WO2023042164A1

WO2023042164A1 - Chimeric proteins for immunization against newcastle disease virus (ndv)

Info

Publication number: WO2023042164A1
Application number: PCT/IB2022/058800
Authority: WO
Inventors: Ali Hatef SALMANIAN; Jafar AMANI HARONI; Atena MOZAFARI; Shahla SHAHSAVANDI
Original assignee: Salmanian Ali Hatef; Amani Haroni Jafar; Mozafari Atena; Shahsavandi Shahla
Priority date: 2021-09-19
Filing date: 2022-09-18
Publication date: 2023-03-23

Abstract

A chimeric protein for immunization against Newcastle disease virus, including a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli, a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus connected to the truncated LTB via a first linker, a second truncated HN protein of the Newcastle disease virus connected to the first truncated HN protein via a second linker, and a truncated fusion protein (F) of the Newcastle disease virus connected to the second truncated HN protein via a third linker.

Description

CHIMERIC PROTEINS FOR IMMUNIZATION AGAINST NEWCASTLE DISEASE VIRUS (NDV)

STATEMENT REGARDING SEQUENCE LISTING

[0001] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is (303-7313) Sequence Listing.txt. The text file is 15 KB, was created on September 10, 2022, and is being submitted electronically via ePCT.

CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of priority from pending U.S. Provisional Patent Application Serial No. 63/245,852, filed on September 19, 2021, and entitled “THE PROCESS OF USING LHN2F SYNTHETIC PROTEIN FROM NEWCASTLE VIRUS FOR APPLYING IN THE POULTRY INDUSTRY,” which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0003] The present disclosure generally relates to Newcastle disease virus, particularly to vaccine candidates against Newcastle disease virus (NDV), and more particularly to recombinant chimeric proteins for diagnosing and immunizing poultry against Newcastle disease virus.

BACKGROUND ART

[0004] Newcastle disease (ND) is one of the most crucial illnesses in the aviculture industry, in which the vaccine may be essential to preventing and controlling the disease. Conventionally, immunization of chickens against Newcastle disease virus can be done by using the whole virus as an antigen. However, this method carries the risks of mass cultivation of the whole virus and working with it, reversion of vaccine strains to pathogenic strains due to mutations, and simultaneous infection of a bird with the vaccine strain and the wild strain, which increases the possibility of creating new strains due to recombination ((Brown, Vienna R., and Sarah N. Bevins. "A review of virulent Newcastle disease viruses in the United States and the role of wild birds in viral persistence and spread." Veterinary research 48.1 (2017): 1-15), and (Cho, Sun-Hee, et al. "Characterization of a recombinant Newcastle disease virus vaccine strain." Clinical and Vaccine Immunology 15.10 (2008): 1572-1579)).

[0005] Currently, it is impossible to use the conventional method of vaccination in herds of rural poultry or wild birds that live near human communities, such as pigeon herds, sparrows, and starlings. The spreading of herds of rural poultry is sporadic and varied, so it is impossible to transfer the adequate vaccine with the cold chain to all villages and rural areas. Furthermore, the Newcastle virus may naturally infect wild birds without making them sick and spread wherever the chicken is raised. Therefore, the importance of bird safety is evident. [0006] Producing safe vaccines for conventional, reappeared, or newly appeared infection agents are time-consuming and expensive. Recent research has shown that computational methods may be used to develop an effective vaccine, particularly for infectious diseases (Sunita, et al. "Computational tools for modern vaccine development." Human vaccines & immunotherapeutic s 16.3 (2020): 723-735.). Using computational methods for designing new recombinant vaccines may help design several conceptual structures with conserved antigenic epitopes to cover a wide range of Newcastle disease viruses with less time and cost.

[0007] Hence, there is a need for an efficient recombinant protein as a vaccine candidate for immunization against the Newcastle disease virus (NDV). Moreover, there is a need for a recombinant and multi-epitope protein to rapidly detect NDV in biological samples of poultry with high sensitivity.

SUMMARY

[0008] This summary is intended to provide an overview of the subject matter of the present disclosure and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0009] In one general aspect, the present disclosure describes an exemplary chimeric protein for immunization against the Newcastle disease virus. In an exemplary embodiment, the chimeric protein may include a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli, a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus, a second truncated HN protein of the Newcastle disease virus, and a truncated fusion protein (F) of the Newcastle disease virus.

[001] In an exemplary embodiment, the first truncated HN protein of the Newcastle disease virus may be connected to the truncated LTB via a first linker. In an exemplary embodiment, the first truncated HN protein of the Newcastle disease virus may be connected to the first truncated HN protein via a second linker. In an exemplary embodiment, the truncated F protein of the Newcastle disease virus may be connected to the second truncated HN protein via a third linker.

[002] In an exemplary embodiment, the first and the second truncated HN proteins may include SEQ ID NO: 2. In an exemplary embodiment, the truncated F protein may include SEQ ID NO: 3. In an exemplary embodiment, the truncated LTB may include SEQ ID NO: 1. In an exemplary embodiment, the first, the second, and the third linkers may include between 2 and 5 hydrophobic units of SEQ ID NO: 4. In an exemplary embodiment, the first and the third linkers may include SEQ ID NO: 5. In an exemplary embodiment, the second linker may include SEQ ID NO: 6.

[003] In an exemplary embodiment, the chimeric protein may be encoded from a nucleic acid molecule of SEQ ID NO: 7. In an exemplary embodiment, the chimeric protein may include SEQ ID NO: 8. In an exemplary embodiment, the chimeric protein may further include a tag conjugated to the truncated F protein. In an exemplary embodiment, the tag may include at least one of a histidine tag (His-tag), a glutathione S transferase (GST) tag, and a maltose binding protein (MBP) tag.

[004] In another general aspect, the present disclosure describes an exemplary method for vaccination against the Newcastle disease virus. In an exemplary embodiment, the method may include administering a composition including the chimeric protein to a bird. In an exemplary embodiment, administering the composition to the bird may include administering the composition to the bird through at least one of injection, aerosol, drinking water, oral, an eye drop, and an edible vaccine.

[005] In an exemplary embodiment, administering the composition to the bird may include administering the composition with an amount between 30 pg and 50 pg to 3 -week-old chicken. In an exemplary embodiment, the composition may further include a pharmaceutically acceptable carrier. In an exemplary embodiment, the pharmaceutically acceptable carrier may include at least one of nanoparticles, mesoporous silica, chitosan, liposomes, poly lysin, and poly(lactic-co-glycolic acid) (PLGA).

