Paramyxovirus combination vaccine

Abstract

The present disclosure relates to a vaccine composition comprising (a) an inactivated paramyxovirus, and (b) an inactivated influenza virus. The vaccine composition may be used in methods for inducing or enhancing an immune response against an influenza infection, methods of producing the vaccine composition, and vaccine compositions produced by such methods.

Description

PARAMYXOVIRUS COMBINATION VACCINERELATED APPLICATIONS

[0001] The present application claims priority from Australian Provisional Application No.2024903738, 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 inactivated paramyxovirus and a further immunogen. In one form, the present disclosure relates to vaccine compositions comprising inactivated Newcastle Disease Virus and inactivated influenza virus. 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), a paramyxovirus, 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, but 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] Avian influenza, also known as bird flu, is commonly caused by Influenza A viruses (IAV). Avian influenza is a pathogen of both wild birds and poultry, that can cause wide-spread death of birds and in some circumstances. Avian influenza virus strains are described as low pathogenicity (LPAI) or high pathogenicity (HPAI). Most LPAI strains of avian influenza virus cause minimal disease in wild birds and poultry. However, some LPAI strains can evolve intoHPAI strains when they spread among poultry. Avian influenza can also circulate among mammalian species, including pigs, horses, and marine animals. Both low and highly pathogenic avian influenza viruses like the H5, H7, and H9 types can infect humans, thereby demonstrating their pandemic potential. New influenza strains continuously emerge. Commercial influenza vaccines typically provide homologous protection against the strains present within the vaccine preparation.

[0006] A general need exists for agents capable of enhancing immune responses induced by vaccine immunogens.

[0007] Further, effective combination vaccines are highly desirable as they may reduce the burden of both medical practitioners and vaccination subjects by reducing the occasions each vaccination subject attends a vaccination clinic, and may also reduce the number of injections each subject receives. These factors may in turn increase compliance with recommended or desirable vaccination schedules.

[0008] 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

[0009] In a first aspect, the present invention provides a vaccine composition comprising (a) an inactivated paramyxovirus, and (b) an inactivated influenza virus.

[0010] In an embodiment, the inactivated paramyxovirus is irradiation-inactivated paramyxovirus. In an embodiment, the inactivated influenza virus is irradiation-inactivated influenza. In an embodiment, the paramyxovirus is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy. In an embodiment, the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.

[0011] In an embodiment, the composition excludes a supplementary adjuvant. In an embodiment, the inactivated paramyxovirus comprises irradiation-inactivated, protease treated paramyxovirus. In an embodiment, the protease treatment occurred following the irradiation inactivation of the paramyxovirus. In an embodiment, the protease is trypsin.

[0012] In an embodiment, the inactivated paramyxovirus Newcastle Disease Virus (NDV). In an embodiment, the influenza virus is a type A influenza virus.

[0013] In an embodiment, the inactivated paramyxovirus enhances the immune response against influenza virus and / or provides an adjuvant effect for the inactivated influenza virus.

[0014] In a second aspect, the present invention provides a method for inducing or enhancing an immune response against an influenza infection in a subject, the method comprising (i) administering to the subject a therapeutically effective amount of the vaccine composition comprising (a) inactivated paramyxovirus, and (b) inactivated influenza virus; or (ii) coadministering to the subject a therapeutically effective amount of two vaccine compositions, the vaccine compositions comprising a first vaccine composition comprising inactivated paramyxovirus, and a second composition comprising inactivated influenza virus.

[0015] In an embodiment, the paramyxovirus is inactivated by irradiation. In an embodiment, the influenza is inactivated by irradiation. In an embodiment, the paramyxovirus is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy. In an embodiment, the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.

[0016] In an embodiment, the paramyxovirus comprises irradiation-inactivated, protease treated paramyxovirus. In an embodiment, the protease treatment occurred following the irradiation inactivation of the paramyxovirus. In an embodiment, the protease is trypsin.

[0017] In an embodiment, the inactivated paramyxovirus is a NDV. In an embodiment, the inactivated influenza is a type A influenza virus.

[0018] In an embodiment, the administering is selected from the group consisting of mucosal administration, intranasal administration and intramuscular administration.

[0019] In an embodiment, the method induces or enhances a protective immune response that treats, prevents or reduces the severity of an influenza infection in the subject. In an embodiment, the method induces a cross- protective immunity against an influenza strain different to the inactivated influenza virus and / or induces protection against a plurality of influenza strains in the subject. In an embodiment, the method induces increased protection against influenza, increased survival against influenza, decreased influenza symptoms, an increased influenzaspecific antibody response, increased influenza-specific neutralising antibody response, and / or an increased neuraminidase neutralising response compared to administering a therapeutically effective amount of an inactivated influenza virus in the absence of the inactivated paramyxovirus. In an embodiment, the inactivated paramyxovirus enhances the immune response against influenza virus and / or provides an adjuvant effect for the inactivated influenza virus.

[0020] In an embodiment, the method further comprises inducing an immune response against a paramyxovirus infection in the subject or treating, preventing or reducing the severity of a paramyxovirus infection in the subject. In an embodiment, the method induces a paramyxovirusspecific antibody response or a paramyxovirus-specific neutralising antibody response.

[0021] In an embodiment, the subject is an avian subject. In an embodiment, the subject is human.

[0022] In a third aspect, the present invention provides a method of producing a vaccine composition, the vaccine composition comprising (a) an irradiation-inactivated paramyxovirus and (b) irradiation-inactivated influenza virus vaccine composition, the method comprising:(i) obtaining a preparation containing the paramyxovirus, and optionally clarifying and / or washing the paramyxovirus;(ii) irradiating the paramyxovirus preparation to obtain an irradiation-inactivated paramyxovirus preparation; and optionally then protease-treating the irradiation-inactivated paramyxovirus preparation;(iii) obtaining a preparation containing influenza virus, and optionally clarifying and / or washing the influenza virus;(iv) irradiating the influenza preparation to obtain an irradiation-inactivated influenza preparation; and(v) optionally, combining the irradiation-inactivated paramyxovirus preparation obtained in step (ii) with the irradiation-inactivated influenza preparation obtained in step (iv).

[0023] In an embodiment, the irradiation-inactivated paramyxovirus is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy. In an embodiment, the irradiation-inactivated influenza is irradiated with a dose of gamma radiation of between 2 kGy and 50 kGy. In an embodiment, the protease is trypsin. In an embodiment, the paramyxovirus is NDV. In an embodiment, the influenza virus is a type A influenza virus.

[0024] In a fourth aspect, the present invention provides a vaccine composition produced by the method of the third aspect.DEFINITIONS

[0025] 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.

[0026] 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.

[0027] 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.

[0028] As used herein, the term “adjuvant effect” is intended to refer to an enhanced immune response associated with administration of a vaccine composition in the presence of, or in combination with, a particular component. For example, the particular component may enhance the immune response parameter following vaccination against a particular infectious agent (e.g. a pathogen), such as increased protection against infection with the infectious agent, increased survival against infection with the infectious agent, decreased infection symptoms, increased agent-specific antibody response, increased agent-specific neutralising antibody response, and / or an increased neutralising response to the infectious agent, compared to administration of the vaccine composition in the absence of the particular component.

[0029] 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 -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -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.

[0030] 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".

[0031] 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.

[0032] 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 ofelements 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.

[0033] The transitional phrase "consisting of’ excludes any element, step, or ingredient not specified. If in the claim, such 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.

[0034] 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".

[0035] 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 to comprise 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 related 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.

[0036] The “irradiation-inactivated” vaccines of the present invention will be understood to be inactivated by irradiation such as photon irradiation or electron irradiation. The term “photonradiation” or “photon-irradiation” will be understood to encompass both gamma-radiation 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.

[0037] 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.

[0038] 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.

[0039] 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 disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.BRIEF DESCRIPTION OF THE FIGURES

[0040] The aspects described above, 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:

[0041] Figure 1 provides graphical representation of influenza symptoms from BALB / c mice vaccinated intranasally (IN) twice at two-week intervals with either y-Flu or y-NDV alone, or covaccinated with y-Flu+y-NDV or PBS-mock vaccinated; and challenged with influenza A virus pdmH1N1 (A / California / 07 / 2009) three weeks post second vaccination (= Day 0), showing (A) weight loss post challenge; (B) percentage survival post challenge based on 20% body weight loss as a humane end point; and weight loss on days (C) 3; (D) 4: (E) 5; (F) 6, (G) 7; or (H) 8 post-challenge. Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test (* p< 0.05, ** p<0.01 ) , comparing individual weight differences at different days post-challenge. Naive (IN mock vaccinated) animals are represented by circles, IN y-NDV vaccinated animals are represented by squares, IN y-flu (i.e. , y-A / PR8) vaccinated animals arerepresented by triangles (apex up), and IN Y-A / PR8+YNDV vaccinated animals are represented by upside down triangles (apex down).

[0042] Figure 2 provides graphical representation of total A / PR8-specific IgG detected using ELISA testing immune serum from BALB / c mice vaccinated intranasally (IN) twice at two-week intervals with either y-Flu (i.e. , y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after primary vaccination and data presented as (A) absorbance measured at450nm and (B) titres; or serum harvested 13 days after secondary vaccination with data presented as (C) absorbance at 450nm and (D) titres. For (A) and (C), naive (IN mock vaccinated) animals are represented by circles, IN y-NDV vaccinated animals represented by squares, IN y-flu (i.e., y-A / PR8) vaccinated animals represented by triangles (apex up), IN y-A / PR8+yNDV vaccinated animals represented by upside down triangles (apex down). Fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using (B) 1 / 100 dilution for primary response or (D) a 1 / 800 dilution for secondary response.

[0043] Figure 3 provides graphical representation of neutralisation assays measuring infectivity of live A / PR8 H1N1 virus following treatment with serum obtained from BALB / c mice vaccinated intranasally (IN) twice at two-week intervals with either y-Flu (i.e., y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 2 weeks after second vaccination was tested for neutralising antibody responses. A / PR8 infected cells were detected using FITC-fluorescence labelled antibody (representative of A / PR8 infection) and florescence intensity was normalized relative to DAPI-fluorescence (cell nuclei) for (A) 1:320 dilution and (B) 1:640 dilution of immune sera.

[0044] Figure 4 provides graphical representation of neuraminidase inhibition against A / PR8 by serum obtained from BALB / c mice vaccinated intranasally (IN) twice at two-week intervals with either y-Flu (i.e., y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after second vaccination. Quantitative data is presented as mean ± SEM (n= 2 technical replicates), and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, **** p < 0.0001).

[0045] Figure 5 provides graphical representation of total (A, B) A / California (H1N1) specific IgG or (C,D) A / PC (H3N2) specific IgG detected using ELISA testing immune serum from BALB / c mice vaccinated intranasally (IN) twice at two-week intervals with either y-Flu (i.e., y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested two weeks after secondary vaccination and data presented as (A, C) absorbance measured at450nm and (B, D) total IgG titre calculated based on a cut-off determined from the mean + 3 x S.D. of the naive control absorbance values at a (B) 1 / 00 dilution or (D) 1 / 200 dilution.

[0046] Figure 6 provides graphical representation of neutralisation of live A / California (H1 N1) virus by serum obtained from BALB / c mice vaccinated intranasally (IN) twice attwo-week intervals with either y-Flu (i.e. , y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu (y-A / PR8)+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after second vaccination was tested for cross-neutralising antibody responses. A / California infected cells were detected using FITC-fluorescence labelled antibody (representative of A / California infection) and fluorescence intensity was normalized relative to DAPI-fluorescence (cell nuclei) and data shown for 1:80 dilution of immune sera.

[0047] Figure 7 provides graphical representation of symptoms from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (i.e., y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated; and challenged with influenza A virus pdmH1N1 (A / California / 07 / 2009) three weeks post second vaccination (= Day 0), showing (A) weight loss post challenge; (B) percentage survival based on 20% body weight loss as a humane end point; or weight loss on days (C) 3; (D) 4 post challenge. Data represented as mean ± SEM of group (n=4-5 per group). Statistical analyses were performed using One-way ANOVA with Tukey’s multiple comparisons test (*p<0.05), comparing individual weight differences of mice from each group at day 3 and 4 post-challenge. Naive (IM mock vaccinated) animals are represented by circles, IN y-NDV vaccinated animals are represented by squares, IN y-flu (i.e., y-A / PR8) vaccinated animals are represented by triangles (apex up), and IN y-A / PR8+yNDV vaccinated animals are represented by upside down triangles (apex down).

[0048] Figure 8 provides graphical representation of total A / PR8-specific IgG detected using ELISA in serum samples obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (i.e., y-A / PR8) or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after primary vaccination and data presented as (A) absorbance measured at 450nm; or (B) titres; or serum harvested 13 days after secondary vaccination with data presented as (C) absorbance at450nm; or (D) titres. For (A) and (C), naive (IM mock vaccinated) animals are represented by circles, IN y-NDV vaccinated animals represented by squares, IM y-flu (i.e., y-A / PR8) vaccinated animals represented by triangles (apex up), IM y-A / PR8+yNDV vaccinated animals represented by upside down triangles (apex down). Fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using (B) 1 / 100 dilution for primary response or (D) a 1 / 200 dilution for secondary response.

[0049] Figure 9 provides graphical representation of neutralization assays performed to measure infectivity of live A / PR8 H 1 N 1 virus following treatment with serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice attwo-week intervals with either y-Flu or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after second vaccination; showing FITC fluorescence (representative of influenza infection) relative to DAPI fluorescence (cell nuclei) at (A) 1:320 and (B) 1:640 serum dilution.

[0050] Figure 10 provides graphical representation of neuraminidase inhibition against A / PR8 in serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after second vaccination. Quantitative data is presented as mean ± SEM (n= 2 technical replicates), and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, **** p < 0.0001).

