# Patent

## Title
In Ovo vaccine against infectious bursal disease

## Abstract

The present invention relates to a non pathogenic vaccine comprising a recombinant Infectious Bursal Disease virus that includes a recombinant Segment A, designated as rD78GLSNSΔ, that includes sequences from D78 and GLS strains and wherein the NS protein is not expressed.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
The subject matter of the present application relates to and encompasses the disclosure of U.S. Provisional Patent Application No. 60/693,505, filed on Jun. 23, 2005 in the name of Vikram Vakharia for “IN OVO VACCINE AGAINST INFECTIOUS BURSAL DISEASE.” The disclosure of such provisional application is hereby incorporated herein by reference in its entirety, for all purposes.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a recombinant non-pathogenic infectious bursal disease virus (IBDV) and methods of delivering via in ova for protection against virulent challenge by classic and variant strains of IBDV.
2. Description of the Related Art
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of a highly immunosuppressive disease in young chickens (Kibenge, et al., 1988). Infectious bursal disease (IBD) or Gumboro disease is characterized by the destruction of lymphoid follicles in the bursa of Fabricius. In a fully susceptible chicken flock of 3-6 weeks of age the clinical disease causes severe immunosuppression, and is responsible for losses due to impaired growth, decreased feed efficiency, and death. Susceptible chickens less than 3 weeks old do not exhibit outward clinical signs of the disease but have a marked infection characterized by gross lesions of the bursa.
IBDV is a pathogen of major economic importance to the nation and world's poultry industries. It causes severe immunodeficiency in young chickens by destruction of precursors of antibody-production B cells in the bursa of Fabricius. Immunosuppression causes increased susceptibility to other diseases, and interferes with effective vaccination against Newcastle disease, Marek's disease and infectious bronchitis disease viruses.
The capsid of the IBDV virion consists of several structural proteins. As many as nine structural proteins have been reported but there is evidence that some of these may have a precursor-product relationship. The designation and molecular weights of the viral proteins (VP) are as shown below.














Viral Protein
Molecular Weight









VP1
90 kDa



VP2
41 kDa



VP3
32 kDa



VP4
28 kDa



VP5 (NS)
17 kDa










The IBDV genome consists of two segments of double-stranded (ds)RNA that vary between 2827 (segment B) to 3261 (segment A) nucleotide base pairs. The larger segment A encodes a 110-kDa precursor protein in a single large open reading frame (polyprotein ORF) which is cleaved by autoproteolysis to form the mature viral proteins VP2, VP3 and VP4 (Hudson, P. J. et al., 1986). VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of IBDV, and contains the antigenic regions responsible for the induction of neutralizing antibodies (Azad, et al., 1987). A second open reading frame (ORF), preceding and partially overlapping the polyprotein gene, encodes a non structural (NS) protein (VP5).
It has been demonstrated that the VP2 protein is the major host protective immunogen of IBDV, and that it contains the antigenic region responsible for the induction of neutralizing antibodies. This region containing the neutralization site has been shown to be highly conformation-dependent. The VP3 protein has been considered to be a group-specific antigen because it is recognized by monoclonal antibodies directed against it from strains of both serotype I and II viruses. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins.
In the past, control of IBDV infection in young chickens has been achieved by live vaccination with avirulent strains, or principally by the transfer of maternal antibodies induced by the administration of live and killed IBDV vaccines to breeder hens. The strategy for the control of IBDV in chicks involves hyperimmunization of breeders, which allows them to transmit high levels of maternal antibodies to progeny during the critical first few weeks of life. Although maternal antibodies provide protection during this period, continued protection against IBDV must be maintained before the maternal immunity reaches sub-protective levels by the administration of live vaccines. However, maternal antibodies can neutralize a vaccine virus and reduce the viral load needed to induce immunity (Sharma et al., 1987). In addition, new antigenic variants of IBDV, which appeared during the 1980's, introduced new problems for poultry production. These new field isolates were able to break through neutralizing maternal antibodies induced by standard IBDV vaccines (Snyder, 1992). Since then, these variant strains have been incorporated into commercial inactivated vaccines for broiler breeders. For example, Mundt et al., 2003 vaccinated broilers, in ovo, with a chimeric IBDV vaccine using epitopes from classic and E/Del variant viruses, however, the challenge induced chronic lesions in BF of the vaccinated broilers.
Unfortunately, despite these vaccination measures, IBDV continues to be a problem. Very virulent strains of IBDV have caused outbreaks of disease with high mortality in Europe and Asia (Nunoya et al., 1992; van den Berg et al., 1991) despite vaccination programs. In addition, live vaccines that are available for mass vaccination of broilers in the first few weeks of life are not suitable for in ovo administration. These vaccines may induce immunosuppression during late stages of incubation, when the embryo is highly susceptible to infection.
Thus, it would be advantageous to develop a vaccine comprising a recombinant virus that can be administered in ovo but is not susceptible to the shortcomings of previous vaccines such as inducing immunosuppression during late stages of incubation or being neutralized by maternal antibodies.
SUMMARY OF THE INVENTION
The present invention relates to a recombinant IBDV virus that is deficient in the expression of VP5 nonstructural protein (NS) and grows one log lower than the parental viruses, exhibits decreased cytotoxic and apoptotic effects in cell culture.
In yet another aspects, the present invention relates to a non-pathogenic vaccine comprising a recombinant IBDV comprising a Segment A designated as rD78GLSNSΔ (SEQ ID NO: 5) that includes nucleotide sequences from D78 and GLS strains and wherein the NS protein is not expressed.
In yet another aspect the present invention relates to an in ovo method of vaccination of poultry in the presence of maternal immunity against IBDV, the method comprising:
introducing in ovo a vaccine comprising a recombinant virus comprising at least Segment A (SEQ ID NO: 5) modified to include nucleotide sequences from D78 and GLS strains and wherein the NS protein is not expressed and the vaccine is delivered in an amount sufficient to protective against STC-IBDV challenge.
A further aspect of the present invention provides for a live, non-pathogenic recombinant IBD virus and method of producing same for use as a vaccine to protect against IBDV, the method comprising the following steps:

