991). They are grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 25 mM HEPES.

DNA sequence In the present invention, the numbering system of Chee et al. (1990) of the DNA sequence of HCMV strain AD169 (Genbank accession number X17403).

Plasmids The plasmids used for the creation of HCMV mutants are constructed using the method described above (Jones et al., 1991, Jones and Muzithras, 1992). In general, the β-glucuronidase indicator gene is surrounded on each side by 1.5 kb of HCMV sequences which flank the gene to be suppressed from the virus. In each case, the plasmid DNA is linearized with a restriction enzyme which cuts into the prokaryotic backbone before the transaction. Genomic DNA fragments of HCMV from strain AD169 are derived from either pHind-G, pHind-X, or pXba-P, which contain the HipdlII-G DNA fragments (bases 176844 through 195837), -X (bases 195837 until 200856), and Xbal-P (bases 200391 to 206314) (Oram et al., 1982; Jones et al., 1991). pUS7 / US3 contains the HCMV fragment of 1.7 kb Ps l-PstI (bases 196447 to 194741 in the vector pIBI30 [International Biotechnologies, Inc.]) derived from pHind-G and pHind-X. To replace the ORF from HCMV USll to IRS1 by β-glucuronidase (ie, RV7186, Figure 3), pBgdUSll / IRSl is constructed. Sequentially, the plasmid containing a 1.8 kb fragment and a Pstl-Xbal fragment (bases 202207 to 200391, containing the promoter sequences US13, US12 and USll of pXba-P), / S-glucuronidase, an SV40 fragment from 288-b containing the early and late polyadenylation signals (from pRcCMV [Invitrogen]), and the 1.7 kb Ncol -Ncol fragment (bases 189763 through 188062, containing the J1I to IRL1 sequences, from pHind-G).

