Patent Application
20190358316
The disclosure generally relates to vaccines. More particularly the disclosure generally relates to modified paramyxoviruses such as recombinant Newcastle disease viruses (rNDVs) expressing proteins of infectious bronchitis virus (IBV) using reverse genetics to form multivalent vaccines.
Infectious Bronchitis (IB) is an acute and highly contagious viral respiratory disease of chickens1,2. IB causes major economic losses in commercial chickens throughout the world1,3. It is one of the most prevalent diseases in the poultry industry. The disease is usually characterized by respiratory signs including gasping, coughing and sneezing. However, the virus can also infect urogenital and reproductive tracts causing renal dysfunction and decreased egg production3,4.
IB is caused by infectious bronchitis virus (IBV), a member of the family Coronaviridae. The genome of IBV is a single stranded, positive-sense RNA of approximately 27.6 kb in length. It encodes a large polyprotein containing spike (S), small envelope (E), membrane (M), nucleocapsid (N) and 15 non-structural proteins. The S glycoprotein is the major antigen against which neutralizing and protective antibodies are produced. The S protein is cleaved into S1 and S2 subunits post translationally4. The S1 subunit is responsible for viral attachment to host cell and contains major neutralizing epitopes. The S2 subunit is highly conserved among IBV strains and contributes to viral fusion activity and elicits some minor but broadly reactive neutralizing antibodies5-9.
Currently IB in commercial chickens is controlled by the use of live attenuated and inactivated IBV vaccines. However, current live attenuated vaccine strains are not genetically stable and frequently undergo reversion to virulence10,11. Furthermore, circulation of live-attenuated viruses in the environment provides a setting in which the viral population may undergo mutations and recombination leading to creation of variant viruses12,13. Sequence analysis of pathogenic IBV strains circulating in the field has found live vaccines as one of the sources of outbreak14. Moreover, live IBV vaccines may causes pathological lesions or secondary bacterial infections in 1-day-old vaccinated chicks15,16. Inactivated IBV vaccines, which usually are administered by injection to layers and breeders at 13 to 18 weeks of age are not an alternative for live-attenuated IBV vaccines. Inactivated vaccines of IBV do not elicit strong immune responses in chickens against circulating virulent IBV strains17,18. The production and administration of inactivated vaccines are also time consuming and costly2,10. Therefore, there is a great need to develop an alternative IBV vaccine. The present disclosure is pertinent to this need.
The present disclosure provides a demonstration of using reverse genetics to generate recombinant Newcastle disease viruses (rNDVs) expressing S1, S2 and S proteins of IBV strain Mass-41. The protective efficacies of rNDVs expressing S1, S2 or S protein of IBV were compared in chickens against a virulent IBV strain Mass-41 challenge. The results demonstrate that including the whole S protein is superior to including either S1 or S2 separately as a protective antigen of IBV. The data also demonstrate that rNDV strain LaSota expressing the S protein of IBV strain Mass-41 protects chickens against both virulent IBV and virulent NDV challenges, thereby demonstrating a bivalent vaccine approach. Thus, in embodiments the disclosure provides a method comprising administering an immunologically effective amount of rNDV particles to avian animals to stimulate a protective immune response against IBV, wherein the rNDV particles comprises a contiguous segment of IBV S protein that spans an IBV cleavage site between IBV S1 and IBV S2 proteins. In an embodiment the contiguous segment of IBV S protein comprises a full length IBV S protein. In certain implementations, a protective immune response stimulated by compositions and methods of this disclosure is greater than a reference value obtained from administration of rNDV particles that comprise only an intact S1 or an intact S2 IBV protein. The protective immune response can be determined by a variety of measures, such as by using a severity score for respiratory clinical signs of IBV infection. In certain embodiments, the protective immune response is serologically distinguishable from an immune response to infection by unmodified IBV. In certain approaches, RNA encoding the IBV S protein is codon optimized for expression of the IBV S protein in chicken cells. Such optimization for translated RNA can be configured by engineering the viral genome to result in codon-optimized mRNA that can be optimized for expression in, for example, chicken cells.
Due to the multivalent characteristic of the rNDV particles, the rNDV particles also stimulate a protective immune response against NDV infection. The diversity of the immune response can be expanded by providing IBV S protein as a chimeric protein that further comprises at least a second polypeptide sequence from a pathogen that is not NDV or IBV. This configuration forms at least a multivalent immunogenic agent, wherein the second polypeptide stimulates a protective immune response to the pathogen that is not NDV or IBV. Accordingly, bivalent, trivalent, and higher numbers of distinct immunogenic determinants can be comprised by the rNDV particles of this disclosure.
In certain approaches the rNDV particles are administered to an avian animal that is an embryo, a fledgling, or an adult avian animal. In embodiments, the avian animal is a chicken, such as Gallus gallus. In embodiments, populations or sub-populations of avian animals are vaccinated to promote, for example, herd immunity.
The disclosure includes a plurality of isolated rNDV particles, and compositions comprising them, such as in a vaccine formulation, which may comprise an adjuvant. The particles may also be present in, for example, an avian embryo. In embodiments, the rNDV particles can be characterized by having a segment of the RNA genome that enables production of the S protein is not mutated over at least five avian embryo passages.
For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.
Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.
Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.
Ranges of values are disclosed herein. Unless otherwise stated, the ranges include all values to the magnitude of the smallest value (either lower limit value or upper limit value) and ranges between the values of the stated range.
The present disclosure relates to modified viral vectors to control infectious bronchitis (IB). Development of viral vectored vaccines to control IB according to this disclosure is not expected to lead to creation of variant viruses, which is a major drawback of current live attenuated IBV vaccines. The present disclosure demonstrates making and using representative modified vectors using Newcastle disease virus (NDV). NDV and several other viruses, such as herpesvirus, fowl pox virus and adenovirus have been evaluated as vaccine vectors for IBV19-23. However, data presented herein unexpectedly demonstrate that although the S1 and S2 proteins of IBV are known to contain virus neutralizing epitopes, the presence of the entire S protein provides a strong protective immune response. Thus, the disclosure provides recombinant NDV (rNDV) vectors that allow for incorporation of intact IBV S protein into rNDV particles.