    
    
        (a) preparing cDNA of infectious bursal disease virus genome segments A and B, wherein the segment A (SEQ ID NO: 5) is modified to prevent expression of NS protein, wherein the cDNA contains epitopic determinants from D78 and/or GLS strains;
        (b) transcribing said cDNA to produce synthetic RNA transcripts,
        (c) transfecting host cells with said synthetic RNA transcripts,
        (d) incubating said host cells in a culture medium, and
        (e) isolating live, nonpathogenic, infectious bursal disease virus from said culture medium.
    
    


Other aspects and features of the invention will be more fully apparent from the ensuing disclosure and appended claims.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows cDNA constructs of D78 and GLS strains used to generate plus-sense RNA transcripts using T7 RNA polymerase. A map of the IBDV genome segment A, with its coding capacity, is shown at the top of the figure. Open boxes depict the coding regions of the D78 strain, whereas, the shaded boxes represent the coding regions of GLS strain. Selected restriction sites, important for the construction of chimeric cDNA clones of segment A, are shown in the figure: B, Bst EII; N, Nar I; Sc, Sac II; S, Spe I; R, Rsr II. All constructs contain a T7 polymerase promoter sequence at their 5′-end.
FIG. 2 shows the ELISA geometric mean titer (GMT) from two-week-old chickens post in ovo vaccination with rD78GLSNSΔ and 10 days post IBDV challenge. A) SPF embryos vaccinated with 5.6×103 pfu/egg of the vaccine; B) SPF embryos vaccinated with 2.3×103 pfu/egg of the vaccine; C)SPF embryos unvaccinated, but challenged at 2 weeks post in ovo vaccination; D) SPF embryos were neither vaccinated, nor challenged; E) broiler embryos vaccinated with 5.6×103 pfu/egg; F) broiler embryos unvaccinated, but challenged at 2 weeks post in ovo vaccination; G) broiler embryos unvaccinated, and non-challenged.
FIG. 3 shows sections of the BF stained by hematoxylin-eosin for histopathological examination 10 days post-challenge with IBDV-STC. Eighteen-day-old embryos were inoculated with either 5.6×103 pfu (full dose) or 2.3×103 pfu (half dose) of rD78GLSNSΔ. A) non-vaccinated, challenged SPF, shows severe lymphocytic necrosis and heterophilic inflammation; B) vaccinated (full dose) and challenged SPF birds shows lymphocytic depletion (indicated by arrows); C) vaccinated (half dose) and challenged SPF shows no visible microscopic lesions; D) non-vaccinated, challenged broiler shows lymphocytic necrosis and loss of follicular structure; E) unvaccinated, and non-challenged control broiler shows no microscopic lesions; F) vaccinated (full dose) and challenged broiler shows no microscopic lesions.
FIG. 4 shows amino acid and nucleotide sequences for Segment B of D78 and GLS strains.
FIG. 5 shows the chimeric nucleotide sequence of the Segment A used in the recombinant virus of the present invention.



