Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-05-06
2001-06-19
Mosher, Mary E. (Department: 1643)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S471000, C435S472000, C435S475000, C435S320100, C536S023720
Reexamination Certificate
active
06248578
ABSTRACT:
BACKGROUND
Recognized in 1956 as a cause of respiratory infection in man, human parainfluenza viruses (HPIV) are believed to account for 4 to 22 percent of the respiratory illnesses in children, second only to respiratory syncytial virus in this regard. HPIV are important causes of the lower respiratory tract diseases such as pneumonia and bronchiolitis, and are the most common cause of croup in young children. Of the four HPIV serotypes, 1-4, type 3 virus (HPIV-3), appears to be the most virulent, frequently causing bronchiolitis and pneumonia during the first month of life.
Unfortunately, effective vaccines or antiviral therapies, which can be used to prevent or treat HPIV-induced infections, are not presently available. Standard methods which are used to produce inactive MAH viruses, such as heat inactivation or chemical treatment of the virus, have been unsuccessful with all HPIV strains and serotypes, including HPIV-3. Moreover, standard methods for producing attenuated viruses produce mutations at random sites and do not allow one to modify the HPIV genome at specific sites or to control the number of mutations that are introduced into genome.
Human parainfluenza viruses are enveloped, single-stranded, negative sense RNA viruses that are members of the paramyxovirus genus within the family Paramyxoviridae. Replication of the human parainfluenza viral genome (vRNA) is similar to that of other members of the Paramyxoviradae family. Upon infection of a cell, transcription is the major RNA synthetic event, resulting in the production of the viral mRNAs from the negative-sense genome, i.e., the vRNA. Later in infection a transition to RNA replication occurs, resulting in synthesis of a full-length, antigenomic, positive-sense RNA, which serves as the template for synthesis of additional negative-sense genomic RNA. Transcription and replication of the genomic RNA is dependent upon formation of a ribonucleoprotein complex (RNP) consisting of the 15462 nucleotide genomic RNA encapsidated by the nucleocapsid protein (NP), and the closely associated phosphoprotein (P), and the large (L) polymerase protein. Several host cell factors are also involved in the replicative cycle of HPIV.
The requirement for an intact RNP for HPIV has hindered analysis of HPIV transcription and replication in a cell-free system. Efforts to encapsidate HPIV-3 vRNA in vitro have failed, and unlike the positive sense RNA viruses, naked HPIV vRNA is not infectious. Moreover, there currently are no known systems for preparing recombinant HPIV, including recombinant infectious HPIV-3.
Accordingly, there is a need for new reagents, systems, and methods that enable one to produce a recombinant HPIV, particularly a recombinant, infectious HPIV-3. Recombinant systems that permit one to introduce one or more site-specific mutations into the genome of HPIV, particularly HPIV-3, are desirable. Recombinant systems which allow one to characterize the effect of site-specific mutations on the transcription or replication of human parainfluenza viral RNA and to identify the site specific mutations which lead to the production of attenuated HPIV are especially desirable.
SUMMARY OF THE INVENTION
In accordance with the present invention a system for generating recombinant, human parainfluenza virus, particularly infectious, recombinant, human parainfluenza virus type 3 (HPIV-3) is provided. In one embodiment, the system comprises a clone comprising a nucleotide sequence that encodes a full-length, positive sense, anti-genome of HPIV, and at least one support clone comprising a nucleotide sequence that encodes the HPIV P protein and the HPIV L protein. In another embodiment, the system further comprises a support clone which comprises a nucleotide sequence that encodes the HPIV NP protein. Preferably, each of the clones in the system comprises an RNA polymerase promoter which is operatively linked to the respective HPIV nucleotide sequence contained within the clone.
The present invention also provides a clone which comprises a nucleotide sequence encoding the full-length, positive sense, anti-genome of HPIV-3. The clone also comprises an RNA polymerase promoter operatively linked to the HPIV-3 antigenome-encoding sequence. In a preferred embodiment, the clone further comprises a nucleotide sequence which encodes a ribozyme immediately downstream from the sequence encoding the HPIV-3 anti-genome.
The present invention also relates to a method of preparing recombinant HPIV-3 virus having site-specific mutations in the HPIV-3 genome. The method comprises preparing a clone comprising a modified HPIV-3 antigenome-encoding sequence; introducing the modified HPIV-3 clone and support clones which comprise nucleotide sequences encoding an HPIV-3 P protein, an HPIV-3 L protein, and, preferably, an HPIV-3 NP protein into host cells.; and culturing the host cells under conditions that allow for synthesis of the modified HPIV-3 antigenome and the L, P, and NP proteins of HPIV-3.
The ability to produce recombinant, HPIV-3 virus genetically engineered to contain site-specific mutations within the HPIV-3 genes and cis-acting elements expedites the study of all aspects of the virus replication cycle. Additionally, a system which permits production of recombinant HPIV that is genetically engineered to contain site-specific mutations within the HPIV-3 genome is useful for identifying attenuating parainfluenza genotypes and for developing a live vaccine for human parainfluenza virus.
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Banerjee Amiya K.
Hoffman Michael A.
Calfee Halter & Griswold LLP
Mosher Mary E.
The Cleveland Clinic Foundation
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