[0051] Figure 11 provides graphical representation of total NDV-specific IgG detected using ELISA in serum samples obtained from BALB / c mice vaccinated intranasally (IN) twice at two-week intervals with either y-Flu or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after secondary vaccination with data presented as (A) absorbance at 450nm; or (B) titres. For (A), naive (IN mock vaccinated) animals are represented by circles, IN y-NDV vaccinated animals represented by squares, IN y-flu (i.e., y-A / PR8) vaccinated animals represented by triangles (apex up), IN y-A / PR8+yNDV vaccinated animals represented by upside down triangles (apex down). For (B), fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using 1 / 200 dilution. Data is presented as mean ± SEM, and was analysed by two-way ANOVA with Tukey’s multiple comparisons test (* p< 0.05, *** p<0.001 , **** p<0.0001).

[0052] Figure 12 provides graphical representation of total NDV-specific IgG detected using ELISA in serum samples obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu or y-NDV alone, or co-vaccinated with y-Flu+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after secondary vaccination with data presented as (A) absorbance at 450nm; or (B) titres. For (A), naive (IM mock vaccinated) animals are represented by circles, IM y-NDV vaccinated animals represented by squares, IM y-flu (i.e., y-A / PR8) vaccinated animals represented by triangles (apex up), IM y-A / PR8+yNDV vaccinated animals represented by upside down triangles (apex down). For (B), fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using 1 / 200 dilution. Data is presented as mean ±SEM, and was analysed by two-way ANOVA with Tukey’s multiple comparisons test (* p< 0.05, *** p<0.001, **** p<0.0001).

[0053] Figure 13 provides graphical representation of neutralization of live NDV by serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (A / PR8) or y-NDV alone, or co-vaccinated with y-Flu(Y-A / PR8)+y-NDV or PBS-mock vaccinated, with serum harvested 13 days after second vaccination; showing FITC fluorescence (representative of NDV infection) normalised relative to DAPI fluorescence (cell nuclei). Serum dilution of 1:80 was used to test neutralization of NDV.

[0054] Figure 14 provides graphical representation of total NDV-specific IgG detected using ELISA in serum samples obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (y-A / PR8) or trypsin-treated y-NDV (y-NDVtrp) alone, or covaccinated with y-Flu+y-NDVP or PBS-mock vaccinated, with serum harvested 2 weeks after (A,B) primary vaccination, or (C,D) secondary vaccination. Data is presented as (A, C) absorbance at 450nm; or (B, D) antibody titres calculated based on a cut-off value determined from the mean + 3x S.D. of the naive control absorbance values at (B) a 1 / 00 dilution or (D) 1 / 200 dilution (D).

[0055] Figure 15 provides graphical representation of ELISA results from serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (Y-A / PR8) or trypsin-treated Y-NDV (y-NDVtrp) alone, or co-vaccinated with y-Flu+y-NDVtrpor PBS-mock vaccinated, with serum harvested 2 weeks after secondary vaccination for (A, B) NDV-specific lgG1, (C, D) NDV-specific lgG2a and (E) NDV-specific lgG1 / lgG2a ratio of the titres of each biological replicate with horizontal lines indicating 0.5 and 2.0 ratios. Titres in (B, D) were calculated based on a cut-off determined from the mean + 3 x S.D. of the naive control absorbance values at a 1 / 200 dilution.

[0056] Figure 16 provides graphical representation of neutralization of live NDV by serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (y-A / PR8) or y-NDVP alone, or co-vaccinated with y-Flu (Y-A / PR8)+v-NDVtrpor PBS-mock vaccinated, with serum harvested 2 weeks after second vaccination; showing FITC fluorescence (representative of NDV infection) relative to DAPI fluorescence (cell nuclei) at (A) 1:20 and (B) 1:40 serum dilution.

[0057] Figure 17 provides graphical representation of A / PR8-specific IgG responses from obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (y-A / PR8) or y-NDVtrpalone, or co-vaccinated with y-Flu (Y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated, with serum harvested 2 weeks after (A,B) primary or (C,D) secondary vaccination;showing (A, C) absorbance at 450nm; or (B, D) titres calculated based on a cut-off determined from the mean + 3 x S.D. of the naive control absorbance values at a (B) 1 / 100 dilution (B) or (D) 1 / 200 dilution (D).

[0058] Figure 18 provides graphical representation of ELISA results from serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (y-A / PR8) or trypsin-treated y-NDV (y-NDVtrp) alone, or co-vaccinated with y-Flu+y-NDVtrp or PBS-mock vaccinated, with serum harvested 2 weeks after secondary vaccination for (A, B) A / PR8-specific lgG1 , (C, D) A / PR8-specific lgG2a and (E) A / PR8-specific lgG1 / lgG2a ratio of the titres of each biological replicate with horizontal lines indicating the 0.5 and 2.0 ratios. Titres in (B, D) were calculated based on a cut-off determined from the mean + 3 x S. D. of the naive control absorbance values at a 1 / 200 dilution.

[0059] Figure 19 provides graphical representation of neutralization of live influenza (A / PR8) by serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (y-A / PR8) or y-NDVP alone, or co-vaccinated with y-Flu (y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated (naive), with serum harvested 2 weeks after second vaccination; showing FITC fluorescence (representative of influenza infection) relative to DAPI fluorescence (cell nuclei) at (A) 1:80 and (B) 1:320 serum dilution.

[0060] Figure 20 provides graphical representation of percentage of inhibition of influenza (A / PR8) neuraminidase activity by serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either Y-FIU (Y-A / PR8) or Y-NDVtrpalone, or co-vaccinated with Y-FIU (Y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated (naive), with serum harvested 2 weeks after second vaccination, relative to neuraminidase activity in the absence of serum.

[0061] Figure 21 provides graphical representation of influenza-specific cross- reactive antibodies in serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either Y-FIU (Y-A / PR8) or Y-NDVtrpalone, or co-vaccinated with Y-FIU (Y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated (naive), with serum harvested 2 weeks after second vaccination, with serum tested against (A) A / Sydney, (B) A / PC and (C) B / Austria influenza strains.

[0062] Figure 22 provides graphical representation of neuraminidase inhibition of (A) A / Sydney, (B) A / PC and (C) B / Austria influenza virus strains by serum obtained from BALB / c mice vaccinated intramuscularly twice at two-week intervals with either Y-FIU (Y-A / PR8) or y-NDVtrpalone, or co-vaccinated with Y-FIU (Y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated, with serum harvested two weeks after second vaccination.

[0063] Figure 23 provides graphical representation of cross-neutralising antibodies in serum obtained from BALB / c mice vaccinated intramuscularly (IM) twice at two-week intervals with either y-Flu (y-A / PR8) or y-NDVtrpalone, or co-vaccinated with y-Flu (y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated (naive), with serum harvested 2 weeks after second vaccination, with serum incubated with live (A) A / Sydney, (B) A / PC and (C) B / Austria influenza virus strains, showing FITC fluorescence (representative of influenza infection) relative to DAPI fluorescence (cell nuclei) at 1:80 serum dilution.

[0064] Figure 24 provides graphical representation of symptoms from BALB / c mice (n=8-10 per group) vaccinated intramuscularly (IM) twice at two-week intervals with either Y-FIU (Y-A / PR8) or Y-NDVP alone, or co-vaccinated with Y-FIU (Y-A / PR8)+Y-NDVtrpor PBS-mock vaccinated; and challenged with influenza A virus pdmH1N1 (A / California / 07 / 2009) three weeks post second vaccination (= Day 0), showing (A) weight loss post challenge; (B) percentage survival based on 20% body weight loss as a humane end point. Statistical analyses was performed using the two-tailed Fisher’s Exact Test (** p < 0.01, compared to PBS-mock control group). Naive (IM mock vaccinated) animals are represented by circles, IN Y-NDV vaccinated animals are represented by squares, IN Y-flu (Y-A / PR8) vaccinated animals are represented by triangles (apex up), and IN y-A / PR8+YNDV vaccinated animals are represented by upside down triangles (apex down).DETAILED DESCRIPTION

[0065] In an aspect, the present disclosure provides a vaccine composition comprising an inactivated paramyxovirus and an inactivated influenza virus. In an embodiment, the paramyxovirus is inactivated by irradiation. In an embodiment, the influenza virus is inactivated by irradiation. In another aspect, the disclosure provides pharmaceutical compositions comprising the vaccine composition.

[0066] In other aspects, the disclosure provides methods and / or uses of the composition in vaccinating a subject against, inducing or enhancing an immune response against, treating or preventing, and / or reducing the symptoms of an influenza infection. In other aspects, the disclosure provides methods and / or uses of the composition in vaccinating a subject against, inducing or enhancing an immune response against, treating or preventing, and / or reducing the symptoms of a paramyxovirus infection.

[0067] 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.

[0068] 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

[0069] In an embodiment, the paramyxovirus is Newcastle Disease Virus (NDV). 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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 mainlyvia 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 viral RNA 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.

[0077] 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

[0078] 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 I FN-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.

[0079] Adaptive immunity relies on both humoral and cell-mediated arm 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 that antibody responses may be associated with survival from NDV infection, whereas T cell responses may be associated with reducing virus shedding.

[0080] 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.

[0081] 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 antibodies 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.

[0082] 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 target 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 to 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

[0083] 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. Mucosal immune responses may be generated when vaccines are administrated via mucosal route such as the conjunctival and intranasal routes of administration.

[0084] 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 short-lived.

[0085] 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 the 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.

[0086] 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

[0087] 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 NDV vaccines 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.

[0088] 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 PARAMYXOVIRUSES

[0089] Paramyxoviruses such as 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.

[0090] 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 paramyxovirus byirradiation. Purification of allantoic fluid may be achieved for example, by temperature-dependent adsorption to chicken red blood cells (CRBC), sucrose gradient, or dialysis.

[0091] Additionally or alternatively, paramyxovirus 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 paramyxovirus 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.

[0092] Propagation of paramyxovirus 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 paramyxovirus and incubated for a period of time sufficient to generate the required numbers 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 paramyxovirus 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.

[0093] The paramyxovirus 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, paramyxovirus 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 viral particles passes through the membrane, whereas particulates and macromolecules 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. Commercial TFF systems may be used such as Repligen KrosFlo® Research 2i TFF System.INFLUENZA VIRUS

[0094] Influenza virus is an enveloped single-stranded RNA virus that causes influenza (flu). There are at least 4 types of influenza viruses, types A, B, C and D. Influenza A and B viruses circulate and cause seasonal epidemics of disease in humans.

[0095] Influenza A viruses are further classified into subtypes according to the antigenic variations in the surface glycoproteins hemagglutinin (HA or H) and neuraminidase (NA or N). About eighteen subtypes of HA (H1-H18) and eleven subtypes of NA (N1-N11) are so far reported, in different combinations, from birds and mammals including humans. Three HA subtypes (H1-H3) and two NA subtypes (N1, N2) have been recovered from humans. Currently circulating in humans are subtype A (H1N1) and A (H3N2) influenza viruses.

[0096] Influenza B viruses are not classified into subtypes but can be broken down into lineages. Influenza type B viruses belong to either B / Yamagata or B / Victoria lineage.

[0097] Influenza C virus is detected less frequently and usually causes mild infections, thus does not present public health importance.

[0098] Influenza D viruses primarily affect cattle and are not known to infect or cause illness in humans.

