Generation of viral protein antigens using fungal constructs

Abstract

The present disclosure relates to the expression of recombinant viral antigens using transgenic fungi. Compositions, production and administration of vaccines comprising those viral antigens are also disclosed. In some embodiments, these viral antigens can be used to formulate vaccines against Newcastle disease. These vaccines may be administered, for example, by intramuscular injection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of an earlier filing date of U.S. Provisional Application No. 63 / 344,252, filed on May 20, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The nucleic acid and amino acid sequences listed in the attached sequence table are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 C.FR § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included in any reference to the displayed strand. The sequence table is submitted as an XML file in the form of a file named "10177-106793-02_ST26.xml" (about 139,557 bytes), which was created on May 11, 2023 and is incorporated herein by reference. In the attached sequence table:

[0005] SEQ ID NO: 1 is the amino acid sequence of the native C1 CBH1ss secretion signal.

[0006] SEQ ID NO: 2 is the amino acid sequence of the C-tag purification tag.

[0007] SEQ ID NO: 3 is the amino acid sequence of the NDV F protein (F [Avian orthoavulavirus 1]) from GenBank AZP53700.1.

[0008] SEQ ID NO: 4 is the amino acid sequence of NDV hemagglutinin-neuraminidase (AA [Avian Orthomopsis Virus 1]) from GenBank AEU17158.1 Accession No. JQ013837.1.

[0009] SEQ ID NO:5 is the nucleic acid sequence that is part of the expression construct pMYT1358 and encodes the synthetic F protein (amino acid sequence) of SEQ ID NO:6.

[0010] SEQ ID NO:6 is the amino acid sequence translation of SEQ ID NO:5 and contains a secretion signal.

[0011] SEQ ID NO:7 is the amino acid sequence of SEQ ID NO:6, wherein the secretion signal is cleaved.

[0012] SEQ ID NO:8 is the nucleic acid sequence that is part of the expression construct pMYT1359 and encodes the synthetic F protein of SEQ ID NO:9.

[0013] SEQ ID NO:9 is the amino acid sequence of the translated synthetic F protein of SEQ ID NO:8 and contains the secretion signal.

[0014] SEQ ID NO: 10 is the mature protein of SEQ ID NO: 9, wherein the secretion signal is cleaved.

[0015] SEQ ID NO:11 is a sequence containing a nucleic acid sequence that is part of the expression construct pMYT1360 and encodes the amino acid sequence of SEQ ID NO:12.

[0016] SEQ ID NO:12 is the amino acid sequence of the synthetic NDV F protein translated from SEQ ID NO:11.

[0017] SEQ ID NO: 13 is the mature protein of SEQ ID NO: 12, wherein the secretion signal is cleaved.

[0018] SEQ ID NO: 14 is the nucleic acid sequence of the synthetic NDV F protein of expression constructs pMYT1361, pMYT1736, pMYT1737 and pMYT1738. It encodes the amino acid of SEQ ID NO: 15.

[0019] SEQ ID NO: 15 is the amino acid sequence of the synthetic NDV F protein encoded by SEQ ID NO: 14 and contains the secretion signal.

[0020] SEQ ID NO: 16 is the amino acid sequence of the mature protein of SEQ ID NO: 15, wherein the secretion signal is cleaved.

[0021] SEQ ID NO: 17 is the nucleic acid sequence of a synthetic NDV HN protein that is part of the expression construct pMYT1362. Translation of SEQ ID NO: 17 produces the amino acid sequence of SEQ ID NO:18.

[0022] SEQ ID NO: 18 is the translated amino acid sequence of SEQ ID NO: 17 of the synthetic NDV HN protein and contains the secretion signal.

[0023] SEQ ID NO: 19 is the mature amino acid sequence of SEQ ID NO: 18, wherein the secretion signal is cleaved.

[0024] SEQ ID NO:20 is a nucleic acid coding sequence that is part of the expression construct pMYT1363 and corresponds to the amino acid sequence of SEQ ID NO:21 of the synthetic NDV HN protein.

[0025] SEQ ID NO:21 is the amino acid sequence of SEQ ID NO:20, and contains a secretion signal which, when cleaved, produces a mature protein of the amino acid sequence of SEQ ID NO:22.

[0026] SEQ ID NO:22 is the mature amino acid sequence of SEQ ID NO:21, wherein the secretion signal is cleaved.

[0027] SEQ ID NO:23 is the nucleic acid coding sequence of the synthetic NDV HN protein, which constitutes a part of the expression construct pMYT1364.

[0028] SEQ ID NO:24 is the amino acid sequence of SEQ ID NO:23 of the synthetic NDV HN protein.

[0029] SEQ ID NO:25 is the amino acid sequence of SEQ ID NO:24, wherein the secretion signal is cleaved.

[0030] SEQ ID NO:26 is the nucleic acid coding sequence of the synthetic NDV HN protein, which is part of the expression constructs pMYT1365, pMYT1741, pMYT1742 and pMYT1743. Translation of this sequence yielded the amino acid sequence of SEQ ID NO:27.

[0031] SEQ ID NO:27 corresponds to the synthetic NDV HN protein containing the secretion signal and encoded by SEQ ID NO:26.

[0032] SEQ ID NO:28 is the amino acid sequence of SEQ ID NO:27, wherein the secretion signal is cleaved.

[0033] SEQ ID NO:29 contains the nucleic acid sequence of the C1 vector pMYT1055.

[0034] SEQ ID NO: 30 is the amino acid sequence of the NDV matrix protein [Avian Orthomumps Virus 1] from NCBI reference sequence YP_009513196.1.

[0035] SEQ ID NO: 31 is the amino acid sequence of NDV phosphoprotein [Avian Orthomumps Virus 1] from NCBI reference sequence YP_009513195.1.

[0036] SEQ ID NO: 32 is the amino acid sequence corresponding to the NDV nucleocapsid protein [Avian Orthomitis Virus 1] from NCBI reference sequence YP_009513194.1.

[0037] SEQ ID NO:33 contains the nucleic acid sequence of C1 vector pMYT1140.

[0038] SEQ ID NO:34 contains the nucleic acid sequence of C1 vector pMYT1727.

[0039] SEQ ID NO:35 contains the nucleic acid sequence of C1 vector pMYT1729.

[0040] SEQ ID NO:36 contains the nucleic acid sequence of the C1 vector pMYT1730.

[0041] SEQ ID NO:37 contains the nucleic acid sequence of the C1 vector pMYT1731.

[0042] SEQ ID NO:38 contains the amino acid sequence of the T4 fibrin trimerization domain.

[0043] SEQ ID NO:39 contains the amino acid sequence of the GCN4 trimerization domain.

[0044] SEQ ID NO:40 contains the nucleic acid sequence of the IgY CH antibody fragment.

[0045] SEQ ID NO:41 contains the amino acid sequence of the IgY CH antibody fragment.

[0046] SEQ ID NO:42 contains the nucleic acid sequence of the IgY CL antibody fragment.

[0047] SEG ID NO:43 contains the amino acid sequence of the IgY CL antibody fragment.

[0048] SEQ ID NO:44 is the NDV nucleic acid sequence encoding the full-length hemagglutinin-neuraminidase (HN) protein (GenBank: MH377274.1).

[0049] SEQ ID NO: 45 is the NDV nucleic acid sequence encoding the full-length NDV fusion (F) protein (GenBank: MH377274.1). Technical Field

[0050] The present disclosure relates to embodiments of methods for producing viral antigens using genetic recombination and fungal expression systems, vaccines comprising the viral antigens, and methods of using the vaccines.

[0051] Parties to the Joint Research Agreement

[0052] Certain of the information disclosed herein resulted from a joint research collaboration between Phibro Animal Health Corporation and Dyadic International, Inc. Background Art

[0053] Newcastle disease (ND) is an economically burdensome disease that occurs in nearly 230 species of poultry. Outbreaks can be particularly devastating in poultry farms. In chickens, symptoms of the disease include respiratory distress, such as wheezing, sneezing and coughing; neurological symptoms as evidenced by wing weakness, neck twisting and muscle tremors; and other signs of discomfort, such as green watery diarrhea, and the production of thin-shelled and / or deformed eggs. The mortality rate can be 90%-100% 8 days after exposure to the avian mumps virus 1 pathogen (which is also known as avian mumps virus, avian paramyxovirus 1 or Newcastle disease virus (NDV)). The virus is included in the Paramyxoviridae family along with the pathogens of measles, mumps and canine distemper. Different strains of the virus can cause different clinical signs of infection. Rapid strains (highly toxic) can cause necrotic hemorrhagic lesions in the intestine, leading to morbidity and mortality rates as high as 100%. Mesogenic strains (moderate virulence) can cause respiratory and neurological symptoms, with infection resulting in moderate morbidity and mortality. Meanwhile, lentogenic strains (low virulence) usually cause only minimal respiratory distress and are sometimes the strains used to prepare attenuated vaccines.

[0054] The genome of NDV strain is generally 14.9 to 17.4 kilobases, and is a negative sense single-stranded RNA. There are at least 6 genes in the genome, which include nucleocapsid, phosphoprotein, matrix, fusion protein, hemagglutinin-neuraminidase attachment protein and RNA dependent RNA polymerase. Partly due to the lack of error detection function to RNA polymerase, these viruses are prone to mutation and can be rapidly differentiated into new strains, which usually frustrates vaccination work. Fusion protein (F) and hemagglutinin-neuraminidase (HN) protein are two necessary glycoproteins for the ability of virus to establish host cell infection.

[0055] The F protein is naturally present on the viral envelope of NDV. The protein is expressed as an inactive F0 protein, which is then cut into F1 and F2 subunits by host proteases. These subunits then promote the fusion event that causes viral infection of the host cell. Specifically, the activated F protein subunit undergoes conformational changes to produce holes in the host cell membrane, through which the virus passes. The HN protein assists this process by initially recognizing and attaching to receptors on the host cell.

[0056] The virulence of a particular NDV strain depends on the amino acid sequence of the cleavage site of the F0 protein. Oligoplasmic cleavage site motifs (RRQR / KR) can be cleaved by more common and promiscuous proteases, and correspond to more toxic strains. In contrast, monobasic cleavage site motifs (GR / KQGR) can only be cleaved by a smaller number of tissue-specific proteases, and correspond to less toxic strains.

[0057] Although commercial NDV vaccines are available, there is still an outbreak of high mortality in poultry farms. The high mutation rate of the virus often leads to new strains, and available vaccines do not always provide immunity for this type of new strain. Therefore, even the colony of vaccination is still susceptible to toxic, differentiated NDV strains. Therefore, there is an urgent need for new effective NDV vaccines and because the NDV strains suddenly change, the method for producing new antigens quickly is needed. Summary of the invention

[0058] Certain disclosed embodiments include transgenic fungal cells comprising a recombinant gene comprising a fungal promoter operably linked to a nucleic acid sequence encoding a Newcastle disease virus antigenic peptide. An example of a suitable transgenic fungus is Thermothelomyces heterothallica (C1). Certain disclosed embodiments use Thermothelomyces heterothallica strains DNL155, DNL157, M5355, M5739, and strains that produce different glycan patterns (such as Man 3-9 , Man3, G0 and / or G2).

[0059] The transgenic fungi may express recombinant antigenic peptides or fragments that elicit an immunogenic response in a subject, such as poultry, such as recombinant antigenic peptides or fragments from Newcastle disease virus. The antigenic peptides may be derived from any viral protein or portion thereof that elicits an immune response, such as NDV F protein, NDV HN protein, or a combination of NDV F protein and NDV HN protein. In some embodiments, the transgenic fungal cell contains a recombinant gene containing a nucleic acid sequence encoding an antigenic peptide containing a sequence having at least 90% similarity to a sequence disclosed herein, such as SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 44, 45, or a combination thereof. Certain disclosed embodiments include the use of transgenic fungal cells comprising a recombinant gene comprising a nucleic acid sequence corresponding to an amino acid sequence having at least 90% similarity to SEQ ID NO:3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32, or a combination thereof. In some embodiments, the episome pMYT1055 (SEQ ID NO:29) can be used as a vector to transform fungi with the antigenic peptide. In some embodiments, the recombinant antigenic peptide comprises a signal sequence, and in specific embodiments, the signal sequence corresponds to the CBH1ss secretion signal sequence (SEQ ID NO:1). In some embodiments, the antigenic peptide sequence can be operably linked to a fungal promoter (e.g., bgl8).

[0060] Other disclosed embodiments include a method for preparing a transgenic fungal cell by inserting an exogenous gene into the chromosome of a fungal cell. The group of exogenous genes may include a nucleic acid sequence encoding an antigenic peptide (such as an antigenic NDV protein or a portion thereof) that is operably connected to a fungal promoter and a fungal terminator.

[0061] Also disclosed are recombinant antigenic peptides produced by transgenic fungi. These recombinant antigenic peptides produce immunogenic responses in subjects (such as poultry). These antigenic peptides may include SEQ ID NO:3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31 or 32 or an amino acid sequence of a combination thereof. It will be appreciated by those of ordinary skill in the art that the number of amino acids forming such peptides may vary, but generally such peptides include 5 to 1000 amino acid residues, more generally include 8 to 800 amino acid residues. In other embodiments, the amino acid residues of any antigenic peptide may be modified. In other embodiments, the antigenic peptide may be conformationally stable by adding a trimerization domain. The antigenic peptide may also be fused with an antibody or antibody fragment to selectively target the antigenic peptide to an immune cell of a subject.

[0062] An embodiment of disclosed method relates to produce recombinant antigen peptides from transgenic fungi by fermenting fungi in bioreactor, and the recombinant antigen peptides produced in the harvest under the situation of purification or non-purification.Purification can comprise filtration, C tag affinity chromatography, histidine tag affinity chromatography, size exclusion chromatography, fast protein liquid chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, reversed phase chromatography, immunoaffinity chromatography, ultracentrifugation, precipitation, and any combination and all combinations thereof.In a specific embodiment, fermentation raw material is filtered to remove non-target culture supernatant particles, and by any suitable method (such as C tag column combination and elution) from gained filtrate, purify target protein.

[0063] The recombinant antigenic peptides can also be harvested from the fermented transgenic fungi and can also be included in the extraction of protein from the fungal mycelium or biomass content of the bioreactor. In another embodiment, the antigenic peptides are harvested from the supernatant of the bioreactor content. In some embodiments, the temperature of the bioreactor fermentation process is controlled within a temperature range of about 20 ℃ to about 40 ℃ and more particularly 25 ℃ to 38 ℃. In other embodiments, the bioreactor comprises a fed-batch technology fermentor.

[0064] Vaccine preparations comprising one or more antigenic proteins are also disclosed.Except the antigenic peptides produced by transgenic fungi, disclosed vaccine preparations generally include other components, such as oil, emulsifiers and / or adjuvants.Some disclosed vaccine embodiments are water-in-oil emulsions, wherein oil is any acceptable oil, such as mineral oil.The vaccine can include a single antigenic peptide, or can include two or more antigenic peptides, such as one or more peptides selected from SEQ ID NO:3,4,6,7,9,10,12,13,15,16,18,19,21,22,24,25,27,28,30,31 or 32.The vaccine preparation can include a combination with other antigens (such as bacterial antigens, inactivated viruses, recombinant proteins, etc.).The vaccine can include a variety of emulsifiers and / or adjuvants, such as sorbitol-T and sorbitan oleate.Disclosed vaccines can be used as a combination with other vaccines or therapeutic agents.

[0065] Also disclosed is an embodiment of a method for preparing a vaccine by recombinant antigenic peptides produced by transgenic fungi. The method includes forming an oil-in-water emulsion comprising one or more antigenic peptides and other suitable vaccine components (including oil, water, emulsifiers and / or adjuvants).

[0066] Also described is a method of administering a disclosed vaccine embodiment to a subject. In one embodiment, the subject may be poultry, particularly chicken. In another embodiment, the subject is a human. The vaccine may be administered by any suitable method, such as spraying, dripping, smearing or injecting the subject with a vaccine formulation through the nose, mouth, skin, muscle or optical contact. The vaccine may be in the form of an aerosol, powder, liquid or solid. The vaccine may also be administered by intramuscular or subcutaneous injection. Another embodiment includes the continuous administration of two or more vaccine doses, such as the first administration to poultry, followed by a second administration 14 to 35 days after the first dose. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] This application contains at least one drawing executed in color. Copies of patent application publications or patents issuing from this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0068] Figure 1 is a schematic diagram illustrating an exemplary method of the present disclosure.

[0069] Figure 2 is a scheme showing the integration of the two arms of the expression cassette designed using the HygR split marker.

[0070] Figure 3 The transmembrane domain prediction of SEQ ID NO: 3 (native NDV F protein) is shown.

[0071] Figure 4 The predicted transmembrane domain of SEQ ID NO: 4 (native NDV HN protein) is shown.

[0072] Figure 5 Images of screening gels based on Western blots of recombinant F proteins expressed in DNL155 derivative strains are provided. Target protein detection was performed using chicken NDV antiserum and biotin-α-C tag conjugates as primary detection reagents and rabbit anti-chicken IgY-IRD680 (shown in red) and streptavidin-IRD800 (shown in green) as secondary detection reagents, respectively. The strain transformed with construct pMYT1358 expressed a full-length F protein with an expected product size of 55 kDa. The strain transformed with construct pMYT1361 expressed a recombinant F protein with a truncated C-terminal transmembrane domain and a modified protease cleavage site. The expected product size of this construct is 50 kDa. The expected product was detected in both the supernatant and the mycelial lysate. The bands marked with asterisks correspond to the transformed strains selected for fermentation and are represented as M5041.

[0073] Figure 6Images of Western blots and stained protein gels are provided for evaluating the optimization of fermentation conditions for expression of F protein in the C1 expression system, where: MT460 refers to fermentation of DNL155 transformed with pMYT1358 carrying full-length recombinant F protein expressed with an expected product size of 55 kDa at +25°C; MT461 refers to fermentation of DNL155 transformed with pMYT1361 carrying a truncated recombinant F protein with an expected product size of 50 kDa at +25°C; and MT455 refers to fermentation of the same construct in the same cell line but at +38°C. Target protein detection was performed with primary detection reagents NDV antiserum and biotin-α-C tag conjugate and secondary detection reagents rabbit anti-chicken IgY-IRD680 (shown in red) and streptavidin-IRD800 (shown in green), respectively. Target protein was produced in all three strains. Lowering the growth temperature from +38°C to +25°C increased the production level of the 50 kDa truncated recombinant F protein.

[0074] Figure 7 Provided is a gel image of the purification of NDV recombinant F protein from the supernatant produced in the MT455 bioreactor run for evaluation. Here, 1.5 mL of supernatant was collected from run MT455-6 after 7 days of fermentation and purified on a CaptureSelect C label XL 1 mL column. The binding buffer contained 1x PBS with 0.5 M NaCl, and the elution buffer was 20 mM Tris-HCl, 2 M MgCl2, with 1 mM EDTA pH 7.5. Target protein detection was shown with red NDV antiserum / rabbit anti-chicken IgY-IRD680 and with green biotin-α-C label / streptavidin-IRD800. Most of the target proteins with a predicted size of 50 kDa were eluted in the third and fourth fractions. The target protein was not detected in the flow-through fraction and the wash fraction. The target protein was co-eluted with the 70 kDa host protein Hsp70.

[0075] FIG8 (A) provides an image of a gel used to screen C1 transformants expressing recombinant HN protein. Transformants were screened for production of target recombinant protein in a 24-well plate culture format. All constructs except pMYT1362 showed positive expression of the target protein in this experiment. Transformants indicated by asterisks were selected for further analysis. Target protein detection was performed using biotin-α-C tag conjugate as primary detection reagent and streptavidin-IRD800 (shown in green) as secondary detection reagent.

[0076] Figure 8 (B) provides a gel image showing screening of recombinant HN protein expressed in C1 transformants. Transformants were screened for production of target recombinant protein in a 24-well plate culture format. Transformants indicated by asterisks were selected for further analysis. Detection was performed using a green biotin-α-C tag / streptavidin-IRD800.

[0077] Fig. 9 Gel images of the output of bioreactor fermentations used to evaluate recombinant HN-producing strains are provided. MT441 is a fermentation run of DNL157 with pMYT1363 corresponding to expression of full-length HN protein of expected size 63 kDa. MT442 is a fermentation run of DNL155 isolate transformed with pMYT1364 corresponding to HN protein with transmembrane domain truncations and expected size of 55 kDa. Protein detection is shown with NDV antiserum / rabbit anti-chicken IgY-IRD680 in red and with biotin-α-C tag / streptavidin-IRD800 in green. Target protein was produced in both setups. Quantitation indicated higher levels of protein production in MT441 with a day 7 yield of 0.94 g / L compared to a day 7 yield of 0.56 g / L using MT442.

