Genomic sequence of avian paramyxovirus type 2 and uses thereof
Inventors
Samal, Siba K. • Collins, Peter L.
Assignees
University of Maryland College Park • US Department of Health and Human Services
Publication Number
US-9937196-B2
Publication Date
2018-04-10
Expiration Date
2030-06-21
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
In this application is described the complete genomic sequence of avian parmyxovirus type 2, strains Yucaipa, England, Kenya and Bangor. The sequences are useful for production of recombinant infective virus, a virus vector, for vaccine development and for therapeutic compositions.
Core Innovation
This invention describes the complete genomic sequences of avian paramyxovirus type 2 (APMV-2), specifically for strains Yucaipa, England, Kenya and Bangor. The sequences enable the production of recombinant infectious viruses, virus vectors, vaccine development, and therapeutic compositions. The genome sequences cover nucleotide and amino acid sequences of viral genes encoding various proteins including nucleocapsid protein (N), phosphoprotein (P), V protein, matrix protein (M), fusion glycoprotein (F), hemagglutinin-neuraminidase protein (HN), large polymerase protein (L), and W protein for each strain.
The problem solved by this invention arises from the significant genetic and biological differences between APMV-2 and other avian paramyxoviruses such as NDV (APMV-1), which impede direct nucleotide-based methods for sequence determination and recombinant virus production. These differences include failure of RNA-RNA hybridization between APMV-2 and NDV, inability to use NDV-based primers in RT-PCR, absence of typical syncytia formation by APMV-2 infection, and lack of virulence in chicken embryos and brain. Consequently, the complete genome sequence of APMV-2 was needed to enable recombinant virus production and the development of novel vaccine vectors and therapeutic agents.
The invention discloses methods for sequencing the APMV-2 genome despite these difficulties by designing consensus primers from various paramyxoviruses and using gene start/gene end consensus sequences along with primer walking techniques. It covers the generation of infectious recombinant and chimeric APMV-2 viruses from the obtained genomic sequences and their use as vectors expressing foreign genes, such as green fluorescent protein (GFP). The invention also encompasses the use of nucleotide sequence variation among APMV-2 strains to distinguish subgroups for diagnostic and therapeutic applications.
Claims Coverage
The patent includes 14 claims with multiple inventive features focusing on isolated nucleic acids, recombinant viruses, support plasmids, and methods of producing infectious APMV-2 virus.
Isolated nucleic acid with specific mutations
An isolated nucleic acid comprising the APMV-2 Yucaipa sequence (SEQ ID NO:1) containing at least one of ten specific nucleotide substitutions (C2923A, G2924A, T2925A, G2926C, G4154C, G5971A, A5973T, T7870C, A111321G, A11322C).
Recombinant infectious APMV-2 virus
A recombinant infectious APMV-2 virus that comprises the isolated nucleic acid of the modified APMV-2 genome with the specified mutations.
Isolated nucleic acid with all ten specific substitutions
An isolated nucleic acid corresponding to a full-length cDNA clone of APMV-2 Yucaipa containing all ten nucleotide substitutions as genetic markers (SEQ ID NO:117 and SEQ ID NO:118).
Recombinant virus expressing non-native antigenic sequences
A recombinant infectious APMV-2 virus comprising the nucleic acid that further includes one or more non-native sequences encoding viral antigens, tumor antigens, or autoantigens involved in autoimmune disorders.
Immunogenic composition with recombinant APMV-2 viruses
Vaccine compositions comprising the recombinant infectious APMV-2 viruses encoding antigenic proteins or heterologous sequences for immunization.
Recombinant cells expressing infectious APMV-2 RNA
Cells that are co-transfected with plasmids encoding the full-length antigenomic cDNA of APMV-2 Yucaipa along with support plasmids expressing viral proteins (N, P, L) necessary for viral replication, enabling production of infectious recombinant virus.
Method for preparing infectious APMV-2 virus
A method for producing infectious recombinant APMV-2 virus by transfecting host cells with the plasmid genome vector encoding APMV-2 full-length antigenome cDNA including the specified mutations along with plasmids expressing viral polymerase complex proteins, culturing the cells, and recovering the infectious virus.
The claims cover isolated nucleic acids of APMV-2 Yucaipa with specific genetic markers, recombinant infectious viruses derived therefrom, recombinant cells engineered to produce infectious virus from cloned cDNA, and methods for preparing these viruses. The claims further extend to recombinant viruses and compositions expressing heterologous antigenic sequences for vaccine and therapeutic uses.
Stated Advantages
APMV-2 differs biologically and genetically from NDV and other paramyxoviruses in ways that offer advantages for vaccine development and virus vector use, such as inability to cause disease in chickens and lack of syncytia formation, reducing concerns about reversion to virulence.
The recombinant APMV-2 viruses are stable, have similar growth characteristics to wild-type virus, and can express foreign proteins such as GFP, demonstrating utility as vaccine vectors.
APMV-2 can infect mammalian tumor cells and effectively kill them, indicating potential applications in cancer therapy.
Documented Applications
Use of complete APMV-2 genomic sequences for production of recombinant infectious virus and virus vectors.
Development of vaccine formulations and immunogenic compositions comprising recombinant or chimeric APMV-2 viruses expressing viral or heterologous antigens.
Diagnostic methods for detection and differentiation of APMV-2 strains and infections using nucleic acid and antibody-based assays.
Treatment and prevention of APMV-2 infections in animals and humans using vaccines based on recombinant or chimeric APMV-2 viruses.
Use of recombinant APMV-2 vectors for expression of foreign antigens to induce immune responses against various pathogens.
Methods and compositions for treatment of cancer using recombinant or chimeric APMV-2 viruses due to their ability to kill tumor cells in vitro.
Interested in licensing this patent?