To replace the ORFs of HCMV USll to US2 with (S-glucuronidase (ie, RV798; Figure 3), pBgdUSll / US2 is constructed.Sequentially, this plasmid contains a 1.8 kb fragment a fragment of Pstl-Xbal (bases 202207 to 200391, which contains the promoter sequences for US13, US12, and USll, from pXba-P), 3-glucuronidase, a fragment of 255-b containing the polyadenylation signal US10 (bases 199276 to 199021, from of pHind-X), and the fragment of 1.3 kb iVhel-Apal (bases 193360 to 192033, containing the C-terminal sequences US2 to IRS1, from pHind-G) To replace the ORF of HCMV USll to US6 by 3 -glucuronidase (ie, RV35; figure 3), pBgdUSll / US6 is constructed sequentially, this plasmid contains the 1.8 kb Pstl-Xbal fragment (bases 202207 to 200391, which contain the promoter sequences for US13, US12 and USll). , from pXba-P), / S-glucuronidase, and the 1.5 kb Hpal-SstII fragment (bases 195589 through 194 062, containing the sequences US6 to US3 C terminal, from pHind-G). The replacement of the ORFs of HCMV US11-US10, or the ORF of USll (uniquely), by / 3-glucuronidase (ie RV67 and RV699, respectively), have been previously described (Jones et al., 1991). To replace the ORFs of HCMV US9 to IRSl by (S-glucuronidase (ie, RV7181; Figure 3), pBgdUS9 / IRSl is constructed.Sequentially, this plasmid contains the 1.1 kb Sall-Apal fragment (bases 200171 through 199021 ), the SV40 early promoter of 351-b (from pRcCMV), (S-glucuronidase, the 288-b polyadenylation signal fragment of SV40, and the 1.7 kb Ncol-Ncol fragment (bases 189763 through 188062 , which contains the sequences J1I to IRL1, from pHind-G) .To replace the ORF of HCMV US6 to IRS1 by / S-glucuronidase (ie RV7177; Figure 3), pBgdUS6 / IRSl is constructed. Sequentially, this plasmid contains the 1.7 kb Ncol-Ncol fragment (bases 188062 to 189763, which contains the promoter sequences for IRL1, Jil, and IRS1, from pHind-G), / S-glucuronidase, the fragment of 255-b containing the polyadenylation signal of US10 (bases 199276 to 199021, from pHind-X), and a 1.8 kb Bsml-Saul fragment (bases 196222 to 198030, containing the sequences US7 to US9 C terminal, from pHind-X). To replace the ORFs of HCMV US3 and US2 by jS-glucuronidase (ie, RV47; Figure 3), pBgdUS3 / US2 is constructed. Sequentially, this plasmid contains the 1.7 kb Pstl-Pstl fragment (bases 196447 to 194741), a 180-b Smal-HaelII fragment containing the gH HSV-1 promoter (McKnight, 1980), / S-glucuronidase, the 255-b US10 polyadenylation signal fragment, and the 1.3 kb Nhel-Apal fragment (bases 193360 through 192033, containing the sequences from US2 C terminal to IRS1, from pHind-G). To replace the ORF of HCMV US1 with 3-glucuronidase (ie, RV5122; Figure 3), pBgdUSl is constructed. Sequentially, this plasmid contains the 1.8 kb Aatll-Sstl fragment (bases 190884 through 192648 containing the IRS1 and US1 C terminal sequences, from pHind-G), a 180-b Smal-HaelII fragment containing the gH promoter of HSV-1 (McKnight, 1980), / S-glucuronidase, the polyadenylation signal fragment of 255-b US10, and the 1.6 kb Sphl -Sphl fragment (bases 192934 through 194544, which contain the sequences US2 and US3 C terminal, from pHind-G). To replace the HCMV IRS1 ORF with / S-glucuronidase (ie, RV46; figure 3), pBgdIRSI is constructed. Sequentially, this plasmid contains the 1.7 kb Ncol-Ncol fragment (bases 188062 to 189763, which contains the promoter sequences of IRL1, J1I and IRS1, from pHind-G), / 3-glucuronidase, the fragment of 255 -b containing the polyadenylation signal of US10 (bases 199276 to 199021, from pHind-X), and the fragment of 1.2 kb Narl -Xhol (bases 191830 to 193003, which contains the sequences IRS1 C terminal and US1, a from pHind-G). To suppress the ORFs of HCMV USll to US2 without insertion of a reporter gene (ie, RV799; Figure 3), pdUSll / US2 is constructed. Sequentially, this plasmid contains the 1.8 kb fragment Pstl-Xbal fragment (bases 202207 to 200391, which contains the promoter sequences of US13, US12 and USll from pXba-P), / 8-glucuronidase, a fragment of 65 -b Nrul-Apal containing the polyadenylation signal US10 (bases 199086 to 199021, from pHind-X), and the fragment of 1.8 or 1.3 kb W? el-Apal (bases 193360 to 192033, which contains the sequences US2 C terminal to IRS1, from pHind-G).

Isolation of recombinant mutant HCMV The creation and isolation of recombinant mutant HCMV is performed as previously described (Jones et al., 1991, Jones and Muzithras, 1992). Cells are divided HFF so that they are 70-80% confluent on the day of transfection. The cells are subjected to trypsinization and suspended up to 5.6 x 10 * cells per ml in DMEM / 10% FCS / 25 mM HEPES. The DNA is transfected by using the modified calcium phosphate coprecipitation technique. 1.5 μg of infectious HCMV DNA and 2.5 μg of lienalized plasmid DNA are mixed in the calcium chloride solution (300 μl containing 10 mM Tris, pH 7.0 / 250 mM calcium chloride) and cooled on ice. To initiate co-precipitation, the DNA is removed from the ice and 300 μl (2X HeBS, pH 6.95 (at room temperature; IX of HeBS is constituted by 19.2 mM HEPES, 137 mM NaCl, 5 mM KCl, phosphate are added dropwise). 0.8 mM sodium and 0.1% dextrose), with gentle agitation After 1.5 minutes, the precipitate is placed on ice (to prevent additional precipitate from forming) The precipitate is mixed with 3 x 10 * cells (in suspension) and placed in a tissue culture plate of 82 mm.After 6 hours at 37 ° C, the medium is removed and the cells are subjected to shock with 20% DMSO in IX of HeBS for 2 minutes. wash twice with PBS and growth medium is added.The medium is changed every 4-7 days.After 14 days, the viral plaques are observed and the cells are coated with 0.5% agarose in DMEM containing 150 μg / ml of X-gluc (5-bromo-4-chloro-3-indol-l-glucuronide; Biosynth) .The blue plaques (ie plaques with mutant virus / S-glucuroni) dasa positives) are taken several days after adding the coating. The recombinant viruses are plaque purified three times. The mutant of HCMV RV799 is (S-glucuronidase negative and is isolated using a modification of the previous procedure.) In this case, the mutant HCMV RV134 / 8-glucuronidase positive is the original virus (Jones et al., 1991). Therefore, RV134 genomic DNA is used instead of wild-type DNA from strain AD169.Primary plates appearing on the primary transfection plates are taken randomly and replated on HFF cells. ten days, the media is removed and the infected cells are coated with agarose containing X-gluc, as described above, in this case, white plates (plaques of mutant virus β-glucuronidase negative) are taken four days later, and plaque purify The appropriate genomic organization of each of the HCMV mutants is verified by hybridization analysis in DNA stain, as previously described (Jones et al., 1991).