[0099] Current human seasonal influenza vaccines are typically either subunit vaccines comprising purified influenza surface antigens (e.g. hemagglutinin and / or neuraminidase) from multiple influenza strains (e.g., quadrivalent) or inactivated split whole virus from multiple strains (e.g., quadrivalent). Such vaccines are typically administered via intramuscular injection. A dose of quadrivalent seasonal flu vaccine is typically calculated by determining the amount of HA from each strain. For example, a quadrivalent vaccine may contains a total of 60 pg of HA that includes 15 pg of HA of each of the 4 influenza strains.[000100] However, the immunity induced by these vaccines is limited and generally restricted to the particular target strains included in the formulation. Although alternative influenza vaccines are available, most provide little or no cross- protective immunity (i.e. immunity to multiple different strains) and consequently are not widely used. In addition, current influenza vaccines provide inadequate protection against secondary infections which are a leading cause of morbidity and mortality in both local influenza outbreaks and major influenza pandemics.[000101] The compositions of the present disclosure may comprise irradiated influenza virus of the of any suitable subtype including influenza A subtypes H1N1, H1N2, H1N7, H2N2, H3N1, H3N2, H3N8, H4N8, H5N1 (e.g. HPA1 A(HSN1)), H5N2, H5N3, H5N8, H5N9, H6N5, H7N1,H7N2, H7N3, H7N4, H7N7, H8N4, H9N2, H10N7, H11N6, H12N5, H13N6, H14N5, and any other virus arising from re-assortment between influenza A subtypes resulting in any possible combination of HA and NA.AVIAN INFLUENZA VIRUS[000102] Avian influenza, also known as bird flu, is commonly caused by Influenza A viruses (IAV) from the family Orthomyxoviridae. Avian influenza mainly spreads amongst wild water birds such as ducks and geese, but they could also circulate among mammalian species, including pigs, horses, and marine animals. Avian influenza viruses are categorised into 2 categories; low pathogenic avian influenza (LPAI), or highly pathogenic avian influenza (HPAI). Classification of a virus strain as either LPAI or HPAI is based on the severity of symptoms in domestic chickens and does not predict severity of symptoms in other species. Chickens infected with LPAI display mild symptoms or are asymptomatic, whereas HPAI causes serious breathing difficulties, significant drop in egg production, and sudden death. Domestic poultry may potentially be protected from specific strains of the virus by vaccination. Both low and highly pathogenic avian influenza viruses like the H5, H7, and H9 types can infect humans, thereby demonstrating their pandemic potential. A particularly virulent strain, influenza A virus subtype H5N1 (A / H5N1) has the potential to decimate domesticated poultry stock. Continuous emergence of new strains and the limited scope of cross- protective immunity associated with the currently available influenza vaccine preparations highlight the need for a better alternative vaccine against flu.PROPAGATION AND PURIFICATION OF INFLUENZA VIRUS[000103] Influenza virus 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, influenza viruses may be derived by serial passaging in embryonated eggs as described, for example, in Coico et al., (Eds) (2007), “Current Protocols in Microbiology”, John Wiley and Sons, Inc. (see in particular Unit 15G.1 entitled “Influenza: Propagation, Quantification, and Storage”). A brief description of this technique is provided below.[000104] Embryonated eggs may be obtained 9-12 days after fertilization and candled to locate the air sac. The egg may then be pierced under aseptic conditions, and the seed-virus inoculated into the air-space 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 theallantoic 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 influenza virus by gammairradiation. Purification of allantoic fluid may be achieved for example, by temperaturedependent adsorption to chicken red blood cells (CRBC), sucrose gradient, or dialysis.[000105] Modifications of the above-mentioned process also suitable for the production of influenza virus are described, for example, in United States Patent No. 7,270,990, PCT publication No. WO 2002 / 067983 and PCT publication No. WO 2005 / 113756.[000106] Additionally or alternatively, influenza virus for use in accordance with the present disclosure may be generated in cell culture (see, for example, Furminger, (1998), “Vaccine production”, in Nicholson et al. (Eds.), “Textbook of Influenza”, Blackwell Science, Oxford, pp.324-332; Merten et al., (1996), “Production of influenza virus in cell cultures for vaccine preparation”, in Cohen & Shafferman (Eds.), “Novel Strategies in Design and Production of Vaccines”, pp. 141-151; United States Patent No. 5,824,536; and United States Patent No. 6,344,354).[000107] Non-limiting examples of suitable cell lines that may be used as substrates for the growth of influenza virus include Vero cells, Madin Darby canine kidney (MDCK) cells, PERC6 cells (see, for example, United States Patent No. 7,192,759), chicken embryo cells (e.g. chicken embryo fibroblasts) and avian embryonic cell lines (see, for example, PCT publication No. WO 2006 / 108846). Variants of these cell lines may be used, including, but not limited to, those described in United States Patent No. 6,825,036, United States Patent No. 6,455,298 and PCT publication No. WO 2006 / 108846.[000108] Propagation of influenza virus 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 influenza virus and incubated for a period of time sufficient to generate the required numbers 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 influenza 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.[000109] Non-limiting examples of processes suitable for the production of influenza virus in cell culture include those described in United States Patent No. 5,698,433, United States PatentNo. 5,753,489, United States Patent No. 6,146,873, United States Patent No. 6,455,298 and United States Patent No. 6,951,752.[000110] The yield of influenza virus production in cell culture may be enhanced, for example, by modifying cellular genes encoding the protein kinase PKR or (2'-5') oligoadenylate (2-5A) synthetase genes (see, for example, United States Patent No. 6,673,591 and United States Patent No. 6,686,190), or modifying the viral backbone with an alternative nonstructural protein 1 (NS1) gene (see, for example, PCT publication No. WO 2005 / 024039). Additionally or alternatively, cell lines utilised for the propagation of influenza virus may over-express sialyltransferase (see, for example, United States Patent No. 7,132,271).[000111] The influenza virus may be purified any / or concentrated by any suitable method known in the art including by sucrose density gradient ultracentrifugation (SDGU) or tangential flow filtration (TFF). For example, influenza virus may be purified by temperature-dependent adsorption to chicken red blood cells using the method described in Laver, (1969), “Purification of influenza virus”, HKaS NP (Ed), New York and London: Academic Press, pp. 82-86.Additionally or alternatively, influenza virus may be purified by density gradient centrifugation (see, for example, Sokolov et al., (1971), “Purification and concentration of influenza virus”, Archiv fiir die gesarate Virusforschung, 35, 356-363).[000112] In certain embodiments, influenza virus 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 viral particles passes through the membrane, whereas particulates and macromolecules 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.[000113] Commercial TFF systems may be used such as Repligen KrosFlo® Research 2i Tangential Flow Filtration (TFF) System.INACTIVATED VIRUSES[000114] The vaccine compositions of the present disclosure may comprise an inactivated whole paramyxovirus and a further immunogen. In an embodiment, the vaccine compositions of the present disclosure may comprise an inactivated whole paramyxovirus and an inactivated influenza virus. In an embodiment, the inactivated paramyxovirus is a whole inactivated NDV. In an embodiment, the inactivated influenza virus is a whole inactivated influenza virus.[000115] Inactivation of the paramyxovirus and / or the influenza virus described herein can be achieved, for example, by use of any one or more of following methods: chemical inactivation exemplified by use of cross-linking agents such as formalin (e.g. 0.2% formalin) or alkylating agents such as beta-propiolactone, thermal (heat) treatment, radiation as exemplified by ultraviolet, photon, proton, heavy ion or low-energy electron irradiation, high hydrostatic pressure, pulsed electric field, ultrashort pulsed laser and ultra sound under pressure. Preferred methods of paramyxovirus inactivation include those which minimise the removal and / or denaturation of surface protein antigens.[000116] Non-limiting examples of suitable techniques for the viral inactivation and / or attenuation are disclosed in: Levinson etal. (1944) Production of potent inactivated vaccines with ultraviolet irradiation. JAMA 125, 532; Hartman FWand Lo Grippo GA (1957) Beta-propiolactone in sterilization of vaccines, tissue grafts and plasma. JAMA. 164, 258-260; Manas P and Pagan R (2005) Microbial inactivation by new methods of food preservation. Delrue I et al. (2012) Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev. Vaccines 11, 695-719; Park JC and Jung MH (2015) Study of the effects of high-energy proton beams on Escherichia coli. J. Korean Physical Soc. 67, 1454-1458; J. Appl. Microbiology.98, 1387-1399; Babb R etal. (2016) Intranasal vaccination with gamma-irradiated Streptococcus pneumoniae whole-cell vaccine provides serotype independent protection mediated by B-cells and innate IL-17 responses. Clin. Sci. 130, 697-710; Fertey J etal. (2016) Pathogens inactivated by low-energy-electron irradiation maintain antigenic properties and induce protective immune responses. Viruses 8, 319 doi: 10.3390 / v8110319; Sabbaghi A etal. (2019) Inactivation methods for whole influenza vaccine production. Rev. Medical Virology. 29 (6) e2074)).[000117] In some embodiments, the paramyxovirus and / or influenza virus are inactivated by exposure to radiation.IRRADIATION-INACTIVATED PARAMYXOVIRUS[000118] In an embodiment, the paramyxovirus in the vaccine compositions of the disclosure may be inactivated by expose to irradiation, that is, irradiation-inactivated paramyxovirus. In anembodiment, inactivation may be determined by measuring the infectiousness of the paramyxovirus following irradiation, for example, by sterility testing or measuring the 50% Tissue Culture Infectious Dose (TCID50) value of paramyxovirus following irradiation as described elsewhere herein. In an embodiment, the paramyxovirus 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 paramyxovirus is considered inactivated when, after irradiation, sterility testing indicates that viral infection is negligible, or undetectable, compared to paramyxovirus prior to inactivation.[000119] In an embodiment, the inactivated paramyxovirus is an irradiation-inactivated paramyxovirus. In an embodiment, the inactivated paramyxovirus is photon-inactivated paramyxovirus. The photon-inactivated paramyxovirus 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” paramyxovirus of the present disclosure may be “gamma-irradiated” by way of exposure to gamma-radiation (i.e., gamma-rays), “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 paramyxovirus is inactivated by exposure to photonradiation at energies of at least 0.01 MeV.[000120] The paramyxovirus 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.[000121] Gamma irradiation of paramyxovirus 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.[000122] Additionally or alternatively, paramyxovirus 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 IBA Industrial (Louvain-la-Neuve, Belgium). Other suitable devices include for example, the RS2400® and RS3400® manufactured by Rad Source Technologies Inc. (Suwanee, Georgia, USA).[000123] In general, the paramyxovirus 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 paramyxovirus is inactivated by photon exposure.[000124] 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.[000125] Paramyxovirus 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 paramyxovirus is exposed to a total dose of X-radiation and / or gammaradiation 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.[000126] In one embodiment, the paramyxovirus 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, theparamyxovirus 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 to 35 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.[000127] 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 paramyxovirus, the quantity of paramyxovirus present to be treated, the temperature of the paramyxovirus 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.[000128] The total dose of photon radiation (e.g., X-radiation and / or gamma-radiation) may be administered to the paramyxovirus of the present disclosure cumulatively over a period of time. For example, photon-radiation may be administered to the paramyxovirus 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.[000129] In one embodiment, preparations of paramyxovirus 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 ofphoton-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 paramyxovirus 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, more than 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 paramyxovirus preparations of the present disclosure.[000130] 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.[000131] In an embodiment, a gamma-irradiation dose of 2-100 kGy or 10-50 kGy may be effective for treating frozen paramyxovirus preparations. In an embodiment, a gamma-irradiation 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 paramyxovirus 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.[000132] In an embodiment, the treatment with gamma-irradiation is sufficient to inactivate the paramyxovirus 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.[000133] 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.[000134] 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.[000135] In an embodiment, the photon-irradiated paramyxovirus is prepared by purification, for example, by tangential flow filtration or sucrose density gradient ultracentrifugation, prior to exposure to photon radiation.[000136] In an embodiment, the irradiation-inactivated paramyxovirus is electron inactivated paramyxovirus. 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 paramyxovirus refers to both photon-inactivated and electron-inactivated paramyxovirus. The electron-inactivated paramyxovirus 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).[000137] 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.[000138] In an embodiment, a paramyxovirus 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 0.2MeV 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.[000139] 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 electrons may 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.[000140] 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 paramyxovirus is inactivated by exposure to electron irradiation at energies of at least 0.01 MeV.[000141] In an embodiment, paramyxovirus could be inactivated using a dose of electron irradiation of 50 kGy, 100 kGy or 200 kGy.[000142] 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.[000143] 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 kGy, 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.[000144] In an embodiment, the paramyxovirus 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.[000145] In an embodiment, the immunogenic composition or vaccine comprising the paramyxovirus 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.[000146] 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 electron radiation 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.[000147] 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.[000148] 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.[000149] 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.[000150] The applicable dose of electron radiation may be influenced by factors such as the purification method used to purify the paramyxovirus, the quantity of paramyxovirus present to be treated, the temperature of the paramyxovirus 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 ofelectron radiation applied over the period of exposure may be optimised to enhance the effectiveness of the treatment.[000151] 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.[000152] In one embodiment, irradiation of paramyxovirus 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, electron 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.[000153] 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.IRRADIATION-INACTIVATED INFLUENZA VIRUS[000154] In an embodiment, the influenza virus in the vaccine compositions of the disclosure may be inactivated by expose to irradiation, that is, irradiation-inactivated influenza. In an embodiment, inactivation may be determined by measuring the infectiousness of the influenza virus following irradiation, for example, by sterility testing or measuring the 50% Tissue Culture Infectious Dose (TCID50) value of influenza virus following irradiation as described elsewhere herein. In an embodiment, the influenza virus 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 influenza virus is considered inactivated when, after irradiation, sterility testing indicates that viral infection is negligible, or undetectable, compared to influenza virus prior to inactivation.