Newcastle disease virus vaccine

Abstract

The present disclosure relates to a vaccine composition comprising irradiation-inactivated Newcastle Disease Virus (NDV). The vaccine composition may be used in methods of inducing a protective immune response to Newcastle Disease Virus (NDV), methods of producing a irradiation-inactivated NDV vaccine composition, and a vaccine composition produced by such methods.

Description

NEWCASTLE DISEASE VIRUS VACCINERELATED APPLICATIONS

[0001] The present application claims priority from Australian Provisional Application No.2024903739, filed on 14 November 2024, the contents of which is incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The present invention relates to the field of vaccines. In one form, the present disclosure relates to vaccine compositions comprising Newcastle Disease Virus inactivated by irradiation. However, it will be appreciated that the invention is not limited to this particular field of use.BACKGROUND OF THE INVENTION

[0003] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0004] Newcastle Disease Virus (NDV) is a pathogen of birds that is associated with widespread livestock losses and a large economic burden. Currently available NDV vaccines are typically chemically inactivated or live attenuated vaccines, but these have limited efficacy. Chemically inactivated vaccines are poorly immunogenic, and are typically co-administered with an adjuvant to individual animals via the subcutaneous or the intramuscular route. Nonetheless, the immune response is restricted to a humoral response. Live-attenuated vaccines are commonly administered via the intranasal route. Although they may induce humoral, cell-mediated and / or mucosal immunity, these vaccines may be associated with undesirable respiratory symptoms, growth retardation, and even increased susceptibility to other pathogens.

[0005] Humoral immunity against NDV is believed to confer protection to an NDV infection, while cell-mediated immunity reduces virus shedding. Neutralising antibodies against the surface glycoproteins fusion (F) and haemagglutinin-neuraminidase (HN) are associated with control of Newcastle disease (ND), with antibodies against F proteins typically providing superior protection compared to antibodies against the HN protein. However, due to the genetic variability between NDV strains, particularly for the two surface glycoproteins, current NDV vaccines have limitedprotective efficacy against presently circulating strains. Moreover, current ND vaccines and vaccine regimes primarily aim to mitigate clinical symptoms and not virus shedding.

[0006] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.SUMMARY OF THE INVENTION

[0007] In a first aspect, the present disclosure provides a vaccine composition comprising irradiation-inactivated Newcastle Disease Virus (NDV). In an embodiment, the vaccine composition further comprises at least one pharmaceutically-acceptable excipient, diluent and / or carrier. In an embodiment, the irradiated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy. In an embodiment, the vaccine composition is an inactivated whole virus vaccine composition.

[0008] In an embodiment, the composition further comprises an adjuvant. In an embodiment, the composition excludes an adjuvant. In an embodiment, the composition excludes an alum adjuvant.

[0009] In an embodiment, the composition comprises irradiation-inactivated, protease treated NDV. In an embodiment, the protease treatment occurred following the irradiation inactivation. In an embodiment, the protease treatment occurred following the irradiation inactivation. In an embodiment, the protease is trypsin.

[0010] In an embodiment, the irradiated NDV is a genotype I strain. In an embodiment, the irradiated NDV is a V4 strain.

[0011] In a second aspect, the present disclosure provides a method for treating or preventing an NDV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition comprising irradiation-inactivated NDV.

[0012] The method of claim 10, wherein the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.

[0013] In an embodiment, the irradiation-inactivated NDV comprises irradiation-inactivated, protease treated NDV. In an embodiment, the protease treatment occurred following the irradiation inactivation. In an embodiment, the protease is trypsin.

[0014] In an embodiment, the administering is by mucosal administration. In an embodiment, the administering is by intranasal administration. In an embodiment, the administering is by intramuscular administration.

[0015] In an embodiment, the method induces a neutralising antibody response. In an embodiment, the method prevents, treats or reduces the severity of an NDV infection in the subject. In an embodiment, the method induces a heterotypic immune response against a NDV genotype different to the irradiation-inactivated NDV in the subject and / or induces protection against a plurality of NDV genotypes in the subject. In an embodiment, the method induces sterilizing immunity against NDV.

[0016] In an embodiment, the subject is an avian subject.

[0017] In a third aspect, the present invention provides a method of producing a protease- treated, irradiation-inactivated NDV vaccine composition, the method comprising:(a) obtaining a NDV preparation, and optionally clarifying and / or washing the NDV preparation;(b) irradiating the NDV preparation to obtain an irradiation-inactivated NDV preparation; and(c) protease-treating the irradiation-inactivated NDV preparation.

[0018] In an embodiment, the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy. In an embodiment, the protease is trypsin. In an embodiment, the irradiation-inactivated NDV is a genotype I strain or a V4 strain.

[0019] In a fourth aspect, the present invention provides a vaccine composition produced by the method of the third aspect.DEFINITIONS

[0020] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0022] The indefinite articles "a" and "an" preceding an element or component of the invention are intended to be non-restrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0023] As used herein, with reference to numbers in a range of numerals, the terms "about," "approximately" and "substantially" are understood to refer to the range of the referenced number, for example, -10% to +10% of the referenced number, -5% to +5% of the referenced number, -1 % to + 1 % of the referenced number, or-0 .1 % to +0 .1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.

[0024] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y". Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "X and Y".

[0025] It will be understood that use of the term “between” herein when referring to a range of numerical values encompasses the numerical values at each endpoint of the range. For example, radiation at energies of between 0.01 MeV and 10MeV is inclusive of energies of 0.01 MeV and 10MeV.

[0026] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “'comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.

[0027] The transitional phrase "consisting of’ excludes any element, step, or ingredient not specified, such that it would close the claim to the inclusion of materials other than those recited (except for impurities ordinarily associated therewith). When the phrase "consisting of' appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0028] The transitional phrase "consisting essentially of' is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term "consisting essentially of" occupies a middle ground between "comprising" and "consisting of".

[0029] While reference may be made in this disclosure to the invention comprising a combination of a plurality of elements, it is also understood that this invention is regarded tocomprise combinations which omit or exclude one or more of such elements, even if this omission or exclusion of an element or elements is not expressly stated herein, unless it is expressly stated herein that an element is essential to the applicant' s combination and cannot be omitted. It is further understood that the prior art may include elements from which this invention may be distinguished by negative claim limitations, even without any express statement of such negative limitations herein. It is to be understood, between the positive statements of applicant's invention expressly stated herein, and the prior art and knowledge of the prior art by those of ordinary skill which is incorporated herein even if not expressly reproduced here for reasons of economy, that any and all such negative claim limitations supported by the prior art are also considered to be within the scope of this disclosure and its associated claims, even absent any express statement herein about any particular negative claim limitations.

[0030] The “irradiation-inactivated” vaccines of the present invention will be understood to be inactivated by irradiation such as photon irradiation or electron irradiation. The terms “photonradiation” or “photon-irradiation” and the like will be understood to encompass both gammaradiation or gamma-irradiation (i.e., gamma-rays) and X-radiation or X-irradiation (i.e., X-rays). The term “electron irradiation” or “electron radiation” and the like will be understood to be referring to a process that involves using electrons to treat materials for various purposes, such as sterilization.

[0031] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0032] A "therapeutically effective amount" is at least the minimum concentration or amount required to affect a measurable improvement of a particular disease or condition, or induce a desired response such as an immune response. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex and weight of the patient. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects.

[0033] Various features of the embodiments of the invention disclosed herein are, for brevity, described in the context of a single embodiment, but may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the illustrative embodiments disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present compositions and are disclosedherein just as if each and every such sub-combination was individually and explicitly disclosed herein.BRIEF DESCRIPTION OF THE FIGURES

[0034] The aspects described herein, as well as other apparent aspects, advantages, and objectives of the present invention are apparent from the detailed description below in combination with the drawing, in which:

[0035] Figure 1 provides photographic images of Vero cell monolayers following three passages with live (virus only) and y-NDV or allantoic fluid from uninfected 12-days old embryonated eggs (mock control). Cells were stained with DAPI and treated with chicken anti-NDV antibodies and anti-chicken IgY FITC-conjugated antibodies to visualise NDV-infected cells.

[0036] Figure 2 provides graphical representation of (A) the hemagglutinin activity of live NDV and y-NDV, and (B) the neuraminidase activity of live NDV and y-NDV; and provides transmission electron microscopy images of (C) live NDV and (D) y-NDV.

[0037] Figure 3 provides graphical representation of (A) NDV-specific IgG responses of BALB / c mice that are naive or intramuscularly vaccinated with one “IM(1)” or two “IM(2)” doses of y-NDV, and (B) lack of neutralising antibody responses in serum samples from BALB / c mice that are naive or intramuscularly vaccinated with one or two dose of y-NDV.

[0038] Figure 4 provides graphical representation of NDV-specific IgG responses of BALB / c mice that are naive or intramuscularly vaccinated with y-NDV with or without adjuvants (y-Flu, incomplete Freund’s adjuvant (IFA), aluminium hydroxide (Alum) or poly(l:C) (P(I:C)) two weeks after (A) first immunisation or (B) second immunisation.

[0039] Figure 5 provides graphical representation showing lack of neutralising antibody responses based on fluorescence microscopy images of Vero cells incubated with live NDV pretreated with serum of naive mice or serum of mice obtained two weeks after the second intramuscular vaccination with y-NDV with or without adjuvants (y-Flu, incomplete Freund’s adjuvant (IFA), aluminium hydroxide (Alum) or poly(l:C) (P(I:C)). Fluorescence levels obtained for Vero cells in the absence of virus or virus alone were used as controls.

[0040] Figure 6 provides graphical representation of NDV-specific IgG responses in serum of naive mice or mice that have been subcutaneously vaccinated with one or two dose of y-NDV two weeks apart, at various serum dilutions for (A) BALB / c mice or (B) C57BL / 6, or (C) BALB / c mice and C57BL / 6 mice; (D) lack of neutralising antibody responses in serum samples from BALB / c and C57BL / 6 mice that are naive or subcutaneously vaccinated twice with y-NDV with serum samples collected two weeks post-secondary vaccination.

[0041] Figure 7 provides graphical representation of the NDV-specific antibody responses of BALB / c mice that have been intramuscularly or intranasally vaccinated with one or two doses of 107TCIDso / mouse y-NDV two weeks apart or intramuscularly with 107TCIDso / mouse live-NDV, with (A) total IgG titres in bronchoalveolar lavage fluid (BAL); (B) IgA titres in BAL; (C) total IgG in serum; (D) lgG1 in serum: (E) lgG2a in serum; (F) lgG2b in serum, (G) lgG3 in serum; and mean titre summaries for all mice following (H) first vaccination and (I) second vaccination.

[0042] Figure 8 provides graphical representation of neutralising antibody responses as measured from fluorescence microscopy images of Vero cells incubated with live NDV pretreated with 1:80 diluted serum of naive mice or serum of mice obtained three weeks after the second intramuscular or intranasal vaccination with y-NDV or intramuscular vaccination with live-NDV without adjuvants. Vero cells treated with virus alone were used as controls.

[0043] Figure 9 provides graphical representation of neutralising antibody responses obtained from fluorescence microscopy images of Vero cells incubated with virus only (vo), or virus treated with 1:2 diluted BAL samples from naive mice, or from mice vaccinated intramuscularly with live NDV or y-NDV, or intranasally vaccinated with y-NDV.

[0044] Figure 10 provides transmission electron microscopy images of live-NDV, y-NDV, formalin treated NDV and formalin treated y-NDV.

[0045] Figure 11 provides graphical representation of (A) the hemagglutinin activity and (B) the neuraminidase activity of live NDV, formalin treated NDV (F-NDV), y-NDV and formalin-treated y-NDV (F-y-NDV).

[0046] Figure 12 provides graphical representation of total NDV-specific serum IgG responses of BALB / c mice immunised intranasally with two doses of 107TCID5o / mouse of either live-NDV, F-NDV, F-y-NDV, or y-NDV two weeks apart, with serum samples were harvested 13 days after (A, B) first and (C, D) second vaccination, with mean absorbance at (A) 1 / 100 dilution or (C) 1 / 200 dilution, and (B, D) titres.

[0047] Figure 13 provides graphical representation of neutralising antibody responses obtained based on fluorescence microscopy images of Vero cells incubated with virus only, or virus treated with 1:80 dilution of serum samples from naive BALB / c mice, or BALB / c mice vaccinated twice intranasally with F-NDV, F-y-NDV, or y-NDV.

[0048] Figure 14 provides graphical representation of total NDV-specific serum IgG responses of BALB / c mice immunised intramuscularly with two doses of 107TCIDso / mouse of either live-NDV, formalin-treated NDV (F-NDV), formalin-treated y-NDV (F-y-NDV), or y-NDV two weeks apart, with serum samples were harvested 13 days after (A, B) first and (C,D) secondvaccination, with mean absorbance at (A) 1 / 100 dilution and (C) 1 / 200 dilution; and titres shown in (B, D).

[0049] Figure 15 provides graphical representation of neutralising antibody responses obtained based on fluorescence microscopy images of Vero cells incubated with a virus only preparation, or virus treated with 1:80 dilution of serum samples from naive BALB / c mice, or BALB / c mice vaccinated twice intramuscularly with two doses of 107TCIDso-equivalent / mouse of either live-NDV, F-NDV, F-y-NDV, ory-NDV.

[0050] Figure 16 provides graphical representation of total NDV-specific serum IgG responses of BALB / c mice immunised intramuscularly with two doses of 107TCIDso / mouse of either trypsin-treated y-NDV (y-NDV + trypsin), y-NDV incubated at 37°C in the absence of trypsin (y-NDV at 37°C), y-NDV incubated at room temperature (y-NDV) and live-NDV two weeks apart, with serum samples were harvested 13 days after (A, B) first and (C,D) second vaccination, with mean absorbance at (A) 1:100 dilution and (C) 1:200 dilution and (B, D) titres.

[0051] Figure 17 provides graphical representation of neutralising antibody responses obtained based on fluorescence microscopy images of Vero cells incubated with virus treated with serum samples at 1:20, 1:40, 1:80, and 1:160 dilutions from naive BALB / c mice, or BALB / c mice vaccinated twice intramuscularly with two doses of 107TCIDso-equivalent / mouse of either trypsin-treated y-NDV (y-NDV + trypsin), y-NDV incubated at 37°C in the absence of trypsin (y-NDV at 37°C), y-NDV incubated at room temperature (y-NDV) and live-NDV.DETAILED DESCRIPTION

[0052] In an aspect, the present disclosure provides a vaccine composition comprising Newcastle Disease Virus (NDV) inactivated by irradiation. In another aspect, the disclosure provides pharmaceutical compositions comprising the vaccine composition. In another aspect, the disclosure provides methods of producing the vaccine composition.

[0053] In other aspects, the disclosure provides methods and / or uses of the composition in vaccinating a subject against, inducing an immune response against, and / or preventing or reducing the symptoms of NDV.

[0054] The skilled addressee will understand that the invention comprises the embodiments and features disclosed herein as well as all combinations and / or permutations of the disclosed embodiments and features.

[0055] Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification.NEWCASTLE DISEASE VIRUS

[0056] NDV is the causative agent of Newcastle disease. It is an avian respiratory virus from the family Paramyxoviridae, sub-family Avulavirinae, and genus Orthoavulavirus. There are 9 serotypes of avian paramyxovirus (APMV), with NDV (also known as APMV-1) being of particular interest as the other 8 serotypes induce only mild or asymptomatic infection in birds. NDV is presently divided into 18 genotypes according to phylogenetic analysis of the F protein. Class I strains are made up of a single genotype; Genotype I (Gl) and includes strains isolated from wild birds. Class I strains are typically of low virulence. Class II strains are further classified into Genotypes II to XXI (Gil to GXXI) and include highly virulent strains that infect both wild birds and domestic poultry.