DETAILED DESCRIPTION OF THE INVENTION
In an effort to aid in the control of this disease, a recombinant IBD vaccine virus that can protect against both classical and variant strains is described herein and which can generated by methods described in Liu, 2003. This recombinant virus, designated as rD78GLSNSΔ, is deficient in the expression of VP5 nonstructural protein (NS). It grows one log lower than the parental viruses, and exhibits decreased cytotoxic and apoptotic effects in cell culture. This virus fails to induce any pathological lesions in the bursa of infected three-week-old chickens. In addition, vaccinated birds challenged with classic (STC) and variant (GLS) strains of IBDV were fully protected.
“Epitopic determinants” as used herein is defined as amino acids or amino acid sequences which correspond to epitopes recognized by one or more monoclonal antibodies.
Briefly, the cDNA clone containing the preferred coding and/or non-coding regions of IBDV-RNA segment A and B can be prepared using standard cloning procedures and methods, as described for IBDV in Mundt, E., 1996, the contents of which are hereby incorporated herein by reference for all purposes. Manipulations of DNAs can be performed according to standard protocols (Sambrook, J., 1989).
To generate cDNA clones of a coding region of the desired structural proteins, the genomic RNA is used as a template for synthesizing and amplifying according to general RT-PCR techniques well known in the art. Specifically, U.S. Pat. No. 5,595,912 provides techniques applicable for cDNA amplifying in the present invention. The desired amplified fragments are then cloned into an expression vector.
Useful vectors for this purpose include plasmids, and viruses such as baculoviruses, herpes virus (HVT) and pox viruses, e.g., fowl pox virus, and the like. The vectors may also expression control sequences including, but not limited to, a promoter, enhancers, operators, inducers, ribosome binding sites, etc.
A promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes. Those promoters most commonly used in recombinant DNA construction include the beta.-lactamase (penicillinase) and lactose promoter systems and a tryptophan (TRP) promoter system. While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to introduce functional promoters into plasmid vectors.
Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike a promoter, an enhancer can function when located at variable distances from transcription start sites so long as a promoter is present.
Also provided herein is a host cell transformed with the recombinant vector of the present invention or a host cell transfected with the synthetic RNA of the present invention. The host cell may be a eukaryotic or a prokaryotic host cell. Suitable examples are E. coli, insect cell lines such as Sf-9, chicken embryo fibroblast (CEF) cells, chicken embryo kidney (CEK) cells, African green monkey Vero cells and the like. Further, the host cell can be an insect cell or yeast cell that is transfected for expression of the desired protein(s).
Means of transforming or transfecting cells with exogenous polynucleotide such as DNA molecules are well known in the art and include techniques such as calcium-phosphate- or DEAE-dextran-mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and adenovirus infection.
The most widely used method is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm of the cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of cultured cells can be transfected at any one time. Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for experiments that require transient expression of the foreign DNA in large numbers of cells. Calcium phosphate-mediated transfection is also used to establish cell lines that integrate copies of the foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.
Electroporation can be extremely efficient and can be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies of the gene of interest. Electroporation, in contrast to calcium phosphate-mediated transfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies of the foreign DNA. The application of brief, high-voltage electric pulses to a variety of mammalian and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closure of the pores.
In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. For example, E. coli can be transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of its own polypeptides. In yet another embodiment, the present invention contemplates a process of preparing a live IBDV of the present invention comprising transfecting cells with a polynucleotide that encodes for the Segment A and B, wherein the NS of Segment A is not expressed to produce transformed host cells; and maintaining the transformed host cells under biological conditions sufficient for expression of the virus. The transformed host cells can be eukaryotic cells or prokaryotic cells. Most preferably, transfection is accomplished using a hereinbefore disclosed expression vector. A variety of cells are amenable to a process of the invention, for instance, yeasts cells, human cell lines, and other eukaryotic cell lines known well to those of the art.
Also part of this invention is a NS protein deficient IBDV vaccine comprising a protecting amount of a recombinantly produced virus or portion of a virus, wherein the virus does not induce pathological lesions. A cDNA clone of IBDV segment A is constructed, in which the first and only initiation codon (ATG) of NS protein A is mutated to a stop codon (TAG).
The virus can be further modified or inactivated by chemical or physical means. Chemical inactivation can be achieved by treating the virus with, for example, enzymes, formaldehyde, .beta.-propiolactone, ethylene-imine or a derivative thereof, an organic solvent (e.g. halogenated hydrocarbon) and or a detergent. If necessary, the inactivating substance can be neutralized after the virus has been inactivated. Physical inactivation can be carried out by subjecting the viruses to radiation such as UV light, X-radiation, or gamma-radiation.
The virus can also be modified by known methods including serial passage, deleting further sequences of nucleic acids and site directed. mutagenesis either before or after production of the infectious virus.