[000155] In an embodiment, the inactivated influenza is photon-inactivated influenza. 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” influenza virus of the present disclosure may be “gamma-irradiated” by way of exposure to gamma-radiation (i.e., gamma-rays), “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 least0.5MeV, 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 influenza virus is inactivated by exposure to photonradiation at energies of at least 0.01 MeV.[000156] The influenza virus in the combination 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.[000157] Gamma irradiation of influenza 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.[000158] Additionally or alternatively, influenza 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 IBA Industrial (Louvain-la-Neuve, Belgium). Other suitable devices include for example, the RS2400® and RS3400® manufactured by Rad Source Technologies Inc. (Suwanee, Georgia, USA).[000159] In general, the influenza 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 influenza virus is inactivated by photon exposure.[000160] 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.[000161] Influenza 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 influenza virus is exposed to a total dose of X-radiation and / or gammaradiation 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.[000162] In one embodiment, the influenza 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 influenza virus of the present disclosure are exposed to a total photon radiation dose of about 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 to 35 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 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 50 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, 1.26 x 106rad (12.6 kGy), a total photon-radiation dose of about 1 x 106rad (about 10 kGy) photon-rays, or a total photon-radiation dose of about 1 x 105rad (1 KGy).[000163] 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 influenza virus, the quantity of influenza virus present to be treated, the temperature of the influenza 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.[000164] The total dose of photon radiation (e.g., X-radiation and / or gamma-radiation) may be administered to the influenza virus of the present disclosure cumulatively over a period of time. For example, photon-radiation may be administered to the influenza 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.[000165] In one embodiment, preparations of influenza 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 influenza types, subtypes and strains. In general, a photon-radiation dose of 10-30 kGy (as described herein) may be effective for treating preparations of frozen and / or lyophilised influenza virus of the present disclosure.Alternatively, a photon radiation dose of 20-40 kGy (e.g., more than 20, more than 22, more than 24, more than 26, more than 28, more than 30, more than 32, more than 34, more than 36, or more than 38 kGy, etc.) may be effective for treating preparations of frozen and / or lyophilised influenza virus preparations of the present disclosure.[000166] 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 against.[000167] In an embodiment, a gamma-irradiation dose of 10-50kGy may be effective for treating frozen influenza virus preparations. In an embodiment, a gamma-irradiation dose of 30-40kGy may be effective for treating frozen influenza virus preparations. In an embodiment, a gamma-irradiation dose of 30-40kGy may be effective for treating frozen influenza virus preparations. In an embodiment, the viral preparations may be on ice. In an embodiment, theviral preparations may be on an ice slurry. In an embodiment, the viral preparations may be on dry ice.[000168] In an embodiment, the treatment with gamma-irradiation is sufficient to inactivate the influenza 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.[000169] The integrity of antigenic determinants can be assessed, for example, by assaying the virus for hemagglutinating activity following gamma-irradiation. Methods of performing hemagglutination assays are known in the art and are described, for example, in Coico et al. (Eds), (2007), “Current Protocols in Microbiology”, John Wiley and Sons, Inc. (see in particular Unit 15G.1 entitled “Influenza: Propagation, Quantification, and Storage”); and Sato et al., (1983), “Separation and purification of the hemagglutinins from Bordetella pertussis”, Infect. Immun., 41, 313-320. 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.[000170] Additionally or alternatively, a neuraminidase assay may be used to assess the integrity of viral antigenic determinants (see, for example, Khorlin etal., (1970), “Synthetic inhibitors of Vibrio cholerae neuraminidase and neuraminidases of some influenza virus strains”, FEBS Lett., 8:17-19; and Van Deusen et al., (1983), “Micro neuraminidase-inhibition assay for classification of influenza A virus neuraminidases”, Avian Dis., 27:745-50).[000171] 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.[000172] In an embodiment, the photon-irradiated influenza virus is prepared by purification by tangential flow filtration prior to exposure to photon radiation.[000173] In an embodiment, the irradiation-inactivated influenza virus is electron inactivated influenza virus. 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 influenza virus refers to both photon-inactivated and electron-inactivated influenza virus. The electron-inactivated influenza virus is inactivated using electron irradiation. Electron irradiation is a process that involves using electrons, usually of highenergy, 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).[000174] 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.[000175] In an embodiment, a influenza virus 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.[000176] 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 electrons may 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.[000177] 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 influenza virus is inactivated by exposure to electron irradiation at energies of at least 0.01 MeV.[000178] In an embodiment, influenza virus could be inactivated using a dose of electron irradiation of 50 kGy, 100 kGy or 200 kGy.[000179] 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.[000180] 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 kGy, 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.[000181] In an embodiment, the influenza virus 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.[000182] In an embodiment, the immunogenic composition or vaccine comprising the influenza virus 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.[000183] 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 electron radiation 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.[000184] 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.[000185] 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.[000186] 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.[000187] The applicable dose of electron radiation may be influenced by factors such as the purification method used to purify the paramyxovirus, the quantity of paramyxovirus present to be treated, the temperature of the paramyxovirus 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.[000188] 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[000189] In one embodiment, irradiation of influenza virus 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, electron beam (e.g., such as Comet, EB-Lab200, Switzerland, and eFIT system or platform, Kyoobe Tech, Germany) at room temperature or cold conditions. The eFIT system may be particularly useful for large scale manufacturing.[000190] 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.[000191] The photon-irradiated influenza viruses of the present disclosure may be of any type, subtype or strain, or a mixture of any number of different types, subtypes and / or strains of influenza virus. In certain embodiments, gamma-irradiated viruses administered in accordance with the methods of the present invention are type A influenza virus, for example, A (H1 N1) or A (H3N2) influenza viruses.PROTEASE TREATMENT OF IRRADIATED PARAMYXOVIRUS[000192] The vaccine compositions of the present disclosure may comprise irradiation inactivated paramyxovirus 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.[000193] 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.[000194] In an embodiment, the paramyxovirus 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 paramyxovirus 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 paramyxovirus may then be administered as a vaccine composition as described herein.[000195]VACCINE COMPOSITIONS[000196] In an embodiment, the vaccine composition of the present disclosure may comprise an inactivated whole paramyxovirus and a further immunogen. In an embodiment, the inactivatedparamyxovirus enhances the immune response to the further immunogen. In an embodiment, the further immunogen is inactivated influenza virus. In an embodiment, the vaccine compositions of the present disclosure may comprise an inactivated paramyxovirus and an inactivated influenza virus. In an embodiment, the vaccine composition is a combination vaccine composition comprising an inactivated paramyxovirus and an inactivated influenza virus. In an embodiment, vaccine compositions of the present disclosure comprise a synergistic combination of inactivated paramyxovirus and inactivated influenza virus. In an embodiment, the inactivated paramyxovirus and inactivated influenza virus may be co-administered. In an embodiment, the inactivated paramyxovirus and inactivated influenza virus may be co-administered at substantially the same time, for example, within 1 day of each other, within 2 hours of each other, within 1 hour of each other, within 30 mins of each other, within 5 minutes of each other, within 1 minute of each other.[000197] In certain embodiments, compositions and vaccines of the present disclosure comprise single or multiple strains of inactivated influenza virus, and / or single or multiple strains of inactivated paramyxovirus. In other embodiments, compositions and vaccines of the present disclosure comprise a single irradiated strain of influenza virus in combination with a single type of irradiated paramyxovirus.[000198] Vaccine compositions of the present invention 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.METHODS AND USES OF VACCINE COMPOSITIONS[000199] The compositions of the present disclosure may be administered to a subject to vaccinate the subject against paramyxovirus and a further infectious agent. In this embodiment, the method comprises administering to the subject a therapeutically effective amount of a vaccine composition, wherein the vaccine composition comprises inactivated paramyxovirus and a further immunogen, and wherein the further immunogen induces a protective immune response against the further infectious agent.[000200] In an embodiment, compositions of the present disclosure may be administered to a subject to vaccinate the subject against paramyxovirus and / or inactivated influenza virus. In an embodiment, the present disclosure provides a method of vaccinating a subject against paramyxovirus infection, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a method of vaccinating a subject against influenza infection, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In anembodiment, the present disclosure provides a method of vaccinating a subject against paramyxovirus infection and influenza infection, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition.[000201] In an embodiment, the compositions of the present disclosure can be used in methods of inducing or enhancing an immune response against paramyxovirus and a further immunogen. In an embodiment, the present disclosure provides method for inducing or enhancing an immune response against an paramyxovirus infection and / or an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a method for inducing or enhancing an immune response against paramyxovirus infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a method for inducing or enhancing an immune response in a subject against influenza infection, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a method for inducing or enhancing an immune response in a subject against paramyxovirus infection and influenza infection, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition.[000202] In an embodiment, the compositions of the present disclosure can be used in methods of treating or preventing an paramyxovirus infection and an infection against a further pathogen, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition, wherein the vaccine composition comprises inactivated paramyxovirus and a further immunogen, and wherein the further immunogen induces a protective immune response against the further pathogen. In an embodiment, the present disclosure provides method for treating or preventing an paramyxovirus infection and / or an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a method for treating or preventing an paramyxovirus infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a treating or preventing an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition. In an embodiment, the present disclosure provides a treating or preventing an paramyxovirus infection and an influenza infection in a subject against, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition.IMMUNE RESPONSE TO VACCINE COMPOSITIONS[000203] Vaccine compositions of the present invention may be administered to naive recipients, being individuals seronegative for particular target strain(s) of influenza and / or seronegative for a particular target strains of paramyxovirus, or seronegative to an agent causative of a different disease or condition targeted by the vaccine composition. Alternatively, the vaccine compositions may be administered to primed recipients, being individuals seropositive for particular target strain(s) of influenza and / or seropositive for a particular agent causative of a different disease or condition targeted by the vaccine composition (e.g. secondary agent(s) associated with influenza infection).[000204] The level of immunogenicity induced by a composition or vaccine 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, 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, NK cell response, oxidative burst assays, and phagocytosis and apoptosis evaluation.[000205] For example, the composition comprising inactivated paramyxovirus and inactivated influenza virus of the present disclosure may induce an immune response to paramyxovirus and / or influenza virus. For example, the compositions of the present invention may induce a systemic and / or a mucosal immune response. The immune response may include the induction of systemic and / or mucosal antibodies that are specific for paramyxovirus and / or influenza. The antibodies induced by the composition may be neutralising antibodies that are protective against paramyxovirus and / or influenza. The compositions may induce increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response, increased influenza-specific neutralising antibody response, and / or an increased neuraminidase neutralising response.[000206] In an embodiment, the compositions may induce increased protection against paramyxovirus, increased survival against paramyxovirus, decreased paramyxovirus symptoms, increased paramyxovirus-specific antibody response, and / or increased paramyxovirus-specificneutralising antibody response in a subject, compared to control subjects that did not receive the vaccine composition.[000207] In an embodiment, the composition induces an immune response to the same type of influenza virus as the type of influenza virus present as inactivated influenza virus within the composition. In an embodiment, the composition induces heterotypic immune response against an influenza strain different to the inactivated influenza virus strain present within the composition. In an embodiment, the composition induces protection against a plurality of influenza strains in the subject.