Title:  Herpes virus complementing cell line

United States Patent:  6,841,373

Issued:  January 11, 2005

Inventors:  Metcalfe; Karen (Wilmington, MA)

Assignee:  AVANT Immunotherapeutics, Inc. (Needham, MA)

Appl. No.:  257229

Filed:  October 10, 2002

PCT Filed:  April 10, 2001

PCT NO:  PCT/US01/11775

371 Date:  October 10, 2002

102(e) Date:  October 10, 2002

PCT PUB.NO.:  WO01/78776

PCT PUB. Date:  October 25, 2001

Abstract

The present invention is directed to a cell line capable of supporting replication of a growth-defective Herpes Simplex Virus strain; specifically a replication-defective HSV-2 double mutant. Particularly disclosed is a cell line that expresses the ICP8 protein and the UL5 protein of Herpes Simplex Virus. This cell line is useful to propagate a replication-defective HSV-2 vaccine strain that contains mutations and/or deletions in the ICP8 and UL5 genes.

Description of the Invention

FIELD OF THE INVENTION

The present invention is in the fields of cellular and molecular biology. Specifically, The present invention is directed to a cell line useful for the growth of a mutant strain of Herpesvirus.

BACKGROUND OF THE INVENTION

Herpesviridae is a large family of enveloped linear dsDNA-containing animal viruses. Herpesviruses are morphologically similar. The virion (.about.120-200 nm diam.) contains a core (DNA wound around a central protein structure) within an icosahedral capsid (.about.100-110 nm diam.) comprising 12 pentameric and 150 hexameric capsomers. The viron is enclosed by a lipoprotein envelope bearing surface projections. The linear dsDNA genome characteristically contains repeated terminal and/or internal sequences.

Herpesviruses have been isolated from a wide range of animals, including mammals, birds, reptiles, amphibians, and fish. Many herpesviruses cause disease in their primary host(s), and may remain latent within the tissue of the host, often for life. Virus transmission commonly occurs by direct contact of mucosal surfaces. Some herpesviruses can be transmitted via body fluids (e.g., milk; via the placenta; etc).

Herpes simplex virus (HSV) type 1 or 2 is a causative agent for serious infections in humans. Herpes simplex diseases are characterized by the formation of thin-walled vesicles, which ulcerate, crust and heal; the vesicles occur, often in clusters, on skin and/or mucous membranes. Transmission occurs as a result of close physical contact; e.g., sexual contact, kissing, close contact sports such as wrestling (Herpes gladiatorum). HSV incubation periods range from 2-12 (average 6) days. The disease state varies from subclinical to severe, and is occasionally fatal. HSV can remain latent in nerve cells near the site of infection. Reactivation may occur spontaneously or in response to other infections (e.g., stress, immunosuppression). In neonates and immunodeficient individuals, HSV may become-disseminated; often affecting the liver, adrenal glands, brain, etc.

HSV-2 is associated with genital, and hence neonatal, infections (Herpes genitalis), and is a disease of significant morbidity in infected individuals (Whitley, 1996). In addition to the conditions described above, other symptoms may include e.g. fever, dysuria, pain, and malaise. In women, the cervix is often the main site of genital infection (herpetic cervicitis). HSV-2 infection in women is associated with an increased risk of abortion and of cervical cancer. Individuals with active HSV-2 have an increased risk of acquiring HIV if exposed to the virus (Augenbraun and McCormack).

Neonatal herpes is usually acquired (during birth) from a mother infected with HSV-2. Fatality rates may be 50% or more in untreated cases. Surviving infants commonly show neurological and/or ocular secondary disorders.

Clinical diagnosis of HSV-2 infection is established by microscopic examination of lesion samples, or biopsies from e.g. skin, brain or liver for multinucleate giant cells with eosinophilic intranuclear inclusion bodies, or by various immunofluorescence techniques (e.g., ELISA).

A number of antiviral agents (e.g. vidarabine, acyclovir, IDU and trifluorothymidine) have activity against HSV and may be effective in some cases (e.g. vidarabine is used against HSV encephalitis). These drugs are not generally effective in preventing recurrence or transmission, however. There remains an unmet need for an effective vaccine against HSV-2 to induce protective immunity and to prevent or to reduce primary infection and, ideally, to reduce recurrent disease and transmission.

Numerous approaches have been attempted to obtain immunization against HSV infection (e.g., glycoprotein subunits, inactivated virus, attenuated virus, and various HSV antigens. See Krause and Straus, 1999; Bernstein and Stanberry, 1999; and Stanberry, 1998). These approaches have shown little to no effectiveness, however (e.g., HSV glycoprotein subunit vaccines, Corey et al., 1999 and Straus et al, 1997; attenuated HSV, Cadoz, et al., 1992).

The use of replication-defective mutant viral strains is a promising avenue of induced immunization against HSV in animal models (Boursnell et al., 1997; Da Costa et al., 1997; Farrell et al., 1994; McLean et al., 1994; McLean, 1996; Morrison and Knipe, 1994; Morrison, Da Costa, and Knipe, 1998; Nguyen et al., 1992; and Stanberry, 1999). Current studies, however, use single mutant viruses, which carry with it the threat of back mutation (reversion to a virulent wild type). Standard vaccine design typically utilizes strains with two or more (non-reverting) mutations to increase safety of the vaccine (Curtiss et al., 1994).