[0057] In addition, strains of NDV are grouped according to their virulence. These groups are asymptomatic, lentogenic (low virulence), mesogenic (medium virulence) and velogenic (highly virulent). Lentogenic strains cause mild respiratory diseases and do not cause any mortality. Mesogenic strains cause acute respiratory disease and mortality rates are less than 10%. Velogenic strains can be further classified into viscerotropic and neurotropic disease, and mortality rates can reach close to 100% in some instances. Infection by viscerotropic velogenic ND strains produce lethal haemorrhagic lesions in the viscera, and are usually characterised by sudden onset of symptoms such as a sharp drop on egg production, marked depression, and often severe diarrhoea that leads to dehydration and collapse. Velogenic strains that cause neurotropic diseases, on the other hand, may result in severe respiratory and nervous signs, including coughing and gasping, head tremors, wings and leg paralysis, and twisted necks.

[0058] In chickens, NDV can cause a range of respiratory and neurological symptoms including coughing, gasping, twisted head or neck, muscle spasms, decrease in egg quality, and swelling around the eyes and neck. There is no treatment for NDV, and infected chickens must be culled to prevent spread to the rest of the flock. Worldwide, NDV is the third highest cause of infectious disease related death in poultry, behind avian influenza and infectious bronchitis. Countries with an outbreak of a virulent form of NDV (i.e., mesogenic or velogenic NDV) are obligated to immediately report to the World Organisation for Animal Health (OIE). As a result, trade restrictions on poultry and poultry products might be imposed by trading partners. In some countries, a strict vaccination regime is adhered to. Experimentally, mice can be infected with NDV, allowing in vivo vaccine testing prior to progressing to avian models.

[0059] NDV has a large (15.2 kb) negative sense single-stranded RNA genome, and some virions carry two or more copies of the genome. The negative sense single-stranded enveloped RNA virus possesses a non-segmented genome that encodes 6 structural proteins (i.e., nucleocapsid protein (NP), phosphoprotein (P), matrix protein (M), fusion protein (F),haemagglutinin-neuraminidase (HN), RNA-dependent RNA polymerase (L)) and 2 non-structural proteins (i.e. , V and W), which are produced by RNA editing during transcription of the viral P gene.

[0060] NP is the most abundant protein present in the virion. It coats the full-length genomic RNA, protecting the genome from nucleases. The genomic RNA in association with the NP, L and P proteins, forms the ribonucleoprotein complex, which acts as a template for RNA synthesis. M protein, a highly conserved protein among paramyxoviruses, is located beneath the envelope, and is responsible for virus assembly and budding. Both M proteins and NP account for approximately 30% of the entire virion. V and W proteins are accessory proteins that are present only in virus- infected cells.

[0061] The F protein spike is approximately 8 nm in length and is presented as a trimer. F protein mediates the fusion of the viral envelope with the host cell membrane, and the amino acid sequence in the F protein cleavage site is the major determinant of the virulence of the NDV isolates. In infected cells, the F protein is synthesised as a non-functional precursor (F0), and may be cleaved by cellular proteases into the activated forms F1 and F2 to render the virion infective.

[0062] HN is approximately 8nm in length. It consists of a cytoplasmic domain, a transmembrane region, a stalk region, and a large globular head domain. The globular head region is associated with recognising sialic acid-containing cell surface receptors and possessing neuraminidase (NA) activity to prevent self-aggregation. HN also promotes the fusion activity of the F protein responsible for virus-cell and cell-cell aggregation. Approximately 10% of the virion comprises HN molecules. The globular head includes 2 sialic acid-binding domains, with the first site being associated with receptor binding and neuraminidase activity, while the second site is involved in receptor binding and fusion. The second site is located at the dimer interface, and is activated after engagement of the first site to its receptor. The stalk domain of HN may promote membrane fusion through its interaction with the F protein.

[0063] Typically, NDV initiates a local infection at the nasal epithelium. NDV may attack respiratory epithelium cells by binding to the sialic acid-containing residues on the cellular surfaces via the HN surface glycoprotein, which in turn may trigger the F protein-mediated fusion of the viral envelope with the plasma membrane of the host cell. NDV infection may occur mainly via a pH-independent manner, and sometimes via receptor- or caveolae-mediated endocytosis. Fusion of the viral membrane with the cellular membrane may result in a large, irreversible conformational change in the F protein. Upon entry, the ribonucleoprotein complex (comprised of the RNA genome encapsidated with NP, and associated with L and P proteins) dissociates from the M protein, where it is released into the cytoplasm of the host cell. The negative sense viralRNA is then subsequently transcribed by the RNA-dependent-RNA-polymerase activity of the L protein to produce structural mRNA that are required for the synthesis of viral proteins.

[0064] When sufficient amounts of viral proteins have accumulated, replication of the viral genome takes place. The positive sense RNA is used as a template to synthesise the negative sense RNA genome. This newly formed genomic RNA is then associated with the NP and polymerase complex to form new ribonucleoprotein complexes. New virions comprising of the ribonucleoprotein complex, matrix, and surface glycoproteins are released from the host cell by budding. Finally, the neuraminidase activity of the HN protein facilitates the detachment of the virion from the cell, and cleaves the sialic acid residues from the progeny virus particles to prevent self-aggregation.IMMUNE RESPONSE TO NDV

[0065] Initial immune responses to NDV infection are fast-acting, non-specific innate responses. In chickens, NDV may induce several anti-viral signalling molecules including nitric oxide (NO), IFN-y, pro-inflammatory cytokines (e.g. IL-6, IL-8, IL-18, IL-1P) and chicken homologues of IFN-a and IFN-p. Similarly, in mouse models NDV is a strong inducer of Type I IFN responses (IFN-I). The V protein of NDV may act as an IFN-suppressor and may suppress apoptosis in infected cells. However, V protein is highly species-specific and has limited function in mammalian cells. The innate immune response plays an important role in priming the adaptive immune response. Low virulence NDV strains have been shown to stimulate a lower innate immune response compared to virulent NDV, and this is attributed to the increased tropism conferred by the polybasic amino acids within the F proteins. The robust innate host response to virulent NDV strains has been suggested as being deleterious to the host, and a possible explanation to the severe pathological damage observed.

[0066] Adaptive immunity relies on both the humoral and cell-mediated immunity to mount a faster response to subsequent challenge. Avian antibodies to NDV may be IgM, IgA, and / or IgY (avian IgG-equivalent) isotypes, and may be detected within 6-8 days after infection, and generally peak at 21-28 days after infection. Vaccine immunogenicity may be measured by antibody responses (Shahar et al., 2018). Passive immunization of birds has been reported to be associated with protection from clinical disease without preventing virus shedding (Reynolds and Maraqa, 2018). In addition, it has been reported that birds with cell mediated immunity (or T cell responses) specific for NDV are not protected from lethal challenge in the absence of antibody responses (Reynolds and Maraqa, 2000). Further, a larger population of T cells, CD8+ T cells particularly, may be associated with mediating a faster clearance of NDV in infected birds through target killing of NDV infected cells (Russell, P.H et al 1997). Prior investigations indicated thatantibody responses may be associated with survival from NDV infection, whereas T cell responses may be associated with reducing virus shedding.

[0067] A protective immune response will be understood by those skilled in the art as an immune response that is associated with prevention of infection with a pathogen, survival of infection with a pathogen, or lessening of symptoms of infection with a pathogen. The induction of a neutralising antibody response to NDV is indicative of induction of a protective immune response against NDV. Neutralising antibodies formed against the viral haemagglutinin (HN) and fusion (F) glycoproteins may be associated with survival and / or control of ND. Antibodies targeting both HN and F proteins have previously been shown to be more protective than antibodies targeting either of those surface glycoproteins. Antibodies against the F protein may be associated with higher protection than those of HN in terms of inducing a neutralising antibody response. There have been 15 reported neutralising epitopes found within both the HN and F proteins: 7 in HN and 8 in F.

[0068] Mammals and chickens have a well-developed mucosal immune system that is characterised by the production and secretion of slgA, as well as the presence of IgA- and IgG-secreting plasma cells at the mucosal surface. Local antibody on the mucosal surface of the respiratory tracts, primarily IgA, may play an important role not only in the protection of the challenged birds, but also in limiting primary replication and virus shedding at the site of infection.

[0069] Cell-mediated responses may be detected as early as 2-3 days after NDV infection and the cell-mediated response may be proportionate to the virulence of the NDV strain. Clonal expansion following vaccination with a live-attenuated vaccine has been reported for both CD4+ and CD8+ T cell populations. However, studies have reported that birds with cell mediated immunity (CMI) specific for NDV may not be protected from lethal challenge in the absence of antibody responses. Nonetheless, CD8+ T cells may play an important role in reducing virus shedding through targeted killing of NDV infected cells. Of interest, NDV possesses the ability to suppress the adaptive immune response by dampening the proliferation of T cells via IL-10-associated Th2 immune responses. Overall, it has been shown that a strong Th1 -mediated immune response combined with a humoral response is more protective than a Th2-oriented response. Although T cells do not appear to play a role in NDV-protection and the development of neutralising antibody responses, CD8+ T cells may be associated with reducing virus shedding.EXISTING NDV VACCINES

[0070] Currently, live-attenuated, inactivated, and viral-vectored NDV vaccines are commercially available. Since infections occur on or through the mucosal surface, mucosal immunity is thought to be associated with controlling an initial infection with NDV. Mucosalimmune responses may be generated when vaccines are administrated via mucosal route such as the conjunctival and intranasal routes of administration.

[0071] Live-attenuated vaccines are typically formulated with avirulent or lentogenic NDV strains, for example, the La Sota strain, which is a Gil lentogenic strain. This vaccine may be administered in drinking water or as an aerosol to allow vaccination of large number of birds quickly and easily. However, while live vaccines provide both mucosal and humoral immunity, they may cause undesirable symptoms following vaccination such as respiratory signs and growth retardation. Moreover, protection induced by the live-attenuated vaccine may be shortlived.

[0072] Infection with lentogenic strain La Sota is thought to induce Th2-biased responses characterised by the production of the anti-inflammatory cytokine IL- 10 and attenuated T cell responses. Considering that La Sota strain has been widely used as live attenuated NDV vaccines, the limited induction of T cell responses may explain the reduced efficacy of existing vaccines against highly virulent NDV genotypes. Overall, while T cell responses induced by live attenuated vaccine appear to contribute to protection, vaccine efficacy could be affected by the ability of V protein to inhibit IFN-I and the subsequent impact on systemic antibody responses and types. In addition, the side effects, virus shedding and impact on birds’ health would reduce the commercial value of live attenuated vaccines.

[0073] Inactivated vaccines are generally thought to be poorly immunogenic on their own, and may be administered with adjuvants to improve immunogenicity (Russell et al. 1997). Inactivated vaccines are typically prepared by treatment with p-propiolactone (BPL) or formalin, and then administered intramuscularly or subcutaneously with oil-based adjuvants. In general, inactivated NDV vaccines may induce strong, long-lasting antibody responses and reasonable protection against live infection with closely related strains of NDV, but may be poor inducers of cellular or mucosal immune responses. Furthermore, birds vaccinated intramuscularly or subcutaneously with inactivated vaccines have been shown to shed large amounts of virulent challenge virus compared to birds vaccinated with live NDV vaccines (Hu et al. 2022), potentially due to poor mucosal immunity or poor neutralising antibody responses.EFFICACY OF EXISTING NDV VACCINES

[0074] Traditional NDV vaccines can be efficient at providing protection against homotypic (closely matched) NDV challenge with reduced virus shedding. However, genotype V, VI and VII strains of NDV are presently circulating worldwide, while traditional vaccines are based on genotypes I and II. F and HN proteins of genotypes I and II show reduced homology compared to the circulating NDV strains with 87-89% and 87-88% respectively. The substantial genetic diversity among circulating viruses may explain the lack of cross-protection for existing NDVvaccines and the need for close-match between vaccine formulation and circulating viruses. Mutations in the two surface glycoproteins may contribute to the viral escape from antibody immunity. Overall, despite the wide application, the reported limited efficacy of currently used NDV vaccines can highlight the need for alternative vaccines.

[0075] Further, current live-attenuated and inactivated ND vaccines may not provide sterile immunity, despite intense vaccination regimes, as only partial efficacy is achieved in preventing virus infection and shedding. Recurrent outbreaks of fatal ND in commercial poultry flocks indicate that routine vaccinations fail to induce high levels of immunity required to control ND. This may be associated with antigenic divergence between the vaccine strain and the circulating strains, particularly in the HN and F envelope glycoproteins. In addition, there are no requirements for commercial ND vaccines to reduce virus shedding. Accordingly, field viruses can silently disseminate in flocks allowing for emergence of vaccine escape mutants.PROPAGATION AND PURIFICATION OF NDV

[0076] NDV for vaccine production may be propagated in any suitable manner, for example, grown in eggs such as chicken eggs or in cell culture in cells (e.g. mammalian cells, avian cells) according to standard techniques. For example, NDV may be derived by serial passaging in embryonated eggs using methods known to those skilled in the art.

[0077] For example, embryonated eggs may be obtained 9-12 days after incubation and candled to locate the air sac. The egg may then be pierced under aseptic conditions, and the seed-virus inoculated into the allantoic cavity with a syringe. The procedure may be carried out manually or automatically by machines. The inoculated egg may then be incubated for approximately two to three days in a humidified atmosphere. At the end of this period, the egg can be maintained at approximately 4°C if desired in order to terminate the embryo and aid clarification of the allantoic fluid. The top of the egg may then be removed, the membrane pierced, and the allantoic fluid collected. Again this can be achieved manually, or by automated machinery. The allantoic fluid may be clarified, for example, by centrifugation to remove cell debris and / or subjected to further purification prior to or following inactivation of the NDV by irradiation. Purification of allantoic fluid may be achieved for example, by temperature-dependent adsorption to chicken red blood cells (CRBC), sucrose gradient, or dialysis.

[0078] Additionally or alternatively, NDV for use in accordance with the present disclosure may be generated in cell culture using standard techniques. Non-limiting examples of suitable cell lines that may be used as substrates for the growth of NDV may include Vero cells, Madin Darby canine kidney (MDCK) cells, PERC6 cells, chicken embryo cells (e.g. chicken embryo fibroblasts) and avian embryonic cell lines, or variants thereof.

[0079] Propagation of NDV using cell lines will, in general, involve expanding the cells to the desired quantity in a chemically defined medium. Preferably, the medium is a serum free medium. Propagation of the virus can be assisted by the addition of proteases to the medium. Generally, the cells are infected with NDV and incubated for a period of time sufficient to generate the required amount of virus (e.g. several days). Parameters such as multiplicity of infection, incubation time and temperature will generally need to be optimised for the specific cell line used and / or specific NDV strain / s being propagated. The optimisation of growth parameters including those referred to above can be readily determined by a person of ordinary skill in the field without undue experimentation. Following the incubation period, the virus may be harvested and purified if so desired.

[0080] The NDV may optionally be purified any / or concentrated by any suitable method known in the art including by sucrose density gradient ultracentrifugation (SDGLI) or tangential flow filtration (TFF). In certain embodiments, NDV is purified and / or concentrated using tangential / cross-flow filtration. For example, virus-containing fluid may be applied to a filtering device such as a membrane having an appropriate pore size (e.g. less than about 80nm). The fluid is pumped tangentially along the surface of the membrane (i.e. across the surface) and pressure applied to force a portion of the fluid through the membrane to the filtrate side. The applied pressure will generally be of a degree that does not adversely affect virion structure and / or the integrity of viral antigens. Filtrate containing egg proteins contaminants passes through the membrane, whereas particulates and macromolecules including viral particles in the fluid that are too large to pass through the membrane pores are retained on the opposing side. In general, retentate (i.e. retained components) does not build up at the surface of the membrane and is instead swept along by the tangential flow. The retentate may be re-diluted with appropriate media (e.g. PBS containing dextran and / or sucrose) and the filtration process repeated if required.

[0081] Commercial TFF systems may be used such as Repligen KrosFlo® Research 2i TFF System. The use of tangential / cross-flow filtration to purify NDV used for irradiation provides an advantage over purification techniques currently used for NDV vaccine preparation (e.g. ultracentrifugation) as the integrity of viral antigens is better preserved. This in turn enhances the immunogenicity of irradiation-inactivated viral preparations, and in particular their ability to elicit cross-protective immunity against heterologous NDV subtypes and strains.IRRADIATION-INACTIVATED NDV

[0082] The Newcastle Disease virus in the vaccine compositions of the disclosure may be inactivated by expose to irradiation. In an embodiment, inactivation may be determined by measuring the infectiousness of the NDV following irradiation, for example, by sterility testing ormeasuring the 50% Tissue Culture Infectious Dose (TCID50) value of NDV following irradiation as described elsewhere herein. In an embodiment, the NDV is considered inactivated when, after irradiation, the TCID50 value is less than 5%, or less than 1%, or less than 0.1% of the activity prior to irradiation, or undetectable after irradiation. In an embodiment, the NDV is considered inactivated when, after irradiation, sterility testing indicates that viral infection is negligible, or undetectable, compared to NDV prior to inactivation.