[000208] In an embodiment, the composition induces an immune response to the same type of paramyxovirus present as the type of paramyxovirus virus present within the composition. In an embodiment, the composition induces heterotypic immune response against an paramyxovirus strain different to the inactivated paramyxovirus virus present within the composition. In an embodiment, the composition induces protection against a plurality of paramyxovirus strains in the subject.INACTIVATED PARAMYXOVIRUS ENHANCES RESPONSE TO FURTHER IMMUNOGEN[000209] In an embodiment, the vaccine composition of the present disclosure may comprise an inactivated paramyxovirus and a further immunogen. The further immunogen may be an antigen for a particular agent causative of a different disease or condition targeted by the vaccine composition. The data shown here indicates that inactivated paramyxovirus may enhance the immune response raised to a further immunogen. In an embodiment, inactivated paramyxovirus provides an adjuvant effect for a further immunogen. In an embodiment, the further immunogen is a protective antigen for a particular agent causative of a different disease or condition targeted by the vaccine composition[000210] In an aspect, the present invention provides a method for inducing or enhancing an immune response against a further immunogen in a subject, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition comprising inactivated paramyxovirus and the further immunogen. In an aspect, the present disclosure provides a method for treating or preventing an infection with an agent causative of a different disease or condition targeted by the vaccine composition in a subject, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition comprising inactivated paramyxovirus and a further immunogen, wherein the further immunogen is an antigen for the agent causative of a different disease or condition targeted by the vaccine composition. In an embodiment, the method reduces the symptoms of an infection agent causative of a different disease or condition targeted by the vaccine composition.[000211] For example, where the composition comprises inactivated paramyxovirus and inactivated influenza, the inactivated paramyxovirus may enhance the immune response generated against influenza. For example, the data disclosed herein in shows that following intranasal vaccination with a composition comprising inactivated paramyxovirus and inactivated influenza, the inactivated paramyxovirus may induce increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response, increased influenza-specific neutralising antibody response, and / or an increased neuraminidase neutralising response compared to administering a therapeutically effective amount of influenza virus inactivated by irradiation in the absence of paramyxovirus inactivated by irradiation. In an embodiment, the inactivated paramyxovirus provides an adjuvant effect for the inactivated influenza virus.[000212] In a further example, the data disclosed herein shows that following intramuscular administration with a composition comprising inactivated paramyxovirus and inactivated influenza, the inactivated paramyxovirus may induce increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response, increased influenza-specific neutralising antibody response, and / or an increased neuraminidase neutralising response compared to administering a therapeutically effective amount of influenza virus inactivated by irradiation in the absence of paramyxovirus inactivated by irradiation. In an embodiment, the inactivated paramyxovirus provides an adjuvant effect for the inactivated influenza virus.[000213] In a further example, intranasal vaccination with inactivated paramyxovirus may induce increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response compared to naive subjects. In an embodiment, the inactivated paramyxovirus provides an adjuvant effect for the inactivated influenza virus.[000214] In a further example, intramuscular vaccination with inactivated paramyxovirus may induce increased influenza-specific antibody response compared to naive subjects.[000215] While not wanting to be bound by theory, in an embodiment, the inactivated paramyxovirus may provide an adjuvant effect for the inactivated influenza virus. In an embodiment, this effect is more pronounced following intranasal vaccination than following intramuscular vaccination. Alternatively or additionally, in an embodiment, the inactivated paramyxovirus and influenza antigens may cross react to enhance the immune response to influenza.[000216] Accordingly, in an aspect, the present invention provides a method for inducing or enhancing an immune response against an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition comprising inactivated paramyxovirus. In an aspect, the present disclosure provides a method for treating or preventing an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a vaccine composition comprising inactivated paramyxovirus. In an embodiment, the method reduces the symptoms of an influenza infection.SUBJECTS[000217] NDV primarily affects birds. Similarly, avian influenza 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. Accordingly, in an embodiment, the subject is avian. However, in an embodiment, the vaccine may be administered to laboratory animals such as mice, rats, rabbits, etc. In an embodiment, the vaccine composition is formulated for delivery to birds.[000218] Further, avian influenza strains have been reported in humans and animals, particularly humans and mammals that eat birds, or interact with or live in close proximity to and / or interact with birds in the wild, in captivity, commercially or domestically. Such animals may include bats, seals, bears, foxes, skunks, mink, domestic or stray animals (for example, cats, dogs, etc.), zoo animals (for example, monkeys and other primates, tigers, lions, cheetahs, leopards, puma, other big cats, polar bears), and farm animals (for example, goats, cows, sheep, horses, pigs). In an embodiment, the subject is mammal. In an embodiment, the subject is a nonhuman animal. In an embodiment, the subject is a non-human mammal. 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. In an embodiment, the vaccine composition is formulated for delivery to mammals.[000219] In an embodiment, the subject is human.FORMULATIONS[000220] 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 etal. (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.[000221] 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.[000222] 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.[000223] 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.[000224] 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 di stea rate.[000225] 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.[000226] 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.[000227] 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.[000228] 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.[000229] 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 anaqueous 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.[000230] 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.[000231] 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[000232] Adjuvant(s) may be included in compositions of the disclosure. However, experimental data provided herein demonstrates that the compositions disclosed herein can induce an immune response to paramyxovirus and / or influenza virus without requiring such adjuvants. Accordingly, compositions of the disclosure may or may not comprise an adjuvant.[000233] 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 the vaccine (e.g., an inactivated paramyxovirus and an inactivated influenza virus). 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. Alternatively, it may increase the intensity of the immune response induced in response to the immunogenic components in the vaccine (e.g., an inactivated paramyxovirus and an inactivated influenza virus).[000234] 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 other active component(s) (e.g., a lower dose of the irradiated influenza virus).[000235] 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.[000236] 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.[000237] 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.[000238] 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.[000239] 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).[000240] 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 thewater 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).[000241] 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.[000242] 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 etal. (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 etal., (1996), “Immunostimulatory DNA sequences necessary for effective intradermal gene immunization", Science, 273:352-354.[000243] 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-wateremulsion 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.[000244] 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[000245] 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). In an embodiment, the composition is administered via an intramuscular, intranasal or subcutaneous route.[000246] In an embodiment, the compositions may be administered by the intramuscular route.[000247] 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, genital tract (vaginal), rectal, intratracheal, skin, and the gastrointestinal tract.[000248] 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 the compositions may be advantageous for enhancing immunity against influenza virus as, forexample, 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.[000249] 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.[000250] 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.[000251] 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 first immunogen and a irradiated influenza virus 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).[000252] 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, et al., (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 et al., (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 etal., (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).[000253] 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. Intranasal devices may be capable of bi-dose delivery, that is, the delivery of two sub-doses of a single vaccination dose, one sub-dose to each nostril.[000254] 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.DOSAGES[000255] 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).[000256] 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 influenza 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.[000257] 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 first immunogen and a irradiated influenza virus). Dosage in an amount of from about 50 micrograms to about 500 micrograms is also preferred.[000258] One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a first immunogen and a irradiated influenza virus thereof to include in a vaccine of the disclosure for the desired therapeutic outcome.[000259] Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about 1000mg of active component(s) (i.e., a first immunogen and a irradiated influenza virus) 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 I. Omg 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.[000260] Alternatively, an effective dosage may be up to about 500mg / m2of active component(s) (e.g., a first immunogen and a irradiated influenza virus). 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.[000261] 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.[000262] 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 the paramyxovirus and the irradiated influenza virus. Alternatively, the compositions may be administered to primed recipients, being individuals seropositive for the paramxyovirus and the influenza virus.MEDICAMENTS AND KITS[000263] The inactivated paramyxovirus and inactivated influenza virus 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. Thus, “a medicament” as contemplated herein may comprise two ormore components capable of separate administration. Similarly, a kit may comprise irradiated influenza viruses and irradiated paramyxovirus as separate components.[000264] In embodiments where medicaments and kits of the present invention comprise inactivated influenza viruses and the inactivated paramyxovirus as separate components of a medicament or kit, the components will generally be for (i.e. “formulated for”) co-administration to a subject. It will be understood that “co-administration” of the components can occur at the same site or at a different site.EXAMPLES[000265] 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: PREPARATION OF v-FLU AND v-NDVPropagation, concentrating, and titration of NDV[000266] 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.[000267] 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[000268] 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. To ensure sterility, both live and y-NDV were passaged three times in embryonated eggs and tissue culture and shown to be sterile. Structural integrity was tested by hemagglutinin assay, neuraminidase assay and transmission electron microscopy. The results suggest that y-NDV is structurally intact and the surface proteins have normal functions (data not shown).Influenza virus propagation[000269] Influenza strain A / Puerto Rico / 8 / 1934(H 1 N1) (referred to as A / PR8) was used in these studies. A / Puerto Rico / 8 / 1934(H1N1) is a widely available laboratory adapted strain.[000270] The virus 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.Influenza virus purification[000271] Infectious influenza viruses were purified and concentrated from the allantoic fluid 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 was resuspended 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.[000272] The concentrated A / PR8 influenza samples were qualitatively analysed for the removal of egg proteins by SDS-PAGE and albumin-specific ELISA. The results indicate that the purification resulted in removal of allantoic fluid proteins (data not shown).Gamma irradiation of influenza virus[000273] The concentrated influenza samples were gamma-irradiated as follows. Concentrated 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. For this testing, live and y-flu were activated with TPCK-treated trypsin at 37° C for 30 minutes. MDCK cell monolayers were treated with the preparations at multiplicity of infection (MOI) of 40 or saline as a control and incubated for 24 hours. Supernatant was collected and used to infect fresh MDCKcell 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 mouse anti-lAV (in-house produced hyper immune sera specific to A / PR8 (H1N1)), using a dilution of 1:200 in PBS+1%BSA and anti-mouse IgG Alexa-FluorTM488-conjugated antibodies (Thermo fisher scientific, CAT#A-11001, using 1:500 dilution in PBS+1%BSA) to visualise lAV-infected cells. Lack of influenza-infected cells for passage 3 indicated a complete loss of virus infectivity. The results showed the gamma irradiated influenza was sterile (data not shown).Vaccination studies[000274] For vaccination studies, y-NDV and y-flu were separately resuspended in PBS. For covaccination, y-NDV and y-flu were mixed together in the same container prior to administration as one composition. Control mice were PBS-mock vaccinated.EXAMPLE 2: INTRANASAL VACCINATION WITH v-FLU AND y-NDV AND CHALLENGE[000275] BALB / c mice were vaccinated intranasally (IN) twice at two-week intervals with either y-Flu (1 x 107TCIDso-equivalent) or y-NDV (1 x 107TCIDso-equivalent) alone, or co-vaccinated with y-Flu+y-NDV (n= 4-5 per group). Serum samples were collected 2 weeks post primary and secondary vaccination for in vitro analysis. In addition, three weeks post 2nd vaccination, animals were challenged via the intranasal route with influenza A virus pdmH1N1 (A / California / 07 / 2009) using standard techniques. Influenza A virus pdmH1N1 (A / California / 07 / 2009) is a highly drifted influenza A strain obtained from WHO collaborating Centre for Reference and Research on Influenza, The Doherty Institute / Melbourne.Survival and weight loss[000276] Mice were monitored for clinical symptoms and weight loss, and humanely euthanised when weight loss was greater than 20% of their initial body weight.[000277] Weight loss was monitored (Figure 1A and 1C-H), and percentage survival based 20% body weight loss as a humane end point was investigated (Figure 1 B). Data represented as mean SEM of group (n=4-5 per group). Statistical analyses were performed using One-way ANOVA with Tukey’s multiple comparisons test (* p< 0.05, ** p<0.01), comparing individual weight differences at different days post-challenge.[000278] Mice vaccinated with y-Flu alone and the combination of y-Flu+y-NDV were protected from a highly drifted influenza challenge, showing a 100% survival and demonstrating that the coadministration of y-Flu+y-NDV did not negatively impact the protective efficacy of y-Flu. Further,mice vaccinated with y-NDV show transient protection against influenza challenge as observed by reduced weight loss prior to development of clinical symptoms.A / PR8-specific total IqG responses[000279] Serum samples were harvested 2 weeks post primary and secondary vaccination via submandibular bleeding, and tested for A / PR8-specific total IgG via ELISA, with results shown in Figure 2. Titers were calculated relative to naive serum. Here, maxisorp ELISA plates were coated with live A / PR8 (2 x 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.[000280] Fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using 1 / 100 dilution for primary response (Figure 2B) or a 1 / 200 dilution for secondary response (Figure 2D). Data is presented as mean ± SEM and was analysed by two-way ANOVA with Tukey’s multiple comparisons test (** p< 0.01, *** p<0.001, **** p<0.0001).