NDV is an attractive vaccine vector for IBV, because it can be used as a multivalent vaccine. Further, and without intending to be constrained by any particular theory, it is considered that a recombinant NDV vector IBV vaccine of this disclosure will eliminate the emergence of vaccine-derived IBV resulting from mutations and recombination as previously reported12,13. The protective efficacy of NDV-vectored IBV vaccine can be enhanced by employing an appropriate regimen of prime-boost immunization strategy.
NDV belongs to the genus Avulavirus in the family Paramyxoviridae. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. The genus Avulavirus includes at least 13 serotypes of Avian Avulaviruses (AAvV). All strains of NDV belong to Avulavirus-type 1(AAvV-1), but the other serotypes can also be used as modified or unmodified vaccine vectors. The disclosure includes use of other such modified or unmodified avulaviruses to achieve multivalent immunizations that include an immune response to IBV.
With respect to NDV, virulent NDV strains cause a fatal neurological disease in chickens. NDV strain LaSota has been used as a safe and effective live vaccine for more than 60 years24. Recombinant NDV (rNDV) strain LaSota has been evaluated as a vaccine vector against several avian pathogens including IB V25-29. In one study, rNDV expressing S2 protein of IBV was found to induce partial protection against a virulent IBV challenge22. In another study, it was shown that a rNDV expressing S1 protein of IBV resulted in inducing protective immunity against virulent IBV challenge23. However, the present disclosure demonstrates superiority of using an rNDV that includes larger segment of the intact S protein that at a minimum spans the S1/S2 cleavage site, as described further below.
The disclosure includes all polynucleotide and amino acid sequences described herein, and every polynucleotide sequence referred to herein includes its complementary DNA sequence, and also includes the RNA equivalents thereof to the extent an RNA sequence is not given. Every DNA and RNA sequence disclosed herein is encompassed by this disclosure, including but not limited to sequences encoding all viral and recombinant proteins that comprise a segment of an avian paramyxovirus protein and an infectious bronchitis virus S protein, as described further below. Where a DNA sequence is provided it may be a cDNA sequence of a viral negative sense genome; the skilled artisan can from the cDNA sequence readily envision the negative sense strand in its RNA form. The disclosure includes all negative strand viral genome sequences, and all complementary (cRNA) sequences thereof. In embodiments, the RNA sequences can comprise non-templated G residues arising from RNA editing. In embodiments, the disclosure includes viral particles that comprise an engineered paramyxovirus antigenome.
All of the amino acid sequences and nucleotide sequences associated with accession numbers disclosed herein are incorporated herein by reference as they exist in the database as of the effective date of the filing of this application or patent. Representative and non-limiting examples of complete NDV LaSota cDNA sequence and the amino acid sequences of proteins included in NDV viral particles are available under NCBI accession no. AF077761.1. The amino acid of the NDVfusion protein (F) of NDV strain LaSota (AFo77761.1) is also described below.
Thus, the amino acid and nucleotide sequences of a variety of strains of Newcastle and IBV viruses are known in the art, and it is contemplated that the segments of such proteins as described herein can be used and/or modified for use with embodiments of this disclosure. In certain embodiments, a protein or segment thereof of this disclosure may differ from a reference sequence. Thus, in certain examples the disclosure comprises a modified segment of a viral protein that comprises at least one amino acid change relative to the unmodified (wild type) counterpart. In certain examples more than one amino acid change can be included. Such changes can comprise conservative or non-conservative amino acid substitutions, insertions, and deletions, provided the modified sequence retains or improves on the capability to be used to stimulate an immune response. In embodiments an NDV fusion (F), M or P amino acid sequence, or a combination thereof, as used herein is at least 80%-99% similar to a reference sequence. Likewise, in embodiments, an IBV S amino acid sequence used herein is at least 80%-99% similar to a reference sequence. As will be apparent from the description herein and figures of this disclosure, in embodiments, the viral genome comprises the NDV NP, P, M, F, HN and L genes, and include the IBV S gene inserted between the P and M proteins, although other insertion sites are contemplated.
In embodiment, the S protein that is incorporated into the rNDVs of this disclosure comprises a contiguous segment of IBV S protein that spans an IBV cleavage site between IBV S1 and IBV S2 proteins. In one embodiment, the amino acid sequences of an IBV S1 proteins comprises a furin consensus motif of RRFRR (AY851295.1; SEQ ID NO:3 for infectious bronchitis virus strain Mass 41, as present in the spike glycoprotein). In an embodiment the cleavage site comprises RRFRRS SEQ ID NO:4). The contiguous segment of IBV S protein that contains the S1/S2 cleavage site is of an adequate length such that sufficient epitopes of the S protein that can elicit a protective immune response against IBV are included. In embodiments, the segment comprises between 500-1000 amino acids of the S protein, inclusive, including all integers and ranges of integers there between. In embodiments, the segment comprises amino acids 532-538 of representative IBV protein provided in the spike glycoprotein under AY851295.1 within a contiguous segment of between 500-1000 amino acids of AY851295.1. In embodiments, the S protein component of the N-terminal domain (rNTDs) of this disclosure have at from 80%-100% sequence identity to any such segment of AY851295.1. In embodiments, the S protein component of the rNTDs of this disclosure has at least 95% identity across the entire length of the S protein amino acid sequence of spike glycoprotein under AY851295.1 In certain non-limiting embodiments, the disclosure includes using one or more of the NTD proteins or the IBV S protein as a chimeric protein that further comprises at least a second polypeptide sequence from a pathogen that is not NDV or IBV, thereby forming at least a multivalent immunogenic agent, and wherein the second polypeptide stimulates a protective immune response to the pathogen that is not NDV or IBV. Non-limiting examples of sources of the second polypeptide (or more than a second polypeptide, such as a third or fourth polypeptide) include antigens from any avian pathogen, including internal and external pathogens. Thus, in embodiments, the second polypeptide can be derived from any non-NDV and non-IBV virus or other pathogenic agent that causes any disease of poultry, including by not limited to: viral inclusion body hepatitis, haemorrhagic enteritis of turkeys, egg drop syndrome, adenovirus group 1—associated infections, infectious bursal disease (gumboro), laryngotracheitis, swollen head syndrome, infectious encephalomyelitis, leucosis, Marek's disease, and fowl pox and reovirus infections. Specific pathogens that can be a source of the second polypeptide include Escherichia coli, Salmonella Pullorum/Gallinarum, Pasteurella multocida, Avibacterium paragallinarum, Gallibacterium anatis, Ornitobacterium rhinotracheale, Bordetella avium, Clostridium perfringens, Mycoplasma spp., Erysipelothrix rhusiopathiae, and Riemerella anatipestifer. In embodiments, the second polypeptide is present in a chimeric protein that includes the IBV S protein, or is present in an NDV protein. In embodiments, the second protein is a complete protein from a non-IBV and non-NDV protein, or is an immunogenic fragment of such a protein. In embodiments, the second polypeptide is a component a foreign protein is from 10-500 amino acids in length, inclusive, and including all integers and ranges of integers there between.