[0083] In an embodiment, the irradiation-inactivated NDV is photon-inactivated NDV. The photon-inactivated NDV is inactivated using photon radiation. As noted above, the term “photon radiation” will be understood to encompass both gamma radiation (i.e., gamma-rays) and X radiation (i.e., X-rays). Accordingly, “photon-irradiated” Newcastle Disease virus of the present disclosure may be “gamma-irradiated” by way of exposure to gamma-radiation (i.e., gammarays), “X-irradiated” by way of exposure to X-radiation (i.e., X-rays), or both. As known to those of ordinary skill in the art, X-rays are identical to gamma-rays except they are emitted by the passage of electrons through an electric field of a nucleus rather than the nucleus itself upon radioactive decay. By way of non-limiting example only, to become photon-irradiated a material may be subjected to photon-radiation at energies of at least 0.01 MeV, at least 0.1 MeV, at least 0.5MeV, between 0.01 MeV and 0.5MeV, between 0.01 MeV and 1MeV, between 0.01 MeV and 10MeV, between 0.5MeV and 20MeV, between 0.5MeV and 15MeV, between 0.5MeV and 10MeV, between 0.5MeV and 5MeV, between 0.5MeV and 2MeV, or between 1MeV and 2MeV (e.g., 1.25MeV). In an embodiment, the NDV is inactivated by exposure to photon-radiation at energies of at least 0.01 MeV.

[0084] The Newcastle Disease virus in the vaccines of the disclosure may be exposed to gamma radiation. Any suitable source of gamma radiation may be used. Suitable gamma emitters include, but are not limited to Ba137, Co60, Cs137, I r192, II235, Se75and Yb169.

[0085] Gamma irradiation of Newcastle Disease virus may be performed using commercially available devices, for example, a Gammacell irradiator manufactured by Atomic Energy of Canada Ltd., Canada (e.g. Gammacell 40 Irradiator, Gammacell 220 Irradiator, Gammacell 1000 irradiator, Gammacell 3000 irradiator), a gamma-irradiator manufactured by J. L. Shepherd and Associates (San Fernando, California, USA), or a Nordion Gamma Cell-1000 irradiator manufactured by Nordion Inc. (Kanata, Ontario, Canada). Other suitable devices are described, for example, in United States Patent No. 3,557,370 and United States Patent No. 3,567,938.

[0086] Additionally or alternatively, Newcastle Disease virus of the disclosure may be X-irradiated. Any suitable source of X-radiation may be used. Suitable sources of X-radiation include, but are not limited to, the eXelis® sterilization X-ray machine manufactured by I BAIndustrial (Louvain-la-Neuve, Belgium). Other suitable devices include for example, the RS2400® and RS3400® manufactured by Rad Source Technologies Inc. (Suwanee, Georgia, USA).

[0087] In general, the Newcastle Disease virus is exposed to a dose of photon radiation sufficient to inactivate the virus. Preferably, the dose of photon radiation is sufficient to inactivate the virus without substantially disrupting the structure of viral antigens, and in particular, without substantially disrupting the structure of viral surface antigens. The immunogenicity of antigenic determinants may therefore be retained by the photon-irradiated virus. Preferably, the dose of photon radiation is administered to the virus over a period of time and at a level sufficient to ensure that all viruses under treatment are exposed without adversely affecting the structural integrity of viral antigenic determinants. In an embodiment, the Newcastle Disease virus is inactivated by photon exposure.

[0088] As known to those of ordinary skill in the art, a measure for an absorbed dose of radiation is the gray (Gy), which is defined as 1 joule of energy deposited in 1 kilogram of mass. An old unit of measure for this is the rad, which stands for “radiation absorbed dose”, where 1 Gy = 100 rad. Radiation doses can be measured using and suitable method known to hose skilled in the art, such as using calibrated Fricke or ceric cerous dosimeters.

[0089] Newcastle Disease virus for use in accordance with the present disclosure may be exposed to a total dose of photon radiation (e.g., gamma-radiation and / or X-radiation) in the range of about 1 x 103rad and about 2 x 109rad (or about 10 Gy to about 2 x 104kGy). In certain embodiments of the disclosure, the Newcastle Disease virus is exposed to a total dose of X-radiation and / or gamma-radiation of between about 1 x 103rad and about 2 x 109rad, between about 1 x 103rad and about 1 x 109rad, between about 1 x 103rad and about 1 x 108rad, between about 1 Xx103rad and about 1 x 107rad, between about 1 x 103rad and about 1 x 106rad, between about 1 x 103rad and about 1 x 105rad, between about 1 x 103rad and about 1 x 104rad, between about 1 x 103rad and about 2 x 109rad, between about 1 x 104rad and about 2 x 109rad, between about 1 x 105rad and about 2 x 109rad, between about 1 x 106rad and about 2 x 109rad, between about 1 x 107rad and about 2 x 109rad, between about 1 x 108rad and about 2 x 109rad or between about 1 x 109rad and about 2 x 109rad.

[0090] In one embodiment, the Newcastle Disease virus of the present disclosure is exposed to a total dose of photon radiation (e.g., X-radiation and / or gamma-radiation) of between about 6.5 x 104rad and about 2 x 107rad (about 0.65 kGy to about 200 kGy). In other embodiments, the Newcastle Disease virus of the present disclosure are exposed to a total photon radiation dose of about 2 kGy to 100k Gy, 10 kGy to about 12 kGy, about 12 kGy to about 14 kGy, about 14 kGy to about 16 kGy, about 10 kGy to about 20 kGy, about 14 kGy to about 20 kGy, about 20 kGy to about 30 kGy, about 20 kGy to about 25 kGy, about 25 kGy to about 30 kGy, about 30 to35 kGy, about 35 to 40 kGy, about 40 kGy to about 45 kGy, about 50 kGy to about 55 kGy, about 30 to 50 kGy, about 30 to 40 kGy, about 35 to 45 kGy, about 35 to 50kGy, about 45 kGy to 55kGY, about 50 kGy to 60 kGy, about 10 kGy, about 11 kGy, about 12 kGy, about 13 kGy, about 14 kGy, about 15 kGy, about 16 kGy, about 17 kGy, about 18 kGy, about 19 kGy, about 20 kGy, about 21 kGy, about 22 kGy, about 23 kGy, about 24 kGy, about 25 kGy, about 26 kGy, about 27 kGy, about 28 kGy, about 29 kGy, about 30 kGy, about 31 kGy, about 32 kGy, about 33 kGy, about 34 kGy, about 35 kGy, about 36 kGy, about 37 kGy, about 38 kGy, about 39 kGy about 40 kGy, about 41 kGy, about 42 kGy, about 43 kGy, about 44 kGy, about 45 kGy, about 46 kGy, about 47 kGy, about 48 kGy, about 49 kGy about 50 kGy, about 51 kGy, about 52 kGy, about 53 kGy, about 54 kGy, about 55 kGy, about 60 kGy, 65 kGy, about 70 kGy, about 75 kGy, about 80 kGy, about 85 kGy, about 90 kGy, about 95 kGy, about 100 kGy, more than 10 kGy, more than 12kGy, more than 14 kGy, more than 16 kGy, more than 18 kGy, more than 20 kGy, more than 22 kGy, more than 24 kGy, more than 26 kGy, more than 28 kGy, more than 30 kGy, more than 35 kGy, more than 40 kGy, more than 45 kGy, more than 50 kGy, more than 60 kGy, more than 70 kGy, more than 80 kGy, more than 100 kGy.

[0091] The optimal dose of photon radiation (e.g., gamma-radiation and / or X-radiation) may be influenced by factors such as the purification method used to purify the Newcastle Disease virus, the quantity of Newcastle Disease virus present to be treated, the temperature of the Newcastle Disease virus present to be treated (e.g., frozen on dry ice or at room temperature), water availability, oxygen availability and / or the subtype or strain under treatment. Accordingly, the total dose of photon radiation, the exposure time and / or the level of photon radiation applied over the period of exposure may be optimised to enhance the effectiveness of the treatment.

[0092] The total dose of photon radiation (e.g., X-radiation and / or gamma-radiation) may be administered to the Newcastle Disease virus of the present disclosure cumulatively over a period of time. For example, photon-radiation may be administered to the Newcastle Disease virus of the present disclosure at a level lower than that of the total dose, over a time period sufficient to achieve the total dose of photon radiation required.

[0093] In one embodiment, preparations of Newcastle Disease virus of the present disclosure are maintained in a frozen and / or lyophilised state while being exposed to photon radiation (e.g., gamma-radiation and / or X-radiation). This may facilitate the preservation of biological integrity and avoid unnecessary damage of viral antigens thereby enhancing the immunogenicity of photon-irradiated viral preparations, and in particular, their ability to elicit cross- reactive / cross-protective immunity against, for example, multiple Newcastle Disease types, subtypes and strains. A photon-radiation dose of 50 kGy may be effective for treating preparations of frozen and / or lyophilised Newcastle Disease virus of the present disclosure. Alternatively, a photon radiation dose of 10-35 kGy, 35-55 kGy or 20-60 kGy (e.g., more than 20, more than 22, morethan 24, more than 26, more than 28, more than 30, more than 32, more than 34, more than 36, more than 38, more than 40, more than 42, more than 44, more than 46, more than 48, more than 50, more than 52, more than 54, more than 56, more than 58, or more than 60 kGy, etc.) may be effective for treating preparations of frozen and / or lyophilised Newcastle Disease virus preparations of the present disclosure.

[0094] This may facilitate the preservation of biological integrity and avoid unnecessary damage of viral antigens thereby enhancing the immunogenicity of gamma-irradiated viral preparations, and in particular, their ability to elicit cross-reactive / cross-protective immunity.

[0095] In an embodiment, a gamma-irradiation dose of 2-100 kGy or 10-50 kGy may be effective for treating frozen Newcastle Disease virus preparations. In an embodiment, a gammairradiation dose of 30-40 kGy may be effective for treating frozen Newcastle Disease virus preparations. In an embodiment, a gamma-irradiation dose of 40-60 kGy may be effective for treating frozen Newcastle Disease virus preparations. In an embodiment, the viral preparations may be on ice. In an embodiment, the viral preparations may be on an ice slurry. In an embodiment, the viral preparations may be on dry ice.

[0096] In an embodiment, the treatment with gamma-irradiation is sufficient to inactivate the Newcastle Disease virus without substantially disrupting the structure of viral antigens. Inactivation of the virus may be assessed using methods generally known in the art. For example, viral infectivity can be measured following gamma-irradiation by inoculating embryonic eggs and / or cell lines as described in the paragraphs above to determine whether the virus is capable of propagation.

[0097] The integrity of antigenic determinants can be assessed, for example, by assaying the virus for haemagglutinin or neuraminidase activity following gamma-irradiation. Methods of performing hemagglutination and neuraminidase assays are known in the art. The integrity of antigenic determinants can alternatively or additionally be assessed, for example, by reactivity with panels of monospecific antisera raised against purified native antigenic components using Western blotting, FACs analysis, or enzymatic assays of surface components, etc.

[0098] Additionally or alternatively, cytotoxic T cell responses against the internal proteins inducible by the photon-irradiated preparations can be used to as indicator for protein integrity.

