Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or... – Inactivation or attenuation; producing viral subunits
Reexamination Certificate
1995-06-06
2001-11-13
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
Inactivation or attenuation; producing viral subunits
C424S206100, C424S215100, C536S023720
Reexamination Certificate
active
06316243
ABSTRACT:
The work reflected in this application was supported, in part, by a grant from the National Institutes of Health, and the Government may have certain rights in the invention.
1. INTRODUCTION
The present invention relates to engineering attenuated viruses by altering a non-coding region or the coding sequence of a viral gene. Alterations of the non-coding regions which regulate transcription and/or replication are described. These alterations result in the down-regulation of the viral gene and an attenuation of the virus, either by the production of defective particles during replication, or by reducing the number of progeny virions produced during viral replication. Alterations of viral coding sequences are also described which result in a recombinant or chimeric attenuated virus.
2. BACKGROUND OF THE INVENTION
Inactivated virus vaccines are prepared by “killing” the viral pathogen, e.g., by heat or formalin treatment, so that it is not capable of replication. Inactivated vaccines have limited utility because they do not provide long lasting immunity and, therefore, afford limited protection. An alternative approach for producing virus vaccines involves the use of attenuated live virus vaccines. Attenuated viruses are capable of replication but are not pathogenic, and, therefore, provide for longer lasting immunity and afford greater protection. However, the conventional methods for producing attenuated viruses involve the chance isolation of host range mutants, many of which are temperature sensitive; e.g., the virus is passaged through unnatural hosts, and progeny viruses which are immunogenic, yet not pathogenic, are selected.
Recombinant DNA technology and genetic engineering techniques, in theory, would afford a superior approach to producing an attenuated virus since specific mutations could be deliberately engineered into the viral genome. However, the genetic alterations required for attenuation of viruses are not known or predictable. In general, the attempts to use recombinant DNA technology to engineer viral vaccines have mostly been directed to the production of subunit vaccines which contain only the protein subunits of the pathogen involved in the immune response, expressed in recombinant viral vectors such as vaccinia virus or baculovirus. More recently, recombinant DNA techniques have been utilized in an attempt to produce herpes virus deletion mutants or polioviruses which mimic attenuated viruses found in nature or known host range mutants. Until very recently, the negative strand RNA viruses were not amenable to site-specific manipulation at all, and thus could not be genetically engineered.
3. SUMMARY OF THE INVENTION
The present invention relates to the production of attenuated viruses using recombinant DNA techniques. At least two approaches for engineering attenuated viruses are described. One approach involves engineering alterations of a non-coding region of the virus that regulates transcription and/or replication of a viral gene so that at least one of the viral genes is down regulated. This approach may be applied to a number of different viruses and is advantageously used to engineer segmented viruses where down regulation of the synthesis of one viral segment results in the generation of defective particles during each round of viral replication so that the progeny viruses demonstrate attenuated characteristics. In non-segmented viruses, the down regulation of a viral gene can result in a decrease in the number of infectious virions produced during replication, so that the virus demonstrates attenuated characteristics.
A second approach involves engineering alterations of a viral coding region so that the viral protein expressed is altered by the insertion, deletion or substitution of an amino acid residue or an epitope and an attenuated chimeric virus is produced.
The attenuated viruses of the invention may advantageously be used safely in live virus vaccine formulation. As used herein, the term “attenuated” virus refers to a virus which is infectious but not pathogenic; or an infectious virus which may or may not be pathogenic, but which either produces defective particles during each round of replication or produces fewer progeny virions than does the corresponding wild type virus during replication. Pathogenic viruses which are engineered to produce defective particles or a reduced number of progeny virions are “attenuated” in that even though the virus is capable of causing disease, the titers of virus obtained in a vaccinated individual will provide only subclinical levels of infection.
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