[0078] Fig.10 is a gel image of additional output from the analysis of bioreactor fermentation runs containing HN producing strains. MT443 is a fermentation run at +38°C containing the DNL155 line transformed with pMYT1365 corresponding to a truncated HN protein lacking the transmembrane domain and with an expected size of 50 kDa. MT444 is a fermentation run of the same line but at +25°C. Protein detection is shown with NDV antiserum / rabbit anti-chicken IgY-IRD680 in red and with biotin-α-C tag / streptavidin-IRD800 in green. The target protein was produced under both conditions.

[0079] Fig.11Images of Western blots and stained protein gels analyzing the purification of recombinant HN protein from the MT443 run are provided. Here, 1.5 mL of sample MT443-5 supernatant was purified on a CaptureSelect C-Tag XL 1 mL column using 1x PBS binding buffer with 0.5 M NaCl and an elution buffer containing 20 mM Tris-HCl, 2 M MgCl2, and 1 mM EDTA (pH 7.5). Target protein detection was performed using NDV antiserum and biotin-α-C tag conjugate as primary detection reagents and rabbit anti-chicken IgY-IRD680 (shown in red) and streptavidin-IRD800 (shown in green) as secondary detection reagents, respectively. Most of the target protein eluted in fractions 3, 4, and 5. No target protein was detected in the wash fractions.

[0080] Fig.12 Gel images of purified NDV recombinant F and HN proteins expressed using the C1 system are provided for analysis on Western blots and stained protein gels under reducing and non-reducing conditions. Target protein detection was performed using NDV antiserum and biotin-α-C tag conjugate as primary detection reagents and rabbit anti-chicken IgY-IRD680 (shown in red) and streptavidin-IRD800 (shown in green) as secondary detection reagents, respectively. Recombinant proteins HN-55 and HN-63 form dimers under non-reducing conditions.

[0081] Fig.13 is a bar graph comparing treatment groups for HI titers against genotype 7 (rLaSota-HN-G7) antigen following the first vaccine dose of 50 μg / dose in Example 2.

[0082] Fig.14 is a bar graph comparing treatment groups for HI titers against genotype 7 (rLaSota-HN-G7) antigen following the second vaccine dose of 50 μg / dose in Example 2.

[0083] Fig.15 is a bar graph comparing the treatment groups in Example 2 for HI titers against genotype 2 (VH) antigen after the first vaccine dose of 50 μg / dose.

[0084] Fig.16 is a bar graph comparing treatment groups for HI titers against genotype 2 (VH) antigen following the second vaccine dose of 50 μg / dose in Example 2.

[0085] Fig.17 Results of ELISA-NDV-F with titers of the F50 treatment group after two doses in Example 2 are shown.

[0086] Fig.18 Results of ELISA-NDV-F with titers of the HN63+F50 treatment group after two doses according to Example 2 are shown.

[0087] Fig.19 HI titers against genotype 7 (rLaSota-HN-G7) antigen after one therapeutic dose according to Example 3 are shown.

[0088] Fig. 20 HI titers against genotype 2 (VH) antigen after one therapeutic dose according to Example 3 are shown.

[0089] Fig.21 NDV-F titers by F50 ELISA after one therapeutic dose according to Example 3 are shown.

[0090] Fig. 22 Comparison between NDV-F ELISA mean titers and standard deviation (STDEV) of past studies at the time of first bleed and the Example 3 study at the time of second bleed.

[0091] Fig.23 Virus secretion titers after NDV challenge described in Example 3 are shown.

[0092] Fig.24 Provided are images of gels used to analyze additional outputs from the following bioreactor fermentation runs containing F-producing strains: MT642 at +25°C: (M6316(M5824(Δchi1) / 2x NDV F+T4 domains) and MT649 at +32°C: (M6318(M5824Δchi1) / 2x NDV F+T4 domains), where M6316 and M6318 are clones purified from two independent transformants. Target protein detection was performed using NDV antiserum / rabbit anti-chicken Ig-IRD680 (shown in red) and biotin-α-C tag / streptavidin-IRD800 (shown in green). Improved NDV F production levels were achieved in the additional runs, reaching 1.7 g / L after six days and 1.9 g / L after 7 days.

[0093] Fig.25Gel images of purified C1 NDV F and HN antigens expressed using the C1 system are analyzed on Western blots and stained gels under reducing and non-reducing conditions. Target protein detection is performed using NDV antiserum / rabbit anti-chicken IgIRD 680 (shown in red) and biotin-α-C tag / streptavidin-IRD800 (shown in green); and the merging of images in which signals overlap (shown in yellow). The purified protein exhibits good stability, and the addition of a trimerization domain to the NDV F antigen results in an increase in molecular weight of approximately 5 kDa. In addition, putative oligomers (dimers and trimers) can be seen in the non-reducing samples of the NDV F antigen in Phase III. The NDV HN antigens produced in Phase I and Phase III show the same gel mobility. The yield of the NDV F antigen with a trimerization domain is preliminarily estimated to reach about 1.9 g / L, and the NDV HN50 antigen with 4 copies of the HN50 expression cassette indicates that the estimated yield level reaches 10 g / L.

[0094] Fig.26 Size exclusion chromatography (UV) purification of NDV-F50 protein with trimerization domain with an expected monomer mass of 53376.3 Da (average mass calculated by NIST protein calculator) with oxidized disulfide bridges (10 cysteines) as analyzed by UV-DAD chromatography with PBS buffer (12 mM NaH2PO4*H2O, 150 mM NaCl pH 7.3) compared to a standard sample. The F50 sample showed a broad early eluting peak and two sharper peaks eluting at 7.85 minutes and 8.14 minutes.

[0095] Fig. 27 is a graph and table (raw data) showing standard exponential fit of sample calculated mass (kDa) versus retention time (in minutes, RT, min) and standard secondary fit of sample calculated mass (kDa) versus retention time (in minutes, RT, min). The calculated monomer mass is 53,376.3 Da, so the trimer is 106.1 kDa.

[0096] Fig.28Provided are size exclusion chromatograms (UV-MS) of NDV-F50 protein with trimerization domain, with expected monomer mass of 53376.3 Da (average mass calculated by NIST protein calculator) with oxidized disulfide bridges (10 cysteines), as analyzed by UV-DAD chromatography with unbuffered and 40 mM ammonium acetate at pH about 6, compared to untreated samples in PBS buffer and untreated samples in MS buffer. The chromatograms in PBS and MS buffer are highly similar. The PNGaseF treated samples are clearly different (removal of N-glycans). The PNGaseF treatment was performed under minimal denaturing conditions (Rapid PngnaseF buffer 1:5 dilution, +Rapid PngaseF enzyme approximately 1:30 protein ratio, 4 hours at 40°C)

[0097] Fig.29 Combined chromatograms of untreated samples and samples treated with PngaseF containing NDV-F50 protein with a trimerization domain are provided at several time points. The spectrum at 8.5 minutes shows the m / z-dependent presence of monomeric and dimeric forms of a 55 kDa protein and a 110 kDa protein. Deconvolution in the m / z region of approximately 2,000 shows proteins of approximately 50 kDa to 58 kDa, and deconvolution in the m / z region of 4,500 shows proteins of approximately 106 kDa and 110 kDa, which may represent dimers.

[0098] Fig.30 A merged and deconvoluted spectrum of the major peak at 9.1 minutes is presented and shows both dimeric and monomeric forms with evidence of a trimeric protein. The deconvoluted result for the monomer at 53,381 Da is close to the theoretical mass.

[0099] Fig.31 The merged and deconvoluted spectra of the main peak at 10 minutes are shown (SEC predicted mass is 50 kDa). The deconvolution shows good quality masses at 49,445 Da and at 53,880 Da, the latter being very close to the theoretical mass. The +3.7 Da difference from the theoretical value may be caused by, for example, open disulfide pairs and deamidation.

[0100] Fig.32 The results of the immunogenicity evaluation by testing the sera on the NDV-F ELISA using the Ab titers in Example 5 are shown.

[0101] Fig.33 The results of immunogenicity evaluation by testing sera on NDV-F ELISA using the Ab titers in Example 6 are shown. DETAILED DESCRIPTION

[0102] I. Abbreviations

[0103] AAvV Avian mumps virus

[0104] AH Animal House

[0105] C1 Heterothralid Thermophilic Verrucosa

[0106] CBH1ss Cellobiohydrolase 1 secretion signal

[0107] CTL Cytotoxic T lymphocyte

[0108] EID50 50% embryo infectious dose

[0109] ELD50 50% embryo lethal dose

[0110] E-QCM English Quality Control Method

[0111] F fusion protein

[0112] HI hemagglutination inhibition assay

[0113] HN Hemagglutinin-neuraminidase

[0114] MHC major histocompatibility complex

[0115] ND Newcastle disease

[0116] NDV Newcastle Disease Virus

[0117] PCR polymerase chain reaction

[0118] PD50 protection dose 50

[0119] PM Autopsy

[0120] QA Quality Assurance

[0121] QCM Quality Control Method

[0122] IgY Immunoglobulin Y

[0123] SOP Standard Operating Procedure

[0124] SPF No Specified Pathogens

[0125] vvNDV Velocitropic Newcastle disease virus

[0126] vNDV velocigenic Newcastle disease virus

[0127] w / o water-in-oil emulsion

[0128] II. Terms and Definitions

[0129] Unless otherwise indicated, technical terms are used according to conventional usage as understood by those of ordinary skill in the art. Definitions of commonly used terms in molecular biology can be found in Lewin's Genes X, edited by Krebs et al., Jones and Bartlett Publishers, 2009 (ISBN 0763766321); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Publishers, 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and George P. Rédei, Encyclopedic Dictionary of Genetics, Gen omics, Proteomics and Informatics, 3rd edition, Springer, 2008 (ISBN: 1402067534).

[0130] The following explanations of terms and abbreviations are provided to better describe the present disclosure and guide those of ordinary skill in the art to practice the present disclosure. As used herein, "comprising" means "including" and the singular form "a" or "an" or "the" refers to one / a kind or more than one / more than one, unless the context clearly indicates otherwise. The term "or" refers to a single element or a combination of two or more elements in the stated alternative elements, unless the context clearly indicates otherwise.

[0131] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety for all purposes. As of the priority date of this application, all sequences relevant to the GenBank accession numbers mentioned herein are incorporated by reference in their entirety. In the event of a conflict, this specification sheet (comprising the explanation of the term) shall prevail.

[0132] Although methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. These materials, methods and examples are merely illustrative and are not intended to be limiting. Other features of the present disclosure will be apparent to those of ordinary skill in the art from the following detailed description and claims.

[0133] Unless otherwise indicated, all numbers used in the specification or claims to represent the amount of components, molecular weights, percentages, temperatures, times, etc. should be understood to be modified by the term "about". Therefore, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximate values, which may depend on the desired properties sought and / or the detection limits under standard test conditions / methods. When directly and clearly distinguishing an embodiment from the prior art in question, the embodiment numbers are not approximate unless the word "about" is stated.

[0134] The amino acid residues in the disclosed sequence table may be conservatively substituted or replaced by another residue with similar properties and characteristics. Generally, conservative substitutions have little or no effect on the activity of the resulting peptide. In a non-limiting example, a tyrosine residue in a peptide of the composition is replaced by a tryptophan residue. The peptide may be produced by chemical substitution to include one or more conservative amino acid substitutions, or may be produced by manipulating the nucleic acid sequence encoding the peptide using, for example, standard methods such as PCR or site-directed mutagenesis. Table 1 below provides conservative amino acid substitutions of the peptide sequences clearly disclosed within the scope of the present disclosure.

[0135] Table 1

[0136] Conservative amino acid substitution

[0137]

[0138]

[0139] The following explanations of specific terms are provided to facilitate review of the various embodiments of the present disclosure:

[0140] Adjuvant: As used herein, the term "adjuvant" means any substance or vehicle that enhances the effectiveness of the disclosed immunogenic compositions, such as by enhancing the immune response of an animal's immune system (such as a mammal's immune system) to an antigen (e.g., an NDV antigen). Adjuvants can be used to form one or more compositions disclosed herein, for example, as part of an ND vaccine composition. Adjuvants included in some embodiments of the compositions disclosed herein may include, but are not limited to, sorbitan oleate, sorbitol-T, aluminum salts (such as aluminum phosphate or aluminum hydroxide); various types of oils, such as vegetable oils, mineral oils; oil-in-water based adjuvants, such as or TS6; Pluronic polyols; saponin-based adjuvants such as saponin, Quil A and QS-21; nonionic block copolymers; microfluidized emulsions such as MF59; water-in-oil adjuvants such as ISA 720, ISA 71VG, ISA35, ISA 51 or ISA 50V; water-in-oil-in-water-based adjuvants such as ISA206 or ISA201 (such as Montanide ISA 201VG); Freund's complete adjuvant; Freund's incomplete adjuvant; polylactide-co-glycolide (PLGA); adjuvants based on toll-like receptor (TLR) ligands, such as TLR7 / 8 adjuvants, such as R848 (resiquimod); carbomer-based adjuvants, such as those containing 934P or 971P; polymer-based adjuvants, such as CARBIGEN or POLYGEN; immunostimulatory complexes (ISCOM); liposomes; polysaccharides; derivatized polysaccharides; oligonucleotides; cytokines; bacterial derivatives, such as trehalose-6,6-dibehenate (TDB) or cyclic diguanylate monophosphate (c-di-GMP); viral derivatives, such as polyinosinic acid-polycytidylic acid ester (poly (I:C)); or a combination thereof.

[0141] "Mucosal adjuvant" or "mucosal adjuvant" refers to an adjuvant or other compound, such as a polymer, that can interact with the mucosa and can stimulate an immune response. U.S. Pat. No. 10,279,031 (which is incorporated herein by reference) provides additional information about mucosal adjuvants. Mucosa includes eye membranes, oral membranes, nasopharyngeal membranes, anal membranes, or vaginal membranes. The immune response that can be stimulated may include IgM, IgG, IgA, or a combination thereof. Compositions containing such adjuvants can be applied to the mucosa of an animal. Mucosal adjuvants can be "mucoadhesive" because they can adhere (usually non-covalently) to the mucosa. Specific adjuvants with mucoadhesive properties include, but are not limited to, adjuvants containing polymers, such as those containing polyacrylic acid, such as carbomer and carbopol, or adjuvants based on oil-in-water. In addition, adjuvants containing nanoparticles can be used for intranasal administration. One of ordinary skill in the art understands that a mucoadhesive adjuvant can contain one or a combination of any of the above adjuvants.

[0142] Administer, Administering, Administration: As used herein, administering a composition (e.g., an immunogenic composition) to an animal means applying the composition, applying the composition to contact the animal, or contacting the composition with the animal. Administration can be achieved by a variety of routes, such as topical, oral, subcutaneous, transdermal, intrathecal, intramuscular, intravenous, intraperitoneal, intranasal, and the like, or a combination thereof.

[0143] As used herein, transmucosal administration of the composition includes administering the composition directly to the animal, such as by placing the composition directly (e.g., spraying and / or dripping) in the mouth, nasal passages or eyes of the animal. Transmucosal administration of the composition also includes providing the composition so that the animal administers the composition to itself, such as providing a composition for animal intake. Exemplary methods of providing the composition include, but are not limited to, spraying the composition onto the animal and / or otherwise applying the composition topically to the skin, or providing the composition in a form that the animal will eat. It will be appreciated by those of ordinary skill in the art that spraying can also facilitate direct administration because the spray droplets can directly enter the mouth, nasal cavity and / or eyes of the pig. Another exemplary method of administering the composition to the animal is by intramuscular administration, such as by injecting a liquid preparation of the composition. The specific application method of poultry includes intraocular injection, intramuscular injection, subcutaneous injection, intradermal injection. The mucosal application of poultry includes eye drops, coarse sprays, aerosol sprays, gel sprays, drinking water and feed applications.

[0144] The disclosed compositions can be formulated for parenteral administration, for example, by intradermal, intraarterial, intraperitoneal, intramuscular, subcutaneous or intravenous routes or combinations thereof. Examples of parenteral formulations of the compositions include, but are not limited to, injectable suspensions, injectable solutions, emulsions, and dry products that can be dissolved or suspended in an acceptable vehicle for injection. In addition, controlled-release parenteral formulations of the compositions can be prepared or administered (or both). Suitable materials for such administration include alcohols or mixtures of alcohols, such as C1-C 10 Alcohols such as ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol and / or decanol; polyols such as polyethylene glycol; sterile water; glucose solution; saline solution; aqueous vehicles such as but not limited to sodium chloride, glucose, glucose injection, sodium chloride injection, Ringer's injection or lactated Ringer's injection or a combination thereof; non-aqueous vehicles such as but not limited to ethyl oleate, peanut oil, corn oil, cottonseed oil, sesame oil or isopropyl myristate or a combination thereof; aqueous and non-aqueous isotonic sterile injection solutions, which may contain bacteriostatic agents, buffers, antioxidants or solutes that make the preparation isotonic in the recipient's blood or a combination thereof; and non-aqueous and aqueous suspensions that may be sterile and may include solubilizers, stabilizers, thickeners, suspending agents and preservatives or a combination thereof. The preparations of the composition may be present in unit dose or multi-dose containers such as bottles, ampoules, syringes, tubes, capsules and vials.

[0145] Animal: "Animal" refers to a living multicellular vertebrate organism, a class that includes, for example, mammals and birds. The term mammal includes humans and non-human mammals. The term avian includes both wild and domesticated species of birds, including especially chickens, geese, ducks, turkeys, quail, guinea fowl, peacocks, partridges, pigeons, emus, and pheasants.

[0146] Antibodies: An "antibody" is an immunoglobulin molecule produced by B lymphocytes. Antibodies are elicited in humans or other animals by a specific antigen (immunogen). Antibodies are characterized by reacting specifically with the antigen in some demonstrable manner. "Eliciting an antibody response" refers to the ability of an antigen or other molecule to induce the production of antibodies.

[0147] Antigen: "Antigen" refers to a compound, composition or substance that can stimulate the production of antibodies or T cell responses in an animal, including compositions that are injected or absorbed into the animal.

[0148] Viral antigens suitable for the present technology include inactivated (or killed) viruses and / or one or more viral peptides, one or more proteins that can be separated, purified or sourced from viruses. Viral antigens can be derived from viruses bred on substrates (such as cell cultures or other substrates), or they can be derived or recombinantly expressed, or they can be synthesized. Typically, viral antigens include but are not limited to epitopes exposed on the surface of the virus during at least one stage of the life cycle. Viral antigens may be conservative in multiple serotypes or isolates. Viral antigens include antigens derived from one or more viruses disclosed herein.

[0149] Attenuated, attenuated: An "attenuated" virus is a virus that is attenuated and / or less toxic than a non-attenuated form of the virus that may be able to cause disease. An attenuated virus can stimulate an immune response and / or immunity, but cannot cause disease. The replication of an attenuated virus in culture and / or in a recipient can be the same, similar, or different than one or more strains from which the attenuated virus is derived. Attenuation can be achieved by altering the virus using one or more methods involving a single step and / or multiple steps. For example, attenuated genetic modifications (e.g., attenuated mutations and / or genetic reassortment) can be introduced into the coding and / or non-coding regions of the viral genome by site-directed mutagenesis, chemical methods, radiation, and / or recombinant techniques. Such methods are well known to those of ordinary skill in the art. Attenuated forms of viruses that originally cause disease can also be identified by culture techniques (such as passage), and / or can be generated by genetic differences in the viral genome that are not induced, generated, or caused by human intervention. Methods for determining whether an attenuated virus retains similar or reduced antigenicity compared to one or more strains from which the attenuated virus is derived are also well known to those of ordinary skill in the art. Such methods may include, for example, chemical selection and / or nucleic acid screening, such as by probe hybridization or PCR. Attenuated viruses, such as certain embodiments of the viral vectors disclosed herein, can be used to stimulate an immune response and / or induce immunity in a recipient (such as an animal, such as a pig).

[0150] Combination: The combination includes two or more components, which are applied so that the effective time period of at least one component overlaps with the effective time period of at least one other component. The component may be a composition. In some embodiments, the effective time periods of all components applied overlap with each other. In an exemplary embodiment of a combination comprising three components, the effective time period of the first component applied may overlap with the effective time periods of the second and third components, but the effective time period of the second component may independently overlap or not overlap with the effective time period of the third component. In an exemplary embodiment of a combination comprising four components, the effective time period of the first component applied overlaps with the effective time periods of the second, third and fourth components; the effective time period of the second component overlaps with the effective time periods of the first and fourth components, but does not overlap with the effective time period of the third component; the effective time period of the fourth component overlaps only with the effective time periods of the second and third components. The combination may be a composition comprising these components, a composition comprising two or more individual components, or a composition comprising one or more components and another individual component (or multiple components) or a composition comprising the remaining components. In some embodiments, two or more components may comprise two or more different components applied substantially simultaneously or sequentially in any order, the same component applied at two or more different times, or a combination thereof.