[0099] In an embodiment, the photon-irradiated Newcastle Disease virus is prepared by purification, for example, by tangential flow filtration or sucrose density gradient ultracentrifugation, prior to exposure to photon radiation.[000100] In an embodiment, the irradiation-inactivated NDV is electron inactivated NDV. As known to those of ordinary skill in the art, inactivation following an exposure to high energy photons (gamma-rays and X-rays) are mediated either by direct damage caused by photons or indirect damage caused by electrons arising because of an exposure to photons. Consequently, irradiation-inactivated NDV refers to both photon-inactivated and electron-inactivated NDV. The electron-inactivated NDV is inactivated using electron irradiation. Electron irradiation is a process that involves using electrons, usually of high energy, to treat an object for a variety of purposes. Electron energies often vary from the keV to MeV range, depending inter alia on the depth of penetration required. The irradiation dose is usually measured in grays but also in Mrads (1 Gy is equivalent to 100 rad).[000101] In some examples, electron-beam accelerators utilize an on-off technology, with a common design being similar to that of a cathode ray television. The basic components of a typical electron-beam processing device may include an electron gun (including a cathode, grid, and anode), used to generate and accelerate the primary beam; and, a magnetic optical (focusing and deflection) system, used for controlling the way in which the electron beam impinges on the sample undergoing irradiation. In operation, the hot cathode of the electron gun emits electrons that may be accelerated and shaped into a collimated beam by the electrostatic field geometry established by the electrode configuration (grid and anode). The electron beam then emerges from the gun assembly through an exit hole in the ground-plane anode with an energy equal to the value of the negative high voltage (gun operating voltage) being applied to the cathode. This use of a direct high voltage to produce a high-energy electron beam may allow the conversion of input electrical power to beam power at greater than 95% efficiency, making electron-beam material processing a highly energy-efficient technique. After exiting the gun, the beam passes through an electromagnetic lens and deflection coil system. The lens is used for producing either a focused or defocused beam spot on the sample, while the deflection coil is used to either position the beam spot on a stationary location or provide some form of oscillatory motion.[000102] In an embodiment, a NDV preparation may be irradiated with electrons accelerated at low energy, for example, accelerated at energies of less than 0.3 MeV, for example, 0.15 MeV. In embodiment, the electrons may be accelerated at energies in a range between 0.01 MeV and 0.1 MeV, between 0.01 MeV and 0.3 MeV, or between 0.1 MeV and 0.3 MeV, or between 0.15 MeV and 0.25 MeV, or between 0.15 MeV and 0.3 MeV, or between 0.2 MeV and 0.25 MeV, or between 0.2 MeV and 0.3 MeV, or between 0.25 MeV and 0.3 MeV, or between 0.25 MeV and 0.35 MeV.[000103] In an embodiment, the electrons are accelerated at low energy or moderate energy, for example, accelerated with an acceleration energy of between 0.15 MeV and 0.7 MeV, or between 0.2 MeV and 0.5 MeV, or between 0.25 MeV and 0.4 MeV. In embodiment, the electronsmay be accelerated at energies in a range between 0.2 MeV and 0.4 MeV, or between 0.2 MeV and 0.5 MeV, or between 0.2 MeV and 0.6 MeV, or between 0.25 MeV and 0.5 MeV, or between 0.25 MeV and 0.6 MeV, or between 0.3 MeV and 0.4 MeV, between 0.3 MeV and 0.5 MeV, or between 0.3 MeV and 0.6 MeV, or between 0.4 MeV and 0.5 MeV, or between 0.4 MeV and 0.6 MeV.[000104] By way of non-limiting example only, to become electron irradiated a material may be subjected to electron radiation at energies of at least 0.01 MeV, at least 0.1 MeV, at least 0.5MeV, between 0.01 MeV and 0.5 MeV, between 0.01 MeV and 1MeV, between 0.01 MeV and 10 MeV, between 0.5 MeV and 20 MeV, between 0.5 MeV and 15 MeV, between 0.5 MeV and 10 MeV, between 0.5 MeV and 5 MeV, between 0.5 MeV and 2 MeV, or between 1 MeV and 2 MeV (e.g., 1.25MeV). In an embodiment, the NDV is inactivated by exposure to electron irradiation at energies of at least 0.01 MeV.[000105] In an embodiment, NDV could be inactivated using a dose of electron irradiation of 50 kGy, 100 kGy or 200 kGy.[000106] It has been found that a dose of at least 50 kGy is advantageous in order to achieve, as far as possible, complete inactivation of viruses.[000107] In an embodiment, the immunogenic composition or vaccine comprises at least one virus irradiated with an electron radiation dose of at least 1 kGy, at least 2 kGy, at least 3 kGy, at least 4 kGy, at least 5 kGy, at least 6 kGy, at least 7 kGy, at least 8 kGy, at least 9 kGy, at least 10 kGy, at least 11 KGy, at least 12 kGgy, at least 13 kGy, at least 14 Kgy, at least 15 kGy, at least 16 kGy, at least 17 kGy, at least 18 kGy, at least 19 kGy, at least 20 kGy, at least 20-25 kGy, at least 25-30kGy, at least 30-35 kGy, at least 35-40 kGy, at least 40-45 kGy, at least 45-50kGy, at least 50 kGy, at least 60 kGy, at least 70 kGy, at least 80 kGy, at least 90 kGy, at least 100 kGy, at least 110 kGy, at least 120 kGy, at least 130 kGy, at least 140 kGy, at least 150 kGy, at least 160 kGy, at least 170 kGy, at least 180 kGy, at least 190 kGy, at least 200 kGy or at least 250 kGy.[000108] In an embodiment, the NDV preparation is irradiated with an electron radiation dose of between 1-5 kGy, 1-10 kGy, 1-15 kGy,1-20 kGy, 1-25 kGy, 1-30 kGy, 1-35 kGy, 1-40 kGy, 1-45 kGy, 1-50 kGy, 5-10 kGy, 5-15 kGy, 5-20 kGy, 5-25 kGy, 5-30 kGy, 5-35 kGy, 5-40 kGy, 5-45 kGy, 5-50 kGy, 10-15 kGy, 10-20 kGy, 10-25 kGy, 10-30 kGy, 10-35 kGy, 10-40 kGy, 10-45 kGy, 10-50 kGy, 15-10 kGy, 15-15 kGy, 15-20 kGy, 15-25 kGy, 15-30 kGy, 15-35 kGy, 15-40 kGy, 15-45 kGy, 15-50 kGy, 20-25 kGy, 20-30 kGy, 20-35 kGy, 20-40 kGy, 20-45 kGy, 20-50 kGy, 25-30 kGy, 25-35 kGy, 25-40 kGy, 25-45 kGy, 25-50 kGy, 30-35 kGy, 30-40 kGy, 30-45 kGy, 30-50 kGy, 35-40 kGy, 35-45 kGy, 35-50 kGy, 40-45 kGy, 40-50 kGy, or 45-50 kGy.[000109] In an embodiment, the immunogenic composition or vaccine comprising the NDV is irradiated with an electron radiation dose of at most 300 kGy, at most 250 kGy, at most 200 kGy, at most 190 kGy, at most 180 kGy, at most 170 kGy, at most 160 kGy, at most 150 kGy, at most 140 kGy, at most 130 kGy, at most 120 kGy, at most 110 kGy, at most 100 kGy, at most 90 kGy, at most 80 kGy, at most 70 kGy or at most 60 kGy.[000110] In an embodiment, the electron radiation dose is in the range of 50 kGy to 300 kGy. For example, the immunogenic composition or vaccine comprising at least one virus may be irradiated at an electronradiation dose of 50 kGy, 60 kGy, 70 kGy, 80 kGy, 90 kGy, 100 kGy, 110 kGy, 120 kGy, 130 kGy, 140 kGy, 150 kGy, 160 kGy, 170 kGy, 180 kGy, 190 kGy, 200 kGy, 210 kGy, 220 kGy, 230 kGy, 230 kGy, 240 kGy, 250 kGy, 260 kGy, 270 kGy, 280 kGy, 290 kGy or 300 kGy.[000111] In a preferred embodiment, the immunogenic composition or vaccine comprises at least one virus is irradiated with an electron radiation dose in the range of 50 kGy to 300 kGy, such as 50 kGy to 200 kGy, or 50 kGy to 150 kGy, or 50 kGy to 120 kGy, or 50 kGy to 110 kGy.[000112] In another embodiment, the at least one virus is irradiated with an electron radiation dose of 1 to 300 kGy, or with an electron radiation dose of 1 to 150 kGy, or with an electron radiation dose of 10 to 120 kGy, or with an electron radiation dose of 15 to 110 kGy.[000113] In an embodiment, it is possible to operate under standard atmospheric pressure or essentially under standard atmospheric pressure. “Essentially standard atmospheric pressure” is understood to mean 1 bar+ / -0.1 bar. The standard atmospheric pressure can be present, for example, as atmospheric oxygen, nitrogen, or carbon dioxide gas.[000114] The applicable dose of electron radiation may be influenced by factors such as the purification method used to purify the Newcastle Disease virus, the quantity of Newcastle Disease virus present to be treated, the temperature of the Newcastle Disease virus present to be treated (e.g., frozen on dry ice or at room temperature), water availability, oxygen availability and / or the subtype or strain under treatment. Accordingly, the total dose of electron radiation, the exposure time and / or the level of electron radiation applied over the period of exposure may be optimised to enhance the effectiveness of the treatment.[000115] Electron irradiation may be applied using electron beam machines or electron guns, such as Comet, EB-Lab200, Switzerland, and eFIT system or platform, Kyoobe Tech, Germany. The eFIT system may be particularly useful for large scale manufacturing.[000116] In one embodiment, irradiation of NDV may be performed in duplicates or triplicates at different doses of irradiation, for example, 5, 10, 15, 20, 25, or 30 kGy with, for example,anelectron beam (e.g., such as Comet, EB-Lab200, Switzerland, or eFIT system or platform, Kyoobe Tech, Germany) at room temperature or cold conditions. Voltage may be modified as required for a particular system of use.[000117] However, other energies and doses of radiation may be suitable. Sterilisation, morphology and / or vaccination studies as described herein (e.g., antibody responses and neutralising antibody responses, etc) may be used to assess results.PROTEASE TREATMENT OF IRRADIATED NDV[000118] The vaccine compositions of the present disclosure may comprise irradiation inactivated NDV that is then protease treated. Suitable proteases may include trypsin, tryptase, prostasin, human airways trypsin-like protease (HAT), transmembrane serine protease 2 (TMPRSS2), matriptase, chymotrypsin, pepsin, thrombin, elastase, subtilisin, carboxypeptidase, papain, bromelain, caspase, etc. Methods of protease treatment are known to those skilled in the art.[000119] In an embodiment, the protease is trypsin. Trypsin is a serine protease that hydrolyses peptide bonds in proteins, primarily targeting those adjacent to the amino acids lysine and arginine. In an embodiment, the protease is selected from the groups consisting of trypsin, tryptase, prostasin, human airways trypsin-like protease (HAT), transmembrane serine protease 2 (TMPRSS2), matriptase, chymotrypsin, pepsin, thrombin, elastase, subtilisin, carboxypeptidase, papain, bromelain and caspase.[000120] In an embodiment, the NDV is irradiated as described herein, and is subsequently protease treated as described herein. Accordingly, in an embodiment, the protease treatment occurs following the irradiation inactivation. It is envisioned that the NDV may alternatively be protease treated as described herein and then irradiated as described herein. Accordingly, in an embodiment, the irradiation inactivation occurs following the protease treatment. The irradiated, protease-treated NDV may then be administered as a vaccine composition as described herein.VACCINE COMPOSITIONS[000121] The vaccine compositions of the present disclosure may comprise NDV that has been inactivated by irradiation. In an embodiment, the vaccine composition may comprise an immunogenic component that consists of NDV that has been inactivated by irradiation. In embodiments, the irradiated NDV is protease treated. The vaccine may be produced from any type, subtype or strain, or a mixture of any number of different types, subtypes and / or strains of Newcastle Disease virus. For example, the NDV vaccine composition may comprise a strain from at least one of genotypes I, II, III, IV, V, VI, VII, VII, VIII, IX, X, XI, XII, XIII, XIV, XV, XVI, XVII,XVII, XVIII, XIX, XX and XXI. In an embodiment, the NDV composition comprises a strain from Genotype I. In an embodiment, the NDV composition comprises a V4 strain.[000122] Vaccine compositions of the present disclosure may be administered to naive recipients, being individuals seronegative for particular target strain(s) of NDV. Alternatively, the vaccine compositions may be administered to primed recipients, being individuals seropositive for particular target strain(s) of NDV.[000123] Vaccine compositions of the present disclosure may be prepared according to standard methods known to those of ordinary skill in the art. Methods for vaccine preparation are generally described in Voller et al., (1978), “New Trends and Developments in Vaccines”, University Park Press, Baltimore, Maryland, USA.[000124] The level of immunogenicity induced by a vaccine composition of the present disclosure may be determined by measuring an immune response of a subject to which it has been administered. The immune response to a composition of the present disclosure may be measured, for example, by analysis of antibody production, neutralising assays, cellular, proliferative and / or cytotoxic responses, and / or cytokines secretion. Non-limiting examples of specific assays for the measurement of immune responses include solid-phase heterogeneous assays (e.g. enzyme-linked immunosorbent assay), solution phase assays (e.g. electrochemiluminescence assay) and amplified luminescent proximity homogeneous assays. Other non-limiting examples include flow cytometry, intracellular cytokine staining, functional T-cell assays functional B-cell assays, functional monocyte-macrophage assays, dendritic and reticular endothelial cell assays, NKcell response, oxidative burst assays, and phagocytosis and apoptosis evaluation.[000125] Alternatively or additionally, immunogenicity can be measured by measuring survival or reduction of symptoms in vaccinated animals following a challenge with a virulent NDV strain. In an embodiment, the compositions of the disclosure induce a heterotypic immune response against heterologous NDV genotypes.[000126] In certain embodiments, compositions and vaccines of the present disclosure comprise single or multiple strains of irradiated NDV. In other embodiments, compositions and vaccines of the present disclosure comprise a single irradiated strain of NDV.METHODS AND USES OF VACCINE COMPOSITION[000127] The compositions of the present disclosure may be administered to a subject to vaccinate the subject against NDV. In an embodiment, the present disclosure provides a method of vaccinating a subject against NDV infection, the method comprising administering to thesubject a therapeutically effective amount of the vaccine composition. In an embodiment, the composition may be for use in vaccinating a subject against NDV infection. In an embodiment, the present disclosure provides use of a therapeutically effective amount of the composition of the disclosure for vaccinating a subject against NDV infection. In an embodiment, the present disclosure provides use of a therapeutically effective amount of the composition of the present disclosure in the preparation of a medicament for vaccinating a subject against NDV infection.[000128] In an embodiment, the compositions of the present disclosure can be used in methods of inducing or enhancing an immune response against NDV. In an embodiment, the present disclosure provides a method for inducing or enhancing an immune response against an NDV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the composition may be for use in inducing or enhancing an immune response against NDV in a subject. In an embodiment, the present disclosure provides use of a therapeutically effective amount of the composition for inducing or enhancing an immune response in a subject against NDV infection. In an embodiment, the present disclosure provides use of a therapeutically effective amount of the composition in the preparation of a medicament for inducing or enhancing an immune response in a subject against NDV infection.[000129] In an embodiment, the composition may be used in a method of preventing or reducing the symptoms of an NDV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition to the subject. In an embodiment, the composition may be for use in preventing or reducing the symptoms of an NDV infection in a subject. In an embodiment, the present disclosure provides use of a therapeutically effective amount of the composition for preventing or reducing the symptoms of an NDV infection in a subject. In an embodiment, the present disclosure provides use of a therapeutically effective amount of the composition in the preparation of a medicament for preventing or reducing the symptoms of an NDV infection in a subject.SUBJECTS[000130] NDV primarily affects birds. In an embodiment, the vaccine composition is administered to avian subjects. For example, the vaccine composition may be administered to birds of commercial significance, such as chickens, turkeys, quails, ducks, geese, emus, ostriches and other birds bred for their meat, eggs or leather. Alternatively, the vaccine may be administered to birds of veterinary or human significance such as companion birds (e.g., budgerigars, parrots, cockatoos, etc.), racing birds (e.g. pigeons) or those of interest such as birds of prey, penguins, endangered species, etc. However, in an embodiment, the vaccine may also be administered to laboratory animals such as mice, rats, rabbits, etc.[000131] It is also envisioned that the vaccines described herein would also be suitable for administration to other species, such as humans or non-human mammals. In an embodiment, the subject is mammal. In an embodiment, the subject is human. In an embodiment, the subject is a non-human animal. In an embodiment, the subject is an animal selected from mice, rats, rabbits, bats, seals, bears, polar bears, foxes, skunks, mink, cats, dogs, tigers, lions, cheetahs, leopards, puma, goats, cows, sheep, horses and pigs, etc.FORMULATIONS[000132] Compositions of the disclosure may be prepared using methods known to those of ordinary skill in the art. Non-limiting examples of suitable methods are described in Gennaro et al. (Eds), (1990), “Remington’s Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pennsylvania, USA, and methods for vaccine preparation are generally described in Voller et al., (1978), “New Trends and Developments in Vaccines”, University Park Press, Baltimore, Maryland, USA.[000133] The compositions may comprise a pharmaceutically acceptable carrier, excipient, diluent and / or adjuvant. Pharmaceutically acceptable carriers, excipients, diluents and / or adjuvants as contemplated herein are substances which do not produce adverse reaction(s) when administered to a particular recipient such as a human or non-human animal. Pharmaceutically acceptable carriers, excipients, diluents and adjuvants are generally also compatible with other ingredients of the vaccine. Non-limiting examples of suitable excipients, diluents, and carriers can be found in the “Handbook of Pharmaceutical Excipients” 4th Edition, (2003) Rowe et al. (Eds), The Pharmaceutical Press, London, American Pharmaceutical Association, Washington.[000134] Non-limiting examples of pharmaceutically acceptable carriers, excipients or diluents include demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or isopropanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.[000135] Compositions of the present disclosure may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, or in a form suitable for parenteral administration, that is, intradermal, subcutaneous, intramuscular or intravenous injection.[000136] Solid forms of the compositions for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and / or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of Wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.