Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1996-09-09
2001-11-27
Wilson, James O. (Department: 1623)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C424S093210, C435S235100, C435S320100, C536S023720
Reexamination Certificate
active
06322780
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a gene derived from Marek's disease virus having a unique nucleotide sequence, recombinant viruses containing this gene, poultry vaccines utilizing this gene, and recombinant fowlpox vaccines that exhibit a syngeristic effect in protecting against Marek's disease.
2. Description of Related Art
Marek's disease (MD) is a highly contagious neoplastic disease of domestic chickens that affects chickens worldwide and causes high mortality and condemnation if chickens are not vaccinated at one day of age. MD is caused by a highly cell-associated oncogenic herpesvirus known as Marek's disease virus (MDV).
A number of live virus cell-associated vaccines are available that protect chickens against MD. These vaccines are maintained and administered in delicate, cell-associated form. The vaccines need special handling, and must be stored and transported in a frozen state in liquid nitrogen in order to maintain their viability and efficacy. These existing vaccines must be maintained and administered in cell-associated form, a condition that is costly and cumbersome.
The known vaccines contain the entire MDV genome, including sequences related to induction of patho-genesis. Although the existing vaccines against MD are either attenuated or are naturally apathogenic, viral mutation is known to occur in herpesviruses, and there is a possibility that virulent pathogenic mutants may emerge in such vaccines. Such mutants could be less effective and even harmful.
Churchill et al. (
Nature
221:744-747 (1969)) and Okazaki et al. (
Avian Dis.
14:413-429 (1970)) developed the first effective and safe vaccines against MD. These vaccines have been in use for the last 20 years, and have reduced losses to the poultry industry worldwide. Other candidate vaccines based on serotype 2 naturally apathogenic MDV (Schat et al.
J. Natl. Cancer Inst.
60: 1075-1082 (1978)), or newly attenuated serotype 1 MDV (Rispens et al.
Avian Dis.
16:108-125 (1972)), and combinations of these viruses as bivalent vaccines (Witter
Avian Dis.
31:252-257 (1987)), have helped provide better protection against MD. All these vaccines, except the herpesvirus of turkeys (HVT) vaccine, require storage and transportation in a frozen state in liquid nitrogen,and have to be administered as infected cells, which calls for careful procedures to prevent inactivation of the vaccine. Even in the case of HVT vaccine, cell-associated viruses have been most widely used because they are more effective than cell-free virus in the presence of maternal antibodies (Witter et al.
Avian Pathol.
8:145-156 (1978)).
Recombinant DNA technology has facilitated the construction of recombinant vaccines that contain only those desired viral genes or gene products that induce immunity without exposing the animal to genes that may induce pathological disorders. Pox viruses, including avipox virus, especially fowlpox virus (FPV), provide excellent models for such vaccines. These viruses have a large DNA molecule with numerous non-essential regions that permit the insertion of several immunogenic genes into the same virus for the purpose of creating multivalent vaccines. These multivalent vaccines may induce cell-mediated as well as antibody-mediated immune response in a vaccinated host. Vaccinia virus (W) has been used extensively for this purpose, and a number of VV recombinants have been constructed that express a variety of foreign genes, including those that elicit neutralizing antibodies against glycoproteins of herpes simplex virus (HSV) type 1 (Blacklaws et al.
Virology
177:727-736 (1990)). Similarly, there are a number of reports describing the expression of foreign genes by recombinant FPV (Boyle et al.
Virus Res.
10:343-356 (1988) and Ogawa et al.
Vaccine
8:486-490 (1990)). Recently, we demonstrated that the recFPVgB protected chickens against MDV challenge (Nazerian et al.
J. Virol.
66:1409-1413 (1992)).
MDV homologues of the HSV genes coding for glycoproteins B, C, D, H, and I, E, L (gB, gC, gD, gH and gI, gE, gL) have recently been cloned and sequenced (Coussens et al.