[0151] Conditions sufficient for: The term "conditions sufficient for" refers to any environment that allows a desired activity (e.g., allows specific binding or hybridization between two nucleic acid molecules, or allows amplification and / or detection of nucleic acids). Such an environment may include, but is not limited to, specific incubation conditions (such as time and / or temperature) or the presence and / or concentration of specific factors, such as in a solution (such as buffers, salts, metal ions, detergents, nucleotides, enzymes, etc.).

[0152] Effective amount: The term "effective amount" or "therapeutically effective amount" or "immunostimulatory amount" refers to an amount of an agent (such as one or more embodiments provided herein, alone, in combination, or possibly in combination with other therapeutic agents) sufficient to induce a desired biological result. The result may be an improvement or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. The amount may vary depending on the condition being treated, the stage of progression of the condition, and the type and concentration of the formulation being applied. In some embodiments, the effective amount of an immunostimulatory composition is an amount sufficient to cause a detectable immune response when administered to a subject. Such a response may include, for example, the production of antibodies specific to one or more epitopes provided in the immunostimulatory composition. Alternatively, the response may include a T helper cell or CTL-based response to one or more epitopes provided in the immunostimulatory composition. All three responses may originate from naive or memory cells. In other embodiments, a "protective effective amount" of an immunostimulatory composition is an amount sufficient to confer protective immunity to a subject when administered to a subject. In any given case, the appropriate amount will be apparent to one of ordinary skill in the art, or can be determined by routine experimentation, such as vaccination and observing the antibody response or vaccination followed by challenge, where vaccinated animals perform better than similarly challenged unvaccinated animals.

[0153] Coding: "Coding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA or mRNA) to serve as a template for other polymers and macromolecules in the synthesis of biological processes, which have a defined nucleotide sequence (e.g., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Therefore, if the transcription and translation of the mRNA produced by a gene can produce a protein, such as in a cell or other biological system, the gene encodes the protein. The coding strand whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, and the non-coding strand of the gene or cDNA used as a transcription template can both be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise indicated, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may include introns, exons, or both.

[0154] Epitope: An "epitope" is an antigenic determinant. These are chemical groups on a molecule or peptide sequences that are antigenic, i.e., that elicit an immune response. T cell epitopes are presented on the surface of antigen presenting cells, where they are bound to MHC molecules. Specialized antigen presenting cells, such as macrophages, dendritic cells, and B cells, specialize in presenting MHC class II peptides, whereas most nucleated somatic cells present MHC class I peptides. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length. Antibodies specifically bind to a particular antigenic epitope on a peptide, such as one or more immunogenic peptides selected from SEQ ID NOs. 3-28 and 30-32. In some examples, the disclosed peptides are epitopes.

[0155] Expression: "Expression" refers to the transcription and / or translation of a nucleic acid sequence. For example, a gene can be expressed when its DNA is transcribed into RNA or RNA fragments, which are processed in some examples to form mRNA. A gene can also be expressed when its mRNA is translated into an amino acid sequence such as a protein or protein fragment. In one specific example, a heterologous gene is expressed when its RNA is transcribed into RNA. In another specific example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include control of transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or by activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

[0156] Expression control sequence: An "expression control sequence" is a nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operably linked to it. An expression control sequence is operably linked to a nucleic acid sequence when the expression control sequence controls and regulates the transcription and, when appropriate, translation of the nucleic acid sequence. Thus, an expression control sequence may include an appropriate promoter, an enhancer, a transcription terminator, a start codon (ATG) in front of a protein-coding gene, a splicing signal for introns, a correct reading frame to maintain the gene to allow correct translation of the mRNA, and a stop codon. The term "control sequence" is intended to include at least such components, the presence of which may affect expression, and may also include such additional components, the presence of which is advantageous, such as a leader sequence and a fusion partner sequence. An expression control sequence may include a promoter.

[0157] Expression vector: An "expression vector" is a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient elements for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses), which incorporate a recombinant polynucleotide.

[0158] Glycoengineering: Using genetic engineering, artificially manipulating the glycan patterns that a host cell adds to the proteins it produces.

[0159] Host cell: "Host cell" refers to a cell in which a vector can proliferate and express its DNA. The cell can be a eukaryotic cell or a prokaryotic cell. The cell can be a mammalian cell, such as a pig cell. "Host cell" also includes any progeny of the subject host cell. It should be understood that all progeny may or may not be identical to the parent cell, as mutations may occur during replication. When the term "host cell" is used, it should be understood to include such progeny.

[0160] Immune response: An "immune response" is the response of cells of the immune system (such as B cells, T cells, macrophages, or polymorphonuclear cells) to a stimulus (such as an antigenic peptide). The immune response may include any cell of the body that participates in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. Immune responses include, but are not limited to, innate immune responses or inflammation. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of a disease associated with infection). Methods for measuring immune responses are known to those of ordinary skill in the art, and include, for example, measuring the proliferation and / or activity of lymphocytes (such as B cells or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, and the like.

[0161] Immunostimulatory composition: As used herein, the terms "immunostimulatory composition" and "immunogenic composition" refer to compositions that can be used to stimulate or elicit an immune response (or immunogenic response) in a subject. The immunostimulatory composition can be a protein antigen, a nucleic acid molecule (such as a vector) for expressing the protein antigen, or a combination thereof. In some embodiments, the immunogenic response is protective or provides protective immunity because it enables the subject to better resist infection or disease progression by the virus against which the immunostimulatory composition is directed.

[0162] Immunization: Protection of a subject (such as birds, and particularly poultry) from infection with an infectious disease (such as ND) by stimulating the subject's immune system (such as by vaccination).

[0163] Immunogen: A compound, composition or substance capable of stimulating an immune response, such as the production of antibodies or T cell responses in an animal, including compositions that are injected or absorbed into an animal. Specific non-limiting examples of immunogens include immunogenic peptides of SEQ ID NOs. 3, 4, 30, 31 and 32, constructs of SEQ ID NOs. 3, 4, 30, 31 and 32, domains of SEQ ID NOs: 3, 4, 30, 31 and 32, and / or full-length and / or partial-length NDV proteins (e.g., one or more proteins of SEQ ID NOs: 7, 10, 13, 16, 19, 22, 25 and 28), and / or nucleic acids, vectors and / or host cells encoding such peptides, constructs, domains and / or full-length and / or partial-length NDV proteins.

[0164] Inactivation: In the context of the present disclosure, an "inactivated" virus is a virus that has been altered to the extent that it cannot establish an infection in a host or host cell. Viruses can be inactivated using, for example, chemicals, heat, pH changes, and / or irradiation (such as ultraviolet light or gamma irradiation). Inactivated viruses are also referred to as "killed". "Chemically inactivated" viruses are viruses that have been inactivated using chemical methods (such as treatment with β-propiolactone, formaldehyde, glutaraldehyde, 2,2'-dithiodipyridine or diethyleneimine).

[0165] Infection: An infection or challenge means that a subject has been exposed to a potentially disease producing organism, resulting in the subject suffering one or more clinical signs of disease when they are exposed to such organism.

[0166] Isolate, isolated: An "isolated" biological component (such as a nucleic acid) has been substantially separated or purified from an organism or other component (e.g., a biological component in which the component is naturally present, such as chromosomal and extrachromosomal DNA, RNA, and proteins). Nucleic acids that have been "isolated" include nucleic acids purified by standard purification methods. The term also includes nucleic acids prepared by recombinant expression in a host cell and subsequently purified, as well as chemically synthesized and purified nucleic acid molecules. Isolation does not require absolute purity, and may include, for example, nucleic acid molecules in which at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% of the components in the original mixture with the desired material are removed. As another example, an isolated biological component is one in which the biological component is more enriched than the biological component in its natural environment within a cell or other production container. The isolated nucleic acid may be in solution (e.g., water or an aqueous solution) or may be dry.

[0167] Newcastle Disease (ND): A disease that can occur in more than 200 species of birds causing respiratory, gastrointestinal and neurological symptoms. The disease can have a 100% mortality rate and is caused by the Newcastle Disease virus.

[0168] Newcastle Disease Virus (NDV): The causative agent of Newcastle Disease, which belongs to the paramyxovirus (PMV) family. The genus of the virus is avulavirus, and includes 19 serotypes of avian PMV (APMV). NDV is included under APMV serotype 1, a group of 15 genotypes. The enveloped virus has a negative-sense, single-stranded RNA genome structure. Newcastle Disease Virus is also known as avian paramyxovirus type 1, avian orthomopsis virus, and has recently been renamed avian mumps virus.

[0169] Peptide: A "peptide" is a polymer having at least two amino acids linked by a peptide bond, and typically a peptide comprises more than 2 amino acids linked together by an amide bond. Certain peptides, such as peptides having 25 or more amino acids, may be referred to as polypeptides. When the amino acid is an α-amino acid, L-optical isomers, D-optical isomers, or a combination thereof may be used. The term "peptide" as used herein is intended to encompass any amino acid sequence, and includes modified sequences such as glycoproteins, and includes naturally occurring amino acid sequences, as well as recombinant or synthetically produced amino acid sequences. The term "residue" or "amino acid residue" refers to an amino acid incorporated into a peptide. Exemplary peptides disclosed herein include peptides of SEQ ID NO.7, 10, 13, 16, 19, 22, 25, and 28, constructs of SEQ ID NO.3, 4, 7, 10, 13, 16, 19, 22, 25, 28, and 30-32, domains of SEQ ID NO:3, 4, and 30-32, and NDV proteins, such as SEQ ID NO.3, 4, and 30-32.

[0170] Polynucleotide, nucleic acid molecule: Unless otherwise indicated, the term "nucleic acid molecule" or "polynucleotide" refers to a polymeric form of nucleotides of at least two bases in length. Nucleic acid molecules can include both sense and antisense strands of cDNA, genomic DNA, RNA and / or mixed polymers and / or synthetic forms of the above. The term "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise indicated, these terms include single-stranded and double-stranded forms of DNA. A polynucleotide may include either or both of naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds. The nucleotides may be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.

[0171] Recombinant polynucleotides include polynucleotides that are directly adjacent to two coding sequences (one at the 5' end and one at the 3' end) that are not directly adjacent to them in the naturally occurring genome of the organism from which they are derived. A recombinant nucleic acid molecule may also be a nucleic acid molecule that is non-naturally occurring or has a sequence produced by an artificial combination of two originally separated sequence segments. This artificial combination is achieved by chemical synthesis or by artificial manipulation of separated nucleic acid segments, such as by genetic engineering techniques known to those of ordinary skill in the art. Therefore, the term includes, for example, recombinant DNA molecules incorporated into a vector, incorporated into an autonomously replicating plasmid or virus, or incorporated into the genomic DNA of a prokaryote or eukaryote; or it exists as a separate molecule (e.g., cDNA) independent of other sequences.

[0172] Poultry: Any type of fowl maintained in a domestic or agricultural setting, including but not limited to chickens, geese, ducks, turkeys, quail, guinea fowl, peacocks, partridges, pigeons, emus, pet birds, and pheasants.

[0173] Prevention: Prevention of a disease means inhibiting the full development of the disease.

[0174] Treatment: refers to therapeutic intervention to improve the signs or symptoms of a disease or pathological condition after it has begun to develop.

[0175] Improvement: refers to a decrease in the number or severity of one or more signs or symptoms of a disease.

[0176] Promoter: A "promoter" is a minimal nucleic acid sequence sufficient to direct transcription. Promoters are usually located in the 5' region near (and upstream of) the transcription start site of a gene, and usually contain a functional TATA box that directs gene expression. Promoters usually contain both structural and functional elements, and provide control points for regulating transcription of related genes. Also included are those promoter elements that are sufficient to make promoter-dependent gene expression controllable for cell type specificity, tissue specificity, or inducible by external signals or agents; such elements may be located in the 5' or 3' region of a gene. Both constitutive promoters and inducible promoters are included. For example, when cloning in a bacterial system, inducible promoters such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of λ phage can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of mammalian cells (such as metallothionein promoters) or promoters derived from mammalian viruses (such as retroviral long terminal repeats; adenovirus late promoters; vaccinia virus 7.5K promoters) can be used. Constitutive promoters can be used to drive expression of genes of interest in fungal cells, such as bgl8pr of Thermomyces heterothallus C1. Alternatively, synthetic constitutive promoters as described in patent US20180371468A1 "Expression system for eukaryotic organisms" can be used to drive expression of genes of interest in eukaryotic cells. Examples of such promoters suitable for fungal cells include, but are not limited to, AnSESpr and / or TrSESpr. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of nucleic acid sequences.

[0177] The polynucleotide can be inserted into an expression vector containing a promoter sequence that promotes efficient transcription of the inserted gene sequence of the host. The expression vector usually contains a replication origin, a promoter, and a specific nucleic acid sequence that allows phenotypic selection of transformed cells.

[0178] Purification: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein, virus, nucleic acid, or other compound is one that is completely or partially separated from related proteins and other contaminants. In certain embodiments, the term "substantially purified" means that the protein, virus, nucleic acid, or other compound has been isolated from cells, cell culture medium, or other crude preparation and subjected to purification to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0179] Recombinant: A recombinant nucleic acid, protein or virus is a nucleic acid, protein or virus that has a sequence that does not occur naturally or that results from the artificial combination of two otherwise separate sequence segments. This artificial combination is usually achieved by chemical synthesis or, more commonly, by manipulating separate nucleic acid segments (e.g., by genetic engineering techniques). The term recombinant includes nucleic acids, proteins and viruses that have been altered merely by adding, substituting or deleting a portion of a natural nucleic acid molecule, protein or virus.

[0180] Sample: "Sample" (or "biological sample") refers to a specimen obtained from an organism, which in certain embodiments comprises DNA (e.g., genomic DNA or cDNA), RNA (including mRNA), protein, or a combination thereof. Examples include, but are not limited to, isolated nucleic acids, cells, proteins, peptides, cell lysates, chromosome preparations, tissues and body fluids (such as blood, derivatives and fractions of blood (such as serum)), extracted bile, biopsied or surgically removed tissues (including, for example, unfixed, frozen, formalin-fixed and / or paraffin-embedded tissues), autopsy material, tears, breast milk, skin scrapings, surface washes, urine, sputum, cerebrospinal fluid, prostatic fluid, pus, bone marrow aspirates, middle ear fluid, bronchoalveolar lavage fluid, tracheal aspirates, nasopharyngeal swabs or aspirates, oropharyngeal swabs or aspirates, nasal washes, or saliva. In one example, the sample comprises viral peptides, such as viral peptides specific for NDV. In particular examples, the sample is used directly (e.g., fresh or frozen), or may be manipulated prior to use, for example, by extraction (e.g., extraction of nucleic acids), fixation (e.g., using formalin) and / or embedding in wax (such as formalin-fixed paraffin-embedded tissue samples).

[0181] Sequence identity / similarity: Identity / similarity between two or more nucleic acid sequences, or between two or more amino acid sequences, is expressed as the identity or similarity between the sequences. Sequence identity can be measured as a percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured as a percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid sequences or amino acid sequences have a relatively high degree of sequence identity / similarity when aligned using standard methods. In some embodiments, one or more disclosed peptides may comprise one or more amino acid sequences having at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity) to the amino acid sequence of one or more peptides of SEQ ID NO. In some embodiments, one or more disclosed nucleic acid molecules encoding one or more peptides of SEQ ID NO. 5, 8, 11, 14, 17, 20, 23, 26, 44, 45, or a combination thereof may comprise one or more nucleic acid sequences having at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity).

[0182] Methods of alignment of sequences for comparison and for determining sequence identity or similarity are known to those of ordinary skill in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene, 73:237-44, 1988; Higgins and Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. A detailed consideration of sequence alignment methods and homology calculations is presented in Altschul et al., J. Mol. Biol. 215:403-10, 1990.

[0183] NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found on the NCBI website.

[0184] BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. If the two compared sequences share homology, the specified output file will present those homology regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences.

[0185] Subject: A "subject" is any multicellular vertebrate organism, a category that includes humans, non-human mammals (such as mice, rats, rabbits, sheep, pigs, horses, cattle, and non-human primates), and birds (such as chickens, geese, ducks, turkeys, quail, guinea fowl, peacocks, partridges, pigeons, emus, and pheasants). Certain disclosed embodiments of the present invention are particularly directed to birds, especially poultry.

[0186] Transformed: A "transformed" cell is a cell into which a nucleic acid molecule has been introduced using molecular biology techniques known to those of ordinary skill in the art. The term encompasses all techniques by which a nucleic acid molecule can be introduced into a cell, including transfection with a plasmid vector, transformation with a viral vector, and introduction of naked DNA by lipofection, electroporation, and / or particle gun acceleration.

[0187] Transformants: A "transformant" is a transformed clone, cell line, strain or isolate produced by genetic transformation in which the recombinant exogenous nucleic acid has been stably integrated into the host cell genome or is otherwise stably present in the host cell as genetic material capable of expression.

[0188] Vaccine: "Vaccine" refers to an immunogenic substance or a composition containing an immunogenic substance that is capable of stimulating an immune response. Vaccines can be administered to prevent, ameliorate or treat one or more infectious diseases or other types of diseases. Immunogenic substances may include attenuated or inactivated microorganisms (such as bacteria or viruses), or antigenic proteins (including VLPs), peptides, or DNA derived from or encoding them, or a combination thereof. An attenuated vaccine is a toxic organism that has been modified to produce a less toxic form, but still retains the ability to elicit antibodies and immune responses against the toxic form. An inactivated vaccine is a previously toxic microorganism that has been killed by chemicals or heat, but it elicits antibodies against toxic microorganisms. Vaccines can elicit both prophylactic (preventative) responses and therapeutic responses. The method of administration varies depending on the vaccine, but may include vaccination, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to enhance the immune response.

[0189] Vector: A vector is a nucleic acid molecule that allows the insertion of exogenous nucleic acid without destroying the vector's ability to replicate and / or integrate in a host cell. A vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. The inserted vector is capable of inserting itself into the host nucleic acid. The vector may also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains regulatory sequences that allow one or more inserted genes to be transcribed and translated.

[0190] Virus-like particles (VLPs): Virus-like particles are composed of one or more viral proteins but lack the viral genome. Because VLPs lack the viral genome, they are non-infectious.

[0191] III. Overview of Implementation Plan

[0192] Disclosed herein are the production, compositions and uses of immunogenic peptides, such as those from NDV. The production of these peptides involves genetic transformation and growth of fungal expression systems. The compositions of these peptides are supported by disclosures of naturally occurring and / or synthetic nucleic acids and amino acid sequences in the form of plasmids, episomes and / or chromosomally integrated or genomic integrated recombinant transgenics.

[0193] Figure 1 An overview of the disclosed embodiments of the present invention is provided. The fragment 100 carries a target transgene 103 corresponding to the nucleic acid sequence of the target antigen protein. The fragment 100 is transferred into the nucleus 102 of the fungal expression system cell 101. Once the target gene 103 enters the fungal nucleus 102, the target gene is recombined with the fungal expression system genome 104 and stably integrated into the fungal expression system chromosome 105. The transformed fungal cell 106 can then be selected to grow and / or maintain in a defined culture condition 107. The transformed fungal cell 106 can then be cultured as an inoculum 108 for the production of the target antigen protein. After about 48 hours of culture, the inoculum 108 can then be used to inoculate a production inoculation tank 109. After fermentation in the inoculation tank for 24 hours, the expression system cell 106 can then be transferred to a fed-batch technology tank 110 containing a defined culture medium 111. Here, the specific fermentation conditions can be adjusted and controlled according to the specific needs of effectively producing the target protein from the expression system cell 106. After 120 to 168 hours of fermentation in the fed-batch technology tank 110, the contents are transferred to a harvest tank from which the target protein will be purified and / or enriched. Once the target protein is harvested, it can be further processed into a vaccine composition 113. The vaccine composition 113 can then be administered to vaccinate and / or generate an immunogenic response in a subject 114.

[0194] A. Fungal expression system

[0195] Heterothracenic thermophilic wart fungus (C1) is a thermophilic ascomycete in the order Sordariales. It was previously known as Myceliophthora thermophila and Sporotrichum thermophilum. The species was originally isolated from alkaline soils in Russia. The morphology of the fungus is unique and can be characterized as a fragmented, non-sporulated mycelial phenotype. In solid medium culture, the fungus grows as a filament with white to light brown mycelium, depending on the strain.