To produce the viral particles, the viral particles themselves, or DNA/cDNA or RNA or cRNA encoding the required set of proteins can be introduced directly into producer cells, and shed viral particles can be isolated from the cells. In embodiments, one or more expression vectors can be used to produce the viral particles. In this regard, a variety of suitable expression vectors known in the art can be adapted to produce the modified paramyxovirus particles of this disclosure. In general, the expression vector comprises sequences that are operatively linked with the sequences encoding the viral particle proteins that comprise an IBV S gene between the NDV P and M genes, or another suitable insertion site. A particular polynucleotide sequences is operatively-linked when it is placed in a functional relationship with another polynucleotide sequence. For instance, a promoter is operatively-linked to a coding sequence if the promoter affects transcription or expression of the coding sequence. Generally, operatively-linked means that the linked sequences are contiguous and, where necessary to join two protein coding regions, both contiguous and in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operatively-linked even at a distance, i.e., even if not contiguous, and may even be provided in trans. Promoters present in expression vectors that are used in the present disclosure may be endogenous or heterologous to the host cells, and may be constitutive or inducible. Expression vectors can also include other elements that are known to those skilled in the art for propagation, such as transcription and translational initiation regulatory sequences operatively-linked to the polypeptide encoding segment. Suitable expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, an enhancer and other regulatory and/or functional elements, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences, as well as a wide variety of selectable markers.
In certain embodiments, the viral particles can be produced using a set of plasmids, which may be used in conjunction with a cDNA, such as an antigenome of an NDV that is modified to include a polynucleotide sequence that encodes (or can be transcribed to encode) an IBV S protein, the coding sequence for which can be placed between the NDV P and M genes. In embodiments, a full length cDNA of NDV can be co-transfected into suitable cells with one or more plasmids that express, for example, the N, P and L genes of NDV so that recombinant NDV particles can be produced by the cells, and recovered. In this regard, the expression vectors can be introduced into the host producer cells by any method known in the art. These methods vary depending upon the type of cellular host, and include but are not limited to transfection employing cationic liposomes, electroporation, calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances as will be apparent to the skilled artisan. In embodiments the viral particles are produced using chicken embryo fibroblast cells, such as DF-1 cells, or mammalian cells, such as HEp-2 cells. In embodiments, the rNDVs are produced in embryonated, specific-pathogen-free (SPF) eggs. Methods of making the modified virus particles are accordingly included, and generally comprise introducing polynucleotides encoding the viral genome and viral proteins virus into suitable producer cells, and recovering shed virus from them. Thus, cells and cell cultures that harbor polynucleotides encoding the modified paramyxoviruses of this disclosure are included, as are isolated and/or purified modified paramyxovirus viral particle preparations. The particles can be purified to any desired degree of purity using standard approaches, such as density gradient separation or commercially available kits used to purify enveloped viruses or exosomes.
In certain embodiments, the disclosure provides a modified antigenomic RNA of Newcastle disease virus, comprising or consisting of: NDV NP gene, NDV P gene, IBV S gene, NDV M gene, NDV F gene, NDV HN gene and NDV L gene, which can be in this order from a 3′ to 5′ direction. In embodiments, the disclosure includes making such modified RNAs and rNDV particles by adapting approaches described in U.S. Pat. Nos. 9,476,033 and 7,244,558, the entire disclosures of which are incorporated herein by reference. In embodiments, the disclosure includes making and using a cDNA encoding all or a segment of the antigenome of any rNDV disclosed herein.
In certain aspects the disclosure includes a pharmaceutical formulation comprising modified paramyxovirus particles as described herein. The form of pharmaceutical preparation is not particularly limited, but generally comprises modified viral particles and at least one inactive ingredient. In certain embodiments suitable pharmaceutical compositions can be prepared by mixing any one type of the particles, or combination of distinct types of particles, with a pharmaceutically-acceptable carrier, diluent or excipient, or immune response regulator, or an antibiotic, and suitable such components are well known in the art. Some examples of such carriers, diluents and excipients can be found in: Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins, the disclosure of which is incorporated herein by reference. In embodiments, a vaccine formulation is provided. In embodiments, a formulation of this disclosure is provided as an effervescent tablet, a pellet, or in a lyophilized form. The pharmaceutical composition can be also include, for example, any suitable adjuvant.
In embodiments, the rNDV particles are administered to avian animals using any suitable route. In embodiments, the particles are provided as vaccines and can be administered orally, intranasally, intraocularly or parenterally, e.g. by intramuscular or subcutaneous injection. In embodiments, the vaccine formulations are administered via an oculanasal route, thus conjunctival and intranasal routes are included. In embodiments, the vaccine is administered in drinking water, or as an aerosol or a spray. In embodiments, a vaccine formulation of this disclosure is administered in ovo, as an eye drop, or injection, such as a subcutaneous or wing-web injection. In embodiments, the disclosure includes an article of manufacture, such as a kit, the kit comprising rNTDs as described herein in any suitable form, wherein the rNTDs are comprised within one or more containers, and wherein the article or kit comprises printed material, such as a label or insert that includes instructions for using the rNTDs for vaccination of avian animals.