[000137] Liquid forms of the compositions for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.[000138] Suspensions comprising the compositions for oral administration may further comprise dispersing agents and / or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or dioleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.[000139] For preparation of the compositions as injectable solutions or suspensions, non-toxic parenterally acceptable diluents or carriers may be used such as Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.[000140] Vaccine emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.[000141] When formulated as drops, the compositions may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and / or fungicidal agent and / or any other suitable preservative, and optionally including a surface-active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. For example, sterilisation may be achieved by filtration followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.[000142] The compositions may include any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.[000143] The compositions may be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stabilisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.ADJUVANTS[000144] Adjuvant(s) may be included in compositions of the disclosure, although experimental data provided herein demonstrates that irradiated NDV can induce immunity without requiring such adjuvants. Accordingly, compositions of the disclosure may or may not comprise an adjuvant.[000145] In general, adjuvant activity in the context of a vaccine composition includes, but is not limited to, the ability to enhance the immune response (quantitatively or qualitatively) induced by immunogenic components in a vaccine. This may reduce the dose or level of the immunogenic components required to produce an immune response and / or reduce the number or the frequency of immunisations required to produce the desired immune response.[000146] In an embodiment, an adjuvant will enhance the immune response induced and / or enhanced by component(s) of the vaccine thereby improving protective efficacy. In an embodiment, the adjuvant will enable the induction of protective immunity utilising a lower dose of active component(s) (e.g., a irradiated NDV).[000147] Non-limiting examples of adjuvants suitable for inclusion in compositions of the disclosure and methods for their preparation are described in “Vaccine Adjuvants: Preparation Methods and Research Protocols (Methods in Molecular Medicine)”, (2000), Ohagan (Ed), Humana Press Inc. Any suitable adjuvant may be included in a vaccine of the disclosure.[000148] Specific examples of such adjuvants include, but are not limited to, aluminium hydroxide; polypeptide adjuvants including interferons, interleukins, and other cytokines; AMPHIGEN, oil-in-water and water-in-oil emulsions; and saponins such as QuilA.[000149] For example, an aluminium-based adjuvant may be utilised. Suitable aluminium-based adjuvants include, but are not limited to, aluminium hydroxide, aluminium phosphate and combinations thereof. Other specific examples of aluminium-based adjuvants that may be utilised are described in European Patent No. 1216053 and United States Patent No. 6,372,223.[000150] Oil in water emulsions may be utilised as adjuvants in compositions of the disclosure. Oil in water emulsions are well known in the art. In general, the oil in water emulsion will comprise a metabolisable oil, for example, a fish oil, a vegetable oil, or a synthetic oil. Examples of suitable oil in water emulsions include those described in European Patent No. 0399843, United States Patent No. 7,029,678 and PCT Publication No. WO 2007 / 006939. The oil in water emulsion may be utilised in combination with other adjuvants and / or immunostimulants.[000151] Non-limiting examples of other suitable adjuvants include immunostimulants such as granulocyte-macrophage colony-stimulating factor (GM-CSF), monophosphoryl lipid A (MPL), cholera toxin (CT) or its constituent subunit, heat labile enterotoxin (LT) or its constituent subunit, toll-like receptor ligand adjuvants such as lipopolysaccharide (LPS) and derivatives thereof (e.g., monophosphoryl lipid A and 3-Deacylated monophosphoryl lipid A), muramyl dipeptide (MDP), Toll-like receptor (TLR) agonists (e.g., TLR-2, TLR-3 agonists) and F protein of Respiratory Syncytial Virus (RSV).[000152] Adjuvants in compositions of the disclosure may typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents. Another type of “self-adjuvant” is provided by the conjugation of immunogenic peptides to lipids such as the water soluble lipopeptides Pam3Cys or its dipalmitoyl derivative Pam2Cys. Such adjuvants have the advantage of accompanying and immunogenic component into the antigen presenting cell (such as dendritic cells) and thus producing enhanced antigen presentation and activation of the cell at the same time (see, for example, Brown and Jackson, (2005), “Lipid based self adjuvanting vaccines”, Current Drug Delivery, 23:83).[000153] Suitable adjuvants are commercially available such as, for example, Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminium salts such as aluminium hydroxide gel (alum) or aluminium phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.[000154] In certain embodiments, an adjuvant included in a vaccine of the disclosure may induce an immune response predominantly of the Th1 type. Suitable adjuvants for use in eliciting a predominantly Th1-type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt. For example, the composition or vaccine may be formulated with adjuvant AS04 containing aluminium hydroxide (alum) and 3-O-deacylated monophosphorylated lipid A (MPL) such as described in Thoelen et al. (2001), “A prophylactic hepatitis B vaccine with a novel adjuvant system”, Vaccine, 19:2400-2403. Other known adjuvants, which preferentially induce a Th1 type immune response, include CpG containing oligonucleotides. The oligonucleotides are characterised in that the CpG dinucleotide is unmethylated. Such oligonucleotides are known to those of ordinary skill in the field and are described, for example, in PCT Publication No. WO 1996 / 02555. Immunostimulatory DNA sequences are also described, for example, in Sato et al., (1996), “Immunostimulatory DNA sequences necessary for effective intradermal gene immunization”, Science, 273:352-354.[000155] Another example of an adjuvant is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants. For example, an enhanced adjuvant system may be utilised involving the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in PCT Publication No. WO 1994 / 00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in PCT publication No.WO 1996 / 33739. Other alternative formulations comprise an oil-in-water emulsion and tocopherol. An adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in PCT Publication No. WO 1995 / 17210. An adjuvant included in a composition of the disclosure may include a formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion such as described in PCT publication No. WO 1995 / 17210. In one embodiment a composition of the disclosure comprises the adjuvant Montanide ISA720 (M-ISA-720; Seppic, Fairfield, N.J.), an adjuvant based on a natural metabolisable oil.[000156] In an embodiment, the adjuvant is a mucosal adjuvant effective in enhancing mucosal immunity and / or systemic immunity to immunogenic components administered via the mucosal route. Mucosal adjuvants may be broadly classified as those that facilitate vaccine delivery (e.g., liposomes, cochleates, live-attenuated vectors, poly D,L-lactide-co-glycolide or PLGA, chitans, DNA vaccines, mucoadhesives) to enhance the induction of protective immunity induced by other immunogenic components of the vaccine, and those having an immunostimulatory role (e.g., innate immunity associated toxin-based, cytokine-based etc.). Without limitation to a particular mechanism, it is postulated that the advantageous effects of mucosal adjuvants partially derive from an ability to assist the passage of immunogenic components in the vaccine across the mucosal barrier. Upon traversing the mucosal barrier, the mucosal adjuvant may enhance immunity, for example, by complement activation, the induction of cytokines, stimulation of antibody production or antibody type switching, stimulating antigen presenting cells, and / or influencing HLA or MHC class I and / or class II expression.ROUTES OF ADMINISTRATION[000157] Compositions of the disclosure may be administered to a recipient by standard routes, including, but not limited to, parenteral (e.g., intradermal, intravenous, intraspinal, intraperitoneal, subcutaneous or intramuscular), oral, topical, or mucosal routes (e.g., intranasal, ocular, conjunctival). In an embodiment, the composition is administered via an intramuscular, intranasal or subcutaneous route. In an embodiment, the composition is administered via an intranasal, ocular or conjunctival route.[000158] In an embodiment, the compositions may be administered by the intramuscular route.[000159] In an embodiment, the compositions may be administered by a mucosal route. Nonlimiting examples of acceptable routes of mucosal vaccine administration including intranasal, ocular, buccal, conjunctival, genital tract (vaginal), rectal, intratracheal, skin, and the gastrointestinal tract.[000160] In an embodiment, compositions of the disclosure are administered by the intranasal route. Without limitation to theory or particular mode(s) of action, intranasal administration of thecompositions may be advantageous for enhancing immunity against NDV virus as, for example, mucosal vaccination (e.g., intranasal vaccination) may induce mucosal immunity not only in the respiratory tracts but also in distant mucosal sites including the genital mucosa.[000161] Intranasal compositions of the disclosure can be formulated, for example, in liquid form as nose drops, spray, or suitable for inhalation, as powder, as cream, or as emulsion. Nebulised or aerosolised intranasal vaccines may also be utilised. Administration of compositions to mucosa of the upper and / or lower respiratory tract via inhalation of mists, powders, or sprays, or by intranasal administration of nose drops, swabs, powders, sprays, mists, aerosols, and the like is also contemplated.[000162] In one embodiment, the compositions for intranasal administration are provided in a freeze-dried powder form capable of re-constitution immediately prior to use. Powder vaccine formulations of compositions of the present disclosure provide a means of overcoming refrigerated storage and distribution requirements associated with liquid-based vaccine stability and delivery. Dry powder formulations offer the advantage of being more stable and also do not support microbial growth.[000163] The freeze-dried compositions may induce levels of cross- protective immunity similar to that of non-freeze-dried compositions. The compositions may be freeze-dried using any suitable technique known in the art. For example, liquid preparations of a irradiated NDV may be frozen in a dry ice - isopropanol slurry and lyophilized in a freeze Dryer (e.g., Virtis Model 10-324 Bench, Gardiner, NY) for a suitable time period (e.g., 24 hours).[000164] In one embodiment, a dry powder nasal vaccine of the disclosure is produced by generating spray-freeze-drying (SFD) particles (see, for example, Costantino et al., (2002), “Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability’, J Pharm Sci., 91:388-395; Costantino, etal., (2000), “Protein spray-freeze drying. Effect of atomization conditions on particle size and stability”, Pharm Res., 17: 1374-1383; Maa et al., (1999), “Protein inhalation powders: spray drying vs spray freeze drying", Pharm Res, 16:249-254; Carrasquillo etal., (2001); “Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein", J Control Release, 76: 199-208; Carrasquillo et al., (2001), “Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation”, J Pharm Pharmacol., 53:115-120; and United States Patent No. 6,569,458).[000165] Preferred devices for intranasal administration of the compositions are nasal spray devices (e.g., devices available commercially from Pfeiffer GmBH, Valois and Becton Dickinson). Non-limiting examples of suitable devices are described, for example, in Bommer, (1999), “Advances in Nasal drug delivery Technology’, Pharmaceutical Technology Europe, p26-33.Intranasal devices may produce droplets in the range 1 to 500 .m. Preferably, only a small percentage of droplets (e.g., <5%) are below 10 .m to minimise the chance of inhalation.[000166] A composition of the disclosure may be administered to a recipient in isolation or in combination with other additional therapeutic agent(s). In embodiments where the vaccine is administered with therapeutic agent(s), the administration may be simultaneous or sequential (i.e., vaccine administration followed by administration of the agent(s) or vice versa). Thus, where a vaccine of the disclosure is administered to a subject in conjunction with another agent, both may be administered in a single composition at the same time, in separate compositions at the same time, or separately at different times.[000167] The vaccine composition may be delivered ocularly orconjunctivally. For example, the composition may be delivered topically by droplet directly onto the eye or conjunctiva. From here, the composition makes its way into the respiratory tract via the lacrimal duct, where it is able to induce an immune response.DOSAGES[000168] In general, compositions of the disclosure are administered in a manner compatible with the route of administration and physical characteristics of the recipient (including health status) and in such a way that it elicits the desired effect(s) (i.e., therapeutically effective, immunogenic and / or protective).[000169] For example, the appropriate dosage of a given vaccine may depend on a variety of factors including, but not limited to, a subject’s physical characteristics (e.g., age, weight, sex), whether the compound is being used as single agent or adjuvant therapy, the progression (i.e., pathological state) of a given NDV infection, and other factors that may be recognized by one skilled in the art. Various general considerations that may be considered when determining an appropriate dosage of a given vaccine of the disclosure are described, for example, in Gennaro et al. (Eds), (1990), “Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al., (Eds), (1990), “Goodman and Gilman's: The Pharmacological Bases of Therapeutics" , Pergamon Press.[000170] In general, compositions of the disclosure may be administered to a patient in an amount of from about 5 micrograms to about 5 mg of active component(s) (i.e., a irradiated NDV). Dosage in an amount of from about 50 micrograms to about 500 micrograms is also preferred.[000171] One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a irradiated NDV thereof to include in a vaccine of the disclosure for the desired therapeutic outcome.[000172] Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about 1000 mg of active component(s) (i.e., a irradiated NDV) per kg body weight; typically, about 0.001 mg to about 750mg per kg body weight; about 0.01 mg to about 500mg per kg body weight; about 0.1 mg to about 500mg per kg body weight; about 0.1 mg to about 250mg per kg body weight; about 1.Omg to about 250mg per kg body weight . More typically, an effective dose range is expected to be in the range about 1.0mg to about 200mg per kg body weight; about 1.0mg to about 100mg per kg body weight; about 1.0mg to about 50mg per kg body weight; about 1.0mg to about 25mg per kg body weight; about 5. Omg to about 50mg per kg body weight; about 5. Omg to about 20mg per kg body weight; about 5. Omg to about 15mg per kg body weight.[000173] Alternatively, an effective dosage may be up to about 500mg / m2of active component(s) (e.g., a irradiated NDV). Generally, an effective dosage is expected to be in the range of about 25 to about 500mg / m2, preferably about 25 to about 350mg / m2, more preferably about 25 to about 300mg / m2, still more preferably about 25 to about 250mg / m2, even more preferably about 50 to about 250mg / m2, and still even more preferably about 75 to about 150mg / m2.[000174] In many instances, it will be desirable to have several or multiple administrations of a vaccine of present disclosure. For example, compositions of the disclosure may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations may be from about one to about twelve-week intervals, six-month intervals, twelve-month intervals, etc., and in certain embodiments from about one to about four-week intervals. Periodic re-administration may be desirable in the case of recurrent exposure to a particular pathogen targeted by a vaccine of the disclosure.[000175] The methods described herein may comprise administering a priming dose of a vaccine of the disclosure. The priming dose may be followed by a booster dose. The booster may be for the purpose of revaccination. In various embodiments, the vaccine is administered at least once, twice, three times or more. Compositions of the disclosure may be administered to naive recipients, being individuals seronegative for NDV. Alternatively, the compositions may be administered to primed recipients, being individuals seropositive for NDV.MEDICAMENTS AND KITS[000176] The composition of the present disclosure may be provided together as a single formulation (i.e. in a single medicament, or a single composition in a kit), or as separate components.[000177] It will be appreciated by persons of ordinary skill in the art that numerous variations and / or modifications can be made to the present disclosure as disclosed in the specificembodiments without departing from the spirit or scope of the present disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.EXAMPLES[000178] The present invention will now be described with reference to the following examples which should be considered in all respects as illustrative and non-restrictive.EXAMPLE 1: DEVELOPMENT OF AN IRRADIATED NDV VACCINEGrowth of NDV, concentrating, and titration[000179] Newcastle disease virus live-attenuated strain V4, which is an apathogenic genotype I strain, is commercially available (provided by Dr Farhid Hemmatzadeh, The University of Adelaide). The V4 strain was grown in the allantoic cavity of 10-day-old embryonated chicken eggs at 37 °C for 48 hours. Eggs were chilled overnight then infectious allantoic fluid was harvested and clarified by centrifugation at 3272 x g. Clarified NDV was concentrated by ultrafiltration (UF) using Amicon® Ultra-15 centrifugal filter units with a 100 kDa cut off (Merck) and centrifuging at 3272 x g. After 10-fold concentration, the preparation was resuspended in PBS and washed 3x, then resuspended in PBS for the final preparation.[000180] Concentrated NDV vaccine stocks were titrated by TCID50 assay using chicken embryonated fibroblasts (CEF). Cells were plated at 5 x 104cells / well in round-bottom 96-well microtitre plates and allowed to adhere overnight. 10-fold serial dilutions of virus were performed across the plate in DMEM + 8% trypsin for virus activation. Plates were incubated at 37°C for 72 hours, then 0.8% RBCs in saline were added to visualise amplified virus. Plates were scored based on a pellet or a mesh formation, where a mesh was considered positive for NDV. 50% infectious doses were then calculated using the Reed-Muench method