[0196] The unique morphology of this fungus allows low viscosity fermentation, and standard sterilization can be used with bioreactors because the fungus is non-sporulated. In addition, in some embodiments, cultivating fungi only requires simple culture media to produce high growth rates. C1 is also able to grow highly under wide range of environmental conditions (including wide variations of pH and temperature). These attributes make it possible to grow a large number of fungi at low cost.

[0197] The C1 genome has been completely sequenced and annotated. The genome is approximately 38.74Mb. C1 has been studied for its ability to degrade a variety of enzymes that are capable of degrading plant cell wall components. Wild-type C1 naturally produces a variety of proteases that can degrade heterologously expressed recombinant proteins. Therefore, many protease deletion strains for producing recombinant target proteins have been created. One such strain is DNL155, which carries the deletion of the following 14 protease genes: alp1 alp2 pep4 prt1 srp1 alp3 pep1 mtp2 pep5 mtp4 pep6 alp4 alp7 kex2. Another disclosed strain is DNL157, which carries the deletion of the alp1 alp2 pep4 prt1srp1 alp3 pep1 mtp2 pep5 mtp4 pep6 alp4 alp7 srp10 protease gene. The deletion of protease genes helps prevent the degradation of target recombinant proteins produced in these expression system strains. There is also a chitinase deletion strain (Δchi1), which can be used as an expression system strain. These include, but are not limited to, strains M5355, M5739, and M5824.

[0198] An additional feature of the C1 fungal expression system is its potential for glycoengineering. The C1 glycan structure is more mammalian than yeast and other known microbial expression systems. Native C1 glycans are high mannose-type glycans, such as Man3-Man9, and include small amounts of hybrid glycans. Successful glycoengineering of C1 allows the generation of new cell lines with different glycan patterns, including strains that produce GlcNAc2Man3 glycans, G0 glycans (e.g., 84% G0), galactosylated glycans (e.g., 26% G2, 45% G1, and 27% G0), and fucosylated glycans. In some embodiments, different glycoengineering in the C1 system can be used to modify or design target antigen peptides.

[0199] Knowledge of C1's morphology, physiology, and genetics makes it a useful and efficient expression system. Low-cost production and adaptability to a variety of culture conditions allow for rapidly scalable and adaptable bioreactor systems capable of producing high yields of target proteins. Additional information about C1 is provided by WO / 2019 / 038623, which is incorporated herein by reference.

[0200] B. Constructs

[0201] Embodiments of vectors capable of transforming the C1 genome with a target gene of interest include episomes and plasmids. A specific example is the vector backbone pMYT1055 (SEQ ID NO: 29), which contains the native C1 promoter bgl8pr. Other expression vector backbones with bgl8pr include, but are not limited to, pMYT1730 (SEQ ID NO: 37) and pMYT1731 (SEQ ID NO: 38). Additional examples are expression vectors with the synthetic promoter AnSESpr, including, but not limited to, pMYT1727 (SEQ ID NO: 34) and pMYT1728 (SEQ ID NO: 35).

[0202] Antigenic peptides can also be engineered to include trimerization domains for stabilizing certain conformations of the antigen. T4 fibrin provides an example that can be fused to antigenic peptides as trimerization domains to stabilize the pre-fusion configuration of the antigen. The amino acid sequence of T4 fibrin is SEQ ID NO:38. GCN4 trimerization domains can also be used, and include, for example, SEQ ID NO:39. Antigenic peptides can also be optionally fused to antibodies or antibody fragments so that the antigen can selectively target certain immune cells. For example, IgY antibody chains (such as CH or CL) can be conjugated or genetically fused to antigenic peptides. Examples of these antibodies or antibody fragments are provided by SEQ ID NO:40-43.

[0203] To produce recombinant proteins from the C1 expression system for downstream purification and harvesting, the protein can be engineered to be secreted from the expression host. For example, Trichoderma reesei or the native C1 cellobiohydrolase 1 secretion signal sequence (CBH1ss) can be used. Alternatively, the signal peptides of Rhizopus oryzae amylase or Aspergillus awamori amylase or any other compatible fungal-derived or synthetic secretion signal can be used. These signal peptides are cleaved, leaving the mature protein.

[0204] In addition, the target recombinant protein can be designed to include a purification tag. One embodiment uses a C tag with the amino acid sequence EPEA (SEQ ID NO: 2) attached to the C-terminus of the target peptide or protein sequence. Other examples of purification tags include polyhistidine, FLAG (DYKDDDK), albumin binding protein tag, alkaline phosphatase, AU1 epitope (DTYRYI), AU5 epitope (TDFYLK), bacteriophage T7 epitope (T7 tag), biotin-carboxyl carrier protein, blue tongue virus tag, calmodulin binding peptide, chloramphenicol acetyltransferase, fiber binding domain, chitin binding domain, choline binding domain, E2 epitope, galactose binding protein, Glu-Glu, glutathione S-transferase, Histidine affinity tag, HSV epitope, KT3 epitope, LacZ, maltose binding protein, Myc epitope, PDZ domain or ligand, polyarginine (Arg tag), polyaspartic acid, polycysteine, polyphenylalanine, Profinity S1 tag (NANNPDWDF) and streptavidin binding peptide. Many of these purification tags can be attached to the C-terminus or N-terminus of a recombinant protein.

[0205] The selectable marker used for genetic engineering with C1 can be a gene that confers antibiotic resistance (e.g., to hygromycin or phleomycin), a gene that enables the utilization of acetamide or nitrate as the sole nitrogen source (the Aspergillus nidulans amdS gene encoding acetamidase or the heterothallic Thermomycin nia1 gene encoding nitrate reductase, respectively), or a gene that remedies an auxotrophic mutation (e.g., the C1 pyr4 gene). Specifically, the gene HygR encoding an aminoglycoside O-phosphotransferase is a positive selectable marker that allows the selection of hygromycin-resistant transformants on a selective growth medium supplemented with hygromycin B. Alternatively, the Aspergillus nidulans amdS gene encoding acetamidase is a positive selectable marker that allows the selection of transformants on a minimal medium supplemented with acetamide as the sole nitrogen source. Selectable markers are optional elements, and embodiments of the present disclosure also include constructs that do not contain selectable markers. Natural constitutive promoters can drive high expression of transgenes in fungi, although synthetic promoters have also been shown to be successful for transgene expression. For example, the native C1 promoter is bgl8 and can be paired with the downstream chi1 terminator.

[0206] like Figure 2 As seen, the gene of interest (GOI) can be the antigenic region of NDV protein. The bgl8 promoter is a natural C1 promoter, and HygR is a selective marker for hygromycin resistance. Only those transformants with the box stably integrated into the genomic DNA are resistant to hygromycin and express the target protein.

[0207] C1 can also be stably transformed to express recombinant antigenic protein without a selectable marker.

[0208] C. Transformation C1

[0209] Fungal expression systems can be genetically transformed using methods that allow exogenous nucleic acids to enter the nucleus of the host cell so that they can come into contact with the genome of the host cell and stably integrate into the genome of the host cell by recombination. Example 1 provides further details on the genetic transformation process of C1.

[0210] Site-directed integration and selection markers can be used to genetically transform fungi. There are specific genomic integration sites associated with high expression of recombinant genes. However, random recombinant DNA integration methods can also be used to produce non-natural proteins with this fungus.

[0211] In some embodiments, site-directed transformation produces a genetically stable C1 cell line in approximately two months. One or two or more genes can be integrated into the same site. Using multiple genes at multiple integration sites can facilitate manufacturing.

[0212] The method could then be used to rapidly respond to new pathogens and diseases while still producing large therapeutic doses. The approach could be more flexible, scalable and affordable than using other expression systems.

[0213] D. Fermentation process

[0214] The fermentation process is carried out in a bioreactor, where the fungi transformed grow to produce a high level of the desired target peptide. Once the fungal expression host has been transformed with the target gene, the fungal transformation system can grow to produce an inoculum. Once enough inoculum is produced, for example, within 48 hours, the inoculum can be transferred to an inoculation tank. In the inoculation tank, the transgenic fungi grow for 24 hours in some embodiments. Then, the inoculation tank content is used to start a fed-batch technology fermenter tank. The fermenter tank can contain a determined or undetermined culture medium, which can be optimized to support the high-level production of the target protein. Glucose feed and NH4OH input can also be optionally added to control the fermentation pH. Once the fermentation is completed, for example, after 120 to 168 hours, the content can be moved into a harvesting tank for target protein separation. In some embodiments, the production of the target protein can be achieved in 4-7 days using this batch fermentation process of transgenic fungi.

[0215] The system allows for control of a wide range of environmental conditions. For example, the pH can be maintained at a specific value between pH 5 and pH 8 or at different pH values. The temperature can be maintained at a point between, for example, 20°C and 45°C or at higher or lower temperatures. Optionally, the temperature can be fluctuated to various temperatures during the fermentation process to optimize growth conditions for certain transgenic fungal strains and to optimize high target protein production.

[0216] The size of the fermenter tank can range from 1 L to 500,000 L and can be, for example, a single-use or stainless steel bioreactor. In some embodiments, no inducer is required to produce proteins from transgenic fungi. In some embodiments, the target protein is secreted into the culture medium during the fermentation step. At the end of the fermentation process, for example, 30%-40% of fungal cell biomass and 60%-70% of supernatant can be produced.

[0217] Figure 6 Examples of yield results for synthetic F protein produced using transgenic fungal cells using different fermentation conditions are provided.

[0218] E. Harvesting the target recombinant protein

[0219] The target antigen recombinant protein can optionally be purified from the fermentation batch before use in the vaccine. One example involves filtering the fermentation batch supernatant. Another example includes harvesting the fungal mycelium, extracting the protein therein, and purifying or collecting the target antigen therefrom.

[0220] The target recombinant protein can be purified using C-tag affinity chromatography, histidine tag affinity chromatography size exclusion chromatography, fast protein liquid chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, reverse phase chromatography, immunoaffinity chromatography, ultracentrifugation, precipitation, other purification methods, or any and all combinations thereof.

[0221] The target recombinant protein can also be collected as a crude extract and used in a vaccine.

[0222] F. Viral antigens

[0223] Disclosed herein are compositions that elicit an immunogenic response for immunization against viral pathogens. These compositions can be administered, for example, to animal subjects, particularly poultry. These compositions can be used to immunize subjects against ND, improve or eliminate the symptoms of ND, and / or prevent future high mortality outbreaks of ND in agricultural settings.

[0224] In some examples, NDV F and HN proteins can be synthetically designed for expression in the C1 system and elicit strong immunogenic responses from subjects. Since the structure of NDV F and HN proteins is highly conserved between genotypes of APMV1, similar modifications and amino acid changes between genotype homologs can produce similar expression results in C1 and immunogenic responses in subjects. Therefore, this expression system platform is highly adaptable to functional homologs of NDV F and HN proteins of all 15 NDV genotypes.

[0225] G. Methods of producing vaccines

[0226] Embodiments of producing vaccines include optionally purifying the target antigen. Antigens can be collected from batch supernatants or from mycelial tissue extracts or from any other cells or materials contacted with an expression system that produces the target antigen. Optionally, the target antigen can be collected without purification prior to vaccine preparation. The supernatant can be filtered prior to collection or purification.

[0227] Once collected, the antigen can be prepared as an oil-in-water emulsion, for example, comprising an aqueous phase and one or more adjuvants. One, two or more types of emulsifiers or combinations thereof can be used in the vaccine preparation. Any suitable oil can be used to prepare the emulsion, such as mineral oil, optionally DRAKEOL white mineral oil. The emulsifier can be a lipophilic surfactant, such as a sorbitan ester, a lipophilic solubilizing agent, a monoester, triester and triester of lauric acid, stearic acid or oleic fatty acid, or a combination thereof. The lipophilic surfactant can also include polysorbates, hydrophilic solubilizing agents or polyethoxylated sorbitan esters or any combination thereof. A specific example of a lipophilic surfactant based on sorbitan esters is MONTANE. The example of a polyethoxylated sorbitan ester lipophilic surfactant is MONTANOX.

[0228] H. Methods of administering vaccines

[0229] The embodiments of peptides, immunogenic compositions, carriers, cells and / or nucleic acid constructs can be applied to humans or animals by any approach commonly used to introduce one or more pharmaceutical compositions into animals. Methods of administration include, but are not limited to, intramuscular, oral, intravenous, intradermal, intraperitoneal, subcutaneous, parenteral, mucosal, rectal, vaginal, inhalation, intranasal or a combination thereof. Parenteral administration (e.g., intramuscular, intravenous or subcutaneous administration) is typically achieved by injection. Administration can be local or systemic or a combination thereof. Injections can be prepared as, for example, emulsions, solid forms suitable for dissolving or being suspended in a liquid before injection, or liquid suspensions or solutions. Injection suspensions or solutions can be prepared by sterile powders, tablets, granules or the like or a combination thereof.

[0230] One or more compositions administered to humans or animals may be administered with at least one acceptable carrier. An acceptable carrier is determined in part by the specific composition administered and by the specific method used to administer the composition. Therefore, there are a variety of acceptable formulations of the disclosed compositions.

[0231] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and / or emulsions, such as oil-in-water and / or water-in-oil emulsions. Preparations for parenteral administration may also include adjuvants and / or polymers, such as CpG oligodeoxynucleotides (CpG ODN), Carbigen, Polygen, ISA 201 or 206 (such as Montanide ISA 201VG), Quil-A, trehalose-6,6-dibehenate (TBD), adjuvants based on toll-like receptor (TLR) ligands (such as TLR7 / 8 adjuvants, such as R848 (Resiquimod)), cyclic diguanylate monophosphate (c-di-GMP), polyinosinic acid-polycytidylic acid (poly (I:C)), or combinations thereof. Examples of non-aqueous solvents are alcohols or glycols (such as propylene glycol, polyethylene glycol), vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose) and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, inert gases, etc.

[0232] In some examples, the vaccine is administered by intramuscular injection into the breast muscle tissue of poultry or by intranasal administration, wherein the disclosed composition may include one or more biodegradable polymer carriers that interact with one or more mucosa. Polymers such as polylactide-co-glycolide (PLGA), chitosan (e.g., in the form of chitosan nanoparticles, such as nanoparticles based on N-trimethyl chitosan (TMC)), alginates (such as sodium alginate), carbopol, and carbopol-based polymers may be included. The composition may include one or more hydrophilic polymers, such as sodium alginate or carbopol, for example, in combination with starch. The composition may be formulated into a particle delivery system. Therefore, the composition may include liposomes, immunostimulatory complexes (ISCOMs), and / or polymer particles, such as virosomes. The composition may also include one or more bacterial-derived lipopeptides or their synthetic derivatives, such as Pam3Cys, Pam2Cys, single-chain / multi-chain palmitic acid, and lipoamino acids (LAA). The composition may also include one or more adjuvants, such as CpG oligodeoxynucleotides (CpG ODN), Flt3 ligand, Carbigen, c-di-GMP, poly (I:C), and monophosphoryl lipid A (MLA).

[0233] I. Application time

[0234] The disclosed compositions can be administered as a single dose or as multiple doses (e.g., boosters). In some examples, the first administration is followed by a second administration. For example, the second administration can be with a composition that is the same or different from the composition administered for the first time. In a specific non-limiting example, the second administration is with a composition that is the same as the composition administered for the first time. In another specific non-limiting example, the second administration is with a composition that is different from the composition administered for the first time. In a specific example, the first dose is administered three weeks (e.g., 19 to 22 days) before the second dose.

[0235] J. Dosage

[0236] The dose administered to the subject should be sufficient to induce a beneficial therapeutic response in the subject over time, or to inhibit viral infection. The dose may vary from subject to subject, depending on the species, age, weight, and general condition of the subject; the severity of the infection being treated; whether the dose is used to treat, alleviate, or vaccinate against infection; the specific composition used; and / or the mode of administration. A person of ordinary skill in the art can determine the appropriate dose using routine experiments. And different antigens can be combined at different concentrations in the dose. However, for certain disclosed embodiments, for HN protein, a suitable dose range is 5 to 100 μg of protein per dose, more typically 5 to 50 μg of protein per dose. For F protein, a suitable dose range is 5 to 150 μg of protein per dose, more typically 5 to 100 μg of protein per dose. Examples 2 and 3 provide further details on the dose range.

[0237] One of ordinary skill in the art will also appreciate that more than one dose may be beneficial or necessary. For example, it is currently believed that two administrations may be beneficial for HN or F protein immunogenic compositions.

[0238] Example

[0239] The following examples are provided to illustrate certain features and / or embodiments of the present disclosure. These examples should not be construed as limiting the present disclosure to the specific features or embodiments described. One of ordinary skill in the art will appreciate variations and other uses within the spirit of the present invention as defined by the scope of the claims.

[0240] Example 1

[0241] Production of F and HN protein antigens against Newcastle disease in the C1 system

[0242] background

[0243] Constructs were designed to provide optimal expression and yield using the C1 fungal expression system. Protein sequences of F (SEQ ID NO: 3) and HN (SEQ ID NO: 4) proteins of target NDV genotype VII were collected and analyzed. Vector backbones pMYT1055, pMYT1727, pMYT1728, pMYT1730 and pMYT1731 were used to add target cassettes transformed in the C1 expression system. The pMYT1055 vector carries the natural C1 bgl8 promoter and hygromycin resistance selection marker. Vectors pMYT1727 and pMYT1728 carry synthetic AnSES promoters and split amdS selection markers. Vectors pMYT1730 and pMYT1731 carry natural C1 bgl8 promoters and split amdS selection markers. As described below, the recombinant sequences were designed to be compatible with the corresponding vector backbones to produce and secrete target NDV antigens from the C1 system. Table 2 presents a summary of the constructs used.

[0244] F protein construct

[0245] The NDV F protein construct was designed taking into account the unique characteristics of the native protein. For example, there are two transmembrane domains in the native F protein structure, which can be removed from the construct to increase the expression and solubility of the target antigen protein ( Figure 3 ).like Figure 4 As can be seen, there is a transmembrane domain in the native HN protein, which can also be removed to improve the solubility of the recombinant protein.

[0246] The construct pMYT1358 contains a cassette with the CBH1 secretion signal and residues 32-553 of the full-length NDV F protein (SEQ ID NO:5). Residues 1-31 of the full-length protein correspond to the native NDV signal peptide and are removed to allow the attachment of the C1 fungal secretion signal CBH1ss. In addition, a C-tag with the EPEA amino acid sequence was added to the C-terminus of the target protein for purification purposes. The mature target protein of this construct (SEQ ID NO:7) has an expected size of 55 kDa. Expression of the target protein from DNL155 transformed cells corresponding to strain M5038 is shown in Figure 5-6 middle.

[0247] The pMYT1359 construct carries information that provides for the expression of the target 50 kDa F protein fragment (SEQ ID NO: 10). Here, the C-terminal transmembrane domain of the protein has been removed so that only residues 32-499 of the native F protein are included in the expressed recombinant protein (SEQ ID NOs: 8 and 9). There is also a C-terminal C-tag for protein purification methods and the native C1 CBH1ss appended to the N-terminus of the protein (SEQ ID NOs: 8 and 9).

[0248] In another example, the pMYT1360 construct carries a fragment of the F protein with two transmembrane domains removed (SEQ ID NOs: 11 and 12). Here, only residues 146-499 of the native F protein were used in the recombinant protein. The mature recombinant protein had an expected size of 38.5 kDa and was also modified with a C-terminal C-tag and CBH1ss for purification (SEQ ID NO: 13).

[0249] Constructs pMYT1361, pMYT1736, pMYT1737 and pMYT1738 contain recombinant protein sequences carrying mutations in the cleavage site motif (SEQ ID NOs: 14, 15 and 16). Specifically, there is a point mutation (K115G) that replaces the lysine at position 115 with glycine. This mutation changes the protease cleavage site in F, making it less likely to be cleaved and therefore more stable in vivo when administered as an antigenic protein to elicit an immunogenic response. The recombinant protein has the C-terminal transmembrane domain removed and also has an N-terminal CBH1ss and C-terminal C tag modifications. The mature recombinant protein has an expected size of 50 kDa (SEQ ID NO: 16). Figure 5 , 6 , 7 and 12 show the confirmed purification of this recombinant protein from strain M5041 (DNL155 strain transformed with pMYT1361).

[0250] HN protein constructs

[0251] The native HN NDV protein is an external viral membrane localized glycoprotein. Residues 24-46 of the protein amino acid sequence correspond to the N-terminal transmembrane domain ( Figure 4 ). Truncation of the N-terminal transmembrane domain may allow increased recombinant protein expression without affecting the antigenic region of the protein. There is no predicted signal peptide in the sequence, and experimental data have confirmed that the protein lacks a signal peptide. However, for the purpose of recombinant expression, the signal peptide may be modified.