Antibodies Rabbit polyclonal antiserum reactive with USll proteins of HCMV and proteins of HCMVL UL80 are purchased as previously described (Jones et al., 1991; 1994). W6 / 32 mouse monoclonal antibodies specific for a heavy chain conformation-dependent epitope of human MHC class I proteins, and Ber-T9, specific for the human transferrin receptor. The mouse monoclonal antibody TP25.99 (D'Urso et al., 1991), specific for an epitope independent of the heavy chain conformation of human MHC class I proteins, is obtained from Dr. S. Ferrone (Department of microbiology, New York Medical College, Valhalla, NY). The mouse monoclonal antibody 9221, specific for the HCMV IE1 protein, is purchased from Dupont.

Radiolabelling and immunoprecipitation of infected cellular proteins Pulse-voltage radiolabelling by saturation is performed according to the standard protocol (Sambrook et al., 1989). HFF cells infected with HCMV (multiplicity of infection equaled to five) are pulsed with 200 μCi of [35S] methionine and [3SS] cysteine (NEN-DuPont) by me in methionine-free Dulcecco-modified Eagle / cysteine medium ( DMEM) in the period of time indicated post-infection. The radioactive medium is removed, the cells are washed twice in complete DMEM and saturation arrests are made for the indicated time in complete DMEM. The proteins are extracted using a triple detergent lysis buffer (Sambrook et al., 1989). Transparent protein extracts (supernatant after centrifugation for 5 minutes at 15,000 x g and 4 ° C) are retained for immunoprecipitation according to the standard or standard protocol (Sambrook et al., 1989). The proteins that bind to antibodies are pelleted using protein A sepharose (Pharmacia). For immunoprecipitations of the human transferrin receptor, rabbit IgG against mouse (Pierce) is added before protein A sepharose. The washed immunoprecipitates are boiled in the presence of 2-mercaptoethanol and subjected to electrophoresis in denaturing polyacrylamide gels. The gels are fixed and moistened in 1M fluorine and sodium salicylate (Sambrook et al., 1989) before drying and autoradiography.

Immunofluoresceneia Immunofluorescence assays were performed according to the standard protocol. All procedures were performed on 60 mm tissue culture plates. Briefly, infected or uninfected HFF cells are fixed with 4% paraformaldehyde and permeabilized with Triton X-100 ai 0.2% (where indicated). After adding 3% bovine serum albumin in phosphate buffered saline, the cells are maintained overnight at 4 ° C. The cells are treated in frequency with the following antisera, each for 30 minutes at room temperature: human serum negative for HCMV 10% (to block any Fc receptor), • the indicated primary antibody; and IgG against mouse or against rabbit conjugated with FITC, as appropriate.