[0057] , The titre for concentrated NDV preparation was determined to be 2 x 108TCIDso / mL.Gamma-irradiation of NDV[000181] The NDV preparation above was exposed to a radiation dose of 50 kGy of y-radiation from60Co irradiation facility at Australian Nuclear Science and Technology Organisation (ANSTO) while frozen on dry ice.Sterility testing of v-NDV[000182] To ensure sterility, both live and y-NDV were passaged three times in embryonated eggs. Allantoic fluid was tested for virus replication using a haemagglutination assay as detailedfollows. Haemagglutinin activity was measured by the ability of the virus to agglutinate RBCs as detailed in Example 2.[000183] No infectious virus was detected in eggs treated with y-NDV at any of the three passages, in contrast to control live NDV (Table 1). This shows that treatment with 50 kGy of gamma irradiation is sufficient to inactivate NDV.Table 1: Sterility testing in 10-day old embryonated eggsSterility testing of y-NDV in tissue culture[000184] Live and y-NDV were activated with 1pg / mL trypsin at 37°C for 1 hour. Vero cell monolayers were then treated with the preparations at a multiplicity of infection (MOI) of 40 and incubated for 24 hours. As a control, monolayers were also treated with allantoic fluid from uninfected 12-days old embryonated eggs. Supernatant was collected and used to infect fresh Vero cell monolayers and incubated for a further 24 hours. This was then repeated for a third passage, and cells were then fixed and stained with DAPI to visualise cell nuclei and treated with chicken anti-NDV antibodies and anti-chicken IgY FITC-conjugated antibodies to visualise NDV-infected cells. Infection levels of all 3 passages are shown in Figure 1. Images are representative of 3 wells per sample, and 2 independent experiments.[000185] No infectivity was detected when the y-NDV was passaged 3 times in tissue culture in contrast to live NDV, as shown in Figure 1 for passage 3. This confirms that treatment with 50 kGy of gamma irradiation is sufficient to inactivate NDV.Structural integrity of gamma-irradiated NDV - haemagglutinin activity[000186] To assess the structural integrity of gamma-irradiated NDV, the functional haemagglutinin activity of the surface protein HN to agglutinate RBCs was measured. Live and gamma-irradiated NDV was serially diluted in normal saline (0.85%) in 96-well round-bottomed microtitre plates. 0.8% red blood cells (RBCs) were added to each well and plates were scoredfor mesh or pellet formation. The reciprocal of the highest dilution to give a positive reading was considered the haemagglutination units (HAU). Quantitative data is expressed as mean SEM (n = 3, collated from 2 independent experiments). Data was analysed by one-way ANOVA (* p < 0.05).[000187] As shown in Figure 2A, y-NDV retained high functional activity of HA protein as illustrated by the binding to sialic acid receptors on RBCs to cause haemagglutination, but this level of activity was slightly reduced compared to live NDV.Structural integrity of gamma-irradiated NDV - neuraminidase assay[000188] Neuraminidase activity was measured by cleavage of 4-MUNANA into the fluorescent substrate 4-MU. The relative fluorescent intensity was then measured for each sample. Live NDV was used as a positive control.[000189] To test for the neuraminidase activity, live and irradiated virus preparations were serially diluted in PBS and 25 .L of each dilution was added to microtitre plates. 25 .L of 0.125mM 2’-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid (4-MUNANA, Sigma M8639) was added to virus samples. Active neuraminidase cleaves 4-MUNANA into the fluorescent substrate 4-Methylumbelliferyl (4-MU). 4-MU was also added to plates at increasing concentrations to generate standard curves. Plates were incubated in the dark at 37°C for 1 hour with gentle shaking every 15 minutes to facilitate cleavage of 4-MUNANA. Reaction was stopped with ice-cold 0.5M Na2CO3 (pH 10.5) and relative fluorescence was measured using a SpectraMax fluorescent plate reader with an excitation wavelength of 365nm and an emission wavelength of 450nm. Quantitative data is expressed as mean ± SEM (n = 3, collated from 2 independent experiments). Data was analysed by one-way ANOVA (* p < 0.05).[000190] The data illustrates the ability of y-NDV to utilise the neuraminidase activity of HN protein to cleave sialic acids at comparable levels to that observed for live NDV (Figure 2B). Despite the reduced level of haemagglutination, these data indicate that the structure of HN protein is well maintained. The surface proteins of y-NDV have normal functions.Structural integrity of gamma-irradiated NDV - transmission electron microscopy[000191] Finally, transmission electron microscopy (TEM) was used to observe the effects of y-radiation on whole virion structure with approximately 3 .L of irradiated virus was loaded onto formvar / carbon-coated grids and incubated at room temperature for 3-5 minutes to allow attachment. Grids were blotted dry then washed with PBS and stained with 2% uranyl acetate for 3 minutes. Grids were washed with PBS then blotted to dry. Grids were visualised with an FEI Tecnai Spirit TEM (Adelaide Microscopy, University of Adelaide).[000192] TEM images showing the structural integrity of live NDV or y-NDV (Figure 2C and 2D, respectively). For live NDV, the bar represents 100 nm, HV=120kV, direct magnification is 68000x. For y-NDV, the bar represents 50nm, HV = 100kV, direct magnification = 98000x). The images show that y-NDV is structurally intact.EXAMPLE 2: IMMUNE RESPONSE TO INTRAMUSCULARLY ADMINISTERED y-NDVIgG response following intramuscular administration of Y-NDV[000193] Using a prime-boost vaccination strategy with 3 weeks interval between 1st and 2nd vaccine administration, 6-8-week old BALB / c mice were vaccinated intramuscularly with 6.4 x 106TCIDso-equivalent of y-NDV per mouse. Unvaccinated mice were included as a control. Serum samples were collected 20 days after the first immunisation, and again three weeks after the second vaccination.[000194] The presence of NDV-specific IgG was determined by direct ELISA, with titres being calculated relative to naive serum. Here, maxisorp ELISA plates were coated with live whole-NDV (2 106TCIDso / well) in bicarbonate coating buffer (0.6% NaHCOs, 0.303% Na2COs in MilliQ water, pH 9.6) and incubated overnight at room temperature. Plates were washed 3 x with 0.05% tween in PBS then blocked with 2% skim milk for 2 hours. Plates were washed again, then serial dilutions of serum collected from mice were added to the ELISA plate. 2 hours later plates were washed again and horseradish peroxidase conjugated goat-anti mouse IgG secondary antibody (1:10,000 dilution in blocking buffer, Thermo Scientific) was added. After 2 hours at room temperature unbound secondary antibody was washed away, and TMB peroxidase substrate was used to develop colour in the dark for 30 minutes. The reaction was stopped with 2M H2SO4. Absorbance of all ELISA plates were measured at 450nm using a Bio-Tek Instruments plate reader. IgG titres were calculated as the reciprocal of the highest serum dilution that gave absorbance readings higher than those of naive serum ± 3 standard deviations. Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA (** p < 0.01).[000195] Figure 3A indicates that IgG titres are relatively low after one dose of y-NDV. However, after a booster dose of y-NDV, the IgG titres were significantly higher, showing that y-NDV is immunogenic.Neutralising antibody response following intramuscular administration of Y-NDV[000196] Next, virus neutralisation was tested by focus forming inhibition assay. Live virus was pre-treated with pooled immune serum from 5 mice, then added to monolayers of Vero cells at an MOI of 0.1 and allowed to attach for 2 hours. Cells were then washed and incubated with a fresh media for a further 22 hours. Cells were fixed and stained with a chicken anti-NDV antibodyand a FITC-conjugated anti-chicken IgY secondary antibody. A fluorescence microscope was used to quantify FITC-fluorescence (representative of NDV infection) relative to DAPI-fluorescence (cell nuclei).[000197] Briefly, Vero cells were plated in 96-well flat-bottom microtitre plates at 6 x 104cells / well and incubated at 37°C overnight. Live NDV was activated with 10 .g / mL TPCK-trypsin at 37°C for 1 hour. Meanwhile, serum samples were heated-inactivated at 56°C for 30 minutes to inactivate complement. Serum samples were then serially diluted in PBS and added to activated NDV at a 1:1 ratio. Virus and serum mixture was then added to cells at an MOI of 0.1 and incubated at 37°C for 2 hours to allow virus adherence. Plates were then washed 3 x with PBS and fresh DM EM + 1% P / S was added to each well. Plates were incubated at 37°C for 22 hours, then cells were fixed, stained and visualised. To calculate neutralisation, the FITC-specific fluorescence and DAPI-specific fluorescence levels were both quantified using NIS elements software (Tokyo, Japan). Representative fluorescence microscope images were taken of each sample in the neutralising assay at a 1:10 serum dilution (representative of 3 wells tested per serum group). The FITC-specific fluorescence readings were then normalised using the corresponding DAPI-specific readings, to account for small differences in total cell number between sample wells. Quantitative data was presented as mean ± SEM and analysed by oneway ANOVA (“ p < 0.01).[000198] Surprisingly, the high level of NDV-specific IgG responses observed following primeboost vaccination did not correlate with neutralising antibody responses, as monolayers of Vero cells treated with live NDV exposed to immune serum showed no significant reduction in NDV infection compared to Vero cells treated with naive serum (see Figure 3B).EXAMPLE 3: EFFECT OF ADJUVANT ON INTRAMUSCULARLY ADMINISTERED y-NDV[000199] Co-administration of y-NDV with adjuvants as a potential means to enhance vaccine immunogenicity and neutralising antibody responses was investigated. Adjuvants tested were gamma-irradiated influenza virus (y-flu), incomplete Freund’s adjuvant (IFA) (source), aluminium hydroxide (Alum) (source) or poly(l:C) (P(l :C)) (source).[000200] A research grade material of y-flu was prepared as follows: a lab adapted strain A / Puerto Rico / 08 / 1937 (H1N1) (A / PR8) was propagated in the allantoic cavity of 10-day old embryonated chicken eggs. Standard techniques were used for injection into embryonated eggs, incubation of infected eggs at 37°C for 2 days, and harvesting of allantoic fluids. Infected allantoic fluid containing infectious virus was collected. Infectious influenza viruses were concentrated by haemadsorption to chicken red blood cells (cRBCs) on ice followed by centrifugation to pellet virus-cRBCs complexes, and allantoic fluid supernatant was removed. Then, the pellet wasresuspended using 0.85% saline and incubated at 37°C for 1.5 hours to allow for virus release from RBCs. Sample was then centrifuged to pellet RBCs, and virus-containing supernatant was collected. The virus was eluted by adding saline to resuspend the pellet and incubation at room temperature for 30 minutes. Concentrated influenza samples were gamma-irradiated as follows. A / PR8 stock was inactivated by exposure to 35 kGy of gamma-radiation from60Co irradiation facility at Australian Nuclear Science and Technology Organisation (ANSTO) on dry ice. Gamma irradiated influenza samples are interchangeably referred to as y-flu, gamma-flu or y-A / PR8. Sterility was verified via three passages in MDCK monolayers at 24 intervals.[000201] Mice were immunised intramuscularly twice 2 weeks apart with 6.4 x 106TCID50-equivalent of y-NDV per mouse either alone or with 1 x 106TCIDso-equivalent of y-flu, incomplete Fruend’s adjuvant (IFA, 10%), aluminum hydroxide (Alum, 10%) or Poly(l:C) (50 ug / mouse) adjuvants. Serum samples were collected 2 weeks after the first and second vaccinations and tested for NDV-specific antibody responses.IqG response following intramuscular administration of Y-NDV with adjuvants[000202] NDV-specific IgG levels were determined by direct ELISA as described above. Data is presented as mean ± SEM (n = 5, compiled from two independent experiments) and was analysed by one-way ANOVA and adjuvanted IgG responses were compared to IgG responses generated by y-NDV alone (** p < 0.01). No significant change in IgG levels were noted following the first vaccination (Figure 4A). Following the second vaccination, no specific an increase in NDV-specific IgG titres was detected following co-administration of y-NDV with y-Flu or P(I:C) compared to vaccination with y-NDV only (Figure 4B). However, IgG responses were enhanced when y-NDV was co-administered with IFA or alum.Neutralising antibody response following intramuscular administration of y-NDV with adjuvants[000203] Given the enhanced IgG responses, the neutralising antibody responses using Focus Forming Inhibition Assay (FFIA) were measured as described above using serum obtained two weeks after the second immunisation to determine whether co-administration with adjuvants were able to improve neutralising responses. Briefly, live NDV was pre-treated with serum samples in triplicate then added to confluent monolayers of Vero cells. Virus was incubated for 2 hours to attach, and then plates were washed, fresh media was given, and plates were incubated for a further 22 hours. Cells were fixed then stained and visualised using DAPI-fluorescence (cell nuclei) and FITC-fluorescence (NDV infection). Data is presented as mean ± SEM and was analysed by one-way ANOVA. Representative fluorescence microscope images were also taken of each sample in the neutralising assay at a 1:10 serum dilution (Figure 5).[000204] Serum obtained from mice vaccinated twice from the different groups was used to measure neutralisation. Despite the enhanced IgG responses following vaccination with y-NDV + I FA or alum, no significant neutralising antibody responses were detected when comparing live virus only or naive serum to any of the serum groups from vaccinated mice (Figure 5). Infection of monolayers of Vero cells with live virus pre-treated with immune sera from each of those groups demonstrated that there was no significant difference in normalised FITC-fluorescence (indicative of NDV replication) when comparing serum from mice treated with y-NDV only to any of the adjuvanted groups.EXAMPLE 4: IMMUNE RESPONSE IN BALB / C AND C57BL / 6J MICE TO SUBCUTANEOUS VACCINATION WITH y-NDV[000205] BALB / c mice and C57BL / 6 (B6) mice have been reported to be biased towards a Th2 and a Th1 immune response, respectively. To study the differences between those two mice strains in response to y-NDV vaccinations, 6-8 weeks old mice were vaccinated subcutaneously twice with 107equavalent-TCIDso / mouse of y-NDV in a prime-boost strategy two weeks apart.IgG response following subcutaneous administration of Y-NDV[000206] Serum samples were collected two weeks after each vaccination were tested for the presence of NDV-specific IgG by direct ELISA as detailed above. Quantitative data was presented as mean ± SEM.[000207] As shown in Figure 6A, 6B and 6C, both BALB / C and C57BL / 6 (B6) mice responded similarly and the IgG levels are relatively low after one dose of y-NDV, and the IgG levels increased after the second vaccination showing a prime-boosting effect.Neutralising antibody response following subcutaneous administration of y-NDV[000208] Serum samples were collected 2 weeks after each vaccination were tested for the ability of immune sera to neutralise NDV infectivity by focus forming inhibition assay as detailed above. Briefly, live virus was pre-treated with pooled immune serum from 5 mice, then added to monolayers of Vero cells at an MOI of 0.2 and allowed to attach for 2 hours. Cells were then washed and incubated with a fresh media for a further 22 hours. Cells were fixed and stained with a chicken anti-NDV antibody and a FITC-conjugated anti-chicken IgY secondary antibody. A fluorescence microscope was used to quantify FITC-fluorescence (representative of NDV infection) relative to DAPI-fluorescence (cell nuclei). Representative data are shown for 1:80 serum dilution (representative of 4 wells tested per serum group). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA (* < 0.05, ** p < 0.01).[000209] Immune sera from BALB / c mice did not neutralise NDV infectivity as there was no significant reduction in live NDV infection following treatment of NDV with immune sera prior to infecting monolayers of Vero cells compared to NDV treated with naive serum (Figure 6D). Similarly, immune serum from C57BL / 6 mice did not neutralise NDV infectivity as comparable levels of NDV infectivity were obtained following treatment of NDV with immune sera from vaccinated mice compared to immune serum from naive C57BL / 6 mice (Figure 6D), showing lack of virus neutralisation.EXAMPLE 5: IMMUNE RESPONSE IN SERUM FOLLOWING INTRANASAL AND INTRAMUSCULAR VACCINATION WITH y-NDV[000210] BALB / c mice were vaccinated either intramuscularly or intranasally twice two weeks apart, with 1 x 107TCIDso-equivalent of y-NDV per mouse, or were intramuscularly vaccinated twice two weeks apart with 107TCIDso / mouse live-NDV V4 strain (as indicated above). Serum samples were collected 13 days after the first immunisation, and again two weeks after the second vaccination. Bronchoalveolar lavage (BAL) samples were collected two weeks after the second vaccination.IgG and IgA response following intranasal and intramuscular administration of Y-NDV[000211] Serum and BAL samples were collected and tested for NDV-specific IgG responses via indirect ELISA as detailed above.[000212] Direct ELISA for NDV-specific IgA was performed as described above for IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse IgA secondary antibody (1:5,000 dilution in blocking buffer, Sigma Aldrich).[000213] Direct ELISA for NDV-specific lgG1 was performed as described above for IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG1 secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000214] Direct ELISA for NDV-specific lgG2a was performed as described above for IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG2a secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000215] Direct ELISA for NDV-specific lgG2b was performed as described above for IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG2b secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000216] Direct ELISA for NDV-specific lgG3 was performed as described above for IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG3 secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000217] NDV-specific total IgG, lgG1 , lgG2a, lgG2b, and lgG3 titres were calculated as the reciprocal of the highest serum dilution that gave absorbance readings higher than those of naive serum ± 3 standard deviations. Outliers were identified using the Grubb’s test, and were excluded from the analysis. Quantitative data was presented as mean ± SEM (n=5) and analysed by oneway ANOVA (** p < 0.01, *** p < 0.001). Mean titres were summarised for results from the first and second vaccination, respectively. NDV-specific IgG and IgA in the BAL of vaccinated mice was calculated as fold change in antibody titre compared to naive control.[000218] Intranasal vaccination with two doses of y-NDV induced an enhanced total IgG response in BAL compared to intramuscularly administered y-NDV and intramuscularly administered live NDV (Figure 7A). Intranasal vaccination with two doses of y-NDV induced a significant NDV-specific IgA response in BAL, whereas intramuscular vaccination with either live-NDV or y-NDV failed to induce detectable IgA in BAL (Figure 7B). These results indicate that intranasally administered y-NDV enhances mucosal immunity to NDV. Further, intranasally administered y-NDV induces enhanced mucosal immunity compared to intramuscularly administered y-NDV or live NDV.[000219] Further, as shown in Figure 7C, intranasal vaccination with two doses of y-NDV resulted in a significantly higher NDV-specific total IgG levels in the serum compared to either live-NDV or y-NDV via the intramuscular route. Intranasal vaccination with two doses of y-NDV induced significantly higher NDV-specific lgG1 (Figure 7D), lgG2a (Figure 7E) and lgG2b levels (Figure 7F). Meanwhile, vaccination with live-NDV and y-NDV via the intramuscular route induced comparable total IgG titres following first vaccination (Figure 7H) and second vaccination (Figure 7I). These results indicate that intranasally administered y-NDV induced an enhanced systemic immunity compared to intramuscularly administered y-NDV or live NDV.Neutralising antibody response following intranasal and intramuscular administration of Y-NDV[000220] Serum samples were collected and tested neutralising antibody responses by focus forming inhibition assay as described elsewhere herein. Live virus was pre-treated with pooled immune serum from 5 mice, then added to monolayers of Vero cells at an MOI of 0.2 and allowed to attach for 2 hours. Cells were then washed and incubated with a fresh media for a further 22 hours. Cells were fixed and stained with a chicken anti-NDV antibody and a FITC-conjugated anti-chicken IgY secondary antibody. A fluorescence microscope was used to guantify FITC-fluorescence (representative of NDV infection) relative to DAPI-fluorescence (cell nuclei).Representative data are shown for 1 :80 serum dilution (representative of 4 wells tested per serum group). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA (** p < 0.01, *** p < 0.001, ****p < 0.0001).[000221] It was expected that vaccination with live-NDV would induce a neutralising antibody response, however, surprisingly, only intranasal vaccination with y-NDV elicited significant neutralising antibody responses and not intramuscularly administered y-NDV (Figure 8). Infection of monolayers of Vero cells with live virus pre-treated with immune sera from each of those groups demonstrated that there is a significant reduction in normalised FITC-fluorescence (indicative of NDV replication) for mice intranasally vaccinated with y-NDV compared to mice intramuscularly vaccinated with y-NDV (Figure 8).[000222] The neutralisation observed following live-NDV and intranasal y-NDV does not appear to be associated with the presence of mucosal IgA levels, as vaccination with live-NDV via the intramuscular route does not induce a mucosal IgA response (Figure 7B). It also appears that the total IgG and IgG subclass titres are not associated with neutralisation, as vaccination with live-NDV and y-NDV via the intramuscular route induced comparable IgG titres (Figure 7H, 7I).EXAMPLE 6: MUCOSAL IMMUNE RESPONSE FOLLOWING INTRANASAL AND INTRAMUSCULAR VACCINATION WITH y-NDV[000223] BALB / c mice were immunised IN or IM with 2 doses of 107TCID50 equivalent / mouse of y-NDV, following a prime-boost vaccination regimen at a 2-week interval. As a control, 107TCIDso / mouse live-NDV V4 strain (as above) was administered via the IM route. Serum and BAL samples collected 3 weeks after prime-boosting. Analysis of immune responses in serum samples are shown above.Virus neutralisation by immune BAL samples[000224] BAL samples were pooled, and virus neutralisation assays using Focus Forming Inhibition Assay (FFIA) were performed as described elsewhere herein. Groups tested were virus only (vo), and BAL samples from naive mice, or BAL samples obtained from vaccinated animals following intramuscular vaccination with live-NDV, intramuscular vaccination with y-NDV or intranasal vaccination with y-NDV. Representative data of Vero cells infected with NDV virus that was pre-treated with 1:2 dilution of BAL from naive or vaccinated animals (representative of 4 wells tested per group was visualised using Nikon TiE inverted fluorescence microscope, with DAPI (blue) indicating cell nuclei, and FITC (green) indicating NDV). FITC fluorescence relative to DAPI-fluorescence was quantified at a 1:2 BAL dilution using NIS elements software.Quantitative data was presented as mean ± SEM and analysed by two-way ANOVA with T ukey’s multiple comparison (**** p < 0.0001).[000225] As shown in Figure 9, intranasal vaccination with y-NDV resulted in virus neutralisation indicative of neutralizing antibody responses in the BAL, whereas intramuscular vaccination with y-NDV or live-NDV did not.EXAMPLE 7: INACTIVATION OF NDV WITH FORMALIN. STERILITY TESTING & MORPHOLOGYSterility testing:[000226] Live-NDV or y-NDV were treated with 0.1% formaldehyde and incubated for 16 hours at 37°C. Live-NDV and y-NDV were used as respective controls for each of the formalin-inactivated preparations. Live-NDV (as the control), y-NDV, formalin-treated NDV (F-NDV), and formalin-treated y-NDV (F-y-NDV) were activated with 1 pg / mL trypsin at 37°C for 30 minutes. To test sterility, Vero cell monolayers were then treated with the preparations at MOI of 0.1, or saline as a control, and incubated for 24 hours. Supernatant was collected and used to infect fresh Vero cell monolayers and incubated for a further 24 hours. This was then repeated for a third passage, and cells were then fixed and stained with DAPI to visualise cell nuclei and treated with chicken anti-NDV and anti-chicken IgY FITC-conjugated antibodies to visualise NDV-infected cells. Images were taken that were representative of 4 wells per sample for all 3 passages are shown in Figure 10. F-NDV, y-NDV, and F-y-NDV preparations were all sterile, whereas the live-NDV preparations showed NDV infection (data not shown).Morphology[000227] Live-NDV or y-NDV were treated with 0.1% formaldehyde and incubated for 16 hours at 37°C. Live-NDV and y-NDV were used as respective controls for each of the formalin-inactivated preparations. 1 part vaccine preparations were treated with 2 parts transmission electron microscopy (TEM) fixative (4% formaldehyde, 0.210-4% glutaraldehyde, 4% sucrose in 1 x PBS) for at least 30 minutes at room temperature, and were absorbed onto 300 mesh copper grids with a carbon film that has been freshly glow discharged. Grids were negatively stained with 1% uranyl acetate, dried by aspiration, and observed under the Tecnai G2 Spirit electron microscope operated at 120kV, and images were acquired at the Adelaide Microscopy Facility, University of Adelaide, South Australia, Australia as shown in Figure 10. All four preparations have similar structures based on TEM images.EXAMPLE 8: Impact of formalin on surface glycoprotein haemagglutinin-neuraminidase activity[000228] Live-NDV or y-NDV were treated with 0.1% formaldehyde as above and incubated for 16 hours at 37°C. Live-NDV and y-NDV (in the absence of formalin) were used as respective controls for each of the form al in- inactivated preparations. HA activity (Figure 11A) and neuraminidase activity (Figure 11 B) of each preparation was analysed as described elsewhere herein. Data is presented as mean ± SEM (collated from 2 independent experiments), and was analysed by (A) Brown- Forsythe and Welch ANOVA tests, or (B) one-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05, **** p < 0.0001).