Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1995-06-06
2001-01-30
Minnfield, Nita (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023720, C424S204100, C424S230100, C435S069100, C435S070100
Reexamination Certificate
active
06180369
ABSTRACT:
The present invention relates to the construction of a recombinant plasmid which is capable of expressing a secretory truncated glycoprotein (Tgp) of Varicella-zoster virus (VZV) in mammalian cells. The secretory Tgp of the present invention contains at least one epitope capable of inducing antibody response. The present invention contemplates the production and utilization of this secretory Tgp in a vaccine against chickenpox and/or shingles. The present invention is also directed towards the use of the secretory Tgp in diagnostic assays for detection of VZV. The present invention is also directed to first antibodies specific to secretory Tgp and to second antibodies specific to the first antibodies. These second antibodies are also useful in diagnostic assays for VZV.
Varicella-zoster virus is the causative agent of childhood chickenpox (varicella) and shingles (zoster), two distinct clinical manifestations. Varicella is the outcome of the primary encounter (infection) with VZV, whereas zoster is the result of VZV reactivation which occurs predominantly in aging and immunosuppressed individuals, including cancer and AIDS patients. There are 2.5 million estimated cases of chickenpox and 1.2 million cases of shingles per year in the United States. It is expected that the number of shingles patients will increase as the population ages. One of the most common complications of shingles includes postherpetic neuralgia which is characterized by interactable pain lasting for four weeks to several years after the onset of skin rash. Other complications of VZV reactivation (shingles) include encephalitis, pneumonitis and disseminated zoster.
VZV is a member of the alpha herpesvirus family. VZV contains a linear double-stranded DNA genome of approximately 125,000 base pairs and consists of the sequence of a long unique (U1)-inverted short repeat (IRs)-short unique (Us)-terminal short repeat (TRs). VZV DNA encodes five glycoproteins, designated gpI, gpII, gpIII, gpIV and gpV, of which gpI through gpIV are readily detected in infected cells and in VZ virions (Davison and Scott,
J. Gen. Virol.,
67:1759-1816, 1986; Davison, et al.,
J. Virol.
57:1195-1197, 1986). These glycoproteins are highly immunogenic and elicit both neutralizing antibodies and cell-mediated immune response in the infected individuals (Davison, et al., supra, 1986).
VZV gpI, which is the most abundant and immunogenic of the virion envelope glycoproteins, elicits the formation of complement-dependent neutralizing antibodies and also mediates antibody-dependent cellular cytotoxicity. The gene encoding gpI is located in the unique short (Us) region of the VZV genome. One of the major antibody-binding sites (epitope) on a VZV glycoprotein has been identified in VZV gpI (Vafai, et al.,
J. Virol.
62:2544 (1988)). The synthetic peptides (14 amino acid residues) comprising this epitope (designated e1) induced antibody response which was recognized by a high-mannose intermediate (82 kDa) but not the mature form (95 kDa) of VZV gpI (Vafai, et al.,
Virus Res.
13:319-336, 1989). These results along with the lack of VZV neutralizing activity of anti-peptide antibodies suggested that the state and extent of O-linked and/or N-linked glycosylation of e1 epitope affect the conformation of gpI or result in steric hindrances which influence the antigenic determinant recognized by anti-peptide antibodies.
An attenuated varicella-zoster virus vaccine has been used in Japan against chickenpox infection in leukemic children as well as for routine vaccination in early childhood. This vaccine is currently being tested in the United States in children with leukemia and is expected to be used in healthy children and for the prevention of VZV reactivation (shingles) in the elderly population. Although the attenuated varicella vaccine has been shown to be safe and effective in inducing immunity against VZV infection, however, similar to natural infection, attenuated varicella vaccine becomes latent in human dorsal root ganglia and may reactivate to produce shingles with its attendant neurologic complications of postherpetic neuralgia and encephalitis.
Therefore, a subunit vaccine which would avoid and eliminate latency is desirable for immunization of children as well as for boosting immune response in the elderly who are more susceptible to VZV reactivation (shingles). Such subunit vaccine as contemplated by the present invention may be prepared by construction of recombinant viruses (e.g., vaccinia virus) expressing one or more VZV glycoproteins or, as particularly contemplated by the present invention, may be composed of secretory highly immunogenic VZV glycoprotein(s) which can be prepared and purified in large quantities and used for immunization and/or boosting the immune response against VZV infection. In addition, such highly purified VZV glycoproteins can be used as a diagnostic tool for the assessment of the immune status to VZV infection in immunosuppressed individuals (leukemic children, AIDS and cancer patients) as well as in vaccinated individuals and the elderly population.
The gene for VZV gpI has been previously isolated, inserted into a plasmid and incorporated into a vaccinia virus expression system. Although gpI protein was produced by the vaccinia expression system, the product remained within the cells and was therefore unsuitable for eliciting an antigenic response in vivo.
The innovation of the present invention resides in the construction of an expression vector which produces a truncated form of VZV gp which is secreted from mammalian cells.
The applications of the present recombinant vaccinia viruses expressing secretory truncated VZV glycoproteins containing one or more highly immunogenic viral epitopes include: (1) using such recombinant viruses as subunit vaccines against VZV infection, wherein secretion of VZV glycoproteins following vaccination provides a stronger immune response to VZV glycoproteins as well as to VZV infection; (2) using large quantities of highly purified and immunogenic secretory VZV glycoproteins containing one or more epitopes as a subunit vaccine against primary VZV infection (chickenpox) in healthy children as well as in immunocompromised individuals and for boosting immunity against VZV reactivation (shingles) in the elderly; and (3) using purified preparations of secretory truncated VZV glycoproteins in diagnostic kits as highly specific target antigens for the detection and assessment of antibody status to VZV glycoproteins. Since VZV reactivation is common in cancer and AIDS patients, there is also a need for the serological diagnosis of VZV infection in these patients. In addition, since VZV reactivation in the growing population of elderly individuals results in pain prior to the onset of clinical symptoms and may also result in encephalitis, pneumonitis and disseminated zoster, the only hope for an early treatment of these patients lies in a rapid means of diagnosis. Application of the present recombinantly prepared secretory VZV glycoproteins in diagnostic kits can provide a rapid and inexpensive means for diagnosis of VZV infection.
The present invention is directed to an expression vector for secretory truncated VZV gp and construction of said vector which permits extracellular secretion of the VZV protein.
More specifically, the present invention is directed to a recombinant DNA expression vector comprising a nucleotide sequence capable of expressing in an infected, transfected or transformed host a Varicella-zoster virus (VZV) truncated glycoprotein (gp) which is secreted extracellularly from said host and wherein said glycoprotein causes a VZV antibody response in mammals.
Another aspect of this invention contemplates the recombinant production of secretory truncated VZV gpI, II, III, IV or V in mammalian cells.
A further aspect of the present invention is directed to a process for producing secretory truncated Varicella-zoster virus gp.
More specifically, the present invention is directed to a process for producing a secretory truncated Varicella-zoster virus glycoprote
Minnfield Nita
Research Corporation Technologies Inc.
Scully Scott Murphy & Presser
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