EXAMPLE 2 Decreasing regulation class I in human fibroblasts infected with wild-type HCMV We sought to determine the timing and nature of down-regulation of the MHC class I heavy chain in a human foreskin fibroblast cell culture system of the present invention (HFF). By flow cytometry, HFF cells infected with the HCMV strain AD169 wild-type reduced significantly in the expression of the heavy chain class I on its cell surface in late times post-infection (ie 72 hours) by using of the class I monoclonal antibody dependent on conformation W6 / 32 (figure 1). In the Wester analysis using the conformation-independent class I monoclonal antibody (TP25.99), it is shown that the steady-state level of the class I protein is also reduced in the late post-infection times (Figure 2A). Because the viral peptides are presented on the cell surface by complexes class I assembled after infection, we seek to determine the state of the class I proteins synthesized at various post-infection times by immunoprecipitation of radiolabelled proteins metabolically. As shown in Figure 2B, the reduction in the expression of heavy class I chains is detected both in the presence and absence of the inhibitor of viral DNA synthesis, phosphonoformate. This indicates that the functions of the immediate or viral early gene is sufficient for a heavy chain reduction. In addition, it is demonstrated that the decreasing regulation of the chain is detected in very early post-infection times: 3 hours (Figure 2C). Since this effect is observed using conformation-independent antibody, the reduction reflects overall levels of newly synthesized heavy chains.

HCMV mutant test for loss of down regulation of MHC class I Several previously constructed HCMV deletion mutants representing 18 non-essential ORFs (UL33, UL81, IRS1, US1-US13, US27-US28, and TRS1) were examined for heavy chain expression by flow cytometry and immunoprecipitation analysis .

Only RV670, a mutant deleted from a region of 9 kb within the S component of the HCMV genome (Jones and Muzithras, 1992) does not retain the down-regulating phenotype of wild-type (Figure 4A). This mutant has deleted at least 11 ORF, IRS1 to USll (except for US10), which includes the gene family US6 (US6-US11) which has been assumed to encode glycoproteins (Chee et al., 1990). To confirm this observation, two additional independent derivative mutants were tested, which had the same suppression as RV670 and a new mutant, RV7186, suppressed in the entire IRS1-US11 region (figure 3). Each was phenotypically identical to RV670 and stably expressed the class I heavy chains. Previously, we constructed deleted HCMV mutants in the ORFs of the US6 family, either individually or in groups (Jones and Muzithras, 1992), and deletion mutants. similar within the adjacent IRS1-US3 region. By means of immunoprecipitation using the conformation-independent antibody, it was shown that all of these mutants retain the ability to down-regulate the class I heavy chains (FIG. 4A) at late post-infection times in HFF cells. The control experiments indicated that the radiolabelling is equivalent between the different cell cultures (figure 4B) and that the infection proceeds at later times in the same way, judging by the extrusion of the pp65 (figure 4B) and UL80 proteins (figure 4C). These data indicate that: (i) more than one viral gene is sufficient for the reduction in class I heavy chains; or (ii) the gene or genes between US3 and US6, suppressed in RV670 and in RV7186, but not in other mutants, are required for the phenotype.

Identification of a 7 kb region of the HCMV genome necessary for the down regulation of MHC class I To further localize the region containing the gene or genes involved in the down-regulation of the MHC class I heavy chain, additional HCMV substitution mutants containing deletions of multiple genes were generated within the region of the IRS1-US11 gene (figure 3). One of these mutants, RV798, was deleted from the genes from US2-US11. In HFF cells infected by RV798 and analyzed in late post-infection times, the MHC class I heavy chains were not subject to down-regulation as were the cells infected with the wild-type strain AD169 (Figure 4A); in fact, a slight stimulation is observed.