The virus is a chimeric recombinant virus, which contains epitopic determinants for more than one strain of IBDV. Epitopic determinants as discussed in the present document are amino acids or amino acid sequences which correspond to epitopes recognized by one or more monoclonal antibodies. Methods for producing a chimeric virus are disclosed in Vakharia, 1997; Snyder et al., 1994, WO 95/26196, and Liu, 2003 the contents of which are incorporated by reference herein for all purposes.
A recombinant peptide is recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises isolating and purifying the recombinant polypeptide. Isolation and purification techniques for polypeptides are well known in the art and include such procedures as precipitation, filtration, chromatography, electrophoresis and the like. For instance, the immunogenic polypeptides can be harvested using conventional techniques described in Dobos et al., 1979.
These virus polypeptides can be used to prepare vaccines which will confer protection on inoculated poultry, in particular, chickens, and in a preferred embodiment, broiler chickens, protection against challenge from each IBDV bearing an epitope reflected in the plurality of epitopic determinants present in the inoculum. Thus, a single immunogen gives rise to immunity against a variety of IBDV strains while remaining non-pathogenic to poultry.
The administration of the vaccines can be effectively done according to well-established procedures. Preferably, the vaccine is injected in ovo to embryonated eggs, such as 14-18 day-old embryonated eggs.
The vaccine of the present invention is administered to poultry to prevent IBD anytime before or after hatching. Preferably, the vaccine is administered prior to the time of birth and after the animal is about 6 weeks of age. Poultry is defined to include but not be limited to chickens, roosters, hens, broilers, roasters, breeders, layers, turkeys and ducks.
Also included within the scope of the present invention are nucleic acid sequences encoding the recombinant virus of the present invention.
In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with the recombinant virus of the present invention, comprising the steps of (a) transfecting recombinant host cells with polynucleotides that encode the recombinant virus, (b) culturing the host cells under conditions sufficient for expression of the peptides; (c) recovering the peptides; and (d) preparing antibodies to the peptides.
Typically, a monoclonal antibody of the present invention can be readily prepared by a technique which involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide of the present invention) in a manner sufficient to provide an immune response. Rodents such as mice and rats are preferred animals. Spleen cells from the immunized animal are then fused with cells of an immortal myeloma cell. Where the immunized animal is a mouse, a preferred myeloma cell is a murine NS-1 myeloma cell.
The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides. Where azaserine is used, the media is supplemented with hypoxanthine. This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microliter plates, followed by testing the individual clonal supernatants for reactivity with an antigen-polypeptides. The selected clones can then be propagated indefinitely to provide the monoclonal antibody.
By way of specific example, to produce an antibody of the present invention, mice are injected intraperitoneally with between about 1-200 ug of an antigen comprising a polypeptide of the present invention. B lymphocyte cells are stimulated to grow by injecting the antigen in association with an adjuvant such as complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis). At some time (e.g., at least two weeks) after the first injection, mice are boosted by injection with a second dose of the antigen mixed with incomplete Freund's adjuvant. A few weeks after the second injection, mice are tail bled and the sera titered by immunoprecipitation against radiolabeled antigen. Preferably, the process of boosting and titering is repeated until a suitable titer is achieved. The spleen of the mouse with the highest titer is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5×107 to 2×108 lymphocytes.
Mutant lymphocyte cells known as myeloma cells are obtained from laboratory animals in which such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they can be propagated indefinitely in tissue culture, and are thus denominated immortal. Numerous cultured cell lines of myeloma cells from mice and rats, such as murine NS-1 myeloma cells, have been established.
Myeloma cells are combined under conditions appropriate to foster fusion with the normal antibody-producing cells from the spleen of the mouse or rat injected with the antigen/polypeptide of the present invention. Fusion conditions include, for example, the presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like myeloma cells, hybridoma cells grow indefinitely in culture. Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium such as HAT media (hypoxanthine, aminopterin, thymidine). Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells) can grow in the selection media. Each of the surviving hybridoma cells produces a single antibody. These cells are then screened for the production of the specific antibody immunoreactive with an antigen/polypeptide of the present invention. Single cell hybridomas are isolated by limiting dilutions of the hybridomas. The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the supernatant is tested for the presence of the monoclonal antibody. The clones producing that antibody are then cultured in large amounts to produce an antibody of the present invention in convenient quantity.
By use of a monoclonal antibody of the present invention, specific polypeptides and polynucleotide of the invention can be recognized as antigens, and thus identified. Once identified, those polypeptides and polynucleotide can be isolated and purified by techniques such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal antibody is bound to a solid substrate and exposed to a solution containing the desired antigen. The antigen is removed from the solution through an immunospecific reaction with the bound antibody. The polypeptide or polynucleotide is then easily removed from the substrate and purified.
