Recombinant vector containing infectious human cytomegalovirus genome with preserved wild-type characteristics of clinical isolates

Abstract

A recombinant vector containing infectious genome of human cytomegalovirus (HCMV) and being useful for the production of reconstituted HCMV virus retaining phenotypic characteristics of a clinical virus isolate including the ability to grow on endothelial cells and to induce microfusion is characterized in that it is obtainable by inserting DNA from a clinical isolate of HCMV virus into a bacterial cloning vehicle. Such vector can be used e.g. for production of reconstituted HCMV virus retaining the phenotypic characteristics of a parental clinical isolate and for studying genes and functions of genes of HCMV virus. A further aspect are mutant viruses and inter alia their use for studying aspects of infectivity of HCMV virus.

1 . Recombinant vector containing infectious genome of human cytomegalovirus (HCMV) and being useful for the production of reconstituted HCMV virus retaining phenotypic characteristics of a clinical virus isolate including the ability to grow on endothelial cells, to induce microfusion and to spread viral material from HUVEC to PMNL, characterized in that it is obtainable by inserting DNA from a clinical isolate of HCMV virus into a bacterial cloning vehicle.  
     
     
         2 . Recombinant vector according to  claim 1  which contains the complete infectious DNA of HCMV.  
     
     
         3 . Recombinant vector according to claims  1  or  2 , characterized in that the bacterial cloning vehicle contains DNA sequences which are homologous to the HCMV DNA and insertion is effected by homologous recombination.  
     
     
         4 . Recombinant vector according to anyone of  claims 1  to  3 , wherein the bacterial cloning vehicle is a BAC system vector.  
     
     
         5 . Recombinant vector according to anyone of  claims 1  to  4  and designated FIX-Bac-7.  
     
     
         6 . Bacterial cell line containing a recombinant vector according to anyone of  claims 1  to  5 .  
     
     
         7 . Bacterial cell line according to  claim 6  which is an  E.coli  cell line.  
     
     
         8 . Bacterial cell line according to claims  6  or  7 , designated FIX-Bac-7- E.coli  DH10B and deposited as DSM 13958 containing at least one copy of FIX-Bac-7.  
     
     
         9 . Use of a recombinant vector according to anyone of  claims 1  to  5  or a bacterial cell line according to anyone of  claims 6  to  8  for the production of reconstituted HCMV virus retaining the phenotypic characteristics of a parental clinical isolate including the ability to grow on endothelial cells, to induce microfusion and to spread viral material from HUVEC to PMNL.  
     
     
         10 . Use according to  claim 9  wherein a recombinant vector according to anyone of  claims 1  to  5  is transfected into suitable eukaryotic host cells and reconstituted infectious virus is collected after culturing said cells.  
     
     
         11 . Use according to  claim 9  or  10 , wherein the reconstituted HCMV virus further retains HUVEC- and/or PMNL-tropism of a parental clinical virus isolate.  
     
     
         12 . Use of reconstituted infectious HCMV virus produced according to claims  9 ,  10  or  11  for the production of vaccines and/or antibodies against HCMV virus.  
     
     
         13 . Use of reconstituted infectious HCMV virus produced according to claims  9 ,  10  or  11  for the screening of drugs or small molecules for their antiviral activity.  
     
     
         14 . Use of a recombinant vector according to anyone of  claims 1  to  5  for studying genes and function of genes of HCMV virus.  
     
     
         15 . Use of a recombinant vector according to anyone of  claims 1  to  5  for the preparation of virus mutants by modifying, deleting, randomly deleting or substituting parts of the viral DNA.  
     
     
         16 . Use according to  claim 15 , wherein the genes which are responsible for microfusion and/or tissue tropism are modified.  
     
     
         17 . Use of a recombinant vector according to anyone of  claims 1  to  5  for vaccine development.  
     
     
         18 . Use of a recombinant vector according to anyone of  claims 1  to  5  for the development and/or screening of substances which inhibit viral gene production on trancriptional and/or translational level.  
     
     
         19 . HCMV virus mutant, wherein the region UL130 to UL132 is either deleted or mutated in such a way that the ability to induce microfusion and/or to infect endothelial cells is lost.  
     
     
         20 . HCMV virus mutant, wherein the region UL128 to UL132 is either deleted or mutated in such a way that the ability to induce microfusion, PMNL and/or HUVEC tropism is reduced or lost.  
     
     
         21 . Use of RV-FIX or a HCMV virus mutant according to  claim 19  or  20  for studying pathogenicity, and especially studying adapted and innate immune surveillance or immune counterstrategies, studying NK-cell recognition, B-cell recognition, T-cell recognition, complement regulation, studying MHC regulation ((classic (HLA-A,B,C) and nonclassic (HLA-E,G; MIC-A, MIC-B)), studying trophoblast infection for mechanism of fetal loss during pregnancy, studying induction and prevention of apoptosis and cellular suicide, studying and modification of anti-apoptotic and proapoptotic viral genes and cellular genes for virally induced transformation of infected cells, studying soluble viral factors (cytokine-like or chemokine-like) and effects on cell migration and cell signalling, studying of latency and reactivation in virally infected endothelial cells, progenitor cells and hematopoietic stem cells as well as mature cells (dendritic cells, monocytes, macrophages, thrombocytes).  
     
     
         22 . Use of RV-FIX or a HCMV virus mutant according to  claim 19  or  20  for studying cell adherence of wild-type virus in comparison with mutant virus.  
     
     
         23 . Use of RV-FIX or a HCMV virus mutant according to  claim 19  or  20  for studying essential or non-essential genes as targets for chemotherapeutical intervention.  
     
     
         24 . Use of RV-FIX or a HCMV virus mutant according to  claim 19  or  20  for the development of vaccines and/or antibodies and small therapeutic molecules.  
     
     
         25 . Use of RV-FIX or a HCMV virus mutant according to  claim 19  or  20  for diagnostics, for drug screening, as attenuation marker, for the development of modified vectors, for the development of or the screening for peptides or antisense genes or antisense RNA which block the activity of the microfusion gene and/or endothelial cell tropism in wild-type virus.  
     
     
         26 . Use of FIX-Bac-7 as vector for gene therapy.  
     
     
         27 . Process for inducing recovery of microfusion, HUVEC and PMNL tropism as well as possibly other pathogenicity features by transfer of the UL132-128 genetic region or-respective cDNAs from FIX-Bac or RV-FIX and of any HCMV clinical isolate into any other virus strain or Bac cloned virus.  
     
     
         28 . Use of the UL131-128 or UL132-UL128 transcripts of FIX-bac or RV-FIX and of any HCMV clinical isolate for pathogenicity studies, therapeutic intervention, vaccine development, vector development or drug screening.  
     
     
         29 . Use of the genetic region UL132-128 or transcripts /cDNAs of this region of FIX-bac or RV-FIX to construct cell lines to study the mechanism of microfusion or PMNL and HUVEC tropism.  
     
     
         30 . Use of FIX-Bac, Phoebe-Bac Powers-Bac, TB40E-Bac, TowneL-Bac, TowneS-Bac or the respective reconstituted Bac-derived viruses and virus mutants for vaccine development, drug screening, gene therapy, pathogenicity studies of HCMV in vitro and in vivo.