Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1997-10-14
2002-06-11
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S205100, C424S229100, C435S235100, C435S320100, C435S236000
Reexamination Certificate
active
06403097
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the fields of vaccination and diagnostics in connection with diseases which are caused by pathogens and involves the use of both the classic methods to arrive at a live attenuated vaccine or an inactivated vaccine and the modern methods based on DNA recombinant technology.
More specifically, the invention relates to live attenuated vaccines an inactivated vaccines for protecting animals, especially bovines, against bovine herpesvirus type 1 (BHV-1), these vaccines being so designed that they are not only safe and effective, but also create the possibility of distinguishing infected from non-infected animals in a vaccinated population.
Diagnostic kits which can be used for such a test for distinguishing infected from non-infected animals in a vaccinated population are also an aspect of the present invention.
BACKGROUND OF THE INVENTION
BHV-1, including the infectious bovine rhinotracheitis virus (IBRV) and the infectious pustular vulvovaginitis virus (IPVV), plays an important role in the development of respiratory diseases and fertility disorders in bovines. After an acute infection, BHV-1 often remains present in the host in a latent form. Latent virus can be reactivated under the influence of inter alia stress—which may or may not be accompanied by clinical phenomena—and subsequently excreted. As a consequence, infected cattle must be regarded as lifelong potential spreaders of BHV-1. BHV-1 occurs endemically in an estimated 75% of Dutch cattle farms. Especially older cattle are serologically positive.
There are a number of inactivated (“dead”) vaccines and a number of attenuated (“live”) vaccines available for inoculation against BHV-1 infections. Inactivated vaccines are prepared by killing the BHV-1 virus, for instance by heat treatment, irradiation or treatment with ethanol or formalin. However, these often give insufficient protection. Attenuated vaccines are prepared by a large number of passages on homologous (bovine) or on heterologous cells such as porcine or canine cells, and sometimes viruses are also treated physically or chemically then. In this way, unknown mutations/deletions develop in the virus genome, which often reduce the disease-producing properties of the virus. Attenuated live vaccines give better protection than inactivated vaccines, inter alia because they present more viral antigens to the immune system of the host. Another important advantage of live vaccines is that they can be administered intranasally, i.e., at the site where the first multiplication of the wild type virus occurs after infection. Yet, live vaccines leave room for improvement. Some live vaccines still seem to possess their abortogenic ability, which becomes manifest in particular after intramuscular administration. Moreover, probably all live vaccines remain latently present in the vaccinated cow. Also, there is a chance that if the vaccine differs only little from the wild-type virus, reversion to virulence will occur. But one of the major problems is that the BHV-1 vaccines cannot prevent infection by wild-type viruses. The result is that vaccinated cattle can also spread wild-type BHV-1.
For a proper BHV-1 control program, it is necessary to have disposal of an efficacious and safe vaccine that can be distinguished from wild-type virus, since the application of an efficacious vaccine can reduce the circulation of BHV-1 considerably and a test which can distinguish between a vaccine and a wild-type virus makes it possible to detect (and then remove) infected cattle in a vaccinated population.
Meanwhile, BHV-1 vaccines have been developed which seem to be safer than conventional vaccines and are distinguishable from wild-type virus. A thymidine kinase deletion mutant has been isolated which is abortogenic to a lesser degree, becomes latent less frequently and cannot be reactivated. Further, using recombinant DNA techniques, a BHV-1 vaccine has been constructed which has a deletion in the gene for glycoprotein gIII, which makes this vaccine distinguishable from wild-type BHV-1 by means of serological techniques. However, there are still some objections to these vaccines. On the one hand, the thymidine kinase gene is involved in the viral replication and less replication can lead to less protection. On the other hand, the glycoprotein gIII is important for generating protective antibodies, which makes a gIII deletion vaccine less effective. A practical problem is that intranasal administration, which generally gives the best protection, of recombinant vaccines is not allowed in some countries. Accordingly, there is a need for a vaccine which is safe as well as effective and yet can be distinguished from wild-type BHV-1, it being further desirable that at least one of such vaccines is based on a virus attenuated via a conventional route rather than a virus constructed by recombinant DNA techniques.
Now, via passages in cell cultures, a BHV-1 strain has been obtained which lacks the gene for glycoprotein gE. The first results of our research indicate that this gene is quite useful to make a serological distinction with regard to wild-type BHV-1 and that it is involved in the expression of virulence. Therefore, its deletion contributes to safety and may render the use of thymidine kinase deletions superfluous. The glycoprotein gE seems to be less important for induction of protection than the glycoprotein gIII. A conventionally attenuated BHV-1 strain which can be serologically distinguished from wild-type virus is unique. The location and DNA sequence of the gE gene described herein for the first time were not previously known, nor were oligonucleotides, polypeptides and oligopeptides that can be derived therefrom. A test for making a serological distinction on the basis of the gE gene is also unique.
An important advantage of this “conventional” gE deletion mutant (“conventional” refers to the use of a conventional method for isolating an attenuated virus) is that it will be possible to administer it intranasally in countries where this is forbidden as far as recombinant vaccines are concerned. Taking due account of the different views on safety, however, in addition to this conventional gE deletion vaccine, well-defined recombinant versions have been constructed as well. These recombinant vaccines also have a gE deletion—and may or may not have a deletion in the thymidine kinase gene as well—and can also be used as vectors for the expression of heterologous genes. All these recombinant vaccines can be distinguished from wild-type virus with the same gE-specific test. The use of a standard test for a set of different vaccines can be a great advantage in the combat of BHV-1 as an international effort. Such an approach has not been previously described in the field of BHV-1, vaccines.
Serological analysis of the anti BHV-1 response in cattle showed that an important fraction of the anti-gE antibodies are directed against a complex formed by glycoprotein gE and another BHV-1 glycoprotein: glycoprotein gI. Serological tests that can (also) demonstrate the presence of such complex-specific antibodies may therefore be more sensitive than tests that can only detect anti-gE antibodies. Cattle vaccinated with a single gE deletion mutant may produce anti-gI antibodies that can interfere with the detection of anti-gI/gE antibodies. Consequently, this invention also includes a vaccine with a gI/gE double deletion.
SUMMARY OF THE INVENTION
In the first place, this invention provides a deletion mutant of BHV-1 which has a deletion in the glycoprotein gE-gene. The words “a deletion in” intended to cover a deletion of the gene as a whole.
A preferred embodiment of the invention is constituted by a deletion mutant of BHV-1 which has a deletion in the glycoprotein gE-gene which has been caused by an attenuation procedure, such as the deletion mutant Difivac-1 to be described hereinafter.
Other preferred embodiments of the invention consist of a deletion mutant of BHV-1 comprising a deletion in the glycoprotein gE-gene which has been constructed by re
Rijsewijk Franciscus Antonius Maria
van Oirschot Johannes Theodorus
Hoffman & Baron LLP
Mosher Mary E.
Stichting Centraal Diergeneeskundig Instituut
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