[006] In another general aspect, the present disclosure describes an exemplary diagnostic method for detecting Newcastle disease virus infection in biological samples of birds. In an exemplary embodiment, the diagnostic method may include putting a biological sample of a bird in contact with a reagent including the chimeric protein and determining the presence of Newcastle disease virus infection in the biological sample responsive to detecting the chimeric protein bound to antibodies against the Newcastle disease virus in the biological sample.

[007] In an exemplary embodiment, detecting the chimeric protein bound to the antibodies against the Newcastle disease virus may include conducting at least one of a chemiluminescent assay, an immunofluorescent assay, enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assay, radioimmunoassay, a Western blot assay, an enzyme immunoassay, an immunoprecipitation assay, an immunohistochemical assay, an immunochromatographic assay, a dot blot assay, a slot blot assay, confocal imaging, laser scanning microscopy, and flow cytometry.

[008] In an exemplary embodiment, putting the biological sample in contact with the reagent may include putting at least one of a blood sample, a plasma sample, a serum sample, an egg yolk sample, a fecal sample, a cloacal swab, an oropharyngeal swab, a tracheal swab, a biopsy sample, and an autopsy sample in contact with the reagent. In an exemplary embodiment, the tag may include at least one of a radioactive substance, a dye, a contrast agent, a fluorophore molecule, a nanoparticle, a bioluminescent molecule, an_affinity agent, and a magnetic agent.

[009] In an exemplary embodiment, detecting the chimeric protein bound to antibodies against the Newcastle disease virus in the biological sample may include detecting the chimeric protein bound to antibodies against at least one of Exotic, AF2240, B l, Bl- HITCHNER/47, Bl/48, CHE85, chicken/India/UP3/2006, class 1, Connecticut/9- 12-60, goose/GPMV-SH/CHN/2009, isolate RJAZ96, LIP97, Miyadera/51, NDV Ow/Tw/2209/95, Qingdao/S D/1/97, 3/91, AUSTRALIA- VICTORIA/32, Beaudette C/45, BOR74, BOR82, D26/76, F48E9, GAM61, GPMV/QY97-1, H, HER/33, IBA/85, Italien/45, Kansas, LAS/46, Queensland/66, T53, TEXAS, TEXAS G.B./48, VOL95, Ulster/2C, and Ulster/67 strains of Newcastle disease virus the biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The drawing figures depict one or more implementations in accord with the present teachings, by way of example only, not by way of limitation. In the figures, like reference numerals refer to the same or similar elements.

[0011] FIG. 1 illustrates a three-dimensional structure of an exemplary chimeric protein for immunization against the Newcastle disease virus, consistent with one or more exemplary embodiments of the present disclosure.

[0012] FIG. 2A illustrates a result of SDS-PAGE analysis of bacterial proteins expression after purification and dialysis containing exemplary chimeric protein (lane 1), the truncated LTB (lane 2), the first truncated HN protein (lane 3), the second truncated HN protein (lane 4) and the truncated F protein (lane 5), consistent with one or more exemplary embodiments of the present disclosure.

[0013] FIG. 2B illustrates the results of Western blotting analysis of purified exemplary chimeric protein (rLHN2F), the truncated LTB (rLTB), the first truncated HN protein (rHN), the first and the second truncated HN proteins (rHN-HN), and the truncated F protein (rF) with commercial anti-His tag antibody, consistent with one or more exemplary embodiments of the present disclosure. C“: Negative control (a protein without histidine-tag).

[0014] FIG. 3 illustrates a predicted secondary structure of the exemplary chimeric protein by PSIPRED software (C = Coil, H = Alpha-helix, and E = Beta plates), consistent with one or more exemplary embodiments of the present disclosure.

[0015] FIG. 4A illustrates the RMSD plot of the exemplary chimeric protein, consistent with one or more exemplary embodiments of the present disclosure.

[0016] FIG. 4B illustrates RMS fluctuation of the exemplary chimeric protein, consistent with one or more exemplary embodiments of the present disclosure.

[0017] FIG. 5 illustrates the Ramachandran diagram of the exemplary chimeric protein based on the designed model, consistent with one or more exemplary embodiments of the present disclosure.

[0018] FIG. 6A illustrates the prediction of the antigenic regions of the truncated LTB protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure.

[0019] FIG. 6B illustrates the prediction of the antigenic regions of the first truncated HN protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure.

[0020] FIG. 6C illustrates the prediction of antigenic regions of the second truncated HN protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure. [0021] FIG. 6D illustrates the prediction of the antigenic regions of the truncated F protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure.

[0022] FIG. 7 A illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) and its recombinant segments by antisera of mice immunized with the commercial vaccine against Bl strain as a positive control, consistent with one or more exemplary embodiments of the present disclosure.

[0023] FIG. 7B illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) and its recombinant segments by antisera of mice immunized with the exemplary chimeric protein, consistent with one or more exemplary embodiments of the present disclosure. L: protein molecular weight marker (KD), C“: Negative control.

[0024] FIG. 7C illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) by antisera of mice immunized with the exemplary chimeric protein (1), truncated LTB (2), first truncated HN (3), second truncated HN2 (4), and truncated F (5) segments, consistent with one or more exemplary embodiments of the present disclosure. L: protein molecular weight marker (KD).

[0025] FIG. 7D illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) and its recombinant segments by antisera of mice immunized with the exemplary chimeric protein, truncated LTB, first truncated HN, second truncated HN2, and truncated F segments, consistent with one or more exemplary embodiments of the present disclosure. L: protein molecular weight marker (KD), C“: Negative control.

[0026] FIG. 8A illustrates the result of specific serum IgG response to the exemplary chimeric protein (rLHN2F) in mice immunized with rLHN2F and in sera of control mice, consistent with one or more exemplary embodiments of the present disclosure.

[0027] FIG. 8B illustrates the result of IgG titration of sera from mice immunized with the exemplary chimeric protein (rLHN2F) after the last injection in response to 200 ng of the rLHN2F and its recombinant segments, including rLTB, rHN, rHN2, and rF, consistent with one or more exemplary embodiments of the present disclosure.

[0028] FIG. 8C illustrates the result of titration of sera from mice immunized with each of rLTB, rHN, rHN2, rF, rLHN2F, and B l against the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure.