[000229] The surface glycoprotein HN (or haemagglutinin-neuraminidase) of y-NDV has a comparable HA and NA activity to live-NDV. However, the treatment of live-NDV and y-NDV with formalin significantly reduced both the haemagglutination and neuraminidase functions of the HN protein compared to their respective live-NDV and y-NDV controls.EXAMPLE 9: Total serum IgG induced by formalin treatment of y-NDV following intranasal vaccination[000230] Total serum IgG following vaccination is an indicator of immunogenicity. Live-NDV or y-NDV were treated with 0.1% formaldehyde and incubated for 16 hours at 37°C. Live-NDV and y-NDV were used as respective controls for each of the form al in- inactivated preparations. BALB / c mice were immunised intranasally with two doses of 107TCID5o / mouse of either F-NDV, F-y-NDV, or y-NDV two weeks apart. Serum samples were harvested 13 days after the first (Figure 12A and Figure 12B) and second (Figure 12C and 12D) vaccinations, and tested for NDV-specific IgG responses via direct ELISA as described above. Titres were calculated based on a cut-off determined from the mean + 3 x S.D. of the naive control absorbance values at a 1 / 100 (Figure 12B) or a 1 / 200 dilution (Figure 12D). Data is presented as mean ± SEM, and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (**** p < 0.0001).[000231] While F-NDV and F-y-NDV induced detectable total NDV-specific serum IgG responses following intranasal administration, y-NDV induces significantly more total NDV-specific serum IgG and is accordingly more immunogenic than F-NDV and F-y-NDV following intranasal vaccination.EXAMPLE 10: Formalin-treatment prevents neutralising antibody responses following intranasal vaccination[000232] Live-NDV or y-NDV were treated with 0.1% formaldehyde and incubated for 16 hours at 37°C. Live-NDV and y-NDV were used as respective controls for each of the formalin-inactivated preparations. BALB / c mice were immunised intranasally with two doses of 107TCID50equivalent / mouse of either F-NDV, F-y-NDV, or y-NDV two weeks apart. Serum samples collected 3 weeks after prime-boosting were pooled, incubated with live NDV, then added to monolayers of Vero cells at an MOI of 0.1 to assess neutralisation of infection (i.e., the neutralising antibody responses) using Focus Forming Inhibition Assay (FFIA) as described herein. Representative images of infected Vero cells at a 1:20 and 1:80 serum dilution (representative of 4 wells tested per serum group) were visualised using Nikon TiE inverted fluorescence microscope, with DAPI (blue) staining indicating cell nuclei, and FITC (green) staining indicating NDV (data not shown). FITC fluorescence relative to DAPI-fluorescence was quantified for 1:80 sera dilution using NIS elements software as shown in Figure 13. Quantitative data presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparison (* p < 0.05, ** p < 0.01).[000233] As shown in Figure 13, y-NDV was the only vaccine preparation to induce a neutralising antibody response following intranasal vaccination. Formalin-treatment of y-NDV prevented the induction of neutralising antibody responses. No neutralizing antibody responses induced by formalin-treated NDV (F-NDV) following intranasal vaccination.EXAMPLE 11: Formalin-treatment reduces immunogenicity following intramuscular vaccination[000234] Live-NDV or y-NDV were treated with 0.1% formaldehyde and incubated for 16 hours at 37°C. Live-NDV and y-NDV were used as respective controls for each of the formalin-inactivated preparations. BALB / c mice were immunised intramuscularly with two doses of 107TCIDso / mouse of either live-NDV, F-NDV, F-y-NDV, or y-NDV two weeks apart. Serum samples were harvested 13 days after first (Figure 14A, 14B) and second (Figure 14C, 14D) vaccinations, and tested for NDV-specific IgG responses via direct ELISA. Titres were calculated based on a cut-off determined from the mean + 3x S.D. of the naive control absorbance values at a (B) 1 / 100 or a (D) 1 / 200 dilution. Data is presented as mean ± SEM, and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01, **** p < 0.0001).[000235] As shown in Figure 14A, 14B, 14C and 14D, both F-NDV and F-y-NDV are immunogenic and can induce detectable antibody responses following intramuscular administration. However, formalin treatment resulted in reduced antibody responses compared to non-formalin treated preparations. y-NDV is significantly more immunogenic than F-NDV and F-y-NDV following intramuscular vaccination.EXAMPLE 11: Formalin-treatment prevents the induction of neutralising antibody responses following intramuscular vaccination[000236] Live-NDV or y-NDV were treated with 0.1% formaldehyde and incubated for 16 hours at 37°C. Live-NDV and y-NDV were used as respective controls for each of the formalin-inactivated preparations. BALB / c mice were immunised intramuscularly with two doses of 107TCIDsoequivalent / mouse of either live-NDV, F-NDV, F-y-NDV, or y-NDV two weeks apart. Serum samples collected 3 weeks after prime-boosting were pooled, incubated with live NDV, then added to monolayers of Vero cells at an MOI of 0.1 to assess neutralisation of infection. Representative images of infected Vero cells at a 1:20 and 1:80 serum dilution (representative of 4 wells tested per serum group) visualised using Nikon TiE inverted fluorescence microscope. DAPI (blue) indicates cell nuclei, and FITC (green) indicates NDV (data not shown). As shown in Figure 15, FITC fluorescence relative to DAPI-fluorescence was quantified for 1:80 sera dilution using NIS elements software. Quantitative data presented as mean ± SEM and analysed by oneway ANOVA with Tukey’s multiple comparison (* p < 0.05, ** p < 0.01).[000237] Figure 15 shows that live-NDV induces neutralising antibody responses following intramuscular vaccination. Formalin-treatment of live-NDV (F-NDV) prevented the induction of neutralising antibody responses. Intramuscular vaccination with y-NDV does not induce a neutralising antibody response despite being highly immunogenic.[000238] Whilst not wanting to be bound by theory, the data suggests that antigenic epitopes required for the induction of neutralising antibody responses are not visible to B cells when inactivated NDV (i.e., y-NDV , F-NDV, F-y-NDV) preparations are used for intramuscular vaccination. Additionally, formalin-treated preparations show limited functional activities in terms of haemagglutination and neuraminidase activities.EXAMPLE 12: Effect of trypsin on NDV-specific IgG response following intramuscular administration[000239] The effect of trypsin treatment of y-NDV was investigated. y-NDV was treated with 5 .g / mL of TPCK-trypsin, at 37°C for 30 minutes (termed y-NDV + trypsin). y-NDV incubated at 37°C for 30 minutes (in the absence of trypsin) was used as a control (termed y-NDV at 37°C); and compared to y-NDV incubated at room temperature (RT) (in the absence of trypsin) and live-NDV. BALB / c mice were immunised intramuscularly with two doses of 107TCIDso-equivalent / mouse of either y-NDV+trypsin, y-NDV at 37°C, y-NDV at RT, or live-NDV, two weeks apart. Serum samples were collected 13 days after the first (Figure 16A, 16B) and second (Figure 16C, 16D) vaccination, and tested for NDV-specific IgG responses via direct ELISA. NDV-specific total IgG titres were calculated based on a cut-off determined from the mean + 3 x S.D. of the naive control absorbance values at (Figure 16B) 1 / 100 or at (Figure 16D) 1 / 200 dilution. Quantitative data was presented as mean ± SEM (n=4-5) and analysed by one-way ANOVA with Tukey’s multiple comparison.[000240] As shown in Figure 16, antibody levels detected using ELISA in immune sera from mice vaccinated with the different vaccine preparations were comparable after both the priming and boosting for all groups. Trypsin treatment did not affect the immunogenicity of y-NDV.EXAMPLE 13: Effect of trypsin on neutralising antibody response following intramuscular administration of -NDV[000241] y-NDV was treated with 5pg / mL of TPCK-trypsin at 37°C for 30 minutes (termed y-NDV + trypsin). y-NDV incubated at 37°C for 30 minutes was used as a control (termed y-NDV at 37°C). These preparations were compared to y-NDV incubated at room temperature (RT) (in the absence of trypsin) and live-NDV. BALB / c mice were immunised intramuscularly with two doses of 107TCIDso-eguivalent / mouse of either y-NDV+trypsin, y-NDV at 37°C, y-NDV at RT, or live-NDV, two weeks apart. Serum samples collected 13 days after the 2nd vaccination were pooled, incubated with live NDV, then added to monolayers of Vero cells at an MOI of 0.1 to assess neutralisation of infection (i.e., the neutralising antibody responses) using Focus Forming Inhibition Assay (FFIA) as described elsewhere herein. Representative images of infected Vero cells at a 1:20, 1:40, 1:80, and 1:160 serum dilution (representative of 4 wells tested per serum group) were visualised using Nikon TiE inverted fluorescence microscope. DAPI (blue) indicates cell nuclei, and FITC (green) indicates NDV (data not shown). FITC fluorescence relative to DAPI-fluorescence was guantified for each sera dilution using NIS elements software. Quantitative data presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).[000242] As shown in Figure 17, immune sera from mice vaccinated intramuscularly with trypsin-treated y-NDV had a comparable or stronger neutralising potency than mice vaccinated intramuscularly with live-NDV. This data indicates that trypsin-treated y-NDV induces neutralising antibody responses following intramuscular vaccination.DISCUSSION[000243] It has previously been shown that humoral immunity from vaccination may be associated with survival and / or control of ND. Further, the presence of neutralising antibodies are believed to be associated with survival from NDV infection (Reynolds and Maraga, 2018). Accordingly, induction of antibodies, and particularly neutralising antibodies, indicate induction of a protective immune response to NDV infection.[000244] The results shown that whole irradiation-inactivated NDV vaccines are highly immunogenic. Unexpectedly, y-NDV induced neutralising antibody responses following administration via mucosal route in contrast to other routes of administration. While y-NDV appeared to be highly immunogenic in mice and induced high-titre IgG responses, the NDV-specific antibody responses induced were not neutralising if administered via intramuscular or subcutaneous routes. However, administration via the intranasal route was associated with the induction of neutralising antibody responses. The ability of irradiation-inactivated NDV to induce neutralising antibody responses following intranasal administration could not have been predicted based on previous literature.[000245] Following intramuscular administration, y-NDV induced a significant NDV-specific IgG response; however, it did not induce a significantly relevant neutralising response. Further, the inclusion of IFA or alum adjuvants significantly enhanced the NDV-specific IgG response but did not significantly enhance the neutralising response.[000246] Subcutaneous delivery of y-NDV induced NDV-specific IgG responses in C57BL / 6 mice and BALB / c mice; however, subcutaneous delivery of y-NDV did not result in significant NDV neutralization in either C57BL6 or BALB / C mice.[000247] Intranasal delivery of y-NDV induced significant increase in NDV-specific IgA responses in BAL. Intramuscular delivery of y-NDV or live-NDV did not. Further, intranasal vaccination with y-NDV resulted in a significantly higher IgG levels compared to the intramuscular vaccination with y-NDV or live-NDV. Accordingly, mucosal administration, such as intranasal administration, appears to provide a significantly advantageous administration route for y-NDV as it resulted in the induction of a neutralising antibody response.[000248] The results indicate that intranasal vaccination with y-NDV induces a protective immune response against NDV infection and / or disease severity. In addition, y-NDV is expected to induce T cell responses, without inducing the expression of V protein, and such responses are expected to be cross-protective against different NDV strains.[000249] Different results were obtained when formalin was used as an inactivation method as compared to irradiation. Intranasal vaccination with y-NDV is associated with the induction of neutralising antibody responses; however, formalin-treatment of y-NDV (F-y-NDV) prevented the induction of neutralising responses following intranasal administration. Intramuscular vaccination with live-NDV can induce neutralising antibody responses; however, formalin treatment of live-NDV inhibited neutralising antibody response following intramuscular administration. Formalin-inactivated NDV vaccine did not induce neutralising antibody responses regardless of administration routes. Accordingly, irradiation-inactivated NDV provides superior protection compared to chemically-inactivated NDV, such as formalin-inactivated NDV. The superiority of irradiation-inactivated NDV over chemically inactivated NDV vaccines to induce neutralising antibody responses has not been previously reported. Further, while not wanting to be bound by theory, it is thought that antigenic epitopes associated with the induction of neutralising antibodyresponses may not be visible to B cells if inactivated NDV preparations such as y-NDV , f-NDV, F-y-NDV preparations are administered intramuscularly vaccination. This could not have been predicted based on existing literature.[000250] Despite high immunogenicity of y-NDV following intramuscular administration, neutralising antibody responses were not induced. However, intramuscular administration of trypsin-treated y-NDV was associated with the induction of neutralising antibody responses.[000251 ] While not wanting to be bound by theory, it is thought that although functional proteins induce neutralising antibody responses following intranasal vaccination, protease treatment of functional NDV proteins is associated with inducing neutralising antibody responses following intramuscular administration. It is theorized that the enhancement of neutralising responses to protease treated, irradiation inactivated NDV following intramuscular administration may be associated with processing of viral glycoprotein and enhancement of accessibility of antigenic epitopes. The enhancement of neutralising antibody response associated with protease treatment of y-NDV could not have been predicted based on existing literature.REFERENCESCheville, N.F. and C.W. Beard, Cytopathology of Newcastle disease. The influence of bursal and thymic lymphoid systems in the chicken. Lab Invest, 1972. 27(1): p. 129-43.Hu, Z., et al., Current situation and future direction of Newcastle disease vaccines. Veterinary research (Paris), 2022. 53(1): p. 1-99.Reynolds, D.L. and A.D. Maraqa, Protective immunity against Newcastle disease: the role of cell-mediated immunity. Avian Dis, 2000. 44(1): p. 145-54.Russell, P.H., P.N. Dwivedi, and T.F. Davison, The effects of cyclosporin A and cyclophosphamide on the populations of B and T cells and virus in the Harderian gland of chickens vaccinated with the HitchnerBI strain of Newcastle disease virus. Vet Immunol Immunopathol, 1997. 60(1-2): p. 171-85.Shahar, E., et al., Newcastle disease virus: is an updated attenuated vaccine needed? Avian pathology, 2018. 47(5): p. 467-478EMBODIMENTS OF THE INVENTION[000252] Other embodiments of the invention as described herein are defined in the following paragraphs:1. A vaccine composition comprising irradiation-inactivated Newcastle Disease Virus (NDV).2. The vaccine composition of embodiment 1, further comprising at least one pharmaceutically-acceptable excipient, diluent and / or carrier.3. The vaccine composition of embodiments 1 or 2, wherein the irradiation-inactivated NDV is selected from gamma-irradiated NDV, X-irradiated NDV or electron-irradiated NDV.4. The vaccine composition of any one of the preceding embodiments, wherein the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.5. The vaccine composition of any one of the preceding embodiments, wherein the vaccine composition is an inactivated whole virus vaccine composition.6. The vaccine composition of any one of the preceding embodiments, wherein the composition further comprises an adjuvant.7. The vaccine composition of any one of the preceding embodiments, wherein the composition excludes an adjuvant.8. The vaccine composition of any one of the preceding embodiments, wherein the composition excludes an alum adjuvant.9. The vaccine composition of any one of the preceding embodiments, wherein the irradiation-inactivated NDV comprises irradiation-inactivated, protease treated NDV.10. The vaccine composition of embodiment 9, wherein the protease treatment occurred following the irradiation inactivation.11. The vaccine composition of embodiment 9 or embodiment 10, wherein the protease is selected from the group consisting of trypsin, tryptase, prostasin, human airways trypsin-like protease (HAT), transmembrane serine protease 2 (TMPRSS2), matriptase, chymotrypsin, pepsin, thrombin, elastase, subtilisin, carboxypeptidase, papain, bromelain and caspase.12. The vaccine composition of embodiment 9 or embodiment 10, wherein the protease is trypsin.13. The vaccine composition of any one of the preceding embodiments, wherein the irradiation-inactivated NDV is a genotype I strain or a V4 strain.14. The vaccine composition of any one of the preceding embodiments, wherein the photon-irradiated NDV is formulated for intramuscular, subcutaneous, intranasal, ocular or conjunctival delivery.15. The vaccine composition of any one of the preceding embodiments, wherein the photon-irradiated NDV is formulated for mucosal delivery.16. The vaccine composition of any one of the preceding embodiments, wherein the photon-irradiated NDV is formulated for intranasal, ocular or conjunctival delivery.17. The vaccine composition of any one of the preceding embodiments, wherein the composition is formulated for delivery to birds.18. The vaccine composition of any one of the preceding embodiments, wherein the composition induces a heterotypic immune response against heterologous NDV genotypes.19. A method of inducing a protective immune response to Newcastle Disease Virus (NDV), the method comprising administering to a subject a therapeutically effective amount of a vaccine composition comprising any one of the previous embodiments.20. A method of inducing a protective immune response to Newcastle Disease Virus (NDV), the method comprising administering to a subject a therapeutically effective amount of a vaccine composition comprising irradiation-inactivated NDV.21. The method of embodiment 20, wherein the irradiation-inactivated NDV comprises irradiation-inactivated, protease treated NDV.22. The method of embodiment 21, wherein the protease treatment occurred following the irradiation inactivation.23. The method of embodiment 20 or embodiment 21 , wherein the protease is selected from the group consisting of trypsin, tryptase, prostasin, human airways trypsin-like protease (HAT), transmembrane serine protease 2 (TMPRSS2), matriptase, chymotrypsin, pepsin, thrombin, elastase, subtilisin, carboxypeptidase, papain, bromelain and caspase.24. The method of embodiment 20 or embodiment 21 , wherein the protease is trypsin.25. The method of any one of embodiments 19 to 24, wherein the administering is by mucosal administration.26. The method of any one of embodiments 19 to 24, wherein the administering is by intranasal administration.27. The method of any one of embodiments 21 to 24, wherein the administering is by intramuscular administration.28. The method of any one of embodiments 19 to 27, wherein the method induces a neutralising antibody response.29. The method of any one of embodiments 19 to 27, wherein the method prevents, treats or reduces the severity of an NDV infection in the subject.31. The method of any one of embodiments 19 to 27, wherein the method induces a heterotypic immune response against a NDV genotype different to the irradiation-inactivated NDV in the subject and / or induces protection against a plurality of NDV genotypes in the subject.32. The method of any one of embodiments 19 to 27, wherein the method induces sterilizing immunity against NDV.33. The method of any one of embodiments 19 to 32, wherein the subject is an avian subject.34. A method of producing a protease-treated, irradiation-inactivated NDV vaccine composition, the method comprising:(a) obtaining a NDV preparation, and optionally clarifying and / or washing the NDV preparation;(b) irradiating the NDV preparation to obtain an irradiation-inactivated NDV preparation; and(c) protease-treating the irradiation-inactivated NDV preparation.35. The method of embodiments 34, wherein the irradiation-inactivated NDV is selected from gamma-irradiated NDV, X-irradiated NDV or electron-irradiated NDV.36. The method of embodiment 34, wherein the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.37. The method of any one of embodiments 34 to 36, wherein the vaccine composition is an inactivated whole virus vaccine composition.38. The method of any one of embodiments 34 to 37, wherein the composition further comprises an adjuvant.39. The method of any one of embodiments 34 to 37, wherein the composition excludes an adjuvant.40. The method of any one of embodiments 34 to 37, wherein the composition excludes an alum adjuvant.41. The method of any one of embodiments 34 to 40, wherein the protease is selected from the group consisting of trypsin, tryptase, prostasin, human airways trypsin-like protease (HAT),transmembrane serine protease 2 (TMPRSS2), matriptase, chymotrypsin, pepsin, thrombin, elastase, subtilisin, carboxypeptidase, papain, bromelain and caspase.42. The method of any one of embodiments 34 to 40, wherein the protease is trypsin.43. The method of any one of embodiments 33 to 43, wherein the irradiation-inactivated NDV is a genotype I strain or a V4 strain.44. A vaccine composition produced by the method of any one of embodiments 33 to 44.45. A pharmaceutical composition comprising the vaccine composition of any one of embodiments 1 to 18 or 44.46. A method for treating or preventing an NDV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39.47. A method of inducing an immune response against NDV in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39.48. A method of vaccinating a subject against NDV, the method comprising administration of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39.49. A method of preventing or reducing the symptoms of an NDV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39.50. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 in the manufacture of a medicament for treating or preventing an NDV infection in a subject.51. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 in the manufacture of a medicament for inducing an immune response against NDV in a subject.52. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 in the manufacture of a medicament for vaccinating a subject against NDV.53. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 in the manufacture of a medicament for preventing or reducing the symptoms of an NDV infection in a subject.54. A therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for treating or preventing an NDV infection in a subject.55. A therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for inducing an immune response against NDV in a subject.56. A therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for vaccinating a subject against NDV.57. A therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for preventing or reducing the symptoms of an NDV infection in a subject.58. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for treating or preventing an NDV infection in a subject.59. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for inducing an immune response against NDV in a subject.60. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for vaccinating a subject against NDV.61. Use of a therapeutically effective amount of the vaccine composition of any one of embodiments 1 to 18 or 44, or the pharmaceutical composition of embodiment 39 for preventing or reducing the symptoms of an NDV infection in a subject.62. The method, use or composition of any one of embodiments 46-61, wherein the method, use or composition induces a heterotypic immune response against a NDV genotype different to the irradiated NDV in the subject and / or induces protection against a plurality of NDV genotypes in the subject.63. The method, use or composition of any one of embodiments 46-61, wherein the method induces sterilising immunity against NDV.64. The method, use or composition of any one of embodiments 46-61, wherein the method induces increased protection against NDV, increased survival against NDV, decreased NDV symptoms, a mucosal NDV-specific IgA response, systemic NDV-specific IgG response, NDV-specific lgG1 response, NDV-specific lgG2b response and / or a NDV-specific neutralising antibody response.65. The method, use or composition of any one of embodiments 46-61, wherein the method induces a mucosal immune response.66. The method, use or composition of any one of embodiments 46-61, wherein the administration is mucosal administration, such as intranasal, ocular or conjunctival administration.67. The method, use or composition of any one of embodiments 46-61, wherein the administration is intranasal, intramuscular, subcutaneous, intranasal, ocular or conjunctival administration.68. The method, use or composition of any one of embodiments 46-61 , wherein the administration is intranasal administration.69. The method, use or composition of any one of embodiments 46-61 , wherein the administration is intramuscular administration and the vaccine composition comprises irradiation-inactivated, protease treated NDV.70. The method, use or composition of any one of embodiments 46-61, where in the subject is an avian subject.71. A method of producing an NDV vaccine according to any one of embodiments 1 to 18, the method comprising inactivating a preparation of NDV by irradiation.72. The method of embodiment 62 wherein the irradiation comprises treatment with a dose of gamma radiation of between 2-100 kGy.73. The method of embodiment 62 or 63 wherein the method further comprises clarifying or purifying the NDV prior to irradiation.74. A photon-irradiated NDV vaccine produced by the method of any one of embodiments 71-73.[000253] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms, and in particular features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention.