J. Virol.
62:2373-2379 (1988); Ross et al.
J. Gen. Virol.
70:1789-1804 (1989); Ross et al.
J. Gen. Virol.
72:939-947 (1991); Ross et al., International Publication No. WO 90/02803 (1990); Brunovskis and Velicer,
Virology
206:324-338 (1995); and Yoshida et al.
Virology
204:414-419 (1994)).
SUMMARY OF THE INVENTION
The present inventors have shown that gB is an important gene for protective immunity against MD (Nazerian et al.
J. Virol.
66:1409-1413 (1992)). Whittaker et al. (1992) reported that Equine herpesvirus type 1 (EHV-1) gene 28 encodes a glycoprotein, gp300, that is homologous to the HSV-1 UL32, and functions in EHV-1 in cell-to-cell fusion processes. We postulated that if such a homologous gene existed in MDV, it may function in cell to cell fusion since MDV is a cell-associated virus. Recently, we identified and sequenced an MDV gene homologous to HSV-1 UL32, and identified an O-linked glycoprotein, gp82, in MDV-infected cells belonging to a class of membrane proteins (Lee, unpublished data).
The present invention relates to the MDV UL32 gene encoding a membrane glycoprotein. The DNA sequence of the UL32 gene is shown in SEQ ID NO:1 in the attached Sequence Listing. The present invention therefore relates to this sequence, which encodes a protein in accordance with the degeneracy of the genetic code, preferably in a cloned, isolated, or purified form, and biologically functional variants thereof. The present invention also relates to recombinant DNA molecules comprising the UL32 sequence.
The present invention also relates to novel recombinant viral vaccines, such as recombinant FPV, HVT (herpesvirus of turkeys), MDV, and ILTV (infectious laryngotracheitis virus), that contain the novel UL32 gene encoding membrane glycoprotein gp82 of MDV. Preferably, the vaccine is based on a recombinant FPV containing the UL32 gene of MDV. More preferably, the recombinant FPV contains an additional gene, i.e., the gB gene, encoding gp100, gp60 and gp49, which provides a synergistic effect in protecting against MD in chickens. As shown below, recombinant FPV expressing UL32 is effective against MD. The sequence and the effectiveness of gB as a vaccine has been described in U.S. Pat. No. 5,369,025. The expression of these two genes in cells results in an unexpectedly strong synergistic protective effect against MD in the natural host (chickens). In addition, the vaccine also protects against fowlpox.
The present invention also relates to recombinant FPV vaccines against MD in which the gB gene of MDV or the UL32 gene of MDV or other genes such as those coding for glycoprotein E homologue, glycoprotein I homologue, and other glycoproteins from different serotypes of MDV are inserted into FPV for the purpose of creating a broad-spectrum vaccine effective against several isolates of MDV.
The present invention also relates to a cell-free vaccine against MD containing recombinant (rec) FPV that can be lyophilized, stored, and used under normal conditions, thereby obviating costly and laborious procedures of storing the vaccine in liquid nitrogen, delicate handling, and administering which are necessary with existing cell-associated MD vaccines. For example, after lyophilization, the vaccine of the present invention can be stored, handled, and transported at ambient temperature (20-22° C.), and stored at 4° C. for prolonged periods of time. The vaccine can also be stored in a frozen state wherein the cell-free recombinant virus is present in an aqueous solution which is frozen and stored at, for example, −20° C. or −70° C.
Accordingly, it is an object of the present invention to provide an isolated, purified DNA molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a nucleotide sequence biologically functionally equivalent thereto.
Another object of the present invention is to provide an isolated, purified DNA molecule comprising a nucleot
Lee Lucy F.
Nazerian Keyvan
Witter Richard L.
Wu Ping
Yanagida Noboru
Birch & Stewart Kolasch & Birch, LLP
The United States of America as represented by the Secretary of
Wilson James O.
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