Several independently derived suppressive mutants, identical to RV798, were similarly examined: all lack the ability to downregulate class I heavy chains. To further confirm that the US2-US11 region of 7 kb HCMV contains the gene or genes required for the down-regulation of the heavy chain, the RV799 mutant was constructed which presented identical suppression US2-US11 as RV798, but was generated by a different strategy. RV798 was derived from wild-type strain AD169 by inserting a 3-glucuronidase marker gene at the US2-US11 site. In contrast, the origin of RV799 was RV134, a mutant which is / S-glucuronidase positive since it presents the expression cassette for / S-glucuronidase inserted within the intergenic region US9-US10 (Jones et al., 1991). To create RV799, a plasmid was designed, which, when recombined with the RV134 genome, would simultaneously suppress US2-US11 and the expression cassette of / S-glucuronidase (figure 3). The mutant of HCMV, Appropriate RV799 is isolated as a white plate in the presence of a substrate of / S-glucuronidase, since it is / S-glucuronidase negative. The RV799 mutant, but not the original RV134 strain, is phenotypically identical to RV798 (Figure 5). Therefore, since RV798 and RV799 were created by different strategies using origins which retained the ability to down-regulate MHC class I heavy chains, this confirms that the gene or genes required for the phenotype are located within the US2-US11 region of 7 kb (bases 193119-200360). To determine whether the appropriate surface expression of the class I heavy chains was present in late post-infection times in either RV798 or RV799, immunofluorescence assays were performed. Through the use of conformation-dependent (W6 / 32) or conformation-independent monoclonal antibodies (TV25.99), surface expression of MHC class I heavy chains was detected in HFF cells not infected and infected with RV798 and with RF799, but not in HFF cells infected with wild-type AD169. Proper mutation of class I heavy chains in uninfected cells provides molecules resistant to endoglycosidase H. In contrast, class I heavy chains synthesized in cells infected with AD169 are reported to be completely sensitive to endoglycosidase H (Beersma et al., 1993) . As shown in figure 6, heavy class I chains synthesized in HFF cells infected with RV798, either in early or late post-infection times, are converted to the mature form resistant to endoglycosidase H at a rate similar to that synthesized. in uninfected cells. When taken together, these data indicate that the synthesis, processing and surface expression of MHC class I is not damaged in cells infected with these HCMV mutants. In addition, the results indicate that the 7 kb region containing the US2-US11 genes contains one or more genes required for down-regulation of the heavy chain by HCMV.

Two subregions within the region of the US2-US11 gene that contain the genes which are involved in the down regulation of the heavy chain class I The deleted HCMV genome region in RV35 was from US6-US11 and US2-US11 in RV798 (figure 3). In HFF cells infected with RV35, the heavy chains of MHC class I were subject to down regulation, but in cells infected with RV798 this did not happen (figure 4A). These data indicate that one or more genes involved in the heavy chain decreasing regulatory maps within the 2 kb subregion from the ORF US2 to US5 (subregion A, bases 193119-195607). To determine if this subregion is required for the down-regulation of the class I heavy chain, the HCMV substitution mutants RV7181 and RV7177 were examined. The ORFs of HCMV, IRS1-US9 and IRS1-US6, respectively, were deleted in these mutants; therefore, subregion A is absent from both mutants. Experiments on infected HFF cells in late post-infection times indicate that both mutants retain the ability to efficiently down-regulate the expression of the heavy chain class I gene (Figure 7). Therefore, when present in the HCMV genome, the gene or genes within region A are sufficient for reduction of MHC expression (eg, RV35), although their presence is not required for the phenotype. In addition, the cumulative data indicate that there are no HCMV genes within the identified 7 kb US2-US11 region (ie, the suppressed region in RV798), which is absolutely required for efficient poor heavy chain regulation in infected HFF cells, suggesting that the gene or genes from another portion of the US2-US11 gene region are also sufficient for the phenotype in late post-infection times.