[0252] The pMYT1362 construct carries the full-length HN protein under the bgl8 promoter and has a C-tag of EPEA appended to the C-terminus (SEQ ID NOs: 17 and 18). No signal peptide was added at the N-terminus. The expected size of the protein expressed from this construct is 63 kDa (SEQ ID NO: 19).

[0253] Although pMYT1363 produces the same expected size of a mature protein of 63 kDa (SEQ ID NO: 22), the recombinant protein in this construct has CBH1ss (SEQ ID NO: 21) added to the N-terminus. The recombinant protein is also under the control of the bgl8 promoter. Figures 8, 9 and 12 show the confirmed purification of the protein from this construct in C1.

[0254] The pMYT1364 construct carries the HN protein with a truncated N-terminal transmembrane domain so that the recombinant protein starts at residue 78 of the full-length native protein (SEQ ID NOs: 23 and 24). A C tag was attached to the C-terminus, and CBH1ss was added to the N-terminus. The expected mature size of the protein is 55 kDa (SEQ ID NO: 25), and purification was confirmed from fermentations of C1 transformed with this construct, as shown in Figures 8, 9, and 12.

[0255] Constructs pMYT1365, pMYT1741, pMYT1742 and pMYT1743 contain further truncated HN proteins starting at residue 124 of the full-length protein (SEQ ID NOs: 26 and 27). These constructs also include a C tag and CBH1ss for purification. Figures 8, 10, 11 and 12 show confirmed purification of the recombinant protein, which has an expected size of 50 kDa (SEQ ID NO: 28).

[0256] method

[0257] The recombinant NDV F and HN antigens derived from NDV genotype VII were expressed in protease-deficient C1 strains DNL155 and DNL157. Seven expression constructs were designed for each antigen. Codon optimization was performed for the coding sequences of both the full-length and truncated forms (lacking a transmembrane domain) of the F and HN antigens appended with a C-terminal C tag EPEA to allow them to be effectively expressed in the C1 system, these coding sequences were placed in the background of the C1 CBH1 signal sequence and embedded in insertion vectors, which contained appropriate 5' and 3' regulatory sequences to drive constitutive strong expression of the gene of interest, appropriate selection marker gene cassettes and targeting arms for integrating the construct into the clear hotspot of the heterothallic thermophilic warty mold C1 genome.

[0258] A synthetic fragment was designed containing the sequences of the full-length forms of the NDV F and HN antigens (CBH1ss and C tags were attached to their 5' and 3' ends, respectively) and flanked by the recombinant sequences of the appropriate C1 expression vector and the MssI restriction enzyme recognition site. The fragment was synthesized by GENSCRIPT. The codon usage of the protein coding sequence was optimized for efficient expression in the C1 expression system of Thermomyces heterothallicus.

[0259] In one example, the synthesized fragments were released from the GENSCRIPT construct by digestion with the restriction enzyme MssI and cloned into the PacI site of the C1 expression vector pMYT1055 under the endogenous C1 bgl8 promoter and C1 chi1 terminator by the Gibson Assembly (NEBUILD ER HiFi DNA Assembly Cloning Kit, New England Biolabs) method. The correct sequence of the construct was confirmed by sequencing the fragments inserted into the plasmid. Plasmids carrying the correct sequence of the expression cassettes for the full-length NDV F (SEQ ID NO: 5) and NDV HN (SEQ ID NO: 20) proteins were given plasmid numbers pMYT1358 and pMYT1363, respectively.

[0260] In the second example, fragments encoding truncated forms of NDV F and HN antigens were amplified from GenScript constructs by PCR using oligonucleotide primers with recombinant sequences attached to appropriate C1 expression vectors. The obtained PCR fragments were cloned into the PacI site in the C1 expression vector pMYT1055 by the Gibson Assembly method, under the endogenous C1 bgl8 promoter and C1 chi1 terminator. The correct sequence of the construct was confirmed by sequencing the fragment inserted into the vector. Plasmids with the correct sequence carrying the expression cassette of the truncated form of NDV F antigen were given plasmid numbers pMYT1359 (SEQ ID NO: 8), pMYT1360 (SEQ ID NO: 11) and pMYT1361 (SEQ ID NO: 14), respectively. Plasmids with the correct sequences carrying the expression cassettes for different forms of the NDV HN antigen were given plasmid numbers pMYT1362 (SEQ ID NO: 17), pMYT1364 (SEQ ID NO: 23), and pMYT1365 (SEQ ID NO: 26), respectively.

[0261] In a third example, fragments encoding truncated forms of NDV F (SEQ ID NO: 14) and HN (SEQ ID NO: 26) antigens were amplified from GENSCRIPT constructs by PCR using oligonucleotide primers with recombinant sequences appended to appropriate C1 expression vectors. The obtained PCR fragments were cloned into the PacI site in the C1 expression vector pMYT1730 by the Gibson Assembly method, under the control of the endogenous C1 bgl8 promoter and C1 chi1 terminator. The correct sequence of the construct was confirmed by sequencing the fragments inserted into the vector. Plasmids with the correct sequence carrying expression cassettes for truncated forms of NDV F and NDV HN antigens were given plasmid numbers pMYT1736 and pMYT1741, respectively. PCR fragments with appropriate recombinant sequences were also cloned into the PacI site in the C1 expression vector pMYT1731, under the control of the endogenous C1 bgl8 promoter and C1 bgl8 terminator. The correct sequence of the construct was confirmed by sequencing the fragments inserted into the vector. Plasmids with the correct sequences carrying the expression cassettes for the truncated forms of the NDV F and NDV HN antigens were given plasmid accessions pMYT1738 and pMYT1743, respectively.

[0262] In the fourth example, fragments encoding truncated forms of NDV F (SEQ ID NO: 14) and HN (SEQ ID NO: 26) antigens were amplified from GENSCRIPT constructs by PCR using oligonucleotide primers with recombinant sequences attached to appropriate C1 expression vectors. The obtained PCR fragments were cloned into the PacI site in the C1 expression vector pMYT1727 by the Gibson Assembly method, under the control of the strong synthetic AnSES promoter and the C1 chi1 terminator. The correct sequence of the construct was confirmed by sequencing the fragments inserted into the vector. Plasmids with the correct sequence carrying expression cassettes for truncated forms of NDV F and NDV HN antigens were given plasmid numbers pMYT1737 and pMYT1742, respectively.

[0263] Use the expression construct described above to transform protease defective C1 bacterial strain DNL155 and DNL157, from which 14 protease encoding genes have been deleted. Each of the expression constructs pMYT1358, pMYT1359, pMYT1360, pMYT1361, pMYT1362, pMYT1363, pMYT1364 and pMYT1365 is used together with the simulation carrier mate pMYT1140, which is required for completing the hygromycin resistance marker gene and being integrated into the bgl8 locus. Before transformation, the expression construct is digested with MssI. Transform with the protoplast / PEG method, and select transformant according to the nia1+ phenotype and hygromycin resistance.

[0264] Expression constructs pMYT1737 and pMYT1742 were used with the mock vector pMYT1729. The mock vector provided the second part of the split amdS marker, as well as the flanking sequences required for proper integration into the C1 bgl8 locus. Expression vector pMYT1736 was used in combination with the mock vector pMYT0781 or with the expression vector pMYT1738. Expression vector pMYT1741 was used in combination with the mock vector pMYT0781 or with the expression vector pMYT1743. The combination of two split marker vectors is required to provide both parts of the amdS selectable marker and the flanking sequences for proper integration into the C1 bgl8 locus. The combination of pMYT1737 and pMYT1742 with pMYT1729, the combination of pMYT1736 and pMYT1741 with pMYT0781 provided a single copy of the expression cassette, whereas the combination of pMYT1736 with pMYT1738 and the combination of pMYT1741 with pMYT1743 provided two copies of the expression cassette that were placed in opposite (tail-to-tail) orientations when integrated into the C1 genome. Prior to transformation, all expression constructs were digested with MssI. Transformations were performed using the protoplast / PEG method, and transformants were selected based on their ability to grow on selective minimal medium with acetamide as the sole nitrogen source.

[0265] The obtained transformants were streaked onto selective medium plates with appropriate selection (hygromycin resistance or using acetamide) and inoculated from the streaks into liquid cultures in 24-well plates. The medium components were (in g / L) glucose 5, yeast extract 1, (NH4)2SO4 4.6, MgSO4·7H2O 0.49, KH2PO4 7.48; and (in mg / L) EDTA 45, ZnSO4·7H2O 19.8, MnSO4·4H2O 3.87, CoCl2·6H2O 1.44, CuSO4·5H2O1.44, Na2MoO4·2H2O1.35, FeSO4·7H2O 4.5, H3BO4 9.9, D-biotin 0.004, 50U / ml penicillin and 0.05mg streptomycin. 24-well plates were incubated at 35°C with 800RPM shaking for four days. Culture supernatants were collected and analyzed by Western blotting using standard methods using primary detection reagent CaptureSelect biotin anti-C tag conjugate (THERMOFISHER) and chicken NDV antiserum (CHARLES RIVERLABORATORIES) and secondary reagent IRDye800CW streptavidin (LI-COR) and IRDye 680LT donkey anti-chicken secondary antibody (LI-COR). Positive clones producing target NDV antigens have been identified by Western analysis ( Figure 5 and 8).

[0266] Transformants producing NDV F and HN antigens were purified by monoclonal plating and the correct integration of the expression cassette and clonal purity of the purified clones were verified by PCR. Verified transformants were stored as glycerol stocks at -80°C.

[0267] Selected C1 strains producing different forms of NDV F and HN antigens were cultured in a 1 L bioreactor in a fed-batch process in a medium with yeast extract as an organic nitrogen source and glucose as a carbon source. For strains producing HN, the culture was carried out for 7 days at 38°C, and for strains producing F, the culture was carried out for 7 days at 38°C or 25°C. After the culture was completed, the fungal mycelium was removed by centrifugation at 4000×g for 20 minutes, the serine protease inhibitor PMSF was added to a final concentration of 1 mM, and the obtained liquid culture supernatant was stored at -80°C. In order to purify the target antigen by C-tag affinity chromatography, the frozen supernatant was thawed on ice, and after thawing, the sample was clarified by centrifugation at 20000×g for 3×20 min at +4°C and then filtered through a 0.45 μM filter. The clarified supernatant was diluted with 2 volumes of 1xPBS / 0.5M NaCl solution (12mM Na2HPO4, 3mM NaH2PO4, 650mM NaCl pH 7,3). The column was equilibrated with 1 ml CaptureSelect C-label XL column (THERMOFISHER) of the Start Protein Purification System (CYTIVA) and operated at a flow rate of 1 ml / min. Before loading the sample, the column was first equilibrated with 5 column volumes (CV) of 1×PBS / 0.5M NaCl. After loading the sample, the column was washed with 15 CV of 1×PBS / 0.5M NaCl and then eluted with a one-step gradient of 10 CV of 20 mM Tris-HCl, 2 M MgCl2, 1 mM EDTA pH 7.5 with a fraction volume of 1 ml. The column was then eluted with 1 ml of a 1% PBS column containing 1% EDTA. The Unicorn 1.0 software in the Start system integrates the UV trace of the elution peak to quantitatively calculate the amount of F and HN antigens eluted. The amount of F and HN antigens was calculated with extinction coefficients of 0.488 and 0.924, respectively. After elution, the column was regenerated with 5CV of 0.1M glycine (pH 2.3), and the column was washed with 1×PBS until pH 7.3 was reached. The eluted fractions containing the target protein were collected for the dialysis step to replace the elution buffer with 1×PBS buffer. The collected fractions were loaded into a 12ml dialysis cassette, and the dialysis cassette was dialyzed for 1 hour in 1.5L of 1×PBS at +4°C under stirring on a magnetic stirrer. After 1 hour, 1×PBS was replaced with fresh buffer, and dialysis was continued for 2 hours under the same conditions. Finally, 1×PBS was replaced and dialysis was continued overnight. The dialyzed sample was filtered through a 0.45μm filter. The concentration of dialyzed NDV F and HN antigens was determined using a Nanodrop spectrophotometer measuring absorbance at 280 nm and using extinction coefficients of 0.488 and 0.924, respectively. Aliquots (1 ml) of the antigen preparation were stored at -80°C. C-tag affinity purification of recombinant NDV F and HN antigens was performed as Figure 7 and Fig.11 shown.

[0268] Table 2

[0269] Summary of constructs used

[0270]

[0271]

[0272] Example 2

[0273] Evaluation of the efficacy and safety of inactivated Newcastle disease virus antigen (produced in C1 technology) in designated pathogen-free chickens

[0274] Study Approval and Ethics

[0275] The experiment was approved by the Israeli National Animal Use and Care Committee before it began. Adherence to this protocol ensured that due consideration was given to the welfare of the birds participating in the study, the protection of the people involved in the study, and the environment and the human food chain.

[0276] The trial was conducted in accordance with the requirements of the European Pharmacopoeia - Ph. Eur (Edition 10.0) and monograph number 01 / 2017:0870 subtitle 2-4-2-1 Evaluation of efficacy of veterinary vaccine and immune sera and in accordance with the European Guidelines on the design of studies to evaluate the safety and efficacy monograph 07 / 2018:0062 subtitle 20402.

[0277] background

[0278] Newcastle disease (ND) is caused by the virulent strain of avian paramyxovirus type 1, and is also referred to as Newcastle disease virus (NDV). NDV has significant genome variability. At present, genotypes V, VI, VII and VIII (from Class II) are dominant in the world. Inactivated NDV vaccine (NECTIV FORTE) is routinely used to vaccinate chickens and turkeys against ND. The vaccine provides slow release, but the immunity lasts longer. The vaccine formulation is a water-in-oil emulsion with a mineral oil base and two emulsifiers MONTANE and MONTANOX. In order to produce the vaccine, approximately 2,000,000,000 embryonated eggs are used to breed required antigens every year. The method is costly and labor-intensive.

[0279] Embodiments of the present disclosure produce NDV vaccination components without sacrificing the billions of chicken embryos currently required.The specific objective of this example was to evaluate the immunogenicity and protection provided by NDV antigens produced in the C1 system compared to treatment with commercially available inactivated vaccines and negative controls.

[0280] Study Design

[0281] This trial was conducted to evaluate the immune response of a new NDV vaccine formulated with HN and F proteins produced in the C1 fungal expression system. The new vaccine formulation is based on inactivated antigens in an oil emulsion for intramuscular vaccination. Six different formulations were tested as described in Table 3. The test vaccine was prepared at the ABIC R&D laboratory. Specific pathogen-free (SPF) chickens were used, and two intramuscular vaccinations were given to each chicken. The treated chickens were then challenged with vvNDV G7.

[0282] Table 3

[0283] Summary of vaccine formulations

[0284]

[0285]

[0286] In this trial, the first vaccination was completed in SPF chickens at 15 (± 3) days of age. The second booster vaccination was completed three weeks later, when the chicks were 5 weeks old. Throughout the trial, antibody responses were monitored by serological methods (HI test). Three weeks after the second vaccination, the subjects were challenged with vvNDV-G7 (358 / 2015 strain IVS).

[0287] To evaluate efficacy, on the same day, but just before the first vaccination, ten chickens were bled at zero time. Blood was taken from all chickens before the second vaccination at five weeks and three weeks after the second vaccination (eight weeks). These blood draws were completed before the vvNDV attack. These steps are summarized in Table 4. The collected serum was used for HI serological testing and to determine the immunogenicity of the test vaccine. Attacking with vvNDV determined the level of protection provided by the vaccine.

[0288] Animal selection and identification information is provided in Table 5 .

[0289] Table 4

[0290] Action Item Timeline

[0291]

[0292]

[0293] Table 5

[0294] Animal Subject Details

[0295]

[0296] Animal subjects

[0297] Only healthy chicks were included in this study. During the study period, sick or dead birds were thoroughly inspected by the veterinarian and the cause of the event was recorded. If the cause of the event was not related to vaccination, the bird was excluded from the analysis of the results.

[0298] Place male and female one-day-old chicks intended for treatment groups 1-7 in cardboard boxes. Chicks are randomly removed and placed in the room for vaccination. Each chick is uniquely identified by a wing tag on the day of vaccination according to AH-001. Record the wing tag number and treatment group.

[0299] Table 6 summarizes animal management and housing.

[0300] Table 6

[0301] Summary of Animal Management

[0302]

[0303] Testing vaccines

[0304] The vaccines used are described in Table 7. The sources of protein antigens for HN and F proteins are described in Table 8.

[0305] Table 7

[0306] Description of the vaccine or antigen used in the vaccine

[0307] Table 8

[0308] Sources of antigens for vaccine preparations

[0309]

[0310] Vaccine preparation

[0311] Preparation of small-scale test batches

[0312] The protein concentration was adjusted to 50 μg / 0.5 mL / vaccine dose. Each chicken received two doses during the trial - a primary vaccination and a booster vaccination. Emulsion preparation was performed for both immunizations. After preparation, the vaccine was filled into sterile tubes and stored at 2°C-8°C until use.

[0313] Material:

[0314] 1. Mineral oil, such as DRAKEOL (supplier: Gadot; batch number: 10-035431)

[0315] 2. Sorbitan oleate, an adjuvant (supplier: Zefroni sapaki chmicalim Ltd.; batch number: 10-038020)

[0316] 3. PBS (supplier: Biological Industries Ltd.; batch number: 2048951)

[0317] 4. Sorbitol-T, an adjuvant (supplier: Zefroni sapaki chmicalim Ltd.; batch number 10-035482)

[0318] preparation:

[0319] 1. In a sterile laminar flow hood, prepare each formulation individually.

[0320] 2. The oil phase consists of mineral oil (such as DRAKEOL) and emulsifier (such as MONTANE).

[0321] 3. In separate tubes, prepare aqueous phase (30%) from each antigen, sterile PBS and emulsifier (eg MONTANOX).

[0322] 4. Mix the oil phase in a homogenizer at low speed and pour the water phase into the oil phase until homogenized. Then increase the speed and mix the solution for 60 seconds. Table 9 presents the volumes of the components in the final emulsion.

[0323] 5. Each volume was then divided into two equal portions in 15 mL tubes.

[0324] 6. Seal the tube and double check the label.

[0325] 7. The final emulsion prepared in the tube was then stored at 2°C-8°C until use.

[0326] Table 9

[0327] Volumes of components used in vaccine formulations

[0328]

[0329]

[0330] *Antigen concentration (mg / mL): HN63=0.4; HN55=0.48; HN50=0.73; F50=0.52

[0331] Inactivated vaccines

[0332] A commercial inactivated ND (VH strain) vaccine (NECTIV FORTE, batch number A0022111672) was used as a reference for efficacy evaluation.Each chick from group number 2 was vaccinated with 0.3 mL of NECTIV FORTE vaccine into the breast muscle by IM route.

[0333] Application

[0334] Vaccination by IM into the pectoral muscle was done according to the timeline as described in Table 4.

[0335] Attack virus

[0336] 10 mL per 0.5 mL volume by IM injection 5.3 EID 50 The challenge was performed in KVI using genotype VII vND (isolate 358 / 2015IVS).

[0337] Efficacy evaluation

[0338] Evaluation parameters

[0339] Serology of the sera was evaluated by the HI method for the recombinant LaSota-HN-G7 strain developed in the R&D department. Antibody titers induced by NDV were tested after two vaccinations. The commercial vaccine NECTIV FORTE and 5 novel vaccine formulations were compared. All serum samples were evaluated using homologous VH and calibration antigens of the laSota-HN-G7 virus.

[0340] Test limits:

[0341] The HI negative control should be less than 2 units.

[0342] HI positive is a sample with HI 2 units higher than the negative control.

[0343] All relevant sera were also tested by ELISA to detect F protein antibodies using NDV-F ELISA by Biocheck. The test limit was in accordance with the instructions of the commercial kit.

[0344] Virus Attack

[0345] The level of protection of all groups vaccinated with the commercial vaccine NECTIVFORTE and the five new vaccine formulations was evaluated after challenge with velogenic ND virus genotype VII (358 / 2015 IVS). The trial was valid since the mortality of the negative control treatment was 100% after challenge.

[0346] result

[0347] Titer

[0348] For HI titers against genotype 7 antigen, all bird sera from the first bleed were tested with rLaSota-HN-G7 antigen. In this test, 24 serial dilutions were included to have maximum HI potential. The results are shown in Fig.13 The avian sera from the second test were also tested for HI potential against genotype 7 titers using the rLaSota-HN-G7 antigen in a 24-serial dilution format. The results from the second test are shown in Fig.14 The titers after the first dose were tested against genotype 2 using VH antigen, and as Fig.15 The second dose response was also measured by HI titer against genotype 2 (VH) antigen, and the results are shown in Fig.16 shown.