In embodiments, immunologically effective amount rNDV particles are administered. Immunologically effective as used herein means an amount that results in production of neutralizing antibodies against IBV, and/or an results in an improved severity score for respiratory clinical signs of IBV infection, and/or results in reduced shedding of the challenge virus, i.e., shedding of an infectious IBV virus, the avian animal encounters in its environment. An amount of shedding can be determined according to numerous approaches known to those skilled in the art. Likewise, respiratory clinical signs of IBV infection and scoring systems for quantitating or quantifying the clinical signs are known. Such signs include but are not necessarily limited to: young avian animals, such as chickens, are depressed and huddle under a heat source; respiratory signs that include but are not limited to gasping, coughing, tracheal rales and nasal and ocular discharge; birds in lay exhibit a marked drop in egg production and an increased number of poor quality eggs may be produced; the external and internal quality of the eggs can be affected and result in soft-shelled or misshapen eggs misshapen, and/or eggs with an abnormal water content; hatchability rate of the eggs can also be affected. In an embodiment the disclosure comprises determining a severity score of clinical signs of IBV infection that include nasal discharge, ocular discharge and difficulty in breathing. In embodiment, the scoring system comprises the following values: 0=normal, to 3=heavy ocular discharge and heavy nasal discharge with mouth breathing. When avian kidneys are affected, increased water intake, depression, scouring and wet litter may be observed. Any measure of immunologic efficacy of this disclosure can be compared to any suitable reference, examples of which include but are not limited a standardized curve, a titration, an area under a curve, or a comparison to any matched or otherwise suitable control.
In embodiments, an immunologically effective amount results in preventing and/or lessening of clinical disease and/or mortality when challenged with a virulent NDV. Immunological protection can be durable, and last for days, weeks or months, or longer, after vaccination, and such vaccinations can be effective to elicit such protection after a single dose, or multiple doses. Booster vaccinations can be used according to schedules that are known in the art and can be adapted for use with vaccines of this disclosure when provided the benefit of this specification, and include such approaches as a Prime-Boost strategy.
In embodiments, neutralizing antibodies are produced. The term “neutralizing antibody” refers to an antibody or a plurality of antibodies that inhibits, reduces or completely prevents viral infection. Whether neutralizing antibodies are produced can be determined by in vitro assays that are known in the art.
In embodiments, viral load in the vaccinated animals is reduced. Viral load can be determined according to methods known to those skilled in the art, including but not limited to observation of IBV specific lesions on chicken embryo, and PCR amplifications, including but not limited to quantitative real-time polymerase chain reaction assays. In embodiments, viral RNA is determined from a tracheal swab sample.
In embodiments, an embryo infective dose (EID) is used. In embodiments, a dosage of mean embryo infectious vaccine dose (EID50) of 104-106 is used. In embodiments, a dose of at least 106 EID50 is used. In embodiments, any suitable multiplicity of infection (MOI) can be used.
In embodiments, an rNDV is modified such that it is less pathogenic than an un-modified NDV, and thus may comprise or be derived from an avirulent rNDV, or it may be provided as an attenuated or inactivated vaccine. In embodiments, an rNDV for use in vaccines of this disclosure are derived from NDV strains from the class II genotype I (i.e. 12, V4, and PHY-LMV42). In embodiments, an rNDV that is resistant to heat is used. In embodiments, the rNDV is derived from a LaSota, B1, or VG/GA type NDV.
In embodiments, the avian animals to which compositions of this disclosure are administered are any type of poultry. In embodiments, the avian animals are Galliformes and thus include any members of the order of heavy-bodied ground-feeding birds that includes turkey, grouse, chicken, New World quail and Old World quail, ptarmigan, partridge, pheasant, junglefowl and the Cracidae. In embodiments, the avian animals are domesticated fowl, including but not limited to domesticated chickens and turkeys. In embodiments, the chickens are Gallus gallus, such as Gallus gallus domesticus. In embodiments, the chickens are roosters or hens. In embodiments, the avian animals are adults, juveniles, or embryos. In an embodiment, a composition of this disclosure is applied to eggs. In embodiments, vaccines of this disclosure administered to a population of avian animals, i.e., a flock. In embodiments, from 50-85% or more members of the flock are vaccinated to achieve, for example, herd or flock immunity.
The Examples that follow demonstrate generation of recombinant rNDVs expressing the S1, S2 and S proteins of IBV using reverse genetics technology. The results showed that the rNDV expressing the S protein of IBV provided better protection than the rNDV expressing S1 or S2 protein of IBV, indicating that the S protein is the better protective antigen of IBV. Immunization of 4-week-old SPF chickens with the rNDV expressing S protein elicited IBV-specific neutralizing antibodies and provided complete protection against virulent IBV and virulent NDV challenges. These results indicate that the rNDV expressing the S protein of IBV is a safe and effective bivalent vaccine candidate for both IBV and NDV. The Examples are presented to illustrate the present disclosure. They are not intended to limiting in any matter.
Generation of rNDVs expressing S1, S2 and S protein of IBV. The expression cassettes containing the codon optimized S1, S2, S and non-codon optimized S genes of IBV were cloned into the cDNA encoding the complete antigenome of NDV strain LaSota, using the PmeI site, between P and M genes (
Evaluation of the expression of the S1, S2 or S protein of IBV.
The expression of codon optimized S2, and S proteins and non-codon optimized S protein of IBV strain Mass-41 by rNDV constructs was detected by Western blot analysis of DF-1 using a chicken polyclonal anti IBV serum (
The expression of codon optimized S1 protein expressed from four individual rNDV constructs were detected by Western blot analysis in lysates of infected DF-1 cells, using a chicken polyclonal anti IBV serum (
Growth Characteristics of rNDV Constructs.
The recovered rNDVs were passaged four times in 9-day-old embryonated SPF chicken eggs. All the viruses were able to replicate well in eggs (>28 HAU/ml). The multicycle growth kinetics of rNDV/S1 (cs+)+NDV-F-TM&CT, rNDV expressing codon optimized S protein of IBV and empty rNDV vector were evaluated in the presence of exogenous protease in DF-1 cells (
The protective efficacy of rNDVs expressing S1, S2 or S protein of IBV in chickens against a virulent IBV challenge. IBV protection experiment 1.