In an effort to develop a live mutant virus vaccine, while reducing the risk of reversion, Da Costa et al. have developed a double deletion mutant HSV-2 strain. This strain lacks two genes essential for DNA replication, thus rendering the mutant incapable of DNA synthesis and viral replication. This double deletion mutant virus strain fails to form plaques or to give any detectable single cycle yields in normal monkey or human cells, yet it is capable of eliciting an immune response (i.e., it functions as an effective immunogen). This double deletion mutant HSV-2 strain induces antibody titers in mice equivalent to those induced by single deletion mutant viruses (Da Costa et al., manuscript submitted).

Because this double deletion mutant HSV-2 strain is replication defective, the replication gene product components it lacks must be provided. There is a need, therefore, for a cell line capable of complementing this double deletion mutant HSV-2 strain, enabling the propagation of the strain, thus providing vaccine production level growth stock of the mutant strain.

SUMMARY OF THE INVENTION

The present invention is directed to a cell line capable of supporting replication of a growth-defective Herpes Simplex Virus strain; specifically a replication-defective HSV-2 double mutant. This complementing cell line provides a means to propagate a growth defective strain of HSV useful as, for example, stock immunogen for vaccine production.

In one preferred embodiment, the present invention is directed to a cell line that expresses the ICP8 protein and the UL5 protein of Herpes Simplex Virus. This cell line is useful to propagate a replication-defective HSV-2 vaccine strain that contains mutations and/or deletions in the ICP8 and UL5 genes. Most preferably, the complementing cell line exhibits the characteristics of the complementing cell line as deposited with the American Type Cell Culture (ATCC) and assigned the Patent Deposit Designation PTA-2403.

In another embodiment of the invention the UL5/ICP8 expressing cell line contains an ICP8/UL5-defective HSV-2 strain.

Another embodiment of the present invention is directed to a method for producing an ICP8/UL5-defective HSV-2 strain by propagating the mutant virus using the UL5/ICP8 expressing cell line, and harvesting the virus resulting from the cell culture.

In one aspect of the present invention, the complementing cell line reduces the possibility of reversion of the defective virus to its wild type form during replication in the complementing cell line. This is achieved by assuring the complementing cell line contains minimal sequence homology with the replication-defective HSV strain to avoid genetic recombination and to produce only defective virus. This aspect is of added benefit because a complementing cell line with little or no sequence homology with a mutant HSV strain provides further safety during growth of the virus.

DETAILED DESCRIPTION OF THE INVENTION

Vero Cells (African Green Monkey Kidney Cells, ATCC #81-CCL) were used to create a cell line that expresses the UL5 and ICP8 proteins of Herpes Simplex Virus. It is understood, however that any of a variety of suitable cell lines may be used in the practice of this invention.

As an initial step, suitable expression vectors for each of the defective viral genes are created by any of a variety of techniques known in the art and using any of a number of suitable vectors.

Then, a cell line capable of expressing one of the two defective viral genes is created, using any of a variety of genetic engineering techniques known in the art For example, an ICP8 expressing cell line may be generated by transfecting Vero cells with an HSV-1 ICP8 expression plasmid. A modified calcium phosphate method of transfection may be used to create the ICP8 expressing cell line. As described herein, the ICP8 expression plasmid, pRC/CMV-ICP8-A1(RA1) may be used in the construction of this cell line.

Finally, the cell line capable of expressing the first of the two defective viral genes is manipulated to express the second of the two defective viral genes. For example, an ICP8 expressing cell line may be further transfected with a UL5 expression plasmid using, for example, an electroporation method of transfection. As described herein, the UL5 expression plasmid, p70-4 is useful in this transfection.

An ICP8/UL5 expressing cell line is selected on the basis of its ability to support the replication of both an ICP8 mutant HSV-2 strain, and a UL5 mutant HSV-2 strain.

Many component steps of the present invention may be performed by alternative, yet functionally equivalent, biochemical or genetic engineering techniques known in the art without altering the inventive nature of the present disclosure. For example, methods for generating recombinant nucleic acids, vector construction, host cell transformation, polypeptide expression systems, and phage display useful in the practice of this invention can involve a wide variety of modern genetic engineering techniques, tools, and biological sources that are well known in the art and routinely practiced by those skilled in the art. Exemplary techniques and methods are described in detail herein by way of preferred example, but are not limiting to the practice of the invention. The present invention incorporates by reference in their entirety techniques and supplies well known in the field of molecular biology, including, but not limited to, techniques and supplies described in the following publications:

Ausubel, F. M. et al. eds., Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X).

Freshney, R. I. Culture of Animal Cells (1987) Alan R. Liss, Inc.

Old, R. W. & S. B. Primrose, Principles of Gene Manipulation: An Introduction To Genetic Engineering (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).

Sambrook, J. et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).

Winnacker, E. L. From Genes To Clones: Introduction To Gene Technology (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-6144).

Claim 1 of 13 Claims

I claim:

1. A method for producing a replication-defective, ICP8/UL5-defective herpesvirus double mutant comprising the steps of:

(a) propagating said ICP8/UL5-defective herpesvirus using a cell line that is able to support the growth said virus; and

(b) harvesting the virus resulting from step (a).

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