[0029] FIG. 9 illustrates ELISA results of cross -reactivity analysis by titration of sera of mice immunized with commercial B l vaccine and with the exemplary chimeric protein (rLHN2F) versus the exemplary chimeric protein and its recombinant components, consistent with one or more exemplary embodiments of the present disclosure.

[0030] FIG. 10 illustrates the results of serum antibody response of the antibodies from the immunized mice with the exemplary chimeric protein (rLHN2F) and with the commercial vaccine against the B l strain by ELISA at 14-, 21-, and 30-day post first immunization, consistent with one or more exemplary embodiments of the present disclosure.

DESCRIPTION OF EMBODIMENTS [0031] In the following detailed description, numerous specific details are set forth by way of examples in order to provide a thorough understanding of the relevant teachings. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well-known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[0032] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be readily apparent to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

[0033] The most significant viral disease in the poultry industry may be Newcastle disease (ND), in which two glycoproteins of the virus, including hemagglutinin/neuraminidase (HN) and fusion (F), are crucial in its pathogenesis. As a result, immunity induction against these two glycoproteins is anticipated to stop a virus from attaching to the host cells and prevent the transmission of the virus to the mucosal surfaces. The present disclosure is about designing an exemplary chimeric recombinant protein including computationally selected regions of HN and F glycoproteins in addition to an effective bio-adjuvant for vaccinating poultry against NDV and detecting NDV in biological samples of birds.

[0034] The present disclosure describes an exemplary chimeric protein for immunization against the Newcastle disease virus. In an exemplary embodiment, the computationally selected regions of HN and F glycoproteins may include immunogenic, short, and conserved regions of HN and F glycoproteins which may be selected using bioinformatic methods and may be used for detecting and immunizing against a wide range of the Newcastle disease viruses. In an exemplary embodiment, the chimeric protein has two truncated HN proteins, including the first and the second truncated HN proteins, which may cause enhanced immunization against epitopes HN protein of the NDV due to antigen dose amplification. In an exemplary embodiment, the chimeric protein may include immunogenic and conserved epitopes of the heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli as a bio-adjuvant for improving the immunogenicity of the exemplary chimeric protein.

[0035] In an exemplary embodiment, the chimeric protein may include a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli, a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus, a second truncated HN protein of the Newcastle disease virus, and a truncated fusion protein (F) of the Newcastle disease virus. In an exemplary embodiment, the first truncated HN protein of the Newcastle disease virus may be connected to the truncated LTB via a first linker. In an exemplary embodiment, the first truncated HN protein of the Newcastle disease virus may be connected to the first truncated HN protein via a second linker. In an exemplary embodiment, the truncated F protein of the Newcastle disease virus may be connected to the second truncated HN protein via a third linker.

[0036] FIG. 1 illustrates a three-dimensional structure of an exemplary chimeric protein 100 for immunization against the Newcastle disease virus, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 1, the exemplary chimeric protein 100 may include truncated heat-labile enterotoxin B subunit (LTB) 102 of enterotoxigenic Escherichia coli 102 connected to first truncated hemagglutininneuraminidase (HN) protein of the Newcastle disease virus (NDV) 106 via first linker 104, and second truncated HN protein of the NDV 110 connected to the first truncated HN protein of the NDV 106 via second linker 108, and truncated fusion protein (F) of the NDV 114 connected to the second truncated HN protein of the NDV 110 via third linker 112. In an exemplary embodiment, exemplary chimeric protein 100 may also include tag 116 conjugated to the truncated F protein 114.

[0037] In an exemplary embodiment, the first 106 and the second 110 truncated HN proteins may include SEQ ID NO: 2. In an exemplary embodiment, the truncated F protein 114 may include SEQ ID NO: 3. In an exemplary embodiment, the truncated LTB 102 may include SEQ ID NO: 1. In an exemplary embodiment, the first linker 104, the second linker 108, and the third linker 112 may include between 2 and 5 hydrophobic units of SEQ ID NO: 4. In an exemplary embodiment, the first linker 104 and the third linker 112 may include four hydrophobic units of SEQ ID NO: 4. In an exemplary embodiment, the first linker 104 and the third linker 112 may include SEQ ID NO: 5. In an exemplary embodiment, the second linker 108 may include five hydrophobic units of SEQ ID NO: 4. In an exemplary embodiment, the second linker 108 may include SEQ ID NO: 6.

[0038] In an exemplary embodiment, the chimeric protein 100 may be encoded from a nucleic acid molecule of SEQ ID NO: 7. In an exemplary embodiment, the chimeric protein 100 may include SEQ ID NO: 8. In an exemplary embodiment, the chimeric protein 100 may further include the tag 116 conjugated to the truncated F protein 114. In an exemplary embodiment, the tag 116 may include at least one of a histidine tag (His-tag), a glutathione S transferase (GST) tag, and a maltose binding protein (MBP) tag.

[0039] In another general aspect, the present disclosure describes an exemplary method for vaccination against the Newcastle disease virus. In an exemplary embodiment, the method may include administering a composition including the exemplary chimeric protein 100 to a bird. In an exemplary embodiment, administering the composition to the bird may include administering the composition to the bird through at least one of injection, aerosol, drinking water, oral, an eye drop, and an edible vaccine.

[0040] In an exemplary embodiment, administering the composition to the bird may include administering the composition with an amount between 30 pg and 50 pg to 3 -week-old chicken. In an exemplary embodiment, the composition may further include a pharmaceutically acceptable carrier. In an exemplary embodiment, the pharmaceutically acceptable carrier may include at least one of nanoparticles, mesoporous silica, chitosan, liposomes, poly lysin, and poly(lactic-co-glycolic acid) (PLGA).

[0041] In an exemplary embodiment, injecting the composition, including the exemplary chimeric protein, may be done instead of injecting the killed NDV in conventional vaccination. In an exemplary embodiment, the exemplary chimeric protein in an edible form may be used instead with sprays of killed NDV in the inhalation steps of conventional vaccination in chicken farms. In an exemplary embodiment, the exemplary chimeric protein in the edible form may be the exemplary chimeric protein produced in transgenic plants.

[0042] In an exemplary embodiment, the exemplary chimeric protein may be used as a diagnostic for detecting Newcastle disease virus infection in biological samples of birds. In an exemplary embodiment, the diagnostic method may include putting a biological sample of a bird in contact with a reagent including the chimeric protein 100 and determining the presence of Newcastle disease virus infection in the biological sample responsive to detecting the chimeric protein 100 bound to antibodies against the Newcastle disease virus in the biological sample.