[0349] For ELISA-NDV-F titers, sera from birds in the F50 treatment group were tested on the NDV-F ELISA. The test was valid and the results were as follows Fig.17 Finally, the ELISA-NDV-F titer of the HN63+F50 treatment group is shown in Fig.18 This test is also valid.

[0350] HI titers are summarized in Table 10.

[0351] Table 10

[0352] HI titer of the vaccine used

[0353]

[0354] Protect

[0355] Following challenge with velogenic NDV genotype VII (358 / 2015 IVS), mortality among subjects in treatment groups was recorded to determine the level of protection provided by various vaccines, as summarized in Table 11.

[0356] Table 11

[0357] Level of protection of the vaccines tested

[0358] Vaccine treatment group Deaths / total subjects Protection level percentage PBS 10 / 10 0％ NECTIV FORTE 1 / 10 90％ HN63 0 / 10 100％ HN55 0 / 10 100％ HN50 1 / 10 90％ F50 1 / 10 90％ HN63+F50 0 / 10 100％

[0359] in conclusion

[0360] All new recombinant HN antigens produced high levels of antibodies against genotype 7 and genotype 2 NDV strains. In addition, the recombinant F antigen also produced high levels of antibodies. Vaccinated chickens were found to be resistant when challenged with velogenic NDV genotype VII (358 / 2015 IVD). The mixture of HN63 and F50 provided no added value in serological or challenge tests. All recombinant antigens were found to be equivalent to the positive control inactivated vaccine.

[0361] Example 3

[0362] Evaluation of inactivated HN50 and F50 recombinant vaccines (produced with C1 technology) for PD50 and reduction of challenge virus secretion in SPF chickens.

[0363] background

[0364] Newcastle disease (ND) is a highly contagious viral disease that can affect all avian species. It is classified as the 14th most problematic disease of poultry and the eighth cause of loss of wildlife through destruction. This enveloped virus (genus Mumps virus, Paramyxoviridae family, Mononegavirales order, recently renamed Mumps virus or AAvV) has significant genomic variability, and genotypes V, VI, VII and VIII (from Class II) are prevalent in different countries around the world.

[0365] Inactivated ND vaccine (NECTIV FORTE) is routinely used to vaccinate chickens and turkeys against ND. The vaccine provides a slow response, but the immunity lasts longer. The vaccine formulation is an oil-in-water emulsion containing an emulsifier, wherein the oil is mineral oil, and the emulsifier is MONTANE and MONTANOX. The vaccine relies on producing viral antigens in embryonated eggs, which is time-consuming and labor-intensive. Although other commonly used expression systems cannot produce correctly folded or biologically active antigens, C1 is disclosed herein as a compatible expression system to produce biologically active NDV antigens in a more effective manner than using embryonated eggs.

[0366] C1 has a unique morphology, allowing high productivity and growth at low viscosity using a wide range of fermentation conditions at a flexible commercial scale. The expression system provides a high-yield, low-cost, rapidly scalable rVaccine and antibody gene expression platform in a disposable or stainless steel bioreactor. It provides a safe and effective antigen, which is generally recognized as safe (GRAS) certified by the U.S. FDA, and no negative clinical effects have been seen in animal studies. In addition, the natural C1 glycan structure is more like mammals than yeast. Natural C1 glycans are mainly high mannose types (Man3-Man9), including a small amount of hybrid glycans. Example 2 demonstrates the ability of three recombinant HN protein antigens and one F protein antigen to confer high Ab levels and good protection levels in vvNDV attacks in poultry.

[0367] The purpose of this embodiment is to prove the PD50 of the recombinant HN50 and F50 albumen produced in C1 and used for inactivated vaccine.Particularly, this embodiment shows the amount of protective dose and evaluates the PD50 existing in 0.5mL vaccine volume (50 μ g recombinant protein per dose).This embodiment has also established the correlation between protective dose, serological response and protection percentage.Finally, it has also been demonstrated that the minimization of NDV genotype VII excretion after the virus attack.

[0368] Reasonable explanation

[0369] The test system described below is based on the requirements of the European Pharmacopoeia (Ph. Eur) 10.5, Newcastle Disease Vaccine (Inactivated) 07 / 2020:0870: "2-3-2-1. Vaccines for use in chickens.

[0370] However, considering the strains found in Israel, NDV challenge was performed with vNDV gVII (strain 358, recently isolated). 6.0 ELD 50 The birds were observed daily for at least 21 days after challenge, and the number of deaths and the number of surviving birds showing clinical signs of disease were recorded.

[0371] Study Design

[0372] Treatment Group

[0373] 195 SPF chicks were hatched in Har-Tuv animal house and divided into 13 groups. All groups had 15 birds each. Each group was placed in a cage in an inactivation room and inoculated with an inactivated vaccine.

[0374] At day 21, wing numbering and vaccination by intramuscular (IM) route were performed on chicks from groups 1-13, wherein for each chick, different dose volumes of the test vaccine were as follows (summarized in Table 12): Groups 4 and 9 received 1:200 doses of their respective vaccines, Groups 5 and 10 received 1:100 doses of their respective vaccines, Groups 6 and 11 received 1:50 doses of their respective vaccines, Groups 7 and 12 received 1:25 doses of their respective vaccines, and Groups 8 and 13 received 1:12.5 doses of their respective vaccines (Table 12). Chicks from group 1 were not vaccinated and served as unvaccinated controls. Chicks from groups 2 and 3 received full doses of F50 and HN50, respectively.

[0375] 21 days after vaccination, all chicks were bled and HI titers were determined using the HI method BSH-QCM-0000626 (E-QCM-169-03) with VH antigen and rNDV-LS-HN G7 antigen according to the European Pharmacopoeia (EP) 10.5 edition monograph (No. 07 / 2020:0870, subheading 2-5-2-3, Haemagglutination inhibition).

[0376] 21 days (3 weeks) after vaccination, all groups (from 1 to 13) were sent to Kimron Veterinary Institute (KVI) to be challenged by IM route with a dose of 106.0 ELD50 of vNDV genotype VII (strain 358). All chicks were observed during at least 21 days after challenge.

[0377] Tracheal and cloacal swabs were collected from Groups 1-5, 9-10 on days 4, 6, and 8 after challenge, and viral RNA was quantified using the qPCR method.

[0378] At the end of the observation period after challenge, the PD50 was calculated by comparing the number of surviving birds without any signs of Newcastle disease in each group using statistical analysis. The experimental procedures are summarized in Table 12. Blood samples were taken for serological tests before vaccination (time zero) and before challenge.

[0379] Table 12

[0380] Study Groups and Activity Phases

[0381]

[0382]

[0383] Timeline of events

[0384] Table 13

[0385] Timeline of Trial Activities and Events

[0386]

[0387]

[0388] Material

[0389] Test system - animals

[0390] All management and husbandry procedures were performed according to AH-001. Table 14 shows the details of the test system used in this study.

[0391] Table 14

[0392] Details of the test system performed

[0393]

[0394] Inclusion / Exclusion / Withdrawal Criteria

[0395] To be included in this study, chicks had to meet the following requirements: (1) originate from the same SPF colony and batch of eggs, and (2) be healthy chicks with no signs of disease at the time of enrollment, as determined by normal appearance and normal behavior.

[0396] The admission of birds to the study was recorded in AH-004 Form 04. Daily observations were conducted for 21 days after the challenge at the Ministry of Agriculture and Rural Development Veterinary Services. During the study, any sick or dead birds were examined by the responsible veterinary surgeon until the end of the challenge and the reasons were recorded in AH-004 Form 04. Throughout the course of the study, animal withdrawals were made according to AH-016. The number of animals withdrawn prematurely from the study and the reasons for such withdrawals were recorded.

[0397] Animal Management

[0398] Animal management and housing procedures are shown in Table 15.

[0399] Table 15

[0400] Animal Management

[0401]

[0402]

[0403] Animal randomization

[0404] One to 13 day-old chicks expected to enter the treatment group were placed in cardboard boxes from which they were randomly removed and assigned to a group. Both males and females were selected for the experiment. Each chick was identified by wing marking on the day of vaccination.

[0405] Blind

[0406] Only the HI test was blinded. Vaccination was not blinded because the vaccination volume was different for each group and required technician familiarity.

[0407] Test items

[0408] Test item characterization

[0409] Table 16

[0410] Characteristics of vaccine emulsion preparations

[0411]

[0412]

[0413] F50 and HN50 proteins

[0414] To generate the emulsions, the purified F50 and HN50 proteins were not previously inactivated.

[0415] Inactivated F50 and HN50 vaccines

[0416] The method for producing F50 and HN50 protein emulsions is as follows.

[0417] F50 (M-1-2670): Prepare the aqueous phase in a sterile fume hood by diluting 2.88 mL of F50 into 1.52 mL of PBS. Add 0.15 mL of Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL of mineral oil with 0.45 mL of Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0418] HN50 (M-1-2671): Prepare the aqueous phase in a sterile fume hood by diluting 2.1 mL HN50 into 2.3 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0419] As shown in Table 17, 15 mL of emulsions were produced by using Montane (Sorbital-S), Montaneox (Sorbitol-T), mineral oil, and PBS.

[0420] Table 17

[0421] HN50 and F50 emulsion preparations

[0422] formula F50 HN50 DRAKEOL (67%) 10 mL 10mL Montane (3%) 0.45mL 0.45mL *antigen 2.88mL 2.1mL PBS 1.52mL 2.3mL Montaneox (1%) 0.15mL 0.15mL Total volume (mL) 15 15

[0423] *Antigen concentration: HN50 = 0.73 mg / mL; F50 = 0.52 mg / mL.

[0424] Test dose

[0425] At 21 days of age, all chicks from Group 2 to Group 13 were vaccinated IM into the breast muscle. This vaccination was performed using a 10 μl syringe. Each chick from Group 4 and Group 9 received a dose of 40 μl of their respective vaccines; Groups 5 and 10 received a dose of 20 μl of their respective vaccines; Groups 6 and 11 received a dose of 10 μl of their respective vaccines; Groups 7 and 12 received a dose of 5 μl of their respective vaccines; and Groups 8 and 13 received a dose of 2.5 μl of their respective vaccines (Table 12). Groups 2 and 3 received a dose of 500 μl of F50 and HN50 (Table 12). Groups 1 and 15 were not vaccinated.

[0426] At 42 days of age (21 days after vaccination), 15 chicks from all groups 1 to 13 were challenged by the IM route into the breast muscle. Each chick from groups 1 to 13 received a dose of 106.0 ELD50 / dose of challenge material (Table 18).

[0427] Table 18

[0428] Attack Material Characterization

[0429] identity VNDV GVII (strain 358) supplier Kimron Veterinary Institute(KVI) Titer 106.0ELD50 / dose

[0430] method

[0431] Trial Administration Method - Vaccination

[0432] For vaccination, the vaccine was taken out of the refrigerator and warmed using a warm water bath until the vaccine reached approximately 37° C.-39° C. This procedure reduced the viscosity of the emulsion, which facilitated administration and prevented local reactions and pain.

[0433] The vaccine container was gently agitated before and during the vaccination process to homogenize the contents. The required amount of vaccine was manually injected into the chicken using a syringe (10 μL volume capacity).

[0434] The vaccination sequence was carried out starting with all chickens injected with inactivated F50 batch number M-1-2670, starting with group 4, second with group 5, third with group 6, fourth with group 7, and then with group 8. Syringes were changed between groups. Vaccination with inactivated HN50 batch number M-1-2671 started with group 9, followed by group 10, group 11, group 12, and then group 13. Syringes were changed before vaccination of group 9 with HN50.

[0435] Test Methods - Efficacy Evaluation

[0436] a) Serology: Blood samples were collected from each chicken on the day of vaccination (antibody levels at time zero) and 21 days after vaccination. In addition, serum was separated according to E-QCM-192 and antibody levels were measured according to the European Pharmacopoeia (EP) (edition 10.5), monograph (No. 07 / 2020:0870, subheading 2-5-2-3, Haemagglutination inhibition) with VH antigen and rNDV-LS-HN G7 antigen by using the HI test according to BSH-QCM-0000626 (E-QCM-169-03).

[0437] The F50 group was tested in the NDV-F ELISA kit (Biocheck) for specific F protein antibody levels.

[0438] b) Challenge: This procedure was performed at the Veterinary Service by injecting 106.0 ELD50 / dose of vvNDV gVII via the IM route. After challenge, the birds were observed daily for 21 days to detect any morbidity, mortality and reduction in excretion (shedding) of vNDV genotype VII virus.

[0439] c) Virus shedding: Cloacal and tracheal swabs were collected from groups 1-5, 9-10 on days 4, 6, and 8 post-challenge. The swabs were sent to KVI and the viral load was determined using quantitative real-time PCR based on the KVI method.

[0440] Statistical analysis

[0441] PD50 is determined by the dose of vaccine that is expected to protect 50% of chickens against a challenge dose of NDV. PD50 also reflects the correlation between antigen quantity, serological response and protection against challenge.

[0442] By using the European Pharmacopoeia Standard 10.2 Monograph 0870 paragraph 2-3-2 as a guide for vaccine (inactivated) immunogenicity for Newcastle disease vaccines, the vaccine should comply with the PD50 test (50% protective dose) and should not fall below 50% of the recommended dose with a lower confidence limit of 35 PD50 per dose. This means that the NDV vaccine must contain at least 50 PD50 in one recommended dose and is calculated using the Reed and Muench method as presented below:

[0443]

[0444] Logarithm of 50% endpoint = dilution above 50% - (PD * log dilution factor)

[0445] PD50=10-x, where x is the logarithm of the 50% endpoint. The statistical differences between each group based on the PD50 results were determined using JPM 12.0.1 software by using a two-way ANOVA model measurement design.

[0446] Evaluation and evaluable parameters

[0447] Both the tested inactivated F50 and HN50 vaccines (batch numbers M-1-2670 and M-1-2671) were compared in terms of protective efficacy by challenge (vNDV genotype VII) and HI tests.

[0448] In addition, PD50 was calculated to evaluate the number of birds in each vaccinated group that survived without showing any signs of Newcastle disease over a period of 21 days post-challenge.

[0449] Furthermore, by this calculation it is verified whether the vaccine complies with the test if the minimum dose specified in the 0.5 mL dose corresponds to no less than 50 PD50.

[0450] Disease Management

[0451] In this study, care was taken to avoid contamination between the experimental groups and the negative control. When visiting the study animals, either for study procedures or animal care, all researchers always attended to the unvaccinated control group followed by the vaccinated group. The following precautions were also taken.

[0452] Study Animal Disposal: During or at the end of the trial, dead birds were disposed of according to the procedures outlined in AH-016. Briefly, biohazardous waste, including carcasses and excess material, was placed in 2 bags and should be emptied only into the biohazardous waste container bin in the yard. Carcasses that could not be emptied by an authorized company on the same day should be kept in a dedicated refrigerator until the day of emptying.

[0453] Disposal of Investigational Product: Place excess test material into 2 bags and empty into biohazard waste container only.

[0454] Clothing: White coat rule according to AH-017. According to this SOP, at each entry into the inactivated vaccine department, each staff member will take off his personal clothing and put on work clothes and shoes belonging only to that department.

[0455] Protective Measures: The procedures used in this study are considered to pose a low risk to operators, except in the case of accidental injection of oil-emulsion products into a person, which may produce a local reaction resulting in severe injury (necrosis). In this case, immediate medical attention and treatment to remove the oil-emulsified product from the affected area should be administered to the injured person. For safe use, caution when administering the vaccine through proper injection technique and bird handling can reduce the potential for accidents.

[0456] Autopsy: In the case of an autopsy, where sampling is done in the washroom, each staff member must don an additional set of overalls and shoe covers per AH-017. Once the procedure is complete, the overalls and shoe covers in the washroom are disposed of as biohazard waste per AH-016.

[0457] result

[0458] HI titer of genotype 7 (rLaSota-HN-G7) antigen after one dose

[0459] All sera from birds after one dose were tested with rLasota-HN-G7 antigen. Only full doses tested from 24 serial dilutions were included in this test to have the maximum HI potential. Dilution doses were tested through 12 serial dilutions.

[0460] As expected, the F50 group did not give a significant reading by the HI test. As expected, the zero time blood draw gave an average HI score of 1.13, and the negative unvaccinated control gave a low HI average score of 2.8. However, the full dose of HN50 (50 μg) gave an average score similar to that described previously after the first vaccination. Then, the dilution dose showed a decrease in the HI average score associated with the dose dilution. The standard deviation of the HN50 group decreased as the dose was diluted. These results are shown in Table 19. Fig.19 HI titers against genotype 7 (rLaSota-HN-G7) antigen after one dose of treatment are shown.

[0461] Table 19

[0462] HI titer of genotype 7 (rLaSota-HN-G7) antigen after one dose

[0463] Group HI average Standard Deviation ％ Negative control (no vaccination) 2.80 1.207 7.0 F50 positive control (full dose) 2.33 0.724 0.0 HN50 positive control (full dose) 10.0 1.604 100 F50 dilution (1:12.5) 2.73 1.438 13 F50 dilution (1:25) 2.60 1.121 0.0 F50 dilution (1:50) 2.00 1.047 7.0 F50 dilution (1:100) 2.53 0.640 0.0 F50 dilution (1:200) 2.67 0.488 0.0 HN50 dilution (1:12.5) 6.80 1.699 87 HN50 dilution (1:25) 6.47 1.407 80 HN50 dilution (1:50) 5.79 1.051 71 HN50 dilution (1:100) 4.33 0.900 13 HN50 dilution (1:200) 4.13 1.246 13 Time 0 1.33 1.118 0

[0464] HI titers against genotype 2 (VH) antigens after one dose

[0465] All bird sera after one dose were tested with VH antigen. In this test, only the full dose was tested through 24 serial dilutions to have maximum HI potential. The diluted dose was tested through 12 serial dilutions.

[0466] As expected, the F50 group did not give a significant reading by the HI test. The zero time bleed gave an average HI score of 0, and the negative unvaccinated control gave a low HI average score of 0.8. However, the full dose of HN50 (50 μg) gave a lower average score than previously described after the first vaccination. The diluted doses showed a decrease in the HI average score associated with the dose dilution. The standard deviation of the HN50 group decreased as the dose was diluted.

[0467] All bird sera from the first bleed were tested with VH antigen. In this test, 24 serial dilutions were included to have maximum HI potential.

[0468] exist Fig. 20 The level of protection after one dose against velogenic ND virus genotype VII (358 / 2015IVS) is shown in . All birds were challenged with velogenic ND virus genotype VII (358 / 2015IVS). The test was effective. All HN50 groups showed high protection.

[0469] Table 20

[0470] Table of mortality at the end of the PD50 trial

[0471] Group Deaths / Total Protect(%) Not vaccinated 15 / 15 0 F50 Full Dose 15 / 15 0 HN50 Full Dose 0 / 15 100 F50 1:12.5 15 / 15 0 F50 1:25 15 / 14 7 F50 1:50 15 / 15 0 F50 1:100 15 / 15 0 F50 1:200 15 / 15 0 HN50 1:12.5 15 / 1 93 HN50 1:25 15 / 1 93 HN50 1:50 14 / 1 93 HN50 1:100 15 / 1 93 HN50 1:200 4 / 15 73

[0472] HN50 antigen PD50 determination

[0473] Using the World Organization for Animal Health (OIE) Newcastle disease vaccine standard as a guide, the vaccine meets the test if the PD50 (50% protective dose) per recommended dose is not less than 50 and if the lower confidence limit is 35 PD50 per dose. The calculation is performed using the Reed and Muench method, as exemplified below:

[0474]

[0475] Logarithm of 50% endpoint = dilution above 50% - (PD * log dilution factor)

[0476] PD50 = 10-x, where x is the logarithm of the 50% endpoint

[0477] Table 21

[0478] Version A—Scenario where 1:400 of the treated group did not survive

[0479]

[0480] *Theoretical assumptions used for calculation purposes.

[0481] 100% (15 / 15) of the chickens died and 0% of the chickens survived.

[0482] It is close to less than 50%

[0483]

[0484] Log of 50% endpoint = -2.6 - (0.136*0.3) = 10 2.64 = 436.5 PD50 / dose.

[0485] Table 22

[0486] Version B—Scenario where 50% of the treatment group 1:400 do not survive

[0487]

[0488]

[0489] *Theoretical assumptions used for calculation purposes.

[0490] 53% (7 / 15) of the chickens died and 47% of the chickens survived.

[0491] It is a number close to less than 50%.