Yet another aspect of the present invention provides for pharmaceutical compositions comprising the recombinant live nonpathogenic virus of the present invention in combination with a physiologically acceptable carrier.
Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The foregoing aspects and embodiments of the present invention are further described in the following Examples. However, the present invention is not limited by the Examples, and variations will be apparent to those skilled in the art without departing from the scope of the present invention.
EXAMPLES
The potential use of this recombinant attenuated virus was evaluated in ovo to determine ability to protect SPF as well as commercial chicks from IBDV challenges. Fertile eggs were obtained from a local poultry farm that routinely immunizes broiler breeders with live and inactivated vaccines to confer high levels of maternal antibodies to the progeny. The vaccination program for these breeders consisted of one live IBDV vaccination at 4 weeks of age, and booster vaccinations at 10 and 18 weeks of age with an inactivated oil-emulsion vaccine containing standard and variant strains of IBDV.
Two different doses of this recombinant vaccine were evaluated in SPF eggs in the absence of maternal antibodies. A full dose was used to vaccinate broiler embryos with maternal antibodies to verify its ability to break through antibody barrier and generate a protective immune response against IBDV challenges.
Materials and Methods:
Vero cells were maintained in M199 medium supplemented with 5% fetal bovine serum (FBS) at 37° C. in a humidified 5% CO2 incubator, and used for propagation of the virus and transfection experiments. Primary chicken embryo fibroblast (CEF) cells were prepared from 10-day-old embryonated eggs (SPAFAS, Inc., Storrs, Conn.), as described previously in Mundt, et al, 1996. Secondary CEF cells were maintained in a growth medium consisting of M199 and F10 (50%/50% v/v) and 5% FBS, and used for immunofluorescence, virus titration and plaque assays. D78, GLS and the recovered chimeric IBDV were titrated in secondary CEF cells as described by Yao, et al., 1998. A tissue culture adapted GLS-5 strain of IBDV (GLS-TC) was propagated in CEF cells. A panel of MAbs, prepared against various strains of IBDV, was used to characterize IBDV antigens by AC-ELISA, as described previously by Vakharia, et al. 1994.
Construction of Full-Length cDNA Clones:
All manipulations of DNAs were performed according to standard protocols. Construction of full-length cDNA clones of IBDV genomic segments of strain D78 (with sequence tags), pUCD78NSΔ, pUCD78B or GLS strain (pGLS-5), has been described previously by Vakharia, et al. 1994; Yao, et al., 1998; and Mundt, et al., 1996. Plasmid pUC19GLSVP2 was prepared by replacing an RsrI-SacII fragment in plasmid pUC19FLAD78 with respective RsrI-SacII fragment derived from plasmid pGLS-5. To generate an NS-deficient IBDV, plasmid pUCD78NSΔGLSVP2 was first prepared by inserting an RsrI-SacII fragment of GLS in plasmid pUCD78NSΔ. This plasmid was then used as a vector by digesting with BstEII and SacII enzymes. As shown in FIG. 1, the GLS-specific sequences from BstEII-NarI and SpeI-SacII sites were replaced with the D78 sequences to obtain plasmid pUC19D78GLSNSΔ. DNA from these plasmids was sequenced by dideoxy chain termination method, using an Automated DNA Sequencer (Applied Biosystem). The integrity of the full-length constructs was tested by in vitro transcription and translation coupled reticulocyte lysate system using T7 RNA polymerase (Promega Corp.). The resulting labeled products were separated by a 12.5% SDS-PAGE and visualized by autoradiography (data not shown).
Transcription and Transfection of Synthetic RNAs:
Transcription of synthetic RNAs, transfection of Vero cells, and characterization of recovered IBDV was carried out essentially as described earlier by Mundt, et al, 1996; and Yao, et al., 1998. Briefly, plasmid pUC19D78GLSNSΔ and pUCD78B were digested with BsrGI or PstI enzymes, respectively (FIG. 1). The combined RNAs, derived from cDNA clones of segments A and B, were used for transfection in Vero cells. The recovered virus was designated as recombinant D78GLSNSΔ (rD78GLSNSΔ).
Plaque Assays:
Virus stocks propagated in cell culture were titered by plaque assay as described by Mundt, et al. 1996. Briefly, the infected supernatant was diluted in ten-fold increments in MEM without FCS. Confluent monolayers of CEFs were infected with serial dilutions of viruses (10−4 to 10−7, 0.1 mL/well). After 1 h adsorption at RT, the media was removed and the monolayer overlaid with 3 mL of 1% SeaPlaque agarose (Difco) containing 10% tryptose phosphate broth, 2% FCS, 0.112% NaHCO3, 100 unit/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL fungizone. On day 3, the agarose overlay was removed and cells were fixed with formalin. After fixing, the cells were stained with crystal violet and plaques were counted and expressed as plaque forming units (pfu/mL).
Experimental Design:
The experimental groups and controls are shown in Table 1. A larger number of eggs were utilized for treatment groups A, B, and E (vaccinated) because of a possible adverse effect due to vaccination. Sample size was calculated accordingly to protocol previously approved. SPF and commercial fertile broiler eggs of 18 days of embryonation were inoculated using a full dose of rIBDV (group A, and E). Each embryo received 5.6×103 pfu/0.2 mL of rD78GLSNSΔ. Another group of SPF eggs received only half a dose of this virus, which was 2.3×103 pfu/0.2 mL (group B). The negative controls consisted of non-vaccinated, unchallenged SPF and commercial embryos (groups D, and G). The challenge control groups consisted of SPF and commercial eggs, which did not receive the vaccine and were challenged two weeks post-vaccination (groups C, and F). After in ovo vaccination, all eggs were sealed with adhesive tape and re-incubated.







