Title:  Processes for the production of HCMV glycoproteins, antibodies thereto and HCMV vaccines, and recombinant vectors therefor

United States Patent:  6,162,620

Assignee:  Cogent Limited (London, GB)

Filed:  July 20, 1994

HCMV glycoproteins B and H have been identified. The gB protein is encoded by DNA in the HindIII F fragment of the HCMV genome lying between 1378 and 4095 bases from the F/D boundary. The gH protein is encoded by DNA in the HindIII L fragment lying between 228 and 2456 bases from the L/D boundary. The genes have been incorporated in recombinant vaccinia vectors and expressed in host animals to raise HCMV-neutralising antibody, thereby indicating vaccine potential. The glycoproteins can also be used in a variety of different ways, as vaccines or in the production, purification or detection of HCMV antibody.

SUMMARY OF THE INVENTION

The present invention is based on the identification and expression of HCMV DNA encoding two glycoproteins, referred to herein as gB and gH. The gB protein is encoded by DNA in the HindIII F fragment of the HCMV genome lying between 1378 and 4095 bases from the F/D boundary. The gH protein is encoded by DNA in the HindIII L fragment lying between 228 and 2456 bases from the L/D boundary.

According to one aspect of the present invention there is provided a process which comprises expressing from a recombinant DNA vector in a suitable host organism a polypeptide incorporating one or more antigenic determinants capable of raising HCMV-neutralising antibodies in humans, said determinant or determinants corresponding to a portion of the protein encoded by DNA in the HindIII F fragment of the HCMV genome lying between 1378 and 4095 bases from the F/D boundary and/or a portion of the protein encoded by DNA in the HindIII L fragment of the HCMV genome lying between 228 and 2456 bases from the L/D boundary.

A second aspect of the present invention provides a recombinant virus vector containing DNA encoding such a polypeptide, said vector being capable of infecting a human subject and expressing the polypeptide in immunogenic form.

A third aspect of the present invention provides a process which comprises synthesising such a polypeptide.

A fourth aspect of the present invention provides a method of preparing HCMV monospecific antiserum comprising immunising a host animal with such polypeptide or with a recombinant virus vector as described above, and extracting from the host animal antiserum specific to said polypeptide. HCMV-specific monoclonal antibody may be prepared from cells from such immunised animals.

A fifth aspect of the present invention provides a method of purifying HCMV-specific antibodies, which comprises contacting the antibodies with HCMV polypeptide hereof, and separating bound antibody from the polypeptide.

A sixth aspect of the present invention provides a method of detecting HCMV-specific antibody in a clinical sample, which comprises contacting the sample with HCMV polypeptide hereof, and detecting antibody that binds to the polypeptide.

A seventh aspect of the present invention provides a kit for carrying out such a detection method, the kit comprising said polypeptide in a form suitable for contacting with the clinical sample, and means for detecting HCMV-specific anti-body that binds to said polypeptide.

By identifying surface glycoprotein(s) of HCMV that lead to an immune response and incorporating the corresponding sequence of genetic material in a mammalian vector, an immunologically active protein may be produced which can form the basis of a vaccine against HCMV.

For the recombinant virus vaccine the identified HCMV genome fragment may be isolated and introduced into a suitable mammalian virus vector by conventional genetic engineering techniques, and transfecting the plasmid into a mammalian host.

Suitable vectors include mammalian cells and viruses such as poxviruses, with vaccinia virus being particularly preferred, and bovine papilloma virus.

Expression of the foreign DNA can be obtained by infecting cells or animals with recombinant virus vector. For example, a recombinant virus, e.g. vaccinia virus, may be used as a live vaccine. Further, cells infected with the recombinant vector may be used to prepare the product of the foreign DNA for use as a vaccine.

In one preferred technique, a glycoprotein-encoding fragment of the HCMV genome is introduced into plasmid pGS62 and then transferred into vaccinia virus by transfecting the plasmid into mammalian cells infected with vaccinia virus.

It will be apparent that the HCMV DNA may be modified in various ways without significantly affecting the functioning of the protein produced thereby. For example, a transmembrane form of protein may be converted to a secreted form by removing the DNA coding for the C-terminal containing the membrane anchor sequence. Such modifications are to be considered within the scope of the present invention.

Claim 1 of 22 Claims

What is claimed is:

1. An isolated polynucleotide encoding an HCMV glycoprotein B (gB) polypeptide, said gB polypeptide selected from the group consisting of (a) a full-length HCMV gB polypeptide as depicted in FIG. 3, (b) a full-lengh HCMV gB polypeptide from an HCMV strain functionally equivalent to HCMV strain AD 169, and (c) the HCMV gB polypeptide of (a) or (b) lacking the C-terminal membrane anchor sequence, but retaining the remainder of the polypeptide sequence.

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