Evidence indicating that the USll gene product is involved in the down regulation of MHC class I heavy chain In HFF cells infected with the RV7181 mutant, suppressed from IRS1-US9 (figure 3), MHC class I heavy chain expression is subject to down regulation, in contrast to HFF cells infected with RV798 (figure 7) . These data suggest that a second subregion (subregion B), made up of the US10 and USll genes (bases 199083-200360), is involved in the reduction of heavy chain expression. However, the expression of US10 from the HCMV context is not sufficient for a decreasing regulation of the heavy chain. The HCMV RV670 mutant expresses US10 at stable levels similar to those of wild type and is deleted from all other ORFs in the US2-US11 gene region of 7 kb, but does not cause down regulation of MHC class heavy chains I in infected HFF cells (Figures 2B and 4A). USll codes for a 32 kDa glycoprotein (gpUSll) containing carbohydrates bound to N, but not bound to O, which are positive to endoglycosidase H, indicating that they are in the high. GpUSII was suppressed by infection, beginning at very early times (ie 3 hours) and continuing through late post-infection times. However, gpUSll levels in the infected cells were more abundant approximately 8 hours post-infection. To determine their position in the infected cell, rabbit polyclonal antiserum (Jones and Muzithras, 1991) was used in immunofluorescence assays of cells infected with wild-type strain AD169. HFF cells not infected and infected with RV699 were used as negative controls. RV699 is a mutant of HCMV which is isogenic with AD169, except for a deletion of the ORF for USll (Jones et al., 1991). In cells fixed and permeabilized at 8 hours post-infection, cytoplasmic fluorescence was observed, which obscured the definition of the nucleus, in HFF cells infected with AD169, but not in any of the negative control cells (figure 8). In general, the specific fluorescence was more intense in the perinuclear area. No specific fluorescence was detected in non-permeabilized cells (Figure 8). Fluorescence and endoglicosidase H sensitivity data indicate that gpUSll is not a cell surface glycoprotein. From the translated DNA sequence, gpUSll is predicted to have hydrophobic domains near its N and C terminal portions, (Weston and Barrell, 1986) which are putative signal sequences and transmembrane domains, respectively. Therefore, gpUSll is associated with intracytoplasmic membranes, possibly of the endoplasmic reticulum.

Decreasing regulation of MHC class I at early post-infection times by HCMV mutants It is shown that the expression of MHC class I in cells infected with wild-type strain AD169 begins in very early post-infection times (Figure 2C). To determine if any of the mutants is deficient for this early decreasing regulation, immunoprecipitation experiments are performed using extracts from radiolabeled infected HFF cells 6 to 10 hours post-infection. The class I heavy chain level was reduced during this early post-infection period in HFF cells, with each of the mutants, except for RV798, the deleted mutant for the entire 7 kb US2-US11 region (Fig. 9A ). The control experiments demonstrated that the cells infected with different mutants are infected and radiolabelled equally (Figure 9B and D). The expression of another cellular glycoprotein, in transferrin receptor, is not differentially affected by the various mutants (Figure 9C). Therefore, the genes required for the down-regulation of the heavy chain at initial post-infection times are the same as those needed for reduction at late post-infection times. Furthermore, the expression of the gene or genes of any identified sub-region that is involved in the down-regulation of heavy chain expression in late post-infection times is sufficient for reduction in very early post-infection times.

E EMPLO 3 Preparation of recombinant HCMV vaccine (RV798) HCMV vaccines were prepared using a method previously described (Elek and Stern, 1974). The HCMV RV798 mutant is grown in human diploid lung fibroblasts MRC-5 (CCL171 [American Type Culture Collection]) or in human foreskin fibroblasts (MRHF [BioWhittaker]). Cells were infected at a multiplicity of infection equal to one in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum and 5% fetal bovine serum. After 24 hours, the medium is removed and the cells are washed three times with Hank's balanced salt solution, or Dulbecco's phosphate-buffered saline. Fresh DMEM medium is added without serum; the infected cells are incubated four days after the appearance of the late cytopathic viral effect (usually 7 days post-infection). After a pre-clearance centrifugation step (6,000 x g for 20 minutes at 18 ° C), the cell-free virus is pelleted by centrifugation at 15,500 x g for one hour at 18 ° C. The sedimented virus is resuspended in Dulbecco's phosphate-buffered saline containing 25% sorbitol and stored in aliquots at -70 ° C. The title of RV798 vaccine accumulation is determined by using standard or conventional procedures in human foreskin fibroblasts (Wentwork and French, 1970). The vaccine is administered by subcutaneous inoculation of approximately 103-107 plaque-forming units in the deltoid region of the upper arm, as previously described (Elek and Stern, 1974; Gehrz et al., 1980; Starr et al., 1981).