[0043] In an exemplary embodiment, detecting the chimeric protein 100 bound to the antibodies against the Newcastle disease virus may include conducting at least one of a chemiluminescent assay, an immunofluorescent assay, enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assay, radioimmunoassay, a Western blot assay, an enzyme immunoassay, an immunoprecipitation assay, an immunohistochemical assay, an immunochromatographic assay, a dot blot assay, a slot blot assay, confocal imaging, laser scanning microscopy, and flow cytometry.

[0044] In an exemplary embodiment, putting the biological sample in contact with the reagent may include putting at least one of a blood sample, a plasma sample, a serum sample, an egg yolk sample, a fecal sample, a cloacal swab, an oropharyngeal swab, a tracheal swab, a biopsy sample, and an autopsy sample in contact with the reagent. In an exemplary embodiment, the tag may include at least one of a radioactive substance, a dye, a contrast agent, a fluorophore molecule, a nanoparticle, a bioluminescent molecule, an_affinity agent, and a magnetic agent.

[0045] In an exemplary embodiment, detecting the chimeric protein 100 bound to antibodies against the Newcastle disease virus in the biological sample may include detecting the chimeric protein 100 bound to antibodies against at least one of Exotic, AF2240, Bl, Bl- HITCHNER/47, Bl/48, CHE85, chicken/India/UP3/2006, class 1, Connecticut/9- 12-60, goose/GPMV-SH/CHN/2009, isolate RJAZ96, LIP97, Miyadera/51, NDV Ow/Tw/2209/95, Qingdao/S D/1/97, 3/91, AUSTRALIA- VICTORIA/32, Beaudette C/45, BOR74, BOR82, D26/76, F48E9, GAM61, GPMV/QY97-1, H, HER/33, IBA/85, Italien/45, Kansas, LAS/46, Queensland/66, T53, TEXAS, TEXAS G.B./48, VOL95, Ulster/2C, and Ulster/67 strains of Newcastle disease virus the biological sample.

[0046] In an exemplary embodiment, using the exemplary chimeric protein for detecting Newcastle disease virus infection in biological samples of birds may include detecting the NDV infection in the biological samples of chickens, laying hens, broilers, turkeys, pheasant, quail, emu, duck, peacock, pigeon, duck, guinea fowl, and other gallinaceous species. In an exemplary embodiment, the exemplary chimeric protein may be used for serum therapy of the Newcastle disease in birds, including chickens, laying hens, broilers, turkeys, pheasant, quail, emu, duck, peacock, pigeon, duck, guinea fowl, and other gallinaceous species. In an exemplary embodiment, serum therapy of the Newcastle disease in birds may include administering purified blood serum from birds immunized with the exemplary chimeric protein into non-immunized birds.

EXAMPLES

[0047] EXAMPLE 1: DESIGN AND PRODUCTION OF EXEMPLARY CHIMERIC PROTEIN

[0048] In this example, an exemplary chimeric protein with SEQ ID NO: 8 was designed and produced as a recombinant protein in E. coli. Firstly, the exemplary chimeric protein was designed in-silico by considering maximum antigenicity, maximum structure stability, and minimum value of the allergenicity. After designing, a nucleotide sequence of a gene cassette encoding the exemplary chimeric protein was codon-optimized for bacterial expression. Then, the gene cassettes encoding the exemplary chimeric protein and each component of the exemplary chimeric protein were cloned into the pET-28a (+) expression vector individually. The ligation products were transformed into a BL21 (DE3) competent E. coli expression host cell, and the recombinant colonies were stored at -70 °C after confirmation of the presence of each gene by PCR, digestion, and sequencing.

[0049] In the next step, transformed E. coli strains expressing the exemplary chimeric protein, the truncated LTB, the first truncated HN protein, two truncated HN proteins, including the first and the second truncated HN proteins, and the truncated F protein were grown in LB broth at 37 °C. The expression of exemplary chimeric protein and each component was induced by adding 1 mM IPTG to the culture, followed by incubation at 37 °C for further 4 hours. After induction of protein expression, expression of the exemplary chimeric protein and its components was analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Also, exemplary chimeric protein and its components were purified using Nickel-nitrilotriacetic acid (Ni-NTA) resin under denaturing conditions with different pH. Finally, stepwise dialysis against PBS (pH 7.5) was performed to remove urea from the purified recombinant proteins.

[0050] FIG. 2A illustrates a result of SDS-PAGE analysis of bacterial proteins expression after purification and dialysis containing exemplary chimeric protein (lane 1), the truncated LTB (lane 2), the first truncated HN protein (lane 3), two truncated HN proteins, including the first and the second truncated HN proteins (lane 4) and the truncated F protein (lane 5), consistent with one or more exemplary embodiments of the present disclosure. M: Protein molecular weight marker (kDa). Referring to FIG. 2A, molecular weights of the exemplary chimeric peptide, the truncated LTB, the first truncated HN protein, two truncated HN proteins, including the first and the second truncated HN proteins, and the truncated F protein are 52 kDa, 13 kDa, 11 kDa, 22 kDa, and 17 kDa, respectively. [0051] After dialysis, concentrations of purified exemplary chimeric protein and its recombinant components were determined using the Bradford method. After preparing the standard curve using pure bovine serum albumin (BSA), purified exemplary chimeric protein concentration and its recombinant components were evaluated using the standard curve and based on adsorption at 595 nm. Concentrations of the exemplary chimeric peptide, the truncated LTB, the first truncated HN protein, two truncated HN proteins, including the first and the second truncated HN proteins, and the truncated F protein were 1232.65 pg/ml, 302.02 pg/ml, 392.22 pg/ml, 743.91 pg/ml, 297.52 pg/ml, respectively. In the end, the purified proteins were stored in PBS buffer at a pH of 7.2 and about -70 °C.

[0052] Western blotting was also used to ensure the presence and authenticity of recombinant proteins produced in the bacteria system. The chromogenic reaction detected specific protein complexes using 3, 3 '-Diaminobenzidine (DAB) reagent. This procedure was repeated with one-step anti-His antibodies for all recombinant proteins. FIG. 2B illustrates the results of Western blotting analysis of purified exemplary chimeric protein (rLHN2F), the truncated LTB (rLTB), the first truncated HN protein (rHN), the second truncated HN protein (rHN2), and the truncated F protein (rF) with commercial anti-His tag antibody, consistent with one or more exemplary embodiments of the present disclosure. C: Negative control (a protein without histidine-tag). Referring to FIG. 2B, the exemplary recombinant protein and other recombinant segments were detected with anti-His tag antibody, and it confirms the production of each exemplary chimeric protein (rLHN2F), the truncated LTB (rLTB), the first truncated HN protein (rHN), the second truncated HN protein (rHN2), and the truncated F protein (rF) in the bacterial host.

