Altered avian virus for in-ovo inoculation and methods of use thereof
Inventors
Afonso, Claudio L. • Marcano, Valerie C. • Cardenas Garcia, Nany Stivalis • Yu, Qingzhong • Taylor, Tonya L.
Assignees
University of Georgia Research Foundation Inc UGARF • US Department of Agriculture USDA
Publication Number
US-10196616-B2
Publication Date
2019-02-05
Expiration Date
2038-02-07
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Abstract
An altered avian NDV that contains the coding sequence of avian interleukin-4 (IL-4), or a portion thereof, in the reverse orientation suppresses in-ovo IL-4 production via RNAi when administered to embryonic birds. An immunogenic composition containing this altered NDV is included in this invention. The altered avian NDV can, optionally contain a polynucleotide encoding a heterologous antigen from a heterologous avian pathogen and can produce said heterologous antigen in-ovo.
Core Innovation
This invention relates to an altered Newcastle Disease Virus (NDV) that contains the coding sequence of avian interleukin-4 (IL-4), or a portion thereof, in the reverse orientation, thereby producing RNA with the reverse complement sequence or dsRNA of IL-4 after infecting an embryonic bird. This RNAi effect suppresses IL-4 production in-ovo, eliciting a stronger and earlier immune response in hatchling chicks against wild-type NDV. The altered NDV optionally encodes a heterologous antigen from a heterologous avian pathogen and produces that antigen in-ovo. An immunogenic composition containing this altered NDV and optionally an adjuvant or pharmaceutically acceptable carrier is also part of the invention.
The problem solved addresses the shortcomings of current Newcastle disease (ND) vaccines and measures, which fail to provide early protection in hatchlings, cannot be administered in-ovo safely, or do not elicit robust cellular and humoral immunity. Existing live NDV vaccines are lethal to embryos and cause respiratory disease post-hatch, while inactivated vaccines fail to induce strong cell-mediated immunity. In-ovo vaccines available either require long periods to induce protection or show inconsistent efficacy. There is a need for an altered NDV and corresponding immunogenic composition that can be administered safely in-ovo, induces early and strong immune responses reducing viral shedding and transmission, overcomes maternal antibody inhibition, and does so without harming bird health or productivity.
This invention uses RNA interference to reduce IL-4 production in embryonic birds by expressing an anti-sense or double-stranded RNA of IL-4 within the NDV genome, enhancing the immune response against NDV and possibly other pathogens via inclusion of heterologous antigens. The altered NDV can be attenuated and may be based on different NDV strains, including lentogenic and attenuated velogenic strains. Methods of administering the altered NDV in-ovo and/or post-hatch are described, with demonstrated improvement in survival rates, immune responses, and reduced viral shedding.
Claims Coverage
The patent includes eighteen independent inventive features addressing plasmids encoding altered NDV, altered NDV compositions, immunogenic compositions, and methods of protecting and increasing survival rates in hatchling chicks via in-ovo administration.
Plasmid encoding altered NDV with IL-4 reverse complementary sequence
A plasmid encoding an altered NDV comprising an NDV sequence and a first polynucleotide encoding a reverse, complementary sequence of IL-4 or fragment thereof, enabling the altered NDV to produce anti-sense RNA of IL-4 or fragment thereof.
Plasmid with specific IL-4 or reverse complementary sequences
The plasmid comprises IL-4 sequences including SEQ ID NO: 1 or reverse complementary sequences selected from SEQ ID NOs: 2, 3, 5, or 23.
Plasmid producing dsRNA for IL-4
The plasmid further comprises a second polynucleotide encoding IL-4 or fragment thereof complementary to the first, allowing the altered NDV to produce double-stranded RNA of IL-4 or fragment thereof.
Plasmid based on LaSota or attenuated ZJ1 NDV strains
The altered NDV encoded by the plasmid is based on LaSota NDV strain or attenuated ZJ1 NDV strain.
Plasmid encoding altered NDV with specific genome sequences
Plasmid encoding altered NDV genome sequences comprising SEQ ID NO: 17 or SEQ ID NO: 24 that produce anti-sense IL-4 RNA.