[000281] The co-administration of y-Flu with y-NDV resulted in an enhancement of A / PR8-specific response compared to vaccination with y-Flu alone. Further, mice that were vaccinated with y-NDV alone showed cross-reactivity towards A / PR8 antigen, with levels appeared similar to those obtained for serum from animals vaccinated with y-Flu alone. Accordingly, the results indicate that y-NDV enhances the immune response against influenza following intranasal administration.A / PR8-specific neutralising IqG responses[000282] Serum samples harvested two weeks after the second vaccination (termed immune serum) was assessed for A / PR8-specific IgG responses. Briefly, live A / PR8 virus was pre-treated with serially diluted immune serum and then added to monolayers of MDCK cells at a multiplicity of infection (MOI) of 0.1 to assess neutralisation of infection. Cells were fixed and stained withFITC-labelled anti-PR8 antibody and a fluorescence microscope was used to quantify FITC-fluorescence (representative of A / PR8 infection) relative to DAPI-fluorescence (cell nuclei) at 1:320 and 1:640 serum dilution. Representative images were also taken of each sample at a 1:160, 1:320, and 1:640 serum dilution (representative of 4 wells tested per serum group, compiled from 2 independent experiments). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparisons test (** p<0.01, ** p<0.001, **** p<0.0001).[000283] At a serum dilution of 1:160, sera from both y-Flu-vaccinated mice and y-Flu+y-NDV-vaccinated mice showed complete neutralisation of A / PR8 (data not shown). At a serum dilution of 1 :320 and 1 :640, sera from y-Flu+y-NDV vaccinated animals show significantly higher levels of A / PR8 neutralisation compared to sera from animals vaccinated with y-Flu only (Figure 3A, 3B). Accordingly, the results show that the intranasal co-administration of y-Flu with y-NDV resulted in an enhanced neutralising antibody responses against influenza compared to vaccination with y-Flu alone. However, intranasal vaccination with y-NDV alone did not appear to enhance the neutralising antibody response to influenza.Neuraminidase inhibition against A / PR8[000284] Neuraminidase inhibition against A / PR8 in serum samples harvested on day 13 after second intranasal vaccination with y-Flu alone, y-NDV alone, y-Flu+y-NDV, or a PBS-mock vaccine (Naive) was investigated. Briefly, live A / PR8 was serially diluted in PBS, and equal volume of pooled, diluted immune serum from each respective groups were added to each virus dilution. A virus-only control contained live A / PR8 serially diluted in PBS, and equal volume of PBS. 25 .L of 0.125mM 2’-(4-Methylumbelliferyl)-a-D-N-acetylneuraminic acid (4-MUNANA, Sigma M8639) was added to virus samples and allowed to incubate for an hour in the dark at 37°C, with gentle shaking every 15 minutes. 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. The reaction was stopped with the addition of ice-cold 0.5M Na2COs, and fluorescence was measured with a SpectraMax fluorescent plate reader at wavelengths of 365 / 450nm. Activity of neuraminidase was quantified via interpolation of a standard curve generated using serially diluted 4-Methylumbelliferyl (4-Mu). Percentage neutralisation of live virus was quantified by comparing the activity of neuraminidase of the serum from each respective group compared to the virus-only control. Quantitative data was presented as mean ± SEM (n= 2 technical replicates) and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, **** p < 0.0001).[000285] As shown in Figure 4, sera from y-NDV vaccinated animal does not neutralise neuraminidase of A / PR8. While sera from y-A / PR8 vaccinated animals show significant inhibition of neuraminidase activity, a significantly higher level of neuraminidase inhibition was observed for sera from y-Flu+y-NDV co-vaccinated animals. Intranasal vaccination with y-NDV and y-flu resulted in a synergistic increase in neuraminidase inhibition against A / PR8 compared to either y-NDV or y-flu alone.Cross-reactive A / California or A / PC specific IgG responses following intranasal vaccination[000286] As above, BALB / c mice were vaccinated intranasally twice at two-week intervals with either y-Flu (i.e., y-A / PR8 (H1N1) flu at 1 x 107TCID50-equivalent) or y-NDV (1 x 107TCID50-equivalent) alone, or co-vaccinated with y-Flu (A / PR8) +y-NDV as described in Example 2. Control mice were PBS-mock vaccinated (naive).[000287] To assess the breadth of cross-reactive antibody responses induced by y-NDV against different influenza strains, serum samples harvested 2 weeks post-secondary vaccination via submandibular bleeding were tested for A / California (H1N1) and A / PC (H3N2)-specific total IgG via ELISA. Here, maxisorp ELISA plates were coated with A / California or A / PC (2 x 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. Titers were calculated based on a cut-off determined from the mean + 3 x standard deviation of the naive control absorbance values at a 1 / 200 dilution.[000288] In addition to inducing an antibody response that is cross- reactive with A / PR8 influenza as discussed in Example 2, y-NDV induced an antibody response that is cross-reactive against A / California and A / PC influenza strains. Mice vaccinated intranasally with y-NDV alone induced comparable levels of A / California-specific (Figure 5A, 5B) and A / PC-specific (Figure 5 C, 5D) IgG compared to the IgG induced by y-Flu+y-NDV vaccination. Notably, vaccination with y-A / PR8 alone via the intranasal route induces limited cross- reactive antibodies towards other strains of influenza, with antibody responses induced in mice vaccinated with y-NDV alone or y-Flu+y-NDV were significantly higher than the respective antibody responses induced by y-Flualone. This indicates that co-vaccination with y-NDV enhances the breadth of cross-reactive antibody responses towards different influenza strains.A / California specific neutralising responses following intranasal vaccination[000289] Serum samples collected 2 weeks post second intranasal vaccination of BALB / c mice with either y-Flu (y-A / PR8) alone, y-NDV alone, y-Flu+y-NDV, or a PBS-mock vaccine (Naive) were also assessed for neutralisation of infection against A / California (H1 N1) with an A / California-specific neutralisation using Focus Forming inhibition assay (FFIA). Sera were collected 13 days post-secondary vaccination and tested for A / California-specific neutralisation using Focus Forming inhibition assay (FFIA). Live A / California virus was pre-treated with pooled immune serum from 4-5 mice of each respective group, then added to monolayers of MDCK cells at an MOI of 0.1 to assess neutralisation of infection. A fluorescence microscope was used to guantify FITC-fluorescence (representative of A / PR8 infection) relative to DAPI-fluorescence (cell nuclei) at 1:80 serum dilution. Representative images were also taken of each sample at a 1:80 serum dilution (representative of 4 wells tested per serum group, compiled from 2 independent experiments). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparisons test.[000290] Despite detecting high cross-reactive antibody responses, serum samples collected from mice vaccinated intranasally with y-NDV alone, or y-Flu (y-A / PR8)+y-NDV were not neutralising against A / California H1N1. As shown in Figure 6, serum samples from all vaccinated groups had shown eguivalent levels of infection in comparison to the naive control and virus-only control, indicating that the cross- reactive antibodies observed in Figure 5 were not crossneutralising in nature.EXAMPLE 3: INTRAMUSCULAR VACCINATION WITH v-FLU AND y-NDV AND CHALLENGE[000291] BALB / c mice were vaccinated intramuscularly (IM) twice using two-week intervals with either y-Flu (1 x 107TCIDso-eguivalent) or y-NDV (1 x 107TCIDso-eguivalent) alone, or covaccinated with y-Flu+y-NDV. Control mice were PBS-mock vaccinated. Serum samples were collected 2 weeks post primary and secondary vaccination for in vitro analysis. Three weeks post second vaccination, animals were challenged with influenza A virus pdmH1N1 (A / California / 07 / 2009) as above.Survival and weight loss[000292] Mice were monitored for clinical symptoms and weight loss over time (Figure 7A, 7C, 7D), and humanely euthanised when weight loss was greater than 20% of their initial body weight.Survival is shown in Figure 7B. Co-vaccination with y-Flu and y-NDV resulted in an enhanced survival against lethal challenge with drifted strain of influenza A virus following intramuscular challenge, in contrast to vaccination with y-Flu only (Figure 7B).A / PR8-specific IgG responses[000293] BALB / c mice were vaccinated IM twice with either y-Flu or y-NDV alone, or covaccinated with y-Flu+y-NDV. Control mice were PBS-mock vaccinated (naive). Sera were collected 2 weeks post-primary and secondary vaccination via submandibular bleeding and tested for A / PR8-specific total IgG via ELISA as above. Fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using 1 / 100 dilution for primary response (Figure 8B) or a 1 / 200 dilution for secondary response (Figure 8D). Data is presented as mean ± SEM, and was analysed by two-way ANOVA with Tukey’s multiple comparisons test (** p< 0.01, **** p<0.0001).[000294] As shown in Figure 8, co-vaccination with y-Flu and y-NDV induced similar A / PR8-specific total IgG responses compared to vaccination with y-Flu alone. However, sera from mice that were vaccinated with y-NDV alone surprisingly induced significant increase in A / PR8-specific total IgG response compared to naive sera, which was similar to that observed with serum from vaccinated y-flu alone, suggesting cross-reactivity towards A / PR8.A / PR8-specific neutralising IgG responses[000295] Serum samples were harvested 2 weeks post second intramuscular vaccination with either y-Flu alone, y-NDV alone, y-Flu + y-NDV, or a PBS-mock vaccine (Naive). Live A / PR8 virus was pre-treated with pooled immune serum from 4-5 mice of each respective group, then added to monolayers of MDCK cells at an MOI of 0.1 to assess neutralisation of infection. A fluorescence microscope was used to guantify FITC-fluorescence (representative of A / PR8 infection) relative to DAPI-fluorescence (cell nuclei) at 1 :320 and 1 :640 serum dilution. Representative images were also taken of each sample at a 1:160, 1:320, and 1:640 serum dilution (representative of 4 wells tested per serum group, compiled from 2 independent experiments). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparisons test (**** p<0.0001).[000296] As shown in Figure 9A and 9B, , there was a significant reduction in FITC fluorescence following treatment of live A / PR8 with sera from y-Flu-vaccinated relative to sera from PBS-mock vaccinated mice and the virus-only control (at a serum dilution of up to 1:320). However, there were no detectable differences in FITC fluorescence between sera from y-Flu-vaccinated and co-vaccinated mice, indicating that the vaccination regimes were equivalent in their neutralising potency. Accordingly , there was no enhancement in neutralisation of A / PR8 by sera from mice intramuscularly vaccinated with y-NDV and y-flu compared to those intramuscularly vaccinated with y-flu alone.Neuraminidase inhibition against A / PR8[000297] Neuraminidase inhibition against A / PR8 in serum samples harvested 2 weeks post second intramuscular vaccination with either y-Flu alone, y-NDV alone, y-Flu+y-NDV, or a PBS-mock vaccine (Naive) was investigated. Live A / PR8 was serially diluted in PBS, and equal volume of pooled, diluted immune serum from each respective groups were added to each virus dilution.0.125mM substrate was then subsequently added, and allowed to incubate for an hour in the dark at 37°C, with gentle shaking every 15 minutes. Reaction was stopped with the addition of ice-cold 0.5M Na2CO3, and fluorescence was measured with a SpectraMax fluorescent plate reader at wavelengths of 365 / 450nm. Activity of neuraminidase was quantified via interpolation of a standard curve generated using serially diluted 4-Methylumbelliferyl (4-Mu). Percentage of neutralisation of live virus was quantified by comparing the activity of neuraminidase by serum from each respective groups versus that of the virus-only control. Quantitative data was presented as mean ± SEM (n= 2 technical replicates), and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, **** p < 0.0001).[000298] As shown in Figure 10, no inhibition of neuraminidase activity of A / PR8 was detected when the virus was incubated with immune sera from y-NDV vaccinated animals, whereas immune sera from animals vaccinated with y-A / PR8 induced significant levels of neuraminidase inhibition compared to control groups. Interestingly, intramuscular co-vaccination of y-A / PR8 with y-NDV resulted in significant enhancement in the ability of immune sera to inhibit neuraminidase activity compared to sera from animals vaccinated with y-A / PR8 alone.EXAMPLE 4: NDV SPECIFIC IgG RESPONSES[000299] To confirm the immunogenicity of y-NDV following coadministration with y-Flu, serum samples from intranasally and intramuscularly vaccinated animals (discussed above) were tested for NDV-specific responses.NDV-specific IgG responses following intranasal vaccination[000300] BALB / c mice were vaccinated IN twice (2 weeks apart) with either y-Flu or y-NDV alone, or co-vaccinated with y-Flu + y-NDV. Control mice were PBS-mock vaccinated (naive).Sera were collected 2 weeks post-secondary vaccination via submandibular bleeding, and tested for NDV-specific total IgG via direct ELISA.[000301] Briefly, maxisorp ELISA plates were coated with live whole-NDV (2 x 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 times 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. Foldincrease in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using 1 / 200 dilution. Data is presented as mean ± SEM, and was analysed by two-way ANOVA with Tukey’s multiple comparisons test (* p< 0.05, *** p<0.001, **** p<0.0001).[000302] As shown in Figures 11A and 11B, mice vaccinated with y-NDV alone or y-Flu and y-NDV induced comparable levels of NDV-specific IgG. This shows that the immunogenicity of y-NDV was maintained following intranasal co-administration with y-Flu. Vaccination with y-Flu alone via the intranasal route did not induce cross- reactivate antibodies against NDV.NDV-specific IgG responses following intramuscular vaccination[000303] BALB / c mice were vaccinated IM twice (2 weeks apart) with either y-Flu or y-NDV alone, or co-vaccinated with y-Flu + y-NDV. Control mice were PBS-mock vaccinated (naive). Sera were collected 2 weeks post-secondary vaccination via submandibular bleeding, and tested for NDV-specific total IgG via ELISA as described above. Fold-increase in antibody responses was calculated based on absorbance values obtained for immune sera from vaccinated animals relative to the naive sera using 1 / 200 dilution. Data is presented as mean ± SEM, and was analysed by two-way ANOVA with Tukey’s multiple comparisons test (* p< 0.05, *** p<0.001 , **** p<0.0001).[000304] As shown in Figures 12A and 12B, mice vaccinated with y-NDV alone or y-Flu + y-NDV showed comparable levels of NDV-specific IgG. This shows that the immunogenicity of y-NDV was maintained following intramuscular co-administration with y-Flu. Vaccination with y-Flu via IM route did not induce cross-reactivate antibodies against NDV.NDV-specific neutralising IqG response following intramuscular vaccination[000305] BALB / c mice were vaccinated intramuscularly twice at two-week intervals with either y-FIu (i.e., Y-A / PR8 (H1N1) flu at 1 x 107TCID50-equivalent) or Y-NDV (1 x 107TCID50-equivalent) alone, or co-vaccinated with y-Flu (A / PR8) +Y-NDV as described in Example 3. Control mice were PBS-mock vaccinated (naive). Sera collected 13 days post-secondary vaccination were tested for neutralising antibody responses to NDV. Live NDV virus was pretreated with pooled immune serum from 4-5 mice of each respective group, then added to monolayers of MDCK cells at an MOI of 0.1 to assess neutralisation of infection. A fluorescence microscope was used to quantify FITC-fluorescence (representative of NDV infection) relative to DAPI-fluorescence (cell nuclei). Representative images were also taken of each sample at a 1:80 serum dilution (representative of 4 wells tested per serum group, compiled from 2 independent experiments). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparisons test.[000306] As shown in Figure 13, there were no significant differences in FITC fluorescence (i.e., neutralisation of live NDV) with sera from Y-NDV alone, Y-FIU alone, or Y-FIU + Y-NDV- vaccinated mice, relative to sera from PBS-mock vaccinated mice, and the virus-only control. This indicates that intramuscular vaccination with Y-NDV does not induce neutralising antibody responses against NDV, and that co-vaccination with y-NDV + Y-FIU has no impact on the immunogenicity of Y-NDV.EXAMPLE 5: INTRAMUSCULAR VACCINATION WITH y-FLU AND TRYPSIN-TREATED y-NDV[000307] y-NDV was prepared as described above and then treated with 5 .g / mL of TPCK-trypsin at 37°C for 30 minutes. The resulting trypsin-treated y-NDV was termed y-NDVtiyp. y-flu (A / PR8) was prepared as described above. BALB / c mice were vaccinated IM twice (2 weeks apart) with either y-FIu (1 x 107TCIDso-equivalent) ory-NDVtiyp(1 x 107TCIDso-equivalent) alone, or co-vaccinated with y-FIu + y-NDVtryp. Control mice were PBS-mock vaccinated (naive). Sera were collected 2 weeks post primary and secondary vaccination via submandibular bleedingNDV-specific total IqG responses following intramuscular vaccination[000308] Serum samples were tested for NDV-specific total IgG via ELISA as described above. Titers were calculated based on a cut-off determined from the mean + 3 x standard deviation of the naive control absorbance values at a 1 / 100 dilution for post-primary response or a 1 / 200 dilution for post-secondary response.