[0053] EXAMPLE 2: BIOINFORMATIC ANALYSIS OF EXEMPLARY CHIMERIC PROTEIN

[0054] In this example, bioinformatic analysis of exemplary chimeric protein (SEQ ID NO: 8), such as prediction of secondary and tertiary structures, molecular dynamics, physicochemical properties, antigenicity, and allergenicity of the exemplary chimeric protein, were done.

[0055] The secondary structure of rLHN2F was predicted by different online servers, such as GorIV, PSI pre, PHYRE, Porter, SOPMA, YASPI, and SCRATCH. The PSIPRED protein structure visualization server was used to generate and visualize schematic annotations of secondary structures. FIG. 3 illustrates a predicted secondary structure of the exemplary chimeric protein by PSIPRED software (C = Coil, H = Alpha-helix, and E = Beta plates), consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 3, the exemplary chimeric protein is predicted to contain 101(21.17%) beta plates, 160 (34.48%) alpha-helix and 203 (43.75%) random coil structures.

[0056] Also, three-dimensional (3D) structure and homology modeling for the exemplary chimeric protein was performed by LTASSER, RaptorX, Swiss-model, Modfold6, and Modeller 9.18 servers. The D.S. visualizer 1.7 was applied to visualize the modeled 3D structures. FIG. 1 illustrates a three-dimensional structure of an exemplary chimeric protein for immunization against the Newcastle disease virus predicted by PDSV software, consistent with one or more exemplary embodiments of the present disclosure. [0057] All 3D models were estimated using the protein structure validation software (PSvS), ProSA-web, and SuperPose server to validate the structural quality and compare with native structures to identify possible errors in modeled structures. A validation quality process was performed to identify potential errors in the 3D structures, identify better fits of the model in predicted structures, and compare the model before and after the refinement by using score and root mean square deviation (RMSD) (TABLE 1). Referring to TABLE 1, comparing the structure of the exemplary chimeric protein with the native structure of each protein segment showed that the alpha-helix and beta sheets formed in the exemplary chimeric protein are consistent with the native structures of each protein segment.

[0058] TABLE 1 : Structural comparison of components of the exemplary chimeric protein with natural proteins by SuperPose software

Protein PDB RMSD Identity Similarity Gaps Score

I I B 1644 L21 22.0% 22.0% 77.2% 515.0

HN le8t 9.817 19.6% 26.6% 48.7% 362.5

F lg5g 35.87 10.6% 11.5% 86.6% 322.5

[0059] Also, molecular dynamics simulation prediction was applied to determine the dynamic stability of the best-designed model by the GROMACS package. In this method, the interaction between atoms and molecules over time is simulated by a computer based on physics laws. The RMSD index is used to evaluate the simulated structure's stability. FIG. 4A illustrates the RMSD plot of the exemplary chimeric protein, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 4A, based on RMSD analyses of the exemplary chimeric protein by molecular dynamics, the exemplary chimeric protein is in the normal range of structural stability and has no structural changes during the study (100 ns) and reaches a steady situation at 20 ns.

[0060] FIG. 4B illustrates RMS fluctuation of the exemplary chimeric protein, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 4B, in the RMS diagram, scores of amino acid residues between zero and 0.2 nm showed normal vibration and fluctuation in the exemplary chimeric protein.

[0061] Moreover, various physicochemical properties of the exemplary chimeric protein were assessed using Expassy ProtParam (TABLE 2). Referring to TABLE 2, the exemplary chimeric protein has a molecular weight of about 49.7 kDa with 458 amino acids. The exemplary chimeric protein has an instability index of about 35.7, which indicates that the exemplary chimeric protein (rLHN2F) can be classified as a stable protein. Also, the aliphatic index and grand average hydrophobicity (GRAVY) of the exemplary chimeric protein are 77.35 and -0.378, respectively. In addition, the half-life of the exemplary chimeric protein in mammalian reticulocytes (in-vitro), in yeast, and in E. coli (in-vivo) are estimated to be about 30 hours, >20 hours, and >10 hours, respectively.

[0062] TABLE 2: Physical and chemical properties of the exemplary chimeric protein using Expassy ProtParam. No. Molecular Isoelectric Instability Aliphatic Grand average of amino weight(Da) pH index: index hydropathicity acids

458 49722.27 8.84 35.70 77.35 -0.378

[0063] Besides, the chemical characteristics of the exemplary chimeric protein were examined using the Ramachandran analysis. FIG. 5 illustrates the Ramachandran diagram of the exemplary chimeric protein based on the designed model, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 5, according to the Ramachandran diagram, 92.6% of the amino acids of the exemplary chimeric protein (rLHN2F) are in the desired areas, 5.2% of the amino acids are in the allowed areas, and only 2.2% of them are outside the allowed areas, which confirms the correct design of the protein. [0064] The antigenicity of the exemplary chimeric protein (rLHN2F) and its recombinant components (rLTB, rHN, rF) was predicted using different web-based servers, including ANTIGENpro, Vaxijen, and EMBOSS (TABLE 3). Referring to TABLE 3, the results showed that the antigenicity of the exemplary chimeric protein (rLHN2F) and its recombinant components (rLTB, rHN, rF) exceeded the threshold defined by the software. As a result, it is confirmed that the exemplary chimeric protein (rLHN2F) and its recombinant components (rLTB, rHN, rF) are antigenic and could provide good antigenicity. TABLE. 4 represents the allergenicity analysis of the exemplary chimeric protein (rLHN2F), and it can be concluded that the exemplary chimeric protein (rLHN2F) is not classified as an allergen protein.