EXAMPLE 4 gpUSll is sufficient to subject the MHC class I heavy chains to down regulation To determine whether the product of the USll gene, in the absence of any other product of the viral gene, is capable of causing the down-regulation of the heavy chain, the region coding for USll was cloned (bases 200360-199716 [Chee et al., 1990]) and some non-coding flanking sequences spanning bases 200391-199683, in a eukaryotic expression plasmid under the transcriptional control of the constitutive HCMV major early promoter extender. Human astrocytoma cells U373-MG (HTB 17 [American Type Culture Collection]) were transfected with this plasmid (Sambrook et al, 1989) and the stably transformed cells were selected in the presence of 0.375 μg / ml puromycin, since the plasmid also codes for the prokaryotic gene that confers resistance to puromycin. The clones were extracted and expanded in cell lines. Those cells expressing gpUSll were identified by Wester spotting analysis; the different cell lines expressed varying amounts of USll. The expression of the MHC class I heavy chain in these cell lines was similarly analyzed. As shown in Figure 11, the expression of USll is inversely correlated with expression of the class I heavy chains. These data demonstrate that the expression of HCMV USll is sufficient for the down regulation of the MHC class I heavy chain expression in the absence of any other viral gene product.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (40)

REIVI DICACIONES

1. A recombinant cytomegalovirus (HCMV) mutant, which does not regulate in a decreasing manner (regulates in a way that decreases its activity) the expression of the cellular major histocompatibility complex (MHC) before infection, characterized in that it comprises a genome from which it has been suppressed a gene sequence capable of down-regulating MHC class I expression, wherein the deleted gene sequence comprises a region containing the open reading frames IRS-1-USll.

2. The recombinant HCMV mutant according to claim 1, characterized in that the deleted gene sequence region comprises open reading frames IRS-1 -US9 and USll.

3. The recombinant HCMV mutant according to claim 1, characterized in that the region of the deleted gene sequence comprises the open reading frames US2-USll.

4. The recombinant HCMV mutant according to claim 1, characterized in that the region of the deleted gene sequence comprises the open reading frame USll.

5. The recombinant HCMV mutant according to claim 1, characterized in that the deleted gene sequence region comprises subregion A, wherein subregion A comprises the open reading frames US2-US5, and sub-region B, wherein subregion B comprises open reading frames US10 - USll.

6. The recombinant HCMV mutant according to claim 5, characterized in that the subregion of the deleted gene sequence consists of the open reading frame USll ..

7. A method for controlling the down regulation of the expression of the major histocompatibility complex (MHC) class I in a cell infected with cytomegalovirus, characterized in that it comprises the steps of: (a) identifying a gene sequence in the region of the cytomegalovirus genome containing the IRS-1-USll open reading frames capable of down-regulating the expression of MHC class I; and (b) deleting the sequence of the identified gene from the cytomegalovirus genome.

8. The method according to claim 7, characterized in that the identified gene sequence comes from the region of the cytomegalovirus genome that contains the open reading frames IRS-1 - US9 and USll.

9. The method according to claim 7, characterized in that the identified gene sequence is from the region of the cytomegalovirus genome that contains the open reading frames US2-USll.

10. The method according to claim 7, characterized in that the sequence of the identified gene is from the region of the cytomegalovirus genome containing the open reading frame USll.

11. The method according to claim 7, characterized in that the identified gene sequence is from the region of the cytomegalovirus genome containing subregion A, in which subregion A comprises the open reading frames US2 - US5, and sub region B , in which subregion B comprises the open reading frames US10 - USll.

12. The method according to claim 11, characterized in that the sequence of the gene identified from subregion B consists of the open reading frame USll.

13. A pharmaceutical composition characterized in that it comprises a recombinant cytomegalovirus (HCMV) mutant, which does not subject to down regulation the expression of the cellular major histocompatibility complex (MHC) to infection, comprising a genome from which a gene sequence capable of being deleted has been deleted of subjecting MHC class I expression to downregulation, wherein the deleted gene sequence comprises a region containing the open reading frames IRS-1-USll.

14. The pharmaceutical composition according to claim 13, characterized in that the region of the deleted gene sequence of recombinant HCMV comprises the open reading frames IRS-l-US9 and USll.

15. The pharmaceutical composition according to claim 13, characterized in that the region of the deleted gene sequence of the mutant. of recombinant HCMV comprises open reading frames US2 - USll.

16. The pharmaceutical composition according to claim 13, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frame USll.