TABLE 1







Experimental design to evaluate live attenuated rIBDV vaccine.










Group
Egg type
Vaccine dosea (pfu)
# of eggs













A
SPFb
5.6 × 103
27


B

2.3 × 103
27


C

NV/CHc
17


D

NV/NCHd
17


E
Broilerse
5.6 × 103
24


F

NV/CH
9


G

NV/NCH
9





a8-day-old embryos received 0.2 mL of rIBDV vaccine containing either 5.6 × 103 or 2.3 × 103 pfu.


bspecific-pathogen-free fertile eggs.


cnon-vaccinated, challenged control group.


dnon-vaccinated, non-challenged control group.


efertile broiler eggs from a poultry farm.






The SPF embryos were free of any other immunosuppressive diseases that could compromise the results, such as adenoviruses, and chicken anaemia virus. The commercial eggs were obtained from Sunrise farms, Catskill, N.Y.
After 21 days of incubation, all hatched chicks were housed in BL-2 isolators at Gudelsky Veterinary Center. Two weeks post-hatch, all birds were bled and challenged with STC strain (0.2 mL by the ocular and nasal route-103 EID50) of IBDV, except group D, and G. At ten days post-challenge, all the remaining birds were anesthetized, bled, and humanely euthanized. Spleen and bursa were collected and bursa/body weight recorded. The bursae were sectioned in half. Spleen and bursa halves were placed in 10% buffered formalin for histology. The other half of the bursa was stored frozen for later testing by antigen capture-ELISA (AC-ELISA).
The antibody levels in serum samples collected at 2 weeks post-vaccination, and 10 days post-challenge, were determined by ELISA and virus neutralization (VN). A commercial ELISA kit was used (Synbiotics, San Diego, Calif.). The frozen bursae were processed as described earlier and probed for antigen detection by AC-ELISA (Synbiotics, San Diego, Calif.). Fixed tissues were sectioned at American Histolabs (Gaithersburg, Md.) and stained by hematoxylin-eosin (HE).
Virus Neutralization Assay (VN):
Serum samples were heat-inactivated at 56° C. for 1 h and diluted in serial two-fold dilutions. Each dilution was mixed with 100 TCID50 of rIBDV and incubated for 1 h at 37° C. The mixture was added to 85-90% confluent monolayer of Vero cells, grown in 96-well tissue culture plates (Costar, Ithaca, N.Y.). All plates were incubated for 5-6 days until the presence of CPE was detected in the virus control wells. Normal serum from SPF birds was used as a negative control and a polyclonal anti-IBDV (SPAFAS) was used as a positive control. Anti-IBDV titers were determined 5-6 days later, averaged, and expressed as log2.
Results:
Vaccine Safety:
Hatchability and first week survival rates are shown in Table 2. The hatchability rates in SPF eggs from group A and B were similar (92.5%), indicating that the vaccine is safe even when higher doses were administered. The percentages of hatched birds and first week survival were not significantly different from vaccinated groups and control non-vaccinated. The hatchability rates in broilers were lower than SPF embryos and this is evident in both vaccinated and non-vaccinated birds (88.8%). This reduction in hatchability is attributed to stress caused by temperature shock during transportation. Embryonated eggs were removed from incubators at 37° C. and transported at 22° C. for 2 h. However, the hatchability in this experiment is much higher than the average observed in the poultry industry (83%) because all unviable eggs were discarded prior to in ovo vaccination.







TABLE 2







Effect of in ovo vaccination of rIBDV on hatchability and


survival of hatched chicks.












# of chicks
1st week


Group
Egg type
hatched (%)
survival rate (%)





A
SPF
25/27 (92.5)a
24 (96)


B

25/27 (92.5)
25 (100)


C

16/17 (94.1)
16 (100)


D

17/17 (100)
17 (100)


E
Broilers
18/24 (75)
17 (94.4)


F

 7/9 (77.8)
 7 (100)


G

 8/9 (88.8)
 8 (100)





apercentage of hatched chicks after in ovo vaccination with rIBDV.






Vaccine protection: Results of the IBDV challenge studies are shown in Table 3. All vaccinated birds were fully protected against IBDV-STC. The bursa/body weight ratio was calculated as bursa weight/body weight×1000. The vaccinated group was considered protected if all bursa/body weight ratios were equal or higher than 2SD (standard deviation) of the non-vaccinated, non-challenged control group. The mean for the SPF control (group D) was 5.35 (2SD=2.48). Thus, all SPF vaccinated birds with either dose were considered protected. Additionally, vaccinated broilers were also considered fully protected.

Table 3 also shows results from antigen detection assessed by AC-ELISA (Synbiotics, San Diego, Calif.) seven days post-challenge. IBDV antigen was detected in two SPF birds that were vaccinated with full dose of the vaccine. Viral antigens could not be detected in the BF of birds that received half dose of the vaccine or in broilers that received full dose. Antigen was detected in SPF challenge control group (11/12). As expected, no IBDV antigen could be detected in the negative control birds (group D, and G), whereas non-vaccinated, challenged broilers (8/9) were positive by AC-ELISA at 10 days post-challenge.







TABLE 3







Protection indices from birds vaccinated with rIBDV-attenuated vaccine and


challenged with the classic STC strain of IBDV.