Altered NDV producing anti-sense IL-4 RNA
An altered NDV comprising RNA encoding a reverse, complementary sequence of anti-sense RNA of IL-4 or fragment thereof, producing said anti-sense IL-4 RNA or fragment thereof.
Altered NDV producing dsRNA of IL-4
The altered NDV further encodes a second RNA complementary to the first, producing dsRNA of IL-4 or fragment thereof.
Altered NDV producing heterologous antigen
The altered NDV further comprises RNA encoding a reverse, complementary sequence of a heterologous antigen from a heterologous avian pathogen, thereby producing that heterologous antigen.
Altered NDV with infectious laryngotracheitis virus (ILTV) antigen
Wherein the heterologous avian pathogen is ILTV, and the produced heterologous antigen is gB, gD, or both proteins from ILTV.
Altered NDV with specific RNA equivalent genomic sequences
The altered NDV genome comprises a reverse, complementary RNA equivalent sequence of SEQ ID NO: 18 or SEQ ID NO: 25.
Immunogenic composition containing altered NDV
An immunogenic composition comprising the altered NDV producing anti-sense RNA of IL-4 or fragment thereof and a pharmaceutically acceptable carrier.
Immunogenic composition with adjuvant
The immunogenic composition further comprises an adjuvant.
Immunogenic composition with heterologous antigen
The altered NDV in the immunogenic composition further encodes RNA for a heterologous antigen from a heterologous avian pathogen and produces that antigen.
Method of protecting in-ovo inoculated hatchlings
A method comprising in-ovo administration of an effective dosage of the immunogenic composition to an embryonic bird and incubation to hatch, wherein the altered NDV produces antigen and anti-sense IL-4 RNA that reduces IL-4 production and causes protective antibodies in the hatchling.
Reduction of IL-4 production with specific RNA sequences
In the method, the anti-sense RNA of IL-4 comprises RNA equivalent sequences selected from SEQ ID NO: 2, 3, 5, 23, or complementary to SEQ ID NO: 1.
Method to increase survival rate after avian pathogen exposure
Method of increasing hatchling survival after exposure by in-ovo inoculation with the immunogenic composition producing antigen and anti-sense IL-4 RNA reducing IL-4 production, resulting in antibody protection and increased survival compared to non-inoculated birds.
Survival increase method with specific RNA sequences
The anti-sense RNA used comprises RNA equivalent sequences selected from SEQ ID NO: 2, 3, 5, 23, or complementary to SEQ ID NO: 1.
The independent claims cover plasmids encoding altered NDV with reverse complementary IL-4 sequences producing anti-sense or double-stranded IL-4 RNA, altered NDVs producing these RNAs and optional heterologous antigens, immunogenic compositions containing such altered NDVs and carriers/adjuvants, and methods of in-ovo administration conferring protection and increased survival in hatchling birds against avian pathogens.
Stated Advantages
The altered NDV safely administered in-ovo induces a stronger and earlier immune response against wild-type NDV in hatchling chicks.
It overcomes maternal antibody inhibition and provides protection at early stages of life.
It induces both strong cell-mediated and humoral immune responses for effective viral clearance and reduced viral shedding and transmission.
The altered NDV does not cause mortality in embryos or hatchlings and does not negatively impact bird growth or productivity.
Compared to prior live NDV vaccines, this altered NDV is safer for in-ovo use and generates protective immunity faster than recombinant herpes virus vaccines.
Documented Applications
In-ovo vaccination of embryonic birds with altered NDV to confer early protection against Newcastle Disease Virus (NDV).
Immunization of hatchling chicks to produce antibodies against NDV and optional heterologous avian pathogens for disease protection and reduced viral shedding.
Use of immunogenic compositions containing altered NDV and optionally heterologous antigens and adjuvants for vaccination against avian diseases.
Method for increasing survival rates in hatchling chicks after exposure to avian pathogens through in-ovo administration of altered NDV or immunogenic compositions.
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