[0492]

[0493] Logarithm of 50% endpoint = -2.6 – (0.49*0.3) = -2.6 – (0.147) = 10 2.747 = 558 PD50 / dose

[0494] ELISA-NDV-F titer of F50 group after one dose

[0495] F50 avian sera from two bleeds were tested on the NDV-F ELISA. The test was valid.

[0496] Table 23

[0497] NDV-F ELISA mean titer and standard deviation

[0498] Group Antibody titer Standard Deviation Not vaccinated 218 318 F50 Full Dose 13,476 7,560 F50 1:12.5 3,250 2,651 F50 1:25 1,607 1,550 F50 1:50 1,137 975 F50 1:100 518 520

[0499] To explain the lack of protection in the F50 full dose group, a comparison between the previous study and the current study was performed (Table 23). The F50 full dose in this study gave an average titer of 13,476 and a standard deviation of 7,560. All birds in this group died after the attack. The F50 full dose in the previous study gave an average titer of 10,116 and a standard deviation of 6,128. No more attacks were performed after the first dose. The booster vaccination was performed 21 hours later. The ELISA NDV-F titers after one dose of F50 were similar between the two studies.

[0500] NECTIV FORTE gave a mean titer of 27,297 and a standard deviation of 4,480 after the initial dose in the previous study. No further challenges were performed after the first dose. The challenge was performed 21 hours after the booster vaccination. After the NECTIV FORTE booster vaccination, the titer was 41,052 with a standard deviation of 2,556. This group gave 90% protection after the challenge.

[0501] In a previous study following F50 booster vaccination, the titer was 32,853 with a standard deviation of 11,967. This group gave 90% protection after challenge.

[0502] The conclusion of this comparison was that one dose of F50 was insufficient to induce high antibody levels and therefore the level of protection was suboptimal.

[0503] NDV secretion in HN50-vaccinated and challenged chickens

[0504] Tracheal and cloacal swabs were collected from Group 3 (HN50 full dose) and Group 9 (HN501:12.5) 4, 6 and 8 days after challenge, and viral RNA was quantified using the qPCR method (KVI method). The results are shown in Table 23.

[0505] Table 24

[0506] ELISA NDV-F raw data

[0507]

[0508]

[0509]

[0510] in conclusion

[0511] The HN50 antigen produced high levels of antibodies against genotype 7 and genotype 2 strains with high percentage protection. The PD50 for HN50 was calculated in two ways and ranged from 436.5 to 558 PD50 per dose.

[0512] Furthermore, the recombinant F50 antigen did not generate high levels of antibodies against challenge with velogenic ND virus genotype VII (358 / 2015IVS).In conclusion, this trial completed a proof-of-concept demonstration of the HN50 antigen.

[0513] Table 25

[0514] HI score before challenge (serology) and mortality score after challenge

[0515]

[0516] *ND = Not Conducted, HI values ​​are the average of each group of 15 birds Example 4

[0517] Expression of NDV antigens in the C1 system

[0518] In the present embodiment, two other strains are constructed.First strain of construction comprises four copies of HN50 expression cassette, two copies of cbh1 and two copies of bgl8 locus.Second strain of construction comprises two copies of expression cassette of F50 albumen with T4 fiber protein trimerization domain at bgl8 locus.The NDV antigen production level of target protein NDV F50-C label is 0.3g / l for the expression cassette of single copy, and is 0.3g / l for the expression cassette of two copies.The NDV production level of NDV HN50-C label is 1.1g / l for the expression cassette of single copy, and is 4.2g / l for the expression cassette of two copies.

[0519] Fig.24 Provided is a gel image of the additional output of the bioreactor fermentation run for analyzing the production of NDV F50 protein production strains.MT642 is performed with the C1 strain M6316 producing NDV F50, which is a clone purified from transformant M5824 (chitinase deletion strain / 2x NDV F50 production strain, with the addition of trimerization domain) at 25°C.MT649 is performed with the C1 strain M6318 producing NDV F50 protein alternatively, which is a clone purified from M5824 transformant (chitinase deletion strain / 2x NDV F50 production strain, with the addition of trimerization domain) at 32°C.The fermentation supernatant is pink, and each well is loaded with 0.5 μL.Target protein detection is carried out with NDV antiserum / rabbit anti-chicken Ig-IRD680 (shown in red) and biotin-α-C label / streptavidin-IRD800 (shown in green). Improved production levels of the NDV F50 producing strain were achieved in additional runs, with the alternative NDV F50 producing strain producing 1.7 g / L after 6 days and 1.9 g / L after 7 days at 32°C.

[0520] Fig.25 Provided are gel images of purified C1 NDV F and HN antigens expressed using the C1 system analyzed on Western blots and stained gels under reducing and non-reducing conditions. Target protein detection is performed using NDV antiserum / rabbit anti-chicken IgIRD 680 (shown in red) and biotin-α-C label / streptavidin-IRD800 (shown in green). The merged (yellow) image indicates where the signal overlaps. The purified protein shows good stability, and the addition of the trimerization domain to the NDV F antigen causes an increase in molecular weight of about 5kDa. In addition, putative oligomers (dimers and trimers) can be seen in the non-reducing samples of the NDV F antigen in phase III. The NDV HN antigen produced in phases I and III shows the same gel mobility. It is preliminarily estimated that the output of the NDV F antigen with the trimerization domain is about 1.9g / L, and it is estimated that the NDV HN50 antigen with 4 copies of the HN50 expression cassette provides a production level of 10g / L. A summary of the strains used in this example is provided in Table 26.

[0521] Table 26

[0522] Summary of strains of Example 4

[0523]

[0524] SEC-UV: A standard protein mixture and a sample containing the NDV-F50 protein with a trimerization domain with an expected monomer mass of 53,376.3 Da (average mass calculated by the NIST protein calculator) with oxidized disulfide bridges (10 cysteines) were analyzed by UV-DAD chromatography using PBS buffer: 12 mM NaH2PO4*H2O, 150 mM NaCl pH 7,3. Fig.26 The SEC-UV chromatogram (202-340 nm) in PBS buffer is shown, which shows that the standard sample is highly similar to the manufacturer's chromatogram, and the F50 sample shows a broad early peak and two sharper peaks eluting at 7.85 minutes and 8.14 minutes. The calculated monomer mass is 53,376.3 Da; therefore, the trimer would be 160.1 kDa. The predicted mass of the main peak based on SEC-UV is approximately 250 kDa. The broad peak eluting early may be due to aggregation and / or oligomerization.

[0525] SEC-UV-MS: Analysis was performed by UV-DAD chromatography with 40 mM unbuffered ammonium acetate and a pH of approximately 6 compared to untreated samples in PBS buffer and compared to untreated samples in MS buffer. Fig.28 Shown are SEC-UV chromatograms of untreated sample (PBS buffer), untreated sample (MS buffer), and MS buffer PngaseF treated using minimal denaturing conditions (1:5 dilution in Rapid PngaseF buffer, approximately 1:30 protein ratio of Rapid PngaseF enzyme, at 40°C for four hours). Fig.28 The combined spectra showing the chromatograms of the untreated sample produced unresolved protein spectra at all time points, and the PngaseF-treated sample produced one spectrum at several time points. Deconvolution in the m / z region of approximately 2000 showed proteins of approximately 50 kDa to 58 kDa. Deconvolution in the m / z region of approximately 4500 showed proteins of approximately 106 kDa and 110 kDa. Given Fig.29 , the spectrum from 8.5 min showed the m / z-dependent presence of approximately 55 kDa and 11 kDa proteins of monomer and dimer, and the SEC-UV exponential fit at 8.5 min predicted a 180 kDa mass (see Fig. 27 ). Fig.30 is the MS spectrum of the PNGase treated sample showing the spectrum merged and deconvoluted from the main peak at 9.1 minutes. In addition, dimers and monomers are seen, with some evidence of trimeric protein. The deconvoluted result for the monomer at 53,381 Da is close to the theoretical mass. Fig.31The MS spectrum of the PNGase treated sample merged from the 10 minute peak is shown (SEC predicted mass is approximately 50 kDa). Deconvolution shows masses at 49,445 Da and 53,880 Da, the latter close to the theoretical mass. The 3.7 Da difference from the theoretical value may be attributed to open disulfide pairs and deamidation.

[0526] SEC-UV-MS results with MS compatible buffer showed UV elution similar to PBS buffer. The MS spectra showed that without PngaseF treatment, the MS spectra were not sufficiently resolved for deconvolution (MW estimation). PngaseF treatment revealed dimer and monomer masses eluting at three to four different time points, because the complexes may have disassembled in the ionization source and / or because mild denaturants were used with PngaseF, resulting in partially interfering complexes.

[0527] The monomeric protein mass was very close to the theoretical mass, so protein expression was successful.

[0528] Example 5

[0529] NDV dose response of HN50 and F50 antigens from the C1 expression system in SPF chickens.

[0530] background

[0531] As previously discussed, Newcastle disease (ND) is a highly contagious viral disease that can affect all avian species. This enveloped virus (genus Mumps virus, family Paramyxoviridae, order Anasenovirales, recently renamed avian mumps virus or AAvV) has significant genomic variability, and genotypes V, VI, VII, and VIII (from class II) predominate in different countries around the world.

[0532] Clinical signs associated with infection are described as respiratory, gastrointestinal and neurological signs, depending on the immune status and susceptibility of the animal and the strain involved. Depending on their pathogenicity, NDV viruses can also be classified as velogenic viscerotropic (ability to cause necrotic hemorrhagic lesions in the intestine, causing up to 100% morbidity and mortality in young chickens, vvNDV); mesotropic (respiratory and neurological signs with moderate morbidity and mortality); and chronic (mild respiratory disease observed, especially when associated with secondary bacterial infection, used as a live vaccine).

[0533] Inactivated ND vaccine (Nectiv Forte TM) is routinely used to vaccinate chickens and turkeys. The vaccine provides a slow response, but the immunity lasts longer. The vaccine formulation is a water-in-oil emulsion and is based on mineral oil plus Montane / Montaneox as an emulsifier. In order to produce NDV antigens for inactivated ND vaccines, the virus is bred in embryonated eggs. The vaccine is based on the LaSota backbone expressing the HN gene of NDV G7. The positive control in this example is the NDV G7 vaccine.

[0534] As disclosed herein, C1 is used to produce immunogenic recombinant antigens (HN or F proteins) in fungal cell lines to replace the production of ND vaccines in embryonated eggs. C1 has a unique morphology that allows high productivity and growth in low-viscosity media using a wide range of fermentation conditions at a flexible commercial scale. The expression system provides a high-yield, low-cost, rapidly scalable rVaccine-antigen and antibody gene expression platform in disposable or stainless steel bioreactors. It provides safe and effective antigens, which are generally recognized as safe (GRAS) certification by the U.S. FDA, and no negative clinical effects have been observed in animal studies.

[0535] In previous studies, the minimum dose of HN protein in the PD50 assay was found to be 13.5 μg to 17 μg per dose (equivalent to 150 PD50 doses). The method for purifying the protein involves centrifugation at 7000 g and then filtering the supernatant using a 0.45 μm filter. The filtrate can be further loaded onto a c-tag affinity column to obtain pure protein.

[0536] Purpose

[0537] The objectives of this study were to (1) test the dose response of HN50 purified protein, 10 kDa ultrafiltered material after centrifugation, and filtrate after filtration at 0.45 μM; and (2) test the dose response of F50 purified protein from Phase 3 in both the initial and boosted maximum doses.

[0538] Study Design

[0539] Treatment Group

[0540] 195 SPF chicks are hatched in Har-Tuv animal house and are divided into 12 groups.The 1-10 group includes 12 chickens separately, and the 11th group and the 12th group include 24 chickens separately.This includes 11 vaccinated groups and an unvaccinated control group.At 21 ages in days, before vaccination (time zero), blood samples are randomly obtained from chicks, the chicken from the 1-12 group is marked with neck, and according to Table 27, the volume of 0.3mL / dosage / chicken is injected by intramuscular (IM) injection with appropriate preparation to the avian in every group is vaccinated.At 42 ages in days (three weeks after vaccination), the chicks in all groups are blooded, and the independent identification sample is used for further testing the HI antibody level (HI titer will use HI method QCM-169 to determine with two antigens: VH and rNDV-LS-HN G7) for NDV.The 1-10 group will be euthanized after first blooding.

[0541] Twelve chickens from Groups 11 and 12 were challenged with NDV on day 43. The remaining 12 chickens from Group 11 were given a booster vaccination and bled on day 63 (along with Group 12). On day 71, the chickens were challenged again with NDV. The event schedule is summarized in Table 28.

[0542] Table 27

[0543] Research Group

[0544]

[0545]

[0546] Timeline of events

[0547] Table 28

[0548] Trial Events

[0549]

[0550] *±3 days deviation is acceptable

[0551] Material

[0552] Test system - animals

[0553] The animals used in this study were day old SPF chicks of the species Gallus gallus White Leghorn breed hatched from SPF eggs from an SPF colony. Both male and female chicks were used.

[0554] Table 29

[0555] Details of the test system performed

[0556]

[0557] Inclusion / Exclusion / Withdrawal Criteria

[0558] To be included in this study, chicks had to meet the following requirements: (1) originate from eggs of the same SPF colony and batch; and (2) be healthy chicks and have no signs of disease at the time of enrollment as determined by normal appearance and normal behavior.

[0559] During the study, any sick or dead birds were examined by the responsible veterinarian and the reasons were recorded in BSH-FRM-0004234. In case of any cause of morbidity / mortality not related to vaccination, these birds were excluded from the analysis of results. Throughout the study period, animal withdrawals were performed according to BSH-SOP-0000231. The number of animals withdrawn prematurely from the study and the reasons for such withdrawals were recorded.

[0560] Animal Management

[0561] Animal management and housing procedures are shown in Table 30.

[0562] Table 30

[0563] Animal Management

[0564]

[0565]

[0566] Animal randomization

[0567] One to twelve day-old chicks expected to enter the treatment group were placed in cardboard boxes from which they were randomly removed and assigned to a test group. Both males and females were selected for the experiment. Each chick was identified by wing tag on the day of vaccination.

[0568] Blind

[0569] Vaccination, blood draw and serum collection were blinded.

[0570] Test items

[0571] The raw materials are as follows.

[0572] NDV genotype 7 inactivated vaccine (Batch A0022131005)

[0573] HN50 purified protein phase 2 (batch MT555-6): centrifuged once after harvest to remove mycelia, PMSF was added to a final concentration of 1 mM; the supernatant was filtered through a 0.45 μm filter before purification on a C-tag column. The HN50 concentration of purified batch number 1 was 5.37 mg / ml.

[0574] HN50 ultrafiltration material phase 2 (batch MT569-6): After harvesting, centrifuge once to remove mycelia and add PMSF to a final concentration of 1 mM. The supernatant is filtered through a 0.45 μm filter and then ultrafiltered against 10 kDa. The HN50 concentration is 0.65 mg / mL.

[0575] HN50 pure protein phase 3 (batch MT641.6.1): After harvesting, centrifuge once to remove mycelia, add PMSF to a final concentration of 1 mM; then filter the supernatant through a 0.45 μm filter and perform C-tag column purification. HN50 concentration equals 1.49 mg / mL.

[0576] F50 pure protein phase 3 (lot MT642.6.1): After harvesting, centrifuge once to remove mycelia, add PMSF to a final concentration of 1 mM; then filter the supernatant through a 0.45 μm filter and perform C-tag column purification. HN50 concentration equals 1.18 mg / mL.

[0577] Other emulsion raw materials are as follows: white mineral oil, such as DRAKEOL; Sorbitol-S; phosphate buffered saline (PBS); Sorbitol-T.

[0578] Vaccine preparations

[0579] The method of producing the emulsion from the raw materials is as follows.

[0580] M-1-3274: In a sterile fume hood, prepare the aqueous phase by diluting 0.14 mL HN50 into 4.26 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37 °C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0581] M-1-3275: In a sterile fume hood, prepare the aqueous phase by diluting 0.47 mL HN50 into 3.93 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0582] M-1-3276: In a sterile fume hood, prepare the aqueous phase by diluting 0.93 mL HN50 into 3.47 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0583] M-1-3277: In a sterile fume hood, prepare an aqueous phase by diluting 0.7 mL HN50 into 2.2 mL PBS. Add 0.1 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 6.7 mL mineral oil with 0.3 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0584] M-1-3278: In a sterile fume hood, prepare the aqueous phase by diluting 2.5 mL HN50 into 0.4 mL PBS. Add 0.1 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 6.7 mL mineral oil with 0.3 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0585] M-1-3279: In a sterile fume hood, prepare the aqueous phase by using 3.8 mL HN50 in 2.2 mL PBS. Add 0.1 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 6.7 mL mineral oil with 0.3 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0586] M-1-3280: In a sterile fume hood, prepare the aqueous phase by diluting 0.5 mL HN50 into 3.9 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0587] M-1-3281: In a sterile fume hood, prepare the aqueous phase by diluting 1.7 mL HN50 into 2.7 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0588] M-1-3282: In a sterile fume hood, prepare the aqueous phase by diluting 3.4 mL HN50 into 1 mL PBS. Add 0.15 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 10 mL mineral oil with 0.45 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0589] M-1-3283: In a sterile fume hood, prepare the aqueous phase by diluting 4.2 mL F50 into 4.6 mL PBS. Add 0.3 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 20 mL mineral oil with 0.9 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0590] A summary of vaccine formulations is provided in Table 31.

[0591] Table 31

[0592] Summary of vaccine formulations.

[0593]

[0594] *HN50 Pure Protein Stage 2 = 5.37 mg / mL, HN50 Ultrafiltration Material = 0.65 mg / mL, HN50 Pure Protein Stage 3 = 1.49 mg / mL, F50 Pure Protein Stage 3 = 1.18 mg / mL.

[0595] Test dose

[0596] At 21 days of age, all chicks from Groups 1 to 11 were vaccinated IM into the breast muscle in a dose volume of 0.3 mL.

[0597] method

[0598] Test Methods - Immunogenicity Evaluation

[0599] a) Serology: On the day of vaccination (antibody levels at time zero), 21 days after vaccination, blood samples were collected from each chicken according to BSH-SOP-0000221. In addition, serum was separated according to BSH-QCM-0001842, and antibody levels were measured in the laboratory using rNDV-LS-ND V-HN G7 and VH antigens according to BSH-FRM-0004280 using the HI test.

[0600] For F50 antigen (Group 11), immunogenicity evaluation was performed by testing sera on NDV-F ELISA kit (Cat. No. CK122 - BioChek)

[0601] b) Challenge: 10 via IM injection 6.0 ELD 50 The procedure was performed at the Veterinary Service Department. After challenge, the birds were observed daily for at least 21 days in order to detect any morbidity and mortality caused by the vvNDV genotype VII virus.

[0602] Disease Management

[0603] Care was taken to avoid contamination between experimental groups and negative controls. When visiting study animals, either for study procedures or animal care, all researchers cared for the unvaccinated control group followed by the vaccinated group. The following precautions were also taken:

[0604] Study Animal Disposal: Disposal of birds that died during or at the end of the trial was performed according to the procedures outlined in BSH-SOP-0000231. Briefly, biohazardous waste, including carcasses and excess material, was placed in 2 bags and emptied only into the biohazardous waste container box in the yard. Carcasses that could not be emptied by an authorized company on the same day were kept in a dedicated refrigerator until the day of emptying.

[0605] Disposal of Investigational Product: Place excess test material into 2 bags and empty into biohazard waste container only.

[0606] Clothing: White coat rules according to BSH-SOP-0000232. According to this SOP, at each entry into the inactivated vaccine department, each staff member will remove personal clothing and put on work clothes and shoes belonging only to that department.

[0607] Autopsy: In case of autopsy, sampling is done in the washroom and each staff member wears another set of coveralls and shoe covers as per BSH-SOP-0000232. Once the procedure is completed, the coveralls and shoe covers belonging to the washroom are disposed of as biohazards as per BSH-SOP-0000231.

[0608] result

[0609] Fig.32 The results of the expression of the dose response of HN50 pure protein, 10 kDa ultrafiltered material after centrifugation and filtrate after filtration at 0.45 μM are demonstrated; and the dose response of F50 pure protein from phase 3 at the initial and boost maximum doses, which shows the ELISA of NDV-F. Table 32 includes the raw ELISA NDV-F data.

[0610] Table 32

[0611] ELISA for NDV-F raw data

[0612]

[0613]

[0614]

[0615]

[0616]

[0617] Example 6

[0618] Dose-response plus stability testing of NDV PD50 and F50 antigens for HN50 batches in SPF chickens

[0619] background

[0620] As previously discussed, Newcastle disease (ND) is a highly contagious viral disease that can affect all avian species. This enveloped virus (genus Mumps virus, family Paramyxoviridae, order Anasenovirales, recently renamed Avian Mumps virus or AAvV) has significant genomic variability. Genotypes V, VI, VII, and VIII (from class II) are prevalent in different countries around the world.