Birds with clinical




Lesion


Group
signs/Challengeda
B/B weightb
AC-ELISAc
VN Log2d

Score
















A
 0/20e (100)f
 6.04 ± 1.2g
 2/12
8.76
7.90
2.0


B
  0/18 (100)
5.27 ± 1.5
 0/11
9.25
5.47
0


C
 12/12 (NA)h
4.31 ± 1.1
11/12
4.00
2.33
5.0


D
  0/12 (NA)
5.35 ± 1.2
 0/12
3.16
4.00
0


E
  0/17 (100)
2.14 ± 0.8
 0/11
9.46
7.58
0


F
 10/12 (NA)
1.48 ± 0.6
8/9
5.3
6.8
4.6


G
  0/12 (NA)
2.31 ± 0.5
0/9
5.4
6.8
0





aat two weeks post-vaccination birds received 0.2 mL of 103EID50% of IBDV-STC challenge.


b(bursa weight/body weight) × 1000.


cnumber of birds that had antigen detected as measured by antigen capture ELISA.


dvirus neutralization results two weeks post-vaccination and ten days post-challenge.


enumber of birds dead/number of birds challenged.


fpercentage of protected birds.


gmean of bursa/body weight rations and standard deviation.


hnot applicable.






Antibody Responses:

The antibody responses as measured by commercial ELISA are shown in FIG. 2. At two weeks post-vaccination, all vaccinated groups had significantly higher antibody titers (p<0.05) than non-vaccinated groups by either test. As expected, all non-vaccinated SPF birds had negative titers for IBDV at 2 weeks post-vaccination. The non-vaccinated, commercial broilers had maternal antibody titers ranging from 455 to 5455 at 2 weeks of age. At 10 days post-challenge, all SPF vaccinated groups (full and half dose) showed significantly (p<0.05) higher titers than the same groups at two weeks post-vaccination. However, broilers receiving a full vaccine dose did not show an antibody increase after challenge as measured by ELISA. Antibody responses at 2 weeks post-vaccination in SPF birds that received half dose of the vaccine were relatively higher than SPF birds vaccinated with a full dose. The level of protective maternal antibodies in the non-vaccinated, unchallenged broilers seemed to wane slightly after challenge. Antibody responses were not detected in SPF non-vaccinated, challenged control birds after 10 days of challenge. This result is expected considering that a primary immune response would take at least 2 weeks to be detected.
The results from virus neutralization assay are expressed as log2 in Table 3. All vaccinated groups showed protective levels (between 4-6 log2) of antibodies 2 weeks post-vaccination. Non-vaccinated control broilers showed maternal antibody levels (4.8 log2) lower than normal for the first few weeks of life. All control groups continued to exhibit only low levels of antibodies 10 days post-challenge. As expected, all vaccinated SPF birds showed higher humoral responses after challenge. Vaccinated broilers also showed higher antibody response 10 days post-challenge (7.5 to 9.46).
Histopathology:
The averages of microscopic lesions are shown in Table 3. At 2 weeks post-vaccination, birds from the control and treatment groups that received either full dose or half dose did not show microscopic lesions in the BF. At 10 days post-challenge, SPF non-vaccinated birds that were challenged showed severe lymphocyte depletion, undulation in the epithelium, intra and interfollicular epithelial cysts, and degeneration of follicular structure (FIG. 3A, Table 3-C). SPF birds that received full dose of the vaccine (FIG. 3B) showed a mild degree of B-cell depletion, localized in a few follicles. The lesion score for this group was 2.0. SPF birds vaccinated with a half dose of the vaccine did not show histopathological lesions in the bursa (FIG. 3C, Table 3-B). The control non-vaccinated, challenged broilers showed bursal lesions. However, they were much milder than SPF challenged controls (FIG. 3D, Table 3-F). In addition, broilers that received a full dose of the vaccine showed normal bursae after 10 days post IBDV challenge (FIG. 3F, Table 3-E).
The spleens of SPF non-vaccinated, challenged birds showed hemorrhages and lymphocytic depletion. Non-vaccinated broilers that were challenged showed a milder but more generalized reduction of lymphocytes throughout the entire organ. No lesions were observed in the spleens from vaccinated groups (data not shown).
Described herein is a recombinant live attenuated vaccine that expresses multiple neutralizing epitopes of classical and variant strain of IBDV. The vaccine was evaluated in 18-day-old SPF and commercial embryos. SPF embryos were injected with a full or half dose of the virus through the amniotic cavity. Two weeks post-vaccination, birds were challenged with an IBDV-STC strain. Commercial broilers vaccinated with a full dose and SPF embryos vaccinated with half dose were fully protected. In addition, no significant microscopic bursal lesions were observed in these groups. Interestingly, SPF birds that received a full dose of the vaccine in ovo exhibited microscopic lesions similar to unvaccinated challenged control group.
No detrimental effects on hatchability with either dosage used were observed. However, histopathological results suggested that a higher dose of the vaccine given to birds that lack sufficient maternal antibody may still be virulent even though clinical signs of IBD were not observed. These findings agree with previous research in SPF and broiler embryos vaccinated with three commercial intermediate vaccines in ovo. Microscopic bursal lesions were observed even when half of the recommended dose was used (Giambrone et al., 2001).
In a previous report, microscopic lesions were not observed when a full dose of rIBDV live attenuated virus was used to vaccinate two-week-old chickens ocularly (Liu, 2003). In the present application, bursal lesions were observed in SPF birds, lacking maternal antibodies that received a full dose of the vaccine when delivered in ovo. At 10 and 15 days of embryonation, prebursal stem cells are migrating via the blood supply from the spleen to the BF (Masteller et al., 1994). Consequently, at eighteen days of incubation, when in ovo vaccination occurred, the avian immune system was not fully developed and a viral infection that targets this organ may have caused irreversible damage.
Notably, vaccinated broilers had significant higher antibody titers than non-vaccinated broiler control group at two weeks post vaccination. According to Lutticken et al., 1994, these findings indicate that the vaccine was able to breakthrough maternal antibody barrier and seroconvert. Similar results were obtained in broilers vaccinated with a chimeric IBDV vaccine 14 days post-vaccination (Mundt et al., 2003). However, in the Mundt, et al. results, the challenge using classic and variant viruses induced chronic lesions in BF of vaccinated broilers with a chimeric virus expressing classic and variant epitopes of E/Del and D78 IBDV strains (Mundt et al., 2003).
Thus, it has been shown herein that use of rD78GLSNSΔ can be used as a vaccine for in ovo delivery, wherein the vaccine is safe, highly immunogenic, and protective against STC-IBDV challenge. Importantly, this recombinant virus was not neutralized by the maternal antibodies present in the embryo because a higher dose of the virus could be used and it replicated efficiently in ovo without affecting the viability of an embryo (since it is attenuated by lack of 17-kDa nonstructural protein expression). Notably, at this dose, the wild type IBD virus would cause mortality of the embryonating egg.
REFERENCES
All reference cited herein are hereby incorporated by reference herein for all purposes.