[000309] As shown in Figures 14A and 14B, mice vaccinated with y-NDVtrypalone or y-Flu+y-NDVtiypshowed comparable levels of NDV-specific IgG post-primary vaccination. As shown in Figures 14C and 14D, mice vaccinated with y-NDVtwalone or Y-Flu+Y-NDVtiypshowed comparable levels of NDV-specific IgG post-secondary vaccination. This shows that the immunogenicity of Y-NDVtiypwas maintained following intramuscular co-administration with Y-FIU. Vaccination with y-FIu via IM route did not induce cross- reactive antibodies against NDV.NDV-specific lgG1 and lgG2a subclass responses following intramuscular vaccination[000310] Immune sera collected 2 weeks post-secondary vaccination were also tested for NDV-specific IgG subclass responses using ELISA.[000311] Direct ELISA for NDV-specific lgG1 was performed as described above for NDV-specific total IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG1 secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000312] Direct ELISA for NDV-specific lgG2a was performed as described above for NDV-specific total IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG2a secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000313] 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 / 200 dilution. lgG1 / lgG2a ratio of the titres of each biological replicate was determined. Horizontal lines at 0.5 and 2.0 defines the patterns of antibody responses. A ratio of < 0.5 indicates a Th1-polarised response, while a ratio of > 2.0 indicates a Th2-polarised response. A ratio between 0.5 and 2.0 indicates a mixed response. Data is presented as mean ± SEM, and was analysed by one-way ANOVA with Tukey’s multiple comparisons test.[000314] As shown in Figures 15A, 15B, 15C and 15D, despite having limited impact on the total IgG responses, intramuscular co-administration of y-NDVtiypwith y-FIu induces a significantly lower NDV-specific lgG1 titre, as well as a significantly higher NDV-specific lgG2a titre than y-NDVtrpalone. This is associated with the change of NDV-specific response from a Th2-type response to a balanced Th1 / Th2 response, with the lgG1 / lgG2a ratio sitting within the 0.5-2 range for intramuscular co-administration of y-NDVtrypwith Y-FIU (Figure 15E).NDV-specific neutralising IgG responses[000315] Serum samples collected 2 weeks post second intramuscular vaccination with either Y-FIU alone, y-NDVtrypalone, Y-FIU + Y-NDVtiyp, or a PBS-mock vaccine (naive) were alsoassessed for neutralisation of infection against NDV. Live NDV virus was pre-treated with pooled immune serum from 4-5 mice of each respective group, then added to monolayers of MDCK cells at an MOI of 0.1 to assess neutralisation of infection. A fluorescence microscope was used to quantify FITC-fluorescence (representative of NDV infection) relative to DAPI-fluorescence (cell nuclei) at a 1:20 and 1:40 serum dilution. Representative images of infected Vero cells at a 1:20 and 1:40 serum dilution (representative of 4 wells tested per serum group compiled from 2 independent experiments) were visualised using Nikon TiE inverted fluorescence microscope. DAPI (blue) indicates cell nuclei, and FITC (green) indicates NDV. FITC fluorescence relative to DAPI-fluorescence was quantified at each serum dilutions using NIS elements software. Quantitative data was presented as mean ± SEM and analysed by two-way ANOVA with Tukey’s multiple comparison (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).[000316] As shown in Figure 16A and 16B, respectively, at serum dilution of 1:20 and 1:40, sera from PBS-mock and y-Flu-vaccinated mice has no impact on NDV infectivity, as illustrated by the comparable FITC fluorescence level compared to the virus-only control. There was a significant reduction in FITC-fluorescence following treatment of live NDV with immune serum collected from mice vaccinated with trypsin-treated NDV (y-NDVtiyp), relative to sera from PBS-mock vaccinated mice and the virus-only control, indicating that y-NDVtrypinduces neutralising antibody responses against NDV following intramuscular administration. However, there were no detectable differences in FITC fluorescence between serum from y-NDVtiypvaccinated mice and serum from y-Flu + y-NDVtrypco-vaccinated mice, indicating that the vaccination regimes were equivalent in their neutralising potency. This suggests that intramuscular vaccination with y-NDVtiypinduces neutralising antibody responses against NDV, and the co-vaccination with y-Flu has no impact on the immunogenicity of y-NDVtiyp.Flu (A / PR8)-specific total IqG responses[000317] Serum samples collected 2 weeks post-primary and post-secondary intramuscular vaccination with either y-Flu (A / PR8) alone, y-NDVtrypalone, y-Flu + y-ND , or a PBS-mock vaccine (Naive) were also assessed for A / PR8-specific total IgG responses. Titres were calculated based on a cut-off determined from the mean + 3 x S.D. of the naive control absorbance values at either a 1 / 100 or a 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)[000318] Here, maxisorp ELISA plates were coated with live A / PR8 (2 x 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 collectedfrom 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. Titers were calculated based on a cut-off determined from the mean + 3 x standard deviation of the naive control absorbance values at a 1 / 100 dilution for post primary response or a 1 / 200 dilution for secondary response. Data is presented as mean ± SEM and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (* p<0.05).[000319] Similar to above, the intramuscular co-administration of y-Flu with y-NDVtiypresulted in an enhancement of A / PR8-specific total IgG response compared to vaccination with y-Flu alone following both the primary and secondary vaccinations (Figures 17A, 17B, 17C, 17D). Further, mice that were vaccinated with y-NDVtiypalone showed cross-reactivity towards A / PR8 antigen. Accordingly, these results indicate that y-NDVtiypenhances the immune response against influenza following intramuscular co-administration with y-Flu.Flu (A / PR8)-specific lgG1 and lgG2a responses[000320] BALB / c mice were vaccinated IM twice with y-Flu, trypsin-treated y-NDV (y- N DVtryp) , or a combination of y-Flu+y-NDVtryp. Control mice were PBS-mock vaccinated (naive). Sera were collected 2 weeks post-secondary vaccination and tested for A / PR8-specific lgG1 and lgG2a responses by ELISA. Direct ELISA for A / PR8-specific IgG 1 was performed as described above for A / PR8-specific total IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG1 secondary antibody (1:10,000 dilution in blocking buffer, Abeam). Direct ELISA for A / PR8-specific lgG2a was performed as described above for A / PR8-specific total IgG with the exception of using horseradish peroxidase conjugated goat-anti mouse lgG2a secondary antibody (1:10,000 dilution in blocking buffer, Abeam).[000321] 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 / 200 dilution. lgG1 / lgG2a ratio of the titres of each biological replicate was determined. Horizontal lines at 0.5 and 2.0 defines the patterns of antibody responses. A ratio of < 0.5 indicates a Th1 -polarised response, while a ratio of > 2.0 indicates a Th2-polarised response. Ratio between 0.5 and 2.0 indicate a mixed response. Data is presented as mean ± SEM, and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01).[000322] The intramuscular co-administration of y-Flu with y-NDVtrypresulted in significantly higher A / PR8-specific lgG2a responses (Figure 18A, 18B) and a trend for a higher A / PR8-specific lgG1 response (Figure 18C, 18D), relative to the group that have received only y-flu. Notably, the cross-reactive antibody responses induced by y-NDVtrypis based on I gG 1 or Th2 responses, and limited lgG2a responses (Figure 18A, 18B, 18C, 18D, 18E).Flu (A / PR8)-specific neutralising IgG responses[000323] Serum samples harvested 2 weeks post second intramuscular vaccination with either y-Flu alone, y-NDVtiypalone, y-Flu + y-NDVtryp, or a PBS-mock vaccine (naive), were tested for the ability to neutralize A / PR8. Live A / PR8 virus was pre-treated with pooled immune serum from 4-5 mice of each respective group, then added to monolayers of MDCK cells at an MOI of 0.1 to assess neutralisation of A / PR8 infectivity. A fluorescence microscope was used to quantify FITC-fluorescence (representative of A / PR8 infection) relative to DAPI-fluorescence (cell nuclei) at 1:80 and 1:320 serum dilution. Representative images were also taken of each sample at a 1:80, and 1:320 serum dilution (representative of 4 wells tested per serum group, compiled from 2 independent experiments). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparisons test (*p<0.05, **p<0.01, **** p<0.0001).[000324] As shown in Figure 19A, at a serum dilution of 1:80, sera from both y-Flu-vaccinated mice and Y-Flu+y-NDVtiyp-vaccinated showed complete neutralisation of A / PR8. As shown in Figure 19B, at a serum dilution of 1:320, sera from v-Flu+v-NDVtrypvaccinated animals show significantly higher levels of A / PR8 neutralisation compared to sera from animals vaccinated with y-Flu only. Accordingly, the results show that the intramuscular co-administration of y-Flu with y-NDVtrypresulted in an enhanced neutralising antibody responses against influenza compared to vaccination with y-Flu alone. However, intramuscular vaccination with y-NDVtrypalone did not appear to induce a neutralising antibody response to A / PR8 as compared to intramuscular vaccination with y-Flu relative to sera from naive (PBS-mock vaccinated) mice and the virus-only control at a serum dilution of 1:80.Inhibition of A / PR8 Neuraminidase activity[000325] Serum samples harvested 2 weeks post second intramuscular vaccination with either y-Flu alone, y-NDVtrypalone, Y-Flu+Y-NDVtiyp, or a PBS-mock vaccine (naive) were tested for the ability to inhibit the neuraminidase enzymatic activity of A / PR8. Live A / PR8 was serially diluted in PBS, and equal volume of pooled, diluted immune serum from each respective groups were added to each virus dilution. 0.125mM substrate was then subsequently added, and allowed to incubate for an hour in the dark at 37°C, with gentle shaking every 15 minutes. Reaction wasstopped with the addition of ice-cold 0.5M Na2COs, and fluorescence was measured with a SpectraMax fluorescent plate reader at wavelengths of 365 / 450nm. Activity of neuraminidase was quantified via interpolation of a standard curve generated using serially diluted 4-Methylumbelliferyl (4-Mu). Percentage of neutralisation of live virus was quantified by comparing the activity of neuraminidase by serum from each respective groups versus that of the virus-only control. Quantitative data was presented as mean ± SEM (n= 2 technical replicates), and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (**** p < 0.0001).As shown in Figure 20, no inhibition of neuraminidase activity of A / PR8 was detected when the virus was incubated with immune sera from y-NDVtiypvaccinated animals, whereas immune sera from animals vaccinated with y-A / PR8 induced significant levels of neuraminidase inhibition compared to control groups. Interestingly, as above, intramuscular co-vaccination of y-A / PR8 with y-NDVtiypresulted in significant increase in inhibition of neuraminidase activity compared to sera from animals vaccinated with y-A / PR8 alone.Influenza-specific cross-reactive IgG responses following intramuscular vaccination[000326] BALB / c mice were vaccinated IM twice with y-Flu (y-A / PR8), trypsin-treated y-NDV (y-NDVtryp), or a combination of v-Flu+v-NDVtryp. Control mice were PBS-mock vaccinated (naive). Serum samples harvested 2 weeks post-secondary intramuscular vaccination with either y-Flu alone, y-NDVtrypalone, Y-Flu+y-NDVtiyp, and naive control group were tested using ELISA for cross-reactive IgG against for influenza A viruses (A / Sydney (H1N1), and A / PC (H3N2)) and influenza B virus (B / Austria / 1359417 / 2021). Titers were calculated based on a cut-off determined from the mean + 3 x standard deviation of the naive control absorbance values at a 1 / 200 dilution. Here, maxisorp ELISA plates were coated with live A / Sydney (H1N1), live A / PC (H3N2) or live B / Austria / 1359417 / 2021 (B / Victoria lineage) at 2 x 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.[000327] As shown in Figures 21A, 21 B and 21C, mice vaccinated with y-NDVtrypalone or y-Flu+Y-NDVtiypinduced detectable levels of A / Sydney-specific, A / PC-specific, and B / Austria-specific IgG. Notably, vaccination with y-A / PR8 alone via the intramuscular route did not induce cross-reactive antibodies towards any strains of influenza. This indicates that co-administration of y-NDVtrypwith y-Flu is associated with inducing cross- reactive antibodies that react with different influenza strains not included in vaccine formulation.Inhibition of Neuraminidase activity of A / Svdney (H1N1), A / PC (H3N2), and B / Austria[000328] Given the cross-reactive antibodies detected above, inhibition of neuraminidase activity of A / Sydney (H1N1), A / PC (H3N2), and B / Austria (Influenza B) were tested. Serum samples harvested 2 weeks post second intramuscular vaccination with either y-Flu alone, y-NDVtiypalone, v-Flu+y-NDVtryp, or a PBS-mock vaccine (naive) were tested for the ability to inhibit neuraminidase activity of different influenza strains. Live viruses (A / Sydney (H1N1), A / PC (H3N2), and B / Austria (B / Victoria lineage) were serially diluted in PBS, and equal volume of pooled, diluted immune serum from each respective groups was added to each virus dilution.0.125mM substrate was then subsequently added, and allowed to incubate for an hour in the dark at 37°C, with gentle shaking every 15 minutes. Reaction was stopped with the addition of ice-cold 0.5M Na2CO3, and fluorescence was measured with a SpectraMax fluorescent plate reader at wavelengths of 365 / 450nm. Activity of neuraminidase was quantified via interpolation of a standard curve generated using serially diluted 4-Methylumbelliferyl (4-Mu). Percentage of neutralisation of live virus was quantified by comparing the activity of neuraminidase by serum from each respective groups versus that of the virus-only control. Quantitative data was presented as mean ± SEM (n= 2 technical replicates), and was analysed by one-way ANOVA with Tukey’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).[000329] As shown in Figures 22A, 22B and 22C, serum samples from mice co-vaccinated with Y-Flu(y-A / PR8) and Y-NDVtiypshow significant inhibition of the neuraminidase activity of A / Sydney (H1N1), A / PC (H3N2) and B / Austria influenza strains compared to sera from the control group and sera from animals vaccinated with y-A / PR8 alone or Y-NDVtiypalone. No inhibition of neuraminidase activity of A / Sydney, A / PC, and B / Austria was detected for sera from y-NDVtrypand Y-A / PR8 vaccinated animals compared to the control group.[000330] Vaccination with y-A / PR8 alone or Y-NDV alone did not induce antibody responses capable of cross-neutralising neuraminidase activity of different influenza strains from influenza A and influenza B. In contrast, co-administration of y-A / PR8 and y-NDV resulted in the induction of cross-reactive immune responses capable of neutralising neuraminidase activity of different influenza strains.Lack of cross-neutralising IqG responses following intramuscular vaccination[000331] Serum samples collected 2 weeks post-secondary intramuscular vaccination with either y-Flu alone, y-NDVtrpalone, y-Flu + y-NDVtrp, or a PBS-mock vaccine (naive) were assessed for the ability to neutralize the infectivity of A / Sydney (H1N1), A / PC (H3N2), and B / Austria (Influenza B). Live virus was pre-treated with pooled immune serum from 4-5 mice of each respective group, then added to monolayers of MDCK cells at an MOI of 0.1 to assess neutralisation of infection. A fluorescence microscope was used to quantify FITC-fluorescence (representative of A / PR8 infection) relative to DAPI-fluorescence (cell nuclei) at 1:80 serum dilution. Representative images were also taken of each sample at a 1:80 serum dilution (representative of 4 wells tested per serum group, compiled from 2 independent experiments). Quantitative data was presented as mean ± SEM and analysed by one-way ANOVA with Tukey’s multiple comparisons test.[000332] Despite inducing cross- reactive antibody responses (discussed above), serum samples collected from mice vaccinated with y-Flu alone, y-NDVtrpalone, or y-Flu+y-NDVP did not neutralise infection with A / Sydney (H1N1), A / PC (H3N2), and B / Austria (Influenza B). As shown in Figure 23A, 23B and 23C, serum samples from all vaccinated groups had shown equivalent levels of infection in comparison to sera from naive control and virus-only control, indicating that the cross-reactive antibodies are not cross-neutralising in nature.Protection against intranasal infection with A / California / 07 / 2009[000333] BALB / c mice were vaccinated IM twice with y-Flu (y-A / PR8), trypsin-treated y-NDV (y-NDVtryp), or a combination of y-Flu+y-NDVtiyp. Control mice were PBS-mock vaccinated (naive). Three weeks post second vaccination, animals were challenged via the intranasal route with influenza A virus pdmH1N1 (A / California / 07 / 2009) using standard techniques. Influenza A virus pdmH1N1 (A / California / 07 / 2009) is a highly drifted influenza A strain obtained from WHO collaborating Centre for Reference and Research on Influenza, The Doherty Institute / Melbourne.