[0065] TABLE 3 : Prediction of antigenicity of the exemplary chimeric protein (rLHN2F) and its recombinant components (rLTB, rHN, rF)

Protein Web-based servers Threshold Antigenicity score

Vaxijen 0.4 0.4508 (Probable ANTIGEN) rLHN2F Antigenpro 0.65 0.928353 (Probable ANTIGEN)

EMBOSS - 21 (Probable ANTIGEN)

Vaxijen 0.4 0.4545 (Probable ANTIGEN). rF Antigenpro 0.65 0.786855 (Probable ANTIGEN)

EMBOSS - 9 (Probable ANTIGEN)

Vaxijen 0.4 0.5816 (Probable ANTIGEN) rHN Antigenpro 0.65 0.814181 (Probable ANTIGEN)

EMBOSS - 4 (Probable ANTIGEN)

Vaxijen 0.4 0.4458 (Probable ANTIGEN). rLTB Antigenpro 0.65 0.925804 (Probable ANTIGEN)

EMBOSS - 8 (Probable ANTIGEN)

[0066] TABLE 4: Allergenicity analysis of the exemplary chimeric protein (rLHN2F)

Software Allergenicity of rLHN2F algpred NON-ALLERGEN

SDAP NON-ALLERGEN

ADFS NON-ALLERGEN farrp NON-ALLERGEN

Allerton NON-ALLERGEN allergome NON-ALLERGEN

[0067] The exposure and accessibility of the epitopes were re-examined using the algorithm of linear and conformational B cell and T cell epitopes servers to ensure that the epitopic regions in the final structure of the exemplary chimeric protein (rLHN2F) are exposed (TABLE 5). Referring to TABLE 5, the exposure and accessibility analysis results confirm the presence and exposure of the desired epitopic regions of the exemplary chimeric protein, which is consistent with the natural epitopic domains of the LTB, HN, and F proteins.

[0068] TABLE 5: Prediction of linear B-cell epitopic regions of the exemplary chimeric protein (rLHN2F) using BCEPRED site

Prediction Parameters Epitope

Hydrophilicity 11-17, 79-85, 90-96, 103-120, 150-162, 167-180, 195-216, 246-258,

263-276, 291-307, 315-321, 322-330, 340-348, 373-386, 414-420

Flexibility 57-63, 87-94, 174-155, 182-188, 243-252, 278-284, 321-328, 363-369,

374-384, 447-454

Accessibility 10-20, 30-37, 57-86, 88-97, 103-120, 148-194, 200-216, 244-290,

296-307, 323-329, 336-348, 350-360, 367-376, 450-461

Exposed surface 58-74, 79-86, 90-96, 150-156, 162,172, 179-182, 246-252, 258-268,

270-278, 337-346, 450-457

Polarity 8-14, 30-41, 58-74, 79-86, 103-120, 162-168, 200-216, 258-262, 296-

307, 338-345, 368-376, 420-429, 431-439, 450-464

Antigenic propensity 39-45, 48-59, 82-89, 139-149, 163-169, 235-245, 259-265, 329-339

Terns 150-157, 246-252, 456-464

[0069] Epitopic regions of the exemplary chimeric protein (rLHN2F) were also predicted using PDSV software. FIG. 6A illustrates the prediction of the antigenic regions of the truncated LTB protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure. FIG. 6B illustrates the prediction of the antigenic regions of the first truncated HN protein in the exemplary chimeric protein

(rLHN2F), consistent with one or more exemplary embodiments of the present disclosure.

[0070] FIG. 6C illustrates the prediction of antigenic regions of the second truncated HN protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure. FIG. 6D illustrates the prediction of the antigenic regions of the truncated F protein in the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure.

[0071] EXAMPLE 3: IN-VIVO IMMUNOGENICITY OF EXEMPLARY CHIMERIC PROTEIN

[0072] In this example, the in-vivo immunogenicity of the exemplary chimeric protein was examined. To evaluate the antigenicity of each segment of the exemplary chimeric protein, thirty-five female Balb/c mice (5-6 weeks old) were randomly divided into seven treatment groups (A to G). The group A, B, C, D, and E were respectively immunized with 15 pg of the purified truncated LTB, the truncated first HN protein, the truncated second HN protein, the truncated F protein, and the exemplary chimeric protein via subcutaneous injection in the back of the neck using complete Freund’s adjuvant.

[0073] The immunization continued with two injections of the same doses (10 pg) via subcutaneous injection with the incomplete Freund’s adjuvant and one booster (2 pg) via intraperitoneal route, without adjuvant. Group F (negative control) was injected with PBS, and the last group G (positive control), was immunized with a commercial vaccine against the Bl strain with the same immunization procedure. Immunizations were performed four times within five weeks. TABLE 7 represents the immunization process of mice with exemplary chimeric protein, different segments of the exemplary chimeric protein, or the commercial vaccine.

[0074] TABLE 7: Procedure of mice immunization with the exemplary chimeric protein

Dose Day No. Amount of the protein (pg) Freund’s adjuvant Injection method

1 0 15 Complete Subcutaneous

2 7 10 Incomplete Subcutaneous

3 15 5 Incomplete Subcutaneous

4 23 2 - Intraperitoneal

[0075] The antisera were prepared from the blood sample of each immunized mouse after the second, third, and final immunization (1^st, 2^nd, and 3^rd booster). The antisera were stored at about -70 °C until further experiments. The serum samples of each mouse were pooled for immunological analyses. The antisera were used for checking the immune cross -reactivity of all segments. The immune cross-reactivity was verified with specific mouse anti-LTB, anti- HN, anti-HN2, anti-F, anti-LHN2F (an antibody against the exemplary chimeric protein), and commercial anti-His antibodies in Western blotting.

[0076] FIG. 7A illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) and its recombinant segments by antisera of mice immunized with the commercial vaccine against Bl strain as the positive control, consistent with one or more exemplary embodiments of the present disclosure. M: protein molecular weight marker (KD), C: Negative control (protein without histidine-tag). Referring to FIG. 7A, Western blotting showed that the serum of the mice immunized with the commercial Newcastle Bl vaccine could detect the exemplary chimeric protein (rLHN2F), the first truncated HN protein, the second truncated HN protein, and the truncated F protein except for the truncated LTB because the LTB is a bacterial protein and is not included in the commercial Newcastle vaccine.

[0077] FIG. 7B illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) and its recombinant segments by antisera of mice immunized with the exemplary chimeric protein, consistent with one or more exemplary embodiments of the present disclosure. M: protein molecular weight marker (KD), C': Negative control (protein without histidine-tag). Referring to FIG. 7B, the exemplary chimeric protein and all its recombinant segments are detected by antisera of mice immunized with the exemplary chimeric protein containing mice anti-LHN2F antibodies.