To evaluate the protective efficacy of rNDVs expressing S1, S2 or S protein of IBV, SPF chicks were immunized at 1-day-old age with each virus via oculanasal route. At three weeks post-immunization, chickens were challenged with virulent IBV strain Mass-41. The severity scores of IBV clinical signs were recorded twice daily for 10 days post-challenge (
IBV protection experiment 2.
To evaluate the protective efficacy of rNDVs expressing codon optimized S protein of IBV in adult chickens, SPF chickens were immunized at 4-week-old age. The protective efficacy of rNDV expressing codon optimized S gene of IBV was determined by challenging the immunized chickens with OIE recommended dose (1031 EID50) of virulent IBV strain Mass-41 at 3 week post-immunization. The severity scores of IBV clinical signs were recorded twice a day for 10 days post-challenge (
IBV protection experiment 3.
To evaluate the protective efficacy of rNDVs expressing codon optimized S protein of IBV in adult chickens against a higher dose of virulent IBV challenge, SPF chickens were immunized at 4-week-old age. The protective efficacy of rNDV expressing codon optimized S gene of IBV was determined by challenging the immunized chickens with 1047 EID50 virulent IBV strain Mass-41 at 3 week post-immunization. The severity scores of IBV clinical signs were recorded twice a day for 8 days post-challenge (
The protective efficacy of rNDVs against a highly virulent NDV challenge.
To evaluate the protective efficacy of rNDV expressing S gene of IBV against a virulent NDV strain, groups of five 4-week-old chickens were immunized with rNDV, rNDV expressing codon optimized S protein, commercial IBV vaccine and PBS. Three weeks after immunization, chickens were challenged with virulent NDV strain Texas GB in our BSL-3 plus facility. Our results showed that chickens immunized with the rNDV and rNDV expressing codon optimized S gene of IBV were fully protected from highly virulent NDV challenge, while all chickens in commercial IBV vaccine and PBS groups died or showed clinical signs of virulent NDV infection (
Antibodies produced against IBV and NDV.
Hemagglutination inhibition (HI) assay using a standard protocol of OIE was used to assess the level of antibodies mounted against NDV in serum samples of chickens 21 days after immunization. HI titers of NDV was detected in serum samples of all chickens. There was no significant differences observed among HI titers against NDV in serum samples of chickens from groups immunized with rNDV and rNDV expressing S protein (
It will be apparent from the foregoing that this disclosure provides a comparison of the protective efficacies of S1, S2 and S proteins of IBV using rNDV as a vaccine vector. The S1, S2 and S genes of IBV strain Mass-41 were individually inserted between the P and M genes of rNDV strain LaSota. Four different versions of IBV S1 gene were used to identify the version that is expressed at the highest level and incorporated into NDV particles. We were able to recover all the recombinant viruses and their growth characteristics were similar to rLaSota. The recombinant viruses containing IBV S genes grew slightly slowly than their parental virus. The viruses were genetically stable after five passages in SPF chicken embryos. Western blot analysis showed that chicken codon optimized S2 and S proteins were expressed at much higher levels and were incorporated into NDV particles. Whereas, all the four versions of S1 protein were detected at very low levels by Western blot analysis. It is noteworthy that the unmodified S1 protein was detected in the infected cell culture supernatant, indicating that the modification of S1 protein probably caused retention of the protein in the cell. These results suggest that the S2 protein acts as a chaperone to assist in the folding of the S1 protein. The S1 protein is folded incorrectly in the absence of S2 protein and the new structure causes loss of some conformational epitopes for IBV antibodies.
In the first IBV protection experiment, we found that 1-day-old chicks immunized with rNDV expressing the S protein of IBV conferred better protection from disease compared to 1-day-old chicks immunized with rNDVs expressing either S1 or S2 protein of IBV. Our results showed that the S protein, which contains both S1 and S2 proteins, is the best protective antigen of IBV. The S2 protein lacks major neutralizing epitopes which are present in the S1 protein, hence it is not an effective antigen. The S1 protein contains major neutralizing epitopes, but it losses some conformational epitopes when expressed separately. In this disclosure we showed that rNDV expressing S protein provided enhanced protection.
In the second IBV protection experiment, we investigated whether age of immunization influences the outcome of IBV challenge. Our results showed that a single immunization of 4-week-old chickens with rNDV expressing S protein completely protected chickens against IBV challenge based on disease and viral load in tracheas. Indeed, the level of protection conferred by rNDV expressing S protein was similar to that of a commercial IBV vaccine. This showed that protection was greater when the chickens were immunized at an age when their immune system is relatively well developed.
In the third IBV protection experiment, we showed that rNDV expressing IBV S protein protects adult chickens against a higher dose of virulent IBV challenge. However, compared to standard challenge dose of virulent IBV, a higher challenge dose of virulent IBV caused mild disease and tracheal viral load in adult chickens immunized with either rNDV expressing IBV S protein or commercial live attenuated IBV vaccine. Our result showed that although both the age of immunization and dose of challenge virus affect the results of IBV challenge, the influence of the age of immunization is greater than the effect of the dose of challenge virus.
The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in embodiments, the method comprises or consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.
Cells and viruses.
Chicken embryo fibroblast (DF-1) cells and human epidermoid carcinoma (HEp-2) cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). They were grown in Dulbecco's minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). The recombinant avirulent NDV strain LaSota was generated previously in our lab using reverse genetics30. The rNDV and rNDVs expressing chicken codon optimized S1, S2 and S genes and non-codon optimized S gene of IBV strain Mass-41 were grown in 9-day-old embryonated SPF chicken eggs at 37° C. The virulent IBV strain Mass-41 was propagated in 10-day-old SPF embryonated chicken eggs and harvested five days after infection. The titer of virus in harvested allantoic fluid was determined by 50% embryo infectious dose (EID50) method. Briefly, ten-fold serial dilutions of IBV strain Mass-41 was inoculated into 10-day-old embryonated SPF chicken eggs. Seven days after inoculation, infected embryos were examined for IBV specific lesions such as stunting or curling. The titer of virus was calculated using Reed and Muench method31. The modified vaccinia virus strain Ankara expressing T7 RNA polymerase (MVA-T7) was propagated in monolayer primary chicken embryo fibroblast cells.