Claims

CLAIMS1. A vaccine composition comprising irradiation-inactivated Newcastle Disease Virus (NDV).

2. The vaccine composition of claim 1, further comprising at least one pharmaceutically- acceptable excipient, diluent and / or carrier.

3. The vaccine composition of claim 1 or claim 2, wherein the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.

4. The vaccine composition of any one of claims 1 to 3, wherein the vaccine composition is an inactivated whole virus vaccine composition.

5. The vaccine composition of any one of claims 1 to 4, wherein the composition excludes an adjuvant.

6. The vaccine composition of any one of claims 1 to 5, wherein the irradiation-inactivated NDV comprises irradiation-inactivated, protease treated NDV.

7. The vaccine composition of claim 6, wherein the protease treatment occurred following the irradiation inactivation.

8. The vaccine composition of claim 6 or claim 7, wherein the protease is trypsin.

9. The vaccine composition of any one of claims 1 to 8, wherein the irradiation-inactivated NDV is a genotype I strain or a V4 strain.

10. A method of inducing a protective immune response to Newcastle Disease Virus (NDV), the method comprising administering to a subject a therapeutically effective amount of a vaccine composition comprising irradiation-inactivated NDV.

11. The method of claim 10, wherein the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.

12. The method of claim 10 or claim 11, wherein the irradiation-inactivated NDV comprises irradiation-inactivated, protease treated NDV.

13. The method of claim 12, wherein the protease treatment occurred following the irradiation inactivation.

14. The method of claim 12 or claim 13, wherein the protease is trypsin.

15. The method of any one of claims 10 to 14, wherein the administering is by mucosal administration.

16. The method of any one of claims 10 to 14, wherein the administering is by intranasal administration.

17. The method of any one of claims 12 to 14, wherein the administering is by intramuscular administration.

18. The method of any one of claims 10 to 17, wherein the method induces a neutralising antibody response.

19. The method of any one of claims 10 to 17, wherein the method prevents, treats or reduces the severity of an NDV infection in the subject.

20. The method of any one of claims 10 to 17, wherein the method induces a heterotypic immune response against a NDV genotype different to the irradiation-inactivated NDV in the subject and / or induces protection against a plurality of NDV genotypes in the subject.

21. The method of any one of claims 10 to 17, wherein the method induces sterilizing immunity against NDV.

22. The method of any one of claims 10 to 21, wherein the subject is an avian subject.

23. A method of producing a protease-treated, irradiation-inactivated NDV vaccine composition, the method comprising:(a) obtaining a NDV preparation, and optionally clarifying and / or washing the NDV preparation;(b) irradiating the NDV preparation to obtain an irradiation-inactivated NDV preparation; and(c) protease-treating the irradiation-inactivated NDV preparation.

24. The method of claim 23, wherein the irradiation-inactivated NDV is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.

25. The method of claim 23 or claim 24, wherein the protease is trypsin.

26. The method of any one of claims 23 to 26, wherein the irradiation-inactivated NDV is a genotype I strain or a V4 strain.

27. A vaccine composition produced by the method of any one of claims 23 to 26.

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