[0078] FIG. 7C illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) by antisera of mice immunized with the exemplary chimeric protein (1), truncated LTB (2), first truncated HN (3), second truncated HN2 (4), and truncated F (5) segments, consistent with one or more exemplary embodiments of the present disclosure. M: protein molecular weight marker (KD), C“: Negative control (protein without histidine-tag). Referring to FIG. 7C, all the antibodies against each component can also detect the complete exemplary chimeric protein (rLHN2F).

[0079] FIG. 7D illustrates the results of Western blotting analysis of the exemplary chimeric protein (rLHN2F) and its recombinant segments by antisera of mice immunized with the exemplary chimeric protein, truncated LTB, first truncated HN, second truncated HN2, and truncated F segments, consistent with one or more exemplary embodiments of the present disclosure. M: protein molecular weight marker (KD), C“: Negative control (protein without histidine-tag). Referring to FIG. 7D, antisera of mice immunized with the exemplary chimeric protein, truncated LTB, first truncated HN, second truncated HN2, and truncated F segments include specific antibodies against each segment which can also detect each segment separately.

[0080] Referring again to FIGs. 7A-7D, Western blotting data indicated that antibodies produced in the serum of the mice immunized with the exemplary chimeric protein could detect the truncated LTB, the first truncated HN protein, the second truncated HN protein, the truncated F protein fragments, and vice versa. The antibody response of each recombinant component against the exemplary chimeric protein (rLHN2F) is detectable.

[0081] Moreover, serum humoral immune responses of mice to the exemplary chimeric protein and each recombinant segment were evaluated by performing an enzyme-linked immunosorbent assay (ELISA). The immune and control mice sera were analyzed by ELISA to identify the quantity of IgG class antibodies raised versus all recombinant proteins. In this experiment, about 200 ng of the purified rLTB, rHN, rHN2, rF, and the exemplary chimeric protein (rLHN2F) were used as antigens. The serial dilution of antiserum against each recombinant protein from the immunized mice (1:6400) and antibodies to mouse IgG labeled with horseradish peroxidase was added to the wells of the ELISA plate as primary and secondary antibodies. The colorimetric reaction was visualized using O -phenylenediamine (OPD), and the reactions were stopped by Sulfuric acid (2.5 M) in each well. Non-immunized mice injected with PBS, and adjuvant sera were used as control (p < 0.05). The error bar is the standard deviation.

[0082] FIG. 8A illustrates the result of specific serum IgG response to the exemplary chimeric protein (rLHN2F) in mice immunized with rLHN2F and in sera of control mice, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 8A, ELISA results indicated appropriate immune system stimulation against each recombinant protein and the highest titer of immune response achieved in mice immunized with the exemplary chimeric protein (rLHN2F) even at 1:409600 dilutions. The specific IgG class antibodies against rLTB, rHN, rHN2, rF, rLHN2F proteins, and Bl strain (as a positive control) were detected after immunization in the sera of the immunized mice. After each injection, a significant increase in antibody titer in comparison with the control was observed, which is noticeable (a significant difference (p < 0.05).

[0083] The cross -reactivity ELISA analyses of sera titration from mice immunized with the exemplary chimeric protein (rLHN2F) against all the rLHN2F and its recombinant segments were compared and shown in FIG. 8B and FIG. 8C. Antibodies titration is calculated at AB492 nm and 1:6400 dilution.

[0084] FIG. 8B illustrates the result of IgG titration of sera from mice immunized with the exemplary chimeric protein (rLHN2F) after the last injection in response to 200 ng of the rLHN2F and its recombinant segments, including rLTB, rHN, rHN2, and rF, consistent with one or more exemplary embodiments of the present disclosure. FIG. 8C illustrates the result of titration of sera from mice immunized with each of rLTB, rHN, rHN2, rF, rLHN2F, and Bl against the exemplary chimeric protein (rLHN2F), consistent with one or more exemplary embodiments of the present disclosure. Referring to FIGs. 8B and 8C, the highest adsorption intensity at 492 nm was shown first against rLHN2F, then rHN2, rF, rHN, and rLTB, respectively. Inversely, the highest adsorption uptake of rLHN2F protein was induced by antibodies against rLHN2F, Bl, rHN2, rF, rHN, and rLTB, respectively. This finding can indicate the immunogenicity of the exemplary chimeric protein and its components.

[0085] Moreover, cross-reactivity analysis of sera of mice immunized against a commercial vaccine against the B 1 strain of NDV and mice immunized against the exemplary chimeric protein was done. Antibodies titrations are calculated at AB492 nm and 1:6400 dilutions. Control mice were injected with PBS and adjuvant (p < 0.05). The error bar is the standard deviation. FIG. 9 illustrates ELISA results of cross -reactivity analysis by titration of sera of mice immunized with commercial B l vaccine and with the exemplary chimeric protein (rLHN2F) versus the exemplary chimeric protein and its recombinant components, consistent with one or more exemplary embodiments of the present disclosure.

[0086] Referring to FIG. 9, the ELISA results confirmed that the higher dilutions of the antiNewcastle Bl antibody could detect the exemplary chimeric protein and its recombinant components except for rLTB, which is not included in the commercial Newcastle vaccine against BL It is shown that the exemplary chimeric protein has the same antigenicity and specific reactivity as the Newcastle B 1 vaccine in mice. As a result, it can be suggested that the exemplary chimeric protein can be used instead of the commercial Newcastle Bl vaccine for vaccination.

[0087] Furthermore, the immunogenicity of the exemplary chimeric protein was assessed by measuring IgG antibody response in mice sera on different days after the first immunization and compared with the commercial vaccine against the Bl strain. A student t-test was performed to analyze and compare treatment groups at the 5% level. The error bar is the standard deviation.

[0088] FIG. 10 illustrates the result of serum antibody response of the antibodies from the immunized mice with the exemplary chimeric protein (rLHN2F) and with the commercial vaccine against the B l strain by ELISA at 14-, 22-, and 30-day post first immunization, consistent with one or more exemplary embodiments of the present disclosure. Referring to FIG. 10, there was a significantly higher antibody response in the sera of mice immunized with the exemplary chimeric protein (anti-LHN2F group) at 14- and 22 days post first immunization compared with the sera of mice immunized with the commercial vaccine against the B l strain (anti-Bl group) (p < 0.05). However, there is the same titer of IgG antibody in both anti-LHN2F and anti-B 1 groups at 30-day post-first immunization.