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## Claims

1. A recombinant Infectious Bursal Disease virus (IBDV) in ovo in a poultry ovum containing poultry maternal antibodies, comprising at least SEQ ID NO: 5 as Segment A of the IBDV, wherein SEQ ID NO: 5 includes nucleotide sequences from D78 of and GLS strains, and wherein SEQ ID NO: 5 does not express a NS protein.

2. A non-pathogenic vaccine in an embryonated poultry egg, said non-pathogenic vaccine comprising a recombinant IBDV comprising at least SEQ ID NO: 5 as Segment A of the IBDV, wherein SEQ ID NO: 5 includes nucleotide sequences from D78 and GLS strains, wherein the recombinant IBDV is not neutralized by maternal antibodies present in an embryo of said embryonated poultry egg, wherein the recombinant IBDV is non-lethal to said embryo, and wherein SEQ ID NO: 5 does not express a NS protein.

3. An in ova method of vaccination of poultry in the presence of maternal immunity against IBDV, the method comprising:
introducing in ovo a vaccine comprising a recombinant Infectious Bursal Disease virus (IBDV) comprising at least Segment A (SEQ ID NO: 5) modified to include nucleotide sequence from D78 and GLS strains, and wherein SEQ ID NO. 5 does not express a NS protein and wherein the vaccaine is delivered in an amount sufficient to protect against STC-IBDV challenge.

4. A live, non-pathogenic recombinant IBD virus in ovo in a poultry ovum containing poultry maternal antibodies, wherein the live, non-pathogenic recombinant IBD virus is present in said ovum in a sufficient amount to protect the ovum against IBD challenge in the presence of said poultry maternal antibodies, and wherein the live, non-pathogenic recombinant IBD virus has been produce by the method comprising the following steps:
(a) preparing cDNA of infectious bursal disease virus genome segments A and B, wherein the segment A comprises SEQ ID NO: 5 wherein the cDNA contains epitopic determinants from D78 and/or GLS strains, and wherein SEQ ID NO. 5 does not express a NS protein;
(b) transcribing said cDNA to produce synthetic RNA transcripts,
(c) transfecting host cells with said synthetic RNA transcripts,
(d) incubating said host cells in a culture medium, and
(e) isolating live, nonpathogenic, infectious bursal disease virus from said culture medium.

5. The recombinant Infectious Bursal Disease virus (IBDV) in ovo in a poultry ovum containing poultry maternal antibodies, according to claim 1, wherein the poultry ovum comprises a chicken egg.

6. The recombinant Infectious Bursal Disease virus (IBDV) in ovo in a poultry ovum containing poultry maternal antibodies, according to claim 1, wherein the poultry ovum comprises an embryonated chicken egg.

7. The recombinant Infectious Bursal Disease virus (IBDV) in ovo in a poultry ovum containing a poultry maternal antibodies, according to claim 1, wherein the poultry ovum comprises a 14-18 day old embryonated chicken egg.

8. The recombinant Infectious Bursal Disease virus (IBDV) in ovo in a poultry ovum containing poultry maternal antibodies, according to claim 6, wherein the recombinant IBDV is not neutralized by maternal antibodies present in said embryonated chicken egg, and the recombinant IBDV is non-lethal to an embryo of said embryonated chicken egg.

9. The recombinant Infectious Bursal Disease virus (IBDV) in ovo in a poultry ovum containing poultry maternal antibodies, according to claim 7, wherein the recombinant IBDV is not neutralized by maternal antibodies present in said embryonated chicken egg, and the recombinant IBDV is non-lethal to an embryo of said enbryonated chicken egg.