[0621] Clinical signs associated with infection are described as respiratory, gastrointestinal and neurological signs, depending on the immune status and susceptibility of the animal and the strain involved. Depending on their pathogenicity, NDV viruses can also be classified as velogenic viscerotropic (ability to cause necrotic hemorrhagic lesions in the intestine, causing up to 100% morbidity and mortality in young chickens, vvNDV), mesotropic (respiratory and neurological signs with moderate morbidity and mortality) and chronic (mild respiratory disease observed, especially when associated with secondary bacterial infections, used as live vaccines).

[0622] Inactivated ND vaccine (Nectiv Forte TM ) is routinely used to vaccinate chickens and turkeys. The vaccine provides a slow response, but the duration of immunity is longer. The vaccine formulation is a water-in-oil emulsion and is based on mineral oil plus Montane / Montaneox as an emulsifier. In order to produce NDV antigens for inactivated ND vaccines, the virus is propagated in embryonated eggs. The vaccine is based on a LaSota backbone expressing the HN gene of NDV G7. The positive control in our trial will be the NDV G7 vaccine.

[0623] C1 has been used to produce immunogenic recombinant antigens (HN or F protein) to replace the production of ND vaccines in embryonated eggs. The expression system provides a high-yield, low-cost, rapidly scalable rVaccine-antigen and antibody gene expression platform in a disposable or stainless steel bioreactor. It provides a safe and effective antigen, which is generally recognized as safe (GRAS) certification by the U.S. FDA, and no negative clinical effects are observed in animal studies. In a previous embodiment, 50 μg / 0.3mL of F50 fused to the T4 trimerization domain gave 100% protection against vvNDV attacks (see Example 5). In addition, the vaccinated chickens gave the same antibody titer as the NDV G7 commercial vaccine.

[0624] Purpose

[0625] The objectives of this study were: (1) to test the dose response and PD50 of F50 purified protein; (2) to test the inhibitory effect of HN50 supernatant using F50 purified protein levels; and (3) to test the stability of HN50 supernatant emulsions.

[0626] Study Design

[0627] Treatment Group

[0628] 195 SPF chicks were hatched in the animal house and divided into 17 groups. Groups 1-17 each included about 12 chickens. This included sixteen vaccinated groups and an unvaccinated control group. Groups 1 and 2 received the NDV G7 positive dose of its 1:50 dilution. Groups 3-7 and 9 received the PD50 test of F50 pure protein (Phase 3). Groups 8-10 were used for the dose-response test of F50 pure protein (Phase 3). Groups 11-13 were used to study the inhibitory effect of HN50 culture medium. Groups 14-16 were used for the stability test of the storage vaccine batch. Finally, Group 17 was an unvaccinated control group. From the first day, each group was assigned in the same rearing unit. At 21 days of age, blood samples were randomly obtained from chicks before vaccination (time zero), neck marks were carried out to the chickens from Groups 1-17, and the birds in each vaccination group were vaccinated with a kind of preparation by intramuscular (IM) injection with different volumes of each chicken according to Table 33. Three weeks after vaccination, at 42 days of age, all chicks in all groups were bled and samples were individually identified for further testing of HI antibody levels against NDV. All chickens were sent for NDV challenge on day 43. The event schedule is summarized in Table 34.

[0629] Table 33

[0630] Research Group

[0631]

[0632]

[0633] Group 1 and Group 2: NDV G7 positive dose at 1:50 dilution.

[0634] Groups 3-7 and 9: PD50 test of F50 purified protein (Phase 3).

[0635] Groups 8-10: Dose response study of F50 purified protein (Phase 3).

[0636] Groups 11-13: The inhibitory effect of HN50 medium was studied.

[0637] Groups 14-16: Stability testing of old batches.

[0638] Group 17: Unvaccinated controls.

[0639] Timeline of events

[0640] Table 34

[0641] Trial Events

[0642]

[0643]

[0644] *Deviation of ±3 days is acceptable.

[0645] Material

[0646] Test system - animals

[0647] The animals used in this study were day-old specific pathogen-free (SPF) chicks of the species Gallus gallus White Leghorn breed hatched from SPF eggs from an SPF colony. Both male and female chicks were used. To qualify for the study, the chicks had to be from the same SPF colony and batch of eggs, healthy and without any signs of disease, and have a normal appearance and normal behavior.

[0648] All management and husbandry procedures were performed according to AH-001. Table 35 shows the details of the test system that will be used in this study.

[0649] Table 35

[0650] Details of the test system performed

[0651]

[0652]

[0653] Inclusion / Exclusion / Withdrawal Criteria

[0654] The admission of birds to the study was recorded in BSH-TMP-0004235. During the study, any sick or dead birds were examined by the responsible veterinarian and the reasons were recorded in BSH-FRM-0004234. In the case of any morbidity / mortality not related to vaccination, these birds were excluded from the results analysis. Animal withdrawal was performed according to BSH-SOP-0000231. The number of animals withdrawn prematurely from the study and the reasons for such withdrawal were recorded and reported in the study report.

[0655] Animal Management

[0656] Animal management and housing procedures are shown in Table 36.

[0657] Table 36

[0658] Animal Management

[0659]

[0660]

[0661] Animal randomization

[0662] One to 17 day-old chicks expected to enter the treatment group will be placed in a cardboard box from which they will be randomly removed and assigned to a group. Both males and females will be selected for the experiment. Each chick will be identified by wing tags on the day of vaccination.

[0663] Blind

[0664] Vaccination, blood draw and serum collection were blinded.

[0665] Test items

[0666] The raw materials are as follows.

[0667] NDV genotype 7 inactivated vaccine (batch 22131006)

[0668] F50 pure protein phase 3 (batch MT642.6.1): After harvesting, centrifuge once to remove mycelia and add PMSF to a final concentration of 1 mM. The supernatant is then filtered through a 0.45 μm filter and then purified on a C-tag column. The F50 concentration is equal to 1.18 mg / ml.

[0669] HN50 supernatant (Batch MT443.6): Sample was passed through a 0.45 μm filter to remove cell debris. Total protein 20.7 mg / ml; HN50 concentration - 1.05 mg / Ml.

[0670] HN50 Supernatant (Batch M-1-3223): HN50 Cl fermentation supernatant (Batch MT569-6) was designed to have 12.5 μg / dose for SP-791 from Phase 2 material.

[0671] HN50 Supernatant (Batch M-1-3227): HN50 Cl fermentation supernatant (Batch MT569-6) was designed to have 95 μg / dose for SP-791 from Phase 2 material.

[0672] HN50 Supernatant (Batch M-1-2921): HN50 Cl fermentation supernatant (Batch MT569-6) was designed to have 15 μg / dose for SP-779 from Phase 2 material.

[0673] Other emulsion raw materials include: white mineral oil, such as DRAKEOL; Sorbitol-S; phosphate buffered saline (PBS); and Sorbitol-T.

[0674] Vaccine preparations

[0675] The vaccine was prepared using the following procedure. Table 37 provides a summary of the vaccination formulations.

[0676] M-1-3402: In a sterile fume hood, prepare the aqueous phase by diluting 0.21 mL F50 into 1.24 mL PBS. Add 0.05 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 3.35 mL mineral oil with 0.15 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0677] M-1-3404: In a sterile fume hood, prepare the aqueous phase by diluting 1.4 mL F50 into 1.6 mL PBS. Add 0.1 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 6.7 mL mineral oil with 0.3 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0678] M-1-3406: In a sterile fume hood, prepare the aqueous phase by diluting 1.2 mL F50 into 0.3 mL PBS. Add 0.05 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 3.35 mL mineral oil with 0.15 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0679] M-1-3407: In a sterile fume hood, prepare the aqueous phase by diluting 0.21 mL F50 into 1.24 mL HN50 supernatant (MT-443.6). Add 0.05 mL Sorbital T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 3.35 mL mineral oil with 0.15 mL Sorbital S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0680] M-1-3409: In a sterile fume hood, prepare the aqueous phase by diluting 0.7 mL F50 into 0.8 mL HN50 supernatant (MT-443.6). Add 0.05 mL Sorbitol T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 3.35 mL mineral oil with 0.15 mL Sorbitol S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0681] M-1-3411: In a sterile fume hood, prepare the aqueous phase by diluting 1.2 mL F50 into 0.3 mL HN50 supernatant (MT-443.6). Add 0.05 mL Sorbitol T and incubate the mixture at 37°C for 30 minutes with occasional mixing / vortexing. Mix 3.35 mL mineral oil with 0.15 mL Sorbitol S. Homogenize the emulsion in a Polytron at maximum speed for 1 minute.

[0682] Table 37

[0683] Vaccine Preparation Summary

[0684]

[0685]

[0686] *F50 pure protein phase 3 = 1.18 mg / mL.

[0687] Test dose

[0688] At 21 days of age, all chicks from Groups 1 to 16 were vaccinated IM into the breast muscle at the dose volume shown in Table 33.

[0689] method

[0690] Test Method: Immunogenicity Evaluation

[0691] a) Serology: Blood samples were collected from each chicken in all groups according to BSH-SOP-0000221 on the day of vaccination (antibody levels at time zero) and 21 days after vaccination.

[0692] In addition, sera were separated according to BSH-QCM-0001842 and antibody levels were measured in the laboratory for Groups 1, 2, 14-17 using the HI assay according to BSH-FRM-0004280 using the rNDV-LS-NDV-HN G7 antigen.

[0693] For F50 antigen in groups 1-13 and 17, immunogenicity evaluation was performed by testing sera on NDV-F ELISA kit (Cat. No. CK122 - BioChek)

[0694] b) Challenge: 10 via IM injection 6.0 ELD 50 The procedure was performed at the Veterinary Service Department. After challenge, the birds were observed daily for at least 14 days in order to detect any morbidity and mortality caused by the vvNDV genotype VII virus.

[0695] Statistical analysis

[0696] PD50 is determined by the dose of vaccine that is expected to protect 50% of chickens against a challenge dose of NDV. PD50 also reflects the correlation between antigen quantity, serological response and protection against challenge.

[0697] By using the European Pharmacopoeia Standard 10.2 Monograph 0870 paragraph 2-3-2 as a guide for vaccine (inactivated) immunogenicity for Newcastle disease vaccines, the vaccine should comply with the PD50 test (50% protective dose) and should not fall below 50% of the recommended dose with a lower confidence limit of 35 PD50 per dose. This means that the NDV vaccine must contain at least 50 PD50 in one recommended dose and is calculated using the Reed and Muench method as presented below:

[0698]

[0699] Log 50% endpoint = dilution above 50% - (PD * log dilution factor) PD50 = 10 - x, where x is the log 50% endpoint. Statistical differences between each group based on PD50 results will be determined using JPM 12.0.1 software by using a two-way ANOVA model measurement design.

[0700] Evaluation and evaluable parameters

[0701] All birds from the negative control (Group 17) should die within 6 days of challenge. The vaccine complies with the test if the minimum dose specified in the 0.3 mL dose corresponds to no less than 50 PD50 and the lower confidence limit is no less than 35 PD50 per dose. If the lower confidence limit is less than 35 PD50 per dose, the test is repeated. The vaccine must contain no less than 50 PD50 in the repeated test.

[0702] Disease Management

[0703] Care was taken to avoid contamination between experimental groups and negative controls according to BSH-SOP-0000231. When visiting study animals, either for study procedures or animal husbandry, all researchers always cared for the unvaccinated control group followed by the vaccinated group.

[0704] Disposal of Investigational Product: Place excess test material into 2 bags and empty into biohazard waste container only.

[0705] Clothing: White coat rules according to BSH-SOP-0000232. According to this SOP, at each entry into the inactivated vaccine department, each staff member will remove personal clothing and put on work clothes and shoes belonging only to that department.

[0706] Autopsy: In case of autopsy, sampling is done in the washroom and each staff member wears another set of coveralls and shoe covers as per BSH-SOP-0000232. Once the procedure is completed, the coveralls and shoe covers belonging to the washroom are disposed of as biohazards as per BSH-SOP-0000231.

[0707] result

[0708] The results of the dose response and stability test of F50 antigen are as follows Fig.33 As shown in, and Table 38 includes raw data for NDV-F ELIA.

[0709] Table 38

[0710] Raw data

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717]

[0718] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are merely preferred examples of the invention and should not be considered to limit the scope of the invention. Instead, the scope of the invention is defined by the following claims. We therefore claim protection for all of our inventions that fall within the scope and spirit of these claims.

Claims

1. A transgenic fungal cell comprising a recombinant gene comprising a fungal promoter operably linked to a nucleic acid sequence encoding an antigenic peptide derived from Newcastle disease virus.

2. The transgenic fungal cell of claim 1, wherein the fungal cell is a heterothallic Thermomyces verrucosum.

3. The transgenic fungal cell of claim 1 or 2, wherein the antigenic peptide is Newcastle Disease Virus F protein, HN protein or any combination thereof.

4. The transgenic fungal cell of any one of claims 1, 2 or 3, wherein the heterothallic Thermomyces verrucosa is a strain selected from DNL155, DNL157, DNL159, M5355, M5739 or a glycoengineered strain producing Man3, G0 or G2 glycans.

5. The transgenic fungal cell of claim 2 or 3, wherein the heterothallic Thermomyces verrucosum strain is glycoengineered.

6. The transgenic fungal cell of claim 1, comprising an antigenic peptide or a fragment thereof from Newcastle Disease Virus.

7. A transgenic fungal cell comprising a recombinant gene having a nucleic acid sequence encoding an antigenic peptide, wherein the nucleic acid sequence comprises a sequence having at least 90% similarity to SEQ ID NO: 5, 8, 11, 14, 17, 20, 23, 26, 44, 45 or a combination thereof.

8. The transgenic fungal cell of any one of claims 1-7, wherein the recombinant gene comprises a nucleic acid sequence corresponding to an amino acid sequence having at least 90% similarity to SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32, or a combination thereof.

9. as described in any one in the transgenic fungal cell of claim 1-8, wherein said recombinant gene is transformed into the described fungal cell by the episomal vector corresponding to nucleotide sequence SEQ ID NO:29,34,36 and / or 37.

10. The transgenic fungal cell of any one of claims 1-9, wherein the antigenic peptide is linked to a signal peptide.

11. The transgenic fungal cell of any one of claims 1 to 10, wherein the signal sequence corresponds to an amino acid sequence having at least 90% similarity to SEQ ID NO:

1.

12. The transgenic fungal cell of claim 1, wherein the fungal promoter is bgl8.

13. A method for preparing a transgenic fungal cell, the method comprising transforming a fungal cell with a recombinant gene comprising a fungal promoter operably linked to a nucleic acid sequence encoding an antigenic peptide selected from Newcastle Disease Virus.

14. A recombinant antigenic peptide produced by the transgenic fungus according to any one of claims 1 to 12, wherein the recombinant antigenic peptide comprises an amino acid sequence capable of generating an immunogenic response in a subject.

15. The antigenic peptide of claim 14, comprising an amino acid sequence from SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31 or 32.

16. The antigenic peptide according to claim 14 or 15, wherein the peptide has a length of 8 to 800 amino acids.

17. The peptide of any one of claims 14 to 16, wherein the amino acid residues of the peptide contain modifications.

18. A method for producing a composition according to any one of claims 14-17, comprising: Fermenting the transgenic fungal cell according to any one of claims 1 to 12 in a bioreactor having a culture medium; as well as The recombinant antigen protein produced during the fermentation is collected. The method according to claim 18 , wherein the recombinant antigen protein is unpurified.

20. The method of claim 18, further comprising purifying the recombinant antigen protein.

21. The method of claim 20, wherein purification comprises filtration, C-tag affinity chromatography, histidine tag affinity chromatography, size exclusion chromatography, fast protein liquid chromatography, high performance liquid chromatography, affinity chromatography, hydrophobic interaction chromatography, ion exchange chromatography, reverse phase chromatography, immunoaffinity chromatography, ultracentrifugation, precipitation, or any combination thereof.

22. The method of claim 20, wherein purification comprises filtration and C-tag affinity chromatography.

23. The method according to any one of claims 18 to 22, comprising extracting the recombinant antigenic protein from mycelial tissue of the transgenic fungus according to any one of claims 1 to 11.

24. The method of any one of claims 18-22, comprising collecting the recombinant antigenic protein from the fermentation supernatant of the transgenic fungal cell of any one of claims 1-12.

25. The method of claim 18, further comprising controlling the bioreactor temperature at 20°C to 40°C during fermentation of the transgenic fungal cell of any one of claims 1-12.

26. The method of claim 18, further comprising controlling the bioreactor temperature at 25°C to 38°C during fermentation of the transgenic fungal cell of any one of claims 1-11.

27. The method of claim 18, wherein the bioreactor comprises a fed-batch fermenter.

28. A vaccine composition comprising the antigenic peptide according to any one of claims 14 to 17.

29. The vaccine composition according to claim 28, further comprising: Oil; emulsifiers; and Adjuvant.

30. The vaccine composition of claim 29, wherein an oil-in-water emulsion is present.

31. The vaccine composition of claim 29, wherein the oil is mineral oil.

32. The vaccine composition according to any one of claims 28-31, further comprising two or more antigenic peptides.

33. The vaccine composition of any one of claims 28-32, wherein the antigenic peptide is selected from SEQ ID NO: 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 32 or any combination thereof.

34. The vaccine composition of any one of claims 28-33, further comprising a second vaccine different from the first vaccine.

35. The vaccine composition of claim 29, further comprising: two or more emulsifiers; and Two or more adjuvants.

36. The vaccine composition of claim 35, wherein the adjuvant comprises sorbitol-T, sorbitan oleate, or a combination thereof.

37. A method of preparing a vaccine, the method comprising mixing one or more peptides according to claim 33 with one or more emulsifiers, one or more adjuvants to form a mineral oil into an aqueous emulsion.

38. A method comprising administering to a subject a vaccine as claimed in any one of the preceding claims.

39. The method of claim 38, wherein the subject is poultry.

40. The method of claim 39, wherein the poultry is chicken.

41. The method of any one of claims 38-40, wherein administering the vaccine composition of any one of claims 28-35 to the subject comprises spraying, dripping, painting or injecting the subject with the vaccine composition through the nose, mouth, skin, muscle or optical contact, wherein the vaccine is in the form of an aerosol, powder, liquid or solid.

42. The method of any one of claims 38-40, comprising administering the vaccine intramuscularly.

43. The method of any one of claims 38-42, further comprising administering the vaccine twice, wherein the first administration occurs at least one day prior to the second administration.

44. The method of claim 42, wherein the second vaccination occurs 20-23 days after the first vaccination.

45. A transgenic fungal cell comprising, consisting of, or consisting essentially of a recombinant gene comprising a fungal promoter operably linked to a nucleic acid sequence encoding an antigenic peptide selected from Newcastle Disease Virus.

46. ​​A transgenic fungal cell comprising, consisting of, or consisting essentially of a recombinant gene having a nucleic acid sequence encoding an antigenic peptide, wherein the nucleic acid sequence comprises a sequence having at least 90% similarity to SEQ ID NO:5, 8, 11, 14, 17, 20, 23, 26, 44, 45, or a combination thereof.

47. A method of preparing a transgenic fungal cell, the method comprising, consisting of, or consisting essentially of the following steps: The fungal cells are transformed with a recombinant gene comprising a fungal promoter operably linked to a nucleic acid sequence encoding an antigenic peptide selected from Newcastle Disease Virus.

48. A recombinant antigenic peptide produced by a transgenic fungal cell as described in any one of claims 1-12, wherein the recombinant antigenic peptide comprises, consists of, or consists essentially of an amino acid sequence capable of generating an immunogenic response in a subject.

49. A method of producing a composition as described in any one of claims 14-17, said method comprising, consisting of, or consisting essentially of the following steps: Fermenting the transgenic fungal cell according to any one of claims 1 to 12 in a bioreactor having a culture medium; as well as The recombinant antigen protein produced during the fermentation is collected.

50. A vaccine emulsion composition comprising, consisting of, or consisting essentially of: The antigenic peptide according to any one of claims 14 to 17; Oil; emulsifiers; and Adjuvant.

51. A method for preparing a vaccine from the composition of claim 22, the method comprising, consisting of, or consisting essentially of the following steps: Mixing one or more peptides as claimed in claim 22 with one or more emulsifiers, one or more adjuvants in a 67% mineral oil solution in water; and The mixture was emulsified.

52. A method comprising, consisting of, or consisting essentially of the vaccine of any one of the preceding claims to a subject.

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