INDUSTRIAL APPLICABILITY

[0089] Applicants have found that exemplary chimeric protein may particularly be utilized for industrial applications. By way of example, industrial applications may include veterinary medicine and poultry industry by using exemplary chimeric protein for vaccination against the Newcastle disease virus (NDV). Also, an exemplary chimeric protein may be suited for industrial purposes like producing diagnostic kits of NDV in biological samples of poultry due to its high sensitivity and rapid detection.

[0090] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[0100] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0101] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[0102] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0103] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "a" or "an" does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0104] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it can be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter.

[0105] While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

WHAT IS CLAIMED IS:

1. A chimeric protein for immunization against Newcastle disease virus, the chimeric protein comprising: a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli; a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus connected to the truncated LTB via a first linker; a second truncated HN protein of the Newcastle disease virus connected to the first truncated HN protein via a second linker; and a truncated fusion (F) protein of the Newcastle disease virus connected to the second truncated HN protein via a third linker.

2. The chimeric protein of claim 1, wherein the first and the second truncated HN proteins comprise SEQ ID NO: 2.

3. The chimeric protein of claim 1, wherein the truncated F protein comprises SEQ ID NO: 3.

4. The chimeric protein of claim 1, wherein the truncated LTB comprises SEQ ID NO: 1.

5. The chimeric protein of claim 1, wherein the first, the second, and the third linkers comprise between 2 and 5 hydrophobic units of SEQ ID NO: 4.

6. The chimeric protein of claim 5, wherein: the first and the third linkers comprise SEQ ID NO: 5, and the second linker comprises SEQ ID NO: 6.

7. The chimeric protein of claim 1, wherein the chimeric protein is encoded from a nucleic acid molecule of SEQ ID NO: 7.

8. The chimeric protein of claim 7, wherein the chimeric protein comprises SEQ ID NO: 8.

9. The chimeric protein of claim 1, wherein the chimeric protein further comprises a tag conjugated to the truncated F protein, the tag comprising at least one of a histidine tag (His- tag), glutathione S transferase (GST) tag, and a maltose binding protein (MBP) tag.

10. A method for vaccination against Newcastle disease virus, the method comprising: administering a composition to a bird, the composition comprising a chimeric protein, the chimeric protein comprising: a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli; a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus connected to the truncated LTB via a first linker; a second truncated HN protein of the Newcastle disease virus connected to the first truncated HN protein via a second linker; and a truncated fusion protein (F) of the Newcastle disease virus connected to the second truncated HN protein via a third linker. The method of claim 10, wherein the chimeric protein comprises: a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli with SEQ ID NO: 1; a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus with SEQ ID NO: 2 connected to the truncated LTB via a first linker; a second truncated HN protein of the Newcastle disease virus with SEQ ID NO: 2 connected to the first truncated HN protein via a second linker; and a truncated fusion protein (F) of the Newcastle disease virus with SEQ ID NO: 3 connected to the second truncated HN protein via a third linker. The method of claim 10, wherein administering the composition to the bird comprises administering the composition to the bird through at least one of injection, aerosol, drinking water, oral, an eye drop, and an edible vaccine. The method of claim 10, wherein administering the composition to the bird comprises administering the composition with an amount between 30 pg and 50 pg to 3 -week-old chicken. The method of claim 10, wherein the composition further comprises a pharmaceutically acceptable carrier, the pharmaceutically acceptable carrier comprising at least one of nanoparticles, mesoporous silica, chitosan, liposomes, poly lysin, and poly(lactic-co-glycolic acid) (PLGA). A method for detecting Newcastle disease virus infection in biological samples of birds, the method comprising: putting a biological sample of a bird in contact with a reagent, the reagent comprising: a chimeric protein comprising: a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli', a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus connected to the truncated LTB via a first linker; a second truncated HN protein of the Newcastle disease virus connected to the first truncated HN protein via a second linker; and a truncated fusion protein (F) of the Newcastle disease virus connected to the second truncated HN protein via a third linker; and a tag conjugated to the truncated F protein; and determining the presence of Newcastle disease virus infection in the biological sample responsive to detecting the chimeric protein bound to antibodies against the Newcastle disease virus in the biological sample. The method of claim 15, wherein the chimeric protein comprises: a truncated heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli with SEQ ID NO: 1; a first truncated hemagglutinin-neuraminidase (HN) protein of a Newcastle disease virus with SEQ ID NO: 2 connected to the truncated LTB via a first linker; a second truncated HN protein of the Newcastle disease virus with SEQ ID NO: 2 connected to the first truncated HN protein via a second linker; and a truncated fusion protein (F) of the Newcastle disease virus with SEQ ID NO: 3 connected to the second truncated HN protein via a third linker. The method of claim 15, wherein detecting the chimeric protein bound to the antibodies against the Newcastle disease virus comprises conducting at least one of a chemiluminescent assay, an immunofluorescent assay, enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI) assay, radioimmunoassay, a Western blot assay, an enzyme immunoassay, an immunoprecipitation assay, an immunohistochemical assay, an immunochromatographic assay, a dot blot assay, a slot blot assay, confocal imaging, laser scanning microscopy, and flow cytometry. The method of claim 15, wherein putting the biological sample in contact with the reagent comprises putting at least one of a blood sample, a plasma sample, a serum sample, an egg yolk sample, a fecal sample, a cloacal swab, an oropharyngeal swab, a tracheal swab, a biopsy sample, and an autopsy sample in contact with the reagent. The method of claim 15, wherein the tag comprises at least one of a radioactive substance, a dye, a contrast agent, a fluorophore molecule, a nanoparticle, a bioluminescent molecule, an affinity agent, and a magnetic agent. The method of claim 15, wherein detecting the chimeric protein bound to antibodies against the Newcastle disease virus in the biological sample comprises detecting the chimeric protein bound to antibodies against at least one of Exotic, AF2240, Bl, Bl-HITCHNER/47, Bl/48, CHI/85, chicken/India/UP3/2006, class 1, Connecticut/9- 12-60, goose/GPMV- SH/CHN/2009, isolate RJAZ96, LIP97, Miyadera/51, NDV Ow/Tw/2209/95, Qingdao/S D/1/97, 3/91, AUSTRALIA- VICTORIA/32, Beaudette C/45, BOR74, BOR82, D26/76, F48E9, GAM61, GPMV/QY97-1, H, HER/33, IBA/85, Italien/45, Kansas, LAS/46, Queensland/66, T53, TEXAS, TEXAS G.B./48, VOL95, Ulster/2C, and Ulster/67 strains of Newcastle disease virus the biological sample.