Generation of rNDVs containing S1, S2 or S gene of IBV.
A plasmid containing full-length antigenomic cDNA of NDV strain LaSota has been constructed previously30. In this study, open reading frames (ORFs) of chicken codon optimized of S2 and S genes, non-codon optimized S gene and four versions of codon optimized S1 gene of IBV strain Mass-41 were constructed in seven individual transcription cassettes. The cassettes contained the following IBV genes; i) codon optimized S1 subunit of S gene (1611 nt), ii) codon optimized S1 gene (1611 nt) fused with N-terminus of transmembrane and cytoplasmic tail of S gene (255 nt), iii) codon optimized S1 subunit of S gene containing S1 protein cleavage site residues (1611 nt) fused with N-terminus of transmembrane and cytoplasmic tail of NDV F gene (171 nt), iv) codon optimized S1 subunit of S gene without S1 protein cleavage site residues (1593 nt) fused with N-terminus of transmembrane and cytoplasmic tail of NDV F gene (171 nt), v) codon optimized S2 subunit of S gene (1878 nt) of IBV fused with C-terminus of signal peptide sequence of S gene (69 nt), vi) codon optimized S gene (3489 nt) and vii) non-codon optimized S gene (3489 nt).
The transcription cassettes were modified to contain PmeI restriction enzyme sequence, 15 nt of untranslated region (UTR) of NDV, NDV gene end (GE) signal, one T nucleotide as intergenic sequence, NDV gene start (GS) signal, extra nucleotides to maintain the rule of six (19, 27), Kozak sequence at the upstream of foreign gene ORFs and PmeI restriction enzyme sequence at downstream of foreign gene ORF. The transcription cassettes were amplified from two plasmids containing commercially synthesized codon optimized and non-codon optimized S genes of IBV strain Mass-41 and cloned into complete antigenomic cDNA of rLaSota at P and M gene junction using PmeI site (
Expression of S1, S2 and S proteins of IBV.
Confluent monolayers of DF-1 cells were infected at a multiplicity of infection (MOI) of 0.01 with rNDV strain LaSota, rNDV/S1, rNDV/S1+IBV-S-TM&CT, rNDV/S1(cs+)+NDV-F-TM&CT, rNDV/S1(cs−)+NDV-F-TM&CT, rNDV/S2, rNDV/codon optimized-S or rNDV/non-codon optimized-S. DF-1 cells were harvested 30 hours post-infection, lysed and analyzed by Western blot. A standard polyclonal chicken anti-IBV serum was used to detect the expression of S1, S2 and S proteins of IBV. To determine the incorporation of IBV proteins into NDV envelope, rNDV, rNDV/S2, rNDV/codon optimized-S and rNDV/non-codon optimized-S were inoculated into 9-day-old embryonated SPF chicken eggs. Three days after incubation, recombinant viral particles from infected allantoic fluids were partially purified by sucrose density gradient centrifugation and analyzed by Western blot analysis.
Growth characteristics of rNDV constructs.
In order to determine the growth kinetics of rNDVs expressing S1, S2 or S protein of IBV, confluent monolayers of DF-1 cells in 6-well tissue culture plates were infected at a MOI of 0.01 with rNDV, rNDV/S1(cs+)+NDV-F-TM&CT, rNDV/S2 or rNDV/codon optimized-S and adsorbed for 90 minutes at 37° C. After adsorption, cells were washed with PBS, then incubated with DMEM containing 2% FBS and 10% fresh SPF chicken egg allantoic fluid at 37° C. in presence of 5% CO2. Aliquots of 200 μL of supernatant from infected cells were collected and replaced with fresh DMEM including FBS at intervals of 8 hours until 56 hours post-infection. The titer of virus in the harvested samples was determined by TCID50 method in DF-1 cells in 96-well tissue culture plates.
The protective efficacy of rNDVs expressing S1, S2 or S protein of IBV against virulent IBV challenge.
Based on the level of expression of S1, S2 and S proteins of IBV from rNDVs, rNDV/S1(cs+)+NDV-F-TM&CT, rNDV/S2, and rNDV/codon optimized-S viruses were selected for animal study to evaluate their protective efficacy against virulent IBV challenge.
IBV protection experiment 1.
In this study, the protective efficacy of rNDVs expressing S1, S2 or S protein of IBV strain Mass-41 were evaluated in 1-day-old specific pathogen free (SPF) chicks. Briefly, a total of eighty 1-day-old chicks were divided into five groups of fifteen each and one group of five. Chicks of the first four groups were inoculated with 107 EID50 of rNDV, rNDV/S1(cs+)+NDV-F-TM&CT, rNDV/S2 and rNDV/codon optimized-S strains via oculonasal route. The fifteen chicks of group five and five chicks of group six were inoculated with PBS. Three weeks after immunization, all immunized chickens, were challenged with 103.1 EID50 of virulent IBV strain Mass-41. This challenge virus dose was determined by an experimental chicken infection study. The severity scores of clinical signs of IBV including, nasal discharge, ocular discharge and difficulty in breathing (0=normal to 3=heavy ocular discharge and heavy nasal discharge with mouth breathing) were recorded twice a day for 10 days post-challenge. In order to evaluate protective efficacy of rNDVs expressing S1, S2 and S genes of IBV in preventing shedding of virulent IBV in immunized chickens, at day five post-challenge, tracheal swab samples were collected from fifteen birds of each group and placed in 2 mL serum free DMEM with 10× antibiotic. The swab samples were analyzed for quantification of viral RNA using an IBV-N gene-specific RT-qPCR.
IBV protection experiment 2.