17. The pharmaceutical composition according to claim 13, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises a subregion A, in which subregion A comprises the open reading frames US2 - US5, and sub region B , in which subregion B comprises the open reading frames US10 - USll.

18. The pharmaceutical composition according to claim 17, characterized in that the sub region B of the deleted gene sequence of the recombinant HCMV mutant consists of the open reading frame USll.

19. A vaccine composition for use in the prevention of cytomegalovirus infections, characterized in that it comprises an effective amount of a recombinant cytomegalovirus (HCMV) mutant which does not subject to decreasing regulation the expression of the cellular major histocompatibility complex (MCH) to infection, comprising a genome from which a gene sequence capable of down-regulating MHC class I expression has been deleted, wherein the deleted gene sequence comprises a region containing the open reading frames IRS-1 - USll, in a pharmaceutically acceptable vehicle.

20. The vaccine composition according to claim 19, characterized in that it additionally comprises an adjuvant.

21. The vaccine composition according to claim 19, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frames IRS-1-US9 and USll.

22. The vaccine composition according to claim 19, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frames US2-USll.

23. The vaccine composition according to claim 19, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frame USll.

24. The vaccine composition according to claim 19, characterized in that the deleted gene sequence region of the recombinant HCMV mutant comprises subregion A, in which subregion A comprises the open reading frames US2-US5, and the subregion B, in which subregion B comprises the open reading frames US10 - USll.

25. The vaccine composition according to claim 24, characterized in that the subregion B of the deleted gene sequence of the recombinant HCMV mutant consists of the open reading frame USll.

26. A composition useful for immunizing an individual against cytomegalovirus, characterized in that it comprises an immunogenic amount of a recombinant cytomegalovirus (HCMV) mutant which does not downregulate the expression of the cellular major histocompatibility complex (MHC) to infection, comprising a genome from which a gene sequence capable of down-regulating MHC class I expression has been deleted, wherein the deleted gene sequence comprises a region comprising the open reading frames IRS-1-USll.

27. The composition according to claim 26, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frames IRS-1-US9 and USll.

28. The composition according to claim 26, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frames US2-USll.

29. The composition according to claim 28, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frame USll.

30. The composition according to claim 26, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises subregion A, in which subregion A comprises the open reading frames US2 - US5, and sub region B, in which subregion B comprises the open reading frames US10 - USll.

31. The composition according to claim 30, characterized in that the sub region B of the deleted gene sequence of the recombinant HCMV mutant consists of the open reading frame USll.

32. A composition useful for preventing or reducing susceptibility to acute cytomegalovirus in an individual, characterized in that it comprises an immunogenic amount of a recombinant cytomegalovirus (HCMV) mutant which does not downregulate the expression of the cellular major histocompatibility complex (MHC). infection, comprising a genome from which a gene sequence capable of down-regulating MHC class I expression has been deleted, wherein the deleted gene sequence comprises a region containing the open reading frames IRS- 1 - USll.

33. The composition according to claim 32, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frames IRS-1-US9 and USll.

34. The composition according to claim 32, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frames US2-USll.

35. The composition according to claim 32, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises the open reading frame USll.

36. The composition according to claim 32, characterized in that the region of the deleted gene sequence of the recombinant HCMV mutant comprises subregion A, in which sub region A comprises the open reading frames US2 - US5, and sub region B, in which subregion B comprises the open reading frames US10 - USll.

37. The composition according to claim 36, characterized in that the sub region B of the deleted gene sequence of the recombinant HCMV mutant consists essentially of the open reading frame USll.

38. A virus-based gene therapy vector, characterized in that it comprises a gene sequence of an open reading frame of USll of the human cytomegalovirus genome.

39. A virus-based gene therapy vector, characterized in that it comprises the sequences of the sub-region A gene of the human cytomegalovirus genome, in which subregion A comprises the open reading frames US2-US5, and subregion B of the genome of human cytomegalovirus, in which subregion B comprises the open reading frames US10-USll.

40. A virus-based gene therapy vector, characterized in that it comprises the gene sequences of subregion A of the human cytomegalovirus genome, wherein subregion A comprises the open reading frames US2-US5.