In this study, the protective efficacy of rNDV expressing codon optimized S protein of IBV was evaluated in 4-week-old SPF chickens against the World Organization for Animal Health (OIE) recommended dose of virulent IBV challenge1. A total of twenty 4-week-old SPF chickens were divided into four groups of five each. Five chickens of groups one and two were inoculated with 107 EID50 of rNDV and rNDV/codon optimized-S, respectively, via oculanasal route. Five chickens of group three were inoculated with recommended dose of a commercial live attenuated Mass-type IBV vaccine via oculanasal route and chickens of group four were inoculated with PBS. Three weeks after immunization, chickens of all groups were challenged with 103.1 EID50 of virulent IBV strain Mass-41 by the oculonasal route. The severity scores of clinical signs of IBV including, nasal discharge, ocular discharge and difficulty in breathing (0=normal to 3=heavy ocular discharge and heavy nasal discharge with mouth breathing) were recorded for 10 days post-challenge. In order to evaluate the efficacy of rNDV expressing S protein of IBV in preventing shedding of virulent IBV in immunized chickens, at day 5 post-challenge, tracheal swab samples were collected from twenty chickens and placed in 2 ml serum free DMEM with 10× antibiotic. The swab samples were analyzed for quantification of viral RNA using an IBV-N gene-specific RT-qPCR.
IBV protection experiment 3.
In this study, the protective efficacy of rNDV expressing codon optimized S protein of IBV was evaluated in 4-week-old SPF chickens against a higher dose of virulent IBV challenge. A total of twenty 4-week-old SPF chickens were divided into four groups of five each. Five chickens of group one and two were inoculated with 107 EID50 of rNDV and rNDV/codon optimized-S, respectively, via oculanasal route. Five chickens of group three were inoculated with recommended dose of a commercial live attenuated Mass-type IBV vaccine via oculanasal route and chickens of group four were inoculated with PBS. Three weeks after immunization, chickens of all groups were challenged with 1047 EID50 of virulent IBV strain Mass-41 by the oculonasal route. The severity scores of clinical signs of IBV including, nasal discharge, ocular discharge and difficulty in breathing (0=normal to 3=heavy ocular discharge and heavy nasal discharge with mouth breathing) were recorded for 10 days post-challenge. In order to evaluate the efficacy of rNDV expressing S protein of IBV in preventing shedding of virulent IBV in immunized chickens, at day 4 post-challenge, tracheal swab samples were collected from twenty chickens and placed in 2 mL serum free DMEM with 10× antibiotic. Each fluid was tested for IBV specific lesions on chicken embryo by inoculation (0.1 ml) of one 10-day-old embryonated SPF chicken egg.
The protective efficacy of rNDV expressing S protein of IBV against virulent NDV challenge.
The protective efficacy of rNDV expressing S protein of IBV strain Mass-41 was evaluated against a virulent NDV strain GB Texas challenge in our biosafety level 3 (BSL-3) plus facility. Briefly, a total of twenty 4-week-old chickens were divided into four groups of five each. Chickens of two groups were inoculated with 107 EID50 of rNDV and rNDV/IBV-codon optimized-S via oculonasal route. The five chickens of group three were inoculated with commercial IBV vaccine. The five chickens of group four were inoculated with PBS. Three weeks after immunization, blood samples of all birds were collected for NDV antibody response analysis and challenged with one hundred 50% chicken lethal dose (CLD50) of the highly virulent NDV strain GB Texas via oculonasal route. The chickens were observed daily for 10 days after challenge for clinical signs of disease and mortality.
Serological analysis.
The level of antibodies induced against NDV and IBV were evaluated. The serum samples were collected three weeks post-immunization. Hemagglutination inhibition (HI) assay using a standard protocol OIE was used to assess the level of antibody titer mounted against NDV in chickens immunized by rNDVs′. The virus neutralization assay according to OIE was used to measure the level of neutralizing antibodies mounted against IBV′. Briefly, serum samples of three birds from the group immunized with rNDV expressing codon optimized S protein of IBV and serum samples of three birds from the group immunized with commercial IBV vaccine group were incubated at 56° C. for 30 minutes. One hundred EID50 of IBV strain Mass-41 was mixed with 2 fold dilutions of antiserum and incubated for 1 hour at 37° C. One hundred μL of each serum and virus mixture was inoculated into three 10-day-old embryonated SPF chicken eggs. To confirm that at least 100 EID50 of virus was inoculated into each egg, three eggs were inoculated with 100 μl of PBS containing 100 EID50 of IBV. Three eggs were inoculated with 100 μL of PBS as negative control. Three eggs were inoculated with a mixture of 100 EID50 of IBV and a dilution of 1:8 of a randomly selected serum sample collected from a bird immunized with rNDV strain LaSota as vector control. The eggs were incubated at 37° C. and were observed daily for dead chicken embryos for 7 days post inoculation. The serum titers were calculated according to the method of Reed and Muench31, based on mortality and IBV specific lesions on chicken embryos.
Quantitative reverse transcription-polymerase chain reaction (RT-qPCR).
RNA was extracted using TRIzol Reagent (Invitrogen) from tracheal swab samples collected from chickens. The first strand cDNA was synthesized using Thermo Scientific RevertAid Reverse Transcriptase (RT). SYBR green RT-qPCR was performed using a specific primer pair set: a) N gene—296 forward primer: 5′ GACCAGCCGCTAACCTGAAT 3′ (SEQ ID NO:5) and b) N gene—445 reverse primer: 5′ GTCCTCCGTCTGAAAACCGT 3′ (SEQ ID NO:6) amplifying 150 nt of N gene of IBV strain Mass-41. PCRs were performed using a Bio-Rad CFX96 Cycler. Each 20 μl reaction was carried out using 5 μl of cDNA, 10 μl of iTaq Universal SYBR Green Supermix (Bio-Rad), 2 μl of forward and reverse primers and 3 μl of nuclease free water. Forty cycles of PCR at 95° C. for 10 s (denaturation), 55° C. for 20 s (annealing), and 72° C. for 30 s (elongation) followed by melting curve analysis that consisted of 95° C. for 5 s and 65° C. for 60 s. A serial 10 fold dilution of cDNA synthesized from extracted RNA of allantoic fluid stock of a virulent IBV strain Mass-41 with 1075 EID50/ml was used to establish the standard curve. The cDNA synthesized from extracted RNA of allantoic fluid stock of a virulent IBV strain Mass-41 and the cDNA synthesized from extracted RNA of swab sample solution were served as positive and negative controls, respectively. Melting point analysis was used to confirm the specificity of the test.
Statistical analysis.
Data were analyzed among groups by One-Way-ANOVA test. The student t-test was used to compare two groups. To avoid bias, all animal experiments were designed as blinded studies.
The following sequences are provided and are used to represent distinct but not limiting embodiments of this disclosure.
In the figures and text of this disclosure the following abbreviations are used: “SP” is the “signal peptide” of S protein of IBV which is involved in secretion of the protein. “GE” (gene end) and “GS” (gene start) are approximately ten conserved sequences, which are transcriptional signals of NDV used to express any foreign gene by NDV polymerase. These sequences are not translated. Before the initiating Met in certain vectors and protein sequences described herein, the expression cassette includes: a PmeI restriction enzyme site sequence (GTTTAAAC), 15 nt of UTR of NDV P gene (tagctacatttaaga SEQ ID NO:7; AF077761.1), gene end (GE) signal of NDV P gene (TTAAGAAAAAA SEQ ID NO:8; AF077761.1), one “t” nucleotide as intergenic sequence (IG), gene start(GS) signal of NDV M gene (ACGGGTAGAA SEQ ID NO:9; AF077761.1), nucleotides for maintaining rule of six (preferable for some expression cassettes), and a Kozak sequence (gccacc).
“TM” is the transmembrane and “CT” is the cytoplasmic tail of F protein of NDV or S protein of IBV, which is exchanged with the corresponding sequences of the foreign protein for incorporation into NDV envelope. “Foreign ORF” is the open reading frame of any foreign gene, which in the embodiments demonstrated herein are the S1, S2 and S gene. The DNA sequences presented below are cDNA sequences of rNDV genome and IBV genes RNA sequences. The “insertion site” is the site in the NDV genome in which the foreign gene (S1, S2 or S as demonstrated herein). In the examples provided herein the insertion site is between the NVD P and M genes.
MLVTPLLLVTLLCVLCSAALYDSSSYVYYYQSAFRPPNGWHLHGGAYAVV
NISSESNNAGSSPGCIVGTIHGGRVVNASSIAMTAPSSGMAWSSSQFCTA
HCNFSDTTVFVTHCYKYDGCPITGMLQKNFLRYSAMKNGQLFYNLTVSVA
KYPTFKSFQCVNNLTSVYLNGDLVYTSNETTDVTSAGVYFKAGGPITYKV
MREVKALAYFVNGTAQDVILCDGSPRGLLACQYNTGNFSDGFYPFINSSL
VKQKFIVYRENSVNTTFTLHNFTFHNETGANPNPSGVQNIQTYQTQTAQS
GYYNFNFSFLSSFVYKESNFMYGSYHPSCNFRLETINNGLWFNSLSVSIA
YGPLQGGCKQSVFSGRATCCYAYSYGGPSLCKGVYSGELDLNFECGLLVY
VTKSGGSRIQTATEPPVITRHNYNNITLNTCVDYNIYGRTGQGFITNVTD
SAVSYNYLADAGLAILDTSGSIDIFVVQGEYGLTYYKVNPCEDVNQQFVV
SGGKLVGILTSRNETGSQLLENQFYIKITNG
SITENVANCPYVSYGKFCIKPDGSIATIVPKQLEQFVAPLLNVTENVLIP
NSFNLTVTDEYIQTRMDKVQINCLQYVCGNSLDCRDLFQQYGPVCDNILS
VVNSIGQKEDMELLNFYSSTKPAGFNTPFLSNVSTGEFNISLLLTTPSSP
RRRSFIEDLLFTSVESVGLPTDDAYKNCTAGPLGFLKDLACAREYNGLLV
LPPIITAEMQTLYTSSLVASMAFGGITAAGAIPFATQLQARINHLGITQS
LLLKNQEKIAASENKAIGRMQEGFRSTSLALQQIQDVVNKQSAILTETMA
SLNKNFGAISSVIQEIYQQLDAIQANAQVDRLITGRLSSLSVLASAKQAE
HIRVSQQRELATQKINECVKSQSIRYSFCGNGRHVLTIPQNAPNGIVFIH
FSYTPDSFVNVTAIVGFCVKPANASQYAIVPANGRGIFIQVNGSYYITAR
DMYMPRAITAGDIVTLTSCQANYVSVNKTVITTFVDNDDFDENDELSKWW
NDTKHELPDFDKFNYTVPILDIDSEIDRIQGVIQGLNDSLIDLEKLSILK
TYIKWPWYVWLAIAFATIIFILILGWVFFMTGC
CGCCCGCFGIMPLMSKCGKKSSYYTTFDNDVVTEQNRPKKSV.
In the following DNA sequences the italicized sequences are the transmembrane (TM) and cytoplasmic tail (CT) sequences. Lower case letters indicate insertion sites. Each sequence is flanked at its ends by a PmeI restriction site. Where indicated in lower case letters the UTR of NDV P gene (tagctacatttaaga) (SEQ ID NO:7) is shown. Also shown in lower case where indicated are Kozak sequence (gccacc). Initiation codons are enlarged.
CTGGTGACTCCACTGCTGCT
CTG
CATTAAATGGCCTTGGTACGTGTGGCTGGCCATCGCTTTTGCAACCATCATTTTCATCCTGATTCTGGGATGGGTGTTCTTT
ATGACAGGGTGCTGCGGCTGCTGCTGCGGATGCTTCGGGATTATGCCACTGATGAGCAAGTGCGGGAAGAAATCCAGCTACT
ATACAACCTTTGACAATGATGTGGTGACAGAGCAGAATCGCCCTAAGAAATCCGTGTGAGTTTAAAC
C
actatcatatctcttgtttttggtatacttagcctgattctagcatgctacctaatgtacaagcaaaaggcgcaacaaaaga
ccttattatggcttgggaataatactctagatcagatgagagccactacaaaaatgtgaGTTTAAAC
CTG
tggtatacttagcctgattctagcatgctacctaatgtacaagcaaaaggcgcaacaaaagaccttattatggcttgggaat
aatactctagatcagatgagagccactacaaaaatgtgaGTTTAAAC
ATCACCGAGAACGTGG
Science. 337, 188(2012).
Although the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.
This application claims priority to U.S. Provisional Application No. 62/455,290, filed on Feb. 6, 2017, the disclosure of which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US18/17029 | 2/6/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62455290 | Feb 2017 | US |