[000334] Mice were monitored for clinical symptoms and weight loss, and humanely euthanised when weight loss was greater than 20% of their initial body weight. Weight loss represented as mean ± SEM for each group (n=8-10 per group) (Figure 24A), and percentage survival based on 20% body weight loss as a humane end point was investigated (Figure 24B). Statistical analyses for survival rates were performed using the two-tailed Fisher’s Exact Test (** p < 0.01, compared to PBS-mock control group).[000335] Co-vaccination with y-Flu+y-NDVtiypresulted in a significantly enhanced survival rate (90%) against lethal challenge with drifted strain of influenza A virus following intramuscularchallenge, in contrast to vaccination with y-Flu only (Figure 24). This demonstrates that covaccination with y-Flu+y-NDVtrpprovides cross-protective immunity.DISCUSSION[000336] The y-NDV vaccine induced a cross- reactive antibody response against Flu antigen and provided transient protection against Flu challenge. The y-NDV provided adjuvant activity to co-administered y-Flu vaccine utilising two different routes of administration.[000337] Mucosal administration of y-Flu+y-NDV was associated with a significant increase in Flu-specific neutralising antibody responses. Intramuscular administration of y-Flu+y-NDV was associated with enhanced cross-protection against highly drifted Flu challenge compared to vaccination with y-Flu only. However, intramuscular administration was not associated with increase in Flu-specific neutralising antibody responses. Co-administration of y-Flu+y-NDV is associated with significant enhancement in the ability of immune sera to neutralise Flu neuraminidase.[000338] Following intranasal vaccination with a combination of y-NDV and y-influenza, increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response, increased influenza-specific neutralising antibody response, and an increased neuraminidase neutralising response were observed compared to intranasal vaccination with y-flu alone. Accordingly, the data indicates that when intranasally administered with y-flu, y-NDV enhanced the immune response against influenza and / or provided an adjuvant effect. Additionally, the immunogenicity of y-NDV was maintained following intranasal co-administration with y-Flu.[000339] Following intramuscular administration with a combination of y-NDV and y-influenza, increased protection against influenza, increased survival against influenza, decreased influenza symptoms, and an increased neuraminidase neutralising response were observed compared to intramuscular vaccination with y-flu alone. Accordingly, the data indicates that when intramuscularly administered with y-flu, y-NDV enhanced the immune response against influenza and / or provided an adjuvant effect. Additionally, the immunogenicity of y-NDV was maintained following intramuscular co-administration with y-Flu.[000340] Intranasal vaccination with y-NDV may induce increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response compared to naive subjects. Accordingly, the data indicates that whenintranasally administered, y-NDV enhanced the immune response against influenza and / or provided an adjuvant effect.[000341] While not wanting to be bound by theory, the enhanced immune response against influenza may be associated with cross-reaction between antigens from influenza and y-NDV. Alternatively or additionally, the enhanced immune response against influenza may be associated with an adjuvant effect provided by the inactivated NDV.[000342] Further, gamma-irradiated Influenza A Virus vaccine (y-FLU) may provide significant cross-protection against seasonal and pandemic influenza virus strains, including avian H5N1, including after intranasal vaccination. Cross-protection mediated by intranasal vaccination with y-FLU is believed to be related to the ability of the vaccine to induce cross-reactive T cell responses. Considering the time required for T cell to be activated and recruited into the lung of vaccinated, intramuscular vaccination with y-FLU was associated with limited cross-protection towards highly drifted strains of flu. It is believed that a vaccination strategy that enhances flu-specific antibodies and enable the induction of cross- reactive responses would have clinical benefits against drifted or heterotypic influenza strains.[000343] Intranasal co-administration of y-NDV + y-Flu (compared to administration of y-Flu alone) is associated with significantly enhanced flu-specific IgG responses (Figure 2), significantly enhanced Flu-specific neutralising antibody responses (Figure 3), and significantly enhanced neuraminidase inhibition (Figure 4).[000344] Intranasal administration of y-NDV induces a transient resistance to lethal challenge with influenza A virus as measured by the reduced weight loss prior to development of clinical symptoms (Figure 1). Cross-reactive antibody responses induced by y-NDV against different influenza A viruses (Figure 2D showing A / PR8, Figure 5A, 5B showing A / California H1N1, and Figure 5C, 5D showing A / PC H3N2). However, cross- reactive antibodies induced by y-NDV against different influenza strains are not neutralising (Figure 3 for A / PR8 and Figure 6 for A / California).[000345] Intramuscular co-administration of y-NDV+y-Flu (compared to administration of y-Flu alone) is associated with enhanced protection against a lethal challenge with a drifted influenza A virus, and significantly enhanced inhibition of influenza neuraminidase activity.[000346] Intramuscular administration of y-NDV induces a transient resistant to a flu challenge as measured by the reduced weight loss prior to development of clinical symptoms. Cross-reactive antibody responses are induced by y-NDV against influenza A virus (Figure 8D).[000347] Intramuscular co-administration of y-NDV+y-Flu (compared to administration of y-NDV alone) has no impact on the immunogenicity of y-NDV in terms of antibody responses and neutralising antibodies.[000348] Intramuscular vaccination with trypsin-treated y-NDV and y-flu has no impact on the level of NDV-specific IgG responses. However, co-administration of y-NDV and y-Flu is associated with changing the NDV-specific responses from a Th2-biased into a balanced Th1 / Th2 response. Intramuscular vaccination with trypsin-treated y-NDV with or without y-Flu is associated with the induction of NDV-specific neutralising antibody responses.[000349] Intramuscular vaccination with protease-treated y-NDV + y-Flu (compared to administration of y-Flu alone) is associated with significantly enhanced cross-protection against lethal influenza challenge, significantly enhanced influenza-specific IgG responses, enhanced influenza-specific neutralising antibody responses, and significantly enhanced inhibition of neuraminidase activity of different influenza A viruses (A / Sydney and A / PC). Intramuscular administration of protease-treated y-NDV induces cross-reactive antibody responses against different influenza A viruses (A / PR8 H1N1, A / Sydney H1N1, and A / PC H3N2). However, cross-reactive antibodies induced by y-NDV against different influenza strains are not neutralising.[000350] The ability of y-NDV to enhance the immunogenicity of y-Flu and the cross- protective immunity against different influenza A viruses (following co-administration with y-Flu) could not have been predicted based on existing literature. The ability of y-NDV to induce cross-reactive antibodies against influenza A viruses could not have been predicted based on existing literature. The ability of y-NDV to enhance inhibition of influenza neuraminidase after co-administration of y-NDV with y-Flu could not have been predicted based on existing literature.[000351] Outcomes:1. Co-administration of y-Flu and y-NDV (with or without protease treatment of y-NDV) is associated with enhanced protection against a lethal challenge with a drifted flu virus compared to vaccination with y-Flu alone.2. Co-administration of y-Flu and y-NDV (using different route of administrations, with or without protease treatment of y-NDV) is associated with significantly enhanced influenzaspecific IgG responses, enhanced influenza-specific neutralising antibody responses, and significantly enhanced inhibition of neuraminidase activity of influenza A virus compared to administration of y-Flu alone.3. y-NDV provides transient protection against a lethal influenza challenge4. Co-administration of y-NDV and y-Flu has no impact on NDV-specific total and neutralising antibody responses.REFERENCESHu, 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[000352] Embodiments of the invention as described herein are defined in the following paragraphs:1. A vaccine composition comprising(a) an inactivated paramyxovirus, and(b) an inactivated influenza virus.2. The vaccine composition of embodiment 1, wherein the inactivated paramyxovirus is irradiation-inactivated paramyxovirus.3. The vaccine composition of embodiment 1 or embodiment 2, wherein the inactivated influenza virus is irradiation-inactivated influenza.4. The vaccine composition of any one of embodiments 1 to 3, wherein the paramyxovirus is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy.5. The vaccine composition of any one of embodiments 1 to 4, wherein the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.The vaccine composition of any one of embodiments 1 to 5, wherein the composition excludes a supplementary adjuvant.The vaccine composition of any one of embodiments 1 to 6, wherein the inactivated paramyxovirus comprises irradiation-inactivated, protease treated paramyxovirus.The vaccine composition of embodiment 7, wherein the protease treatment occurred following the irradiation inactivation of the paramyxovirus.The vaccine composition of embodiment 7 or embodiment 8, wherein the protease is trypsin.The vaccine composition of any one of embodiments 1 to 9, wherein the inactivated paramyxovirus is Newcastle Disease Virus (NDV).The vaccine composition of any one of embodiments 1 to 10, wherein the influenza virus is a type A influenza virus.The vaccine composition of any one of embodiments 1 to 11, wherein the inactivated paramyxovirus enhances the immune response against influenza virus and / or provides an adjuvant effect for the inactivated influenza virus.A method for inducing or enhancing an immune response against an influenza infection in a subject, the method comprising(i) administering to the subject a therapeutically effective amount of a vaccine composition comprising (a) inactivated paramyxovirus, and (b) inactivated influenza virus; or(ii) co-administering to the subject a therapeutically effective amount of two vaccine compositions, the vaccine compositions comprising a first vaccine composition comprising inactivated paramyxovirus, and a second composition comprising inactivated influenza virus.The method of embodiment 13, wherein the paramyxovirus is inactivated by irradiation.The method of embodiment 13 or embodiment 14, wherein the influenza is inactivated by irradiation.The method of any one of embodiments 13 to 15, wherein the paramyxovirus is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy.The method of any one of embodiments 13 to 16, wherein the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.The method of any one of embodiments 13 to 17, wherein the paramyxovirus comprises irradiation-inactivated, protease treated paramyxovirus.The method of embodiment 18, wherein the protease treatment occurred following the irradiation inactivation of the paramyxovirus.The method of embodiment 18 or embodiment 19, wherein the protease is trypsin.The method of any one of embodiments 13 to 20, wherein the inactivated paramyxovirus is a NDV.The method of any one of embodiments 13 to 21, wherein the inactivated influenza is a type A influenza virus.The method of any one of embodiments 13 to 22, wherein the administering is selected from the group consisting of mucosal administration, intranasal administration and intramuscular administration.The method of any one of embodiments 13 to 23, wherein the method induces or enhances a protective immune response that treats, prevents or reduces the severity of an influenza infection in the subject.The method of any one of embodiments 13 to 24, wherein the method induces a cross-protective immunity against an influenza strain different to the inactivated influenza virus and / or induces protection against a plurality of influenza strains in the subject.The method of any one of embodiments 13 to 25, wherein the method induces increased protection against influenza, increased survival against influenza, decreased influenza symptoms, an increased influenza-specific antibody response, increased influenzaspecific neutralising antibody response, and / or an increased neuraminidase neutralising response compared to administering a therapeutically effective amount of an inactivated influenza virus in the absence of the inactivated paramyxovirus.The method of any one of embodiments 13 to 26, wherein the inactivated paramyxovirus enhances the immune response against influenza virus and / or provides an adjuvant effect for the inactivated influenza virus.The method of any one of embodiments 13 to 27, wherein the method further comprises inducing an immune response against a paramyxovirus infection in the subject or treating, preventing or reducing the severity of a paramyxovirus infection in the subject.The method of any one of embodiments 13 to 28, wherein the method induces a paramyxovirus-specific antibody response or a paramyxovirus-specific neutralising antibody response.The method of any one of embodiments 13 to 29, wherein the subject is an avian subject.The method of any one of embodiments 13 to 29, wherein the subject is human.A method of producing a vaccine composition, the vaccine composition comprising (a) an irradiation-inactivated paramyxovirus and (b) irradiation-inactivated influenza virus vaccine composition, the method comprising:(i) obtaining a preparation of the paramyxovirus, and optionally clarifying and / or washing the paramyxovirus;(ii) irradiating the paramyxovirus preparation to obtain an irradiation-inactivated paramyxovirus preparation; and optionally protease-treating the irradiation-inactivated paramyxovirus preparation;(iii) obtaining a preparation of influenza virus, and optionally clarifying and / or washing the influenza virus;(iv) irradiating the influenza preparation to obtain an irradiation-inactivated influenza preparation; and(v) optionally, combining the irradiation-inactivated paramyxovirus preparation obtained from step (ii) with the irradiation-inactivated influenza preparation obtained from step (iv).The method of embodiment 32, wherein the irradiation-inactivated paramyxovirus is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.The method of embodiment 32 or 33, wherein the irradiation-inactivated influenza is irradiated with a dose of gamma radiation of between 2 kGy and 50 kGy.The method of any one of embodiments 32 to 34, wherein the protease is trypsin.The method of any one of embodiments 32 to 35, wherein the paramyxovirus is NDV.37. The method of any one of embodiments 32 to 36, wherein the influenza virus is a type A influenza virus.38. A vaccine composition produced by the method of any one of embodiments 32 to 37.Forms of the invention as described herein are defined in the following paragraphs:1. A vaccine composition comprising (a) inactivated Newcastle Disease Virus (NDV), and (b) inactivated influenza virus.2. The vaccine composition of form 1, wherein the inactivated NDV is inactivated by photonirradiation.3. The vaccine composition of form 1 or form 2, wherein the inactivated influenza virus is inactivated by photon-irradiation.4. The vaccine composition of any one of forms 1 to 3, wherein the NDV is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy.5. The vaccine composition of any one of forms 1 to 4, wherein the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.6. The vaccine composition of any one of forms 1 to 5, further comprising a supplementary adjuvant.7. The vaccine composition of any one of forms 1 to 5, wherein the composition excludes a supplementary adjuvant.8. The vaccine composition of any one of forms 1 to 5, wherein the composition excludes an alum adjuvant.9. The vaccine composition of any one of forms 1 to 8, wherein the photon-irradiated NDV is a genotype I strain.10. The vaccine composition of any one of forms 1 to 9, wherein the photon-irradiated NDV is a V4 strain.11. The vaccine composition of any one of forms 1 to 10, wherein the photon-irradiated influenza virus is a type A influenza virus.12. The vaccine composition of any one of forms 1 to 11, wherein the vaccine composition is formulated for intramuscular, subcutaneous, intranasal, ocular or conjunctival delivery.13. The vaccine composition of any one of forms 1 to 12, wherein the vaccine composition is formulated for intranasal, ocular or conjunctival delivery.14. The vaccine composition of any one of forms 1 to 13, wherein the vaccine composition is formulated for mucosal delivery.15. The vaccine composition of any one of forms 1 to 14, wherein the vaccine composition is formulated for delivery to birds.16. The vaccine composition of any one of forms 1 to 15, wherein the inactivated NDV enhances the immune response against influenza virus.17. The vaccine composition of any one of forms 1 to 16, wherein the inactivated NDV provides an adjuvant effect for the inactivated influenza virus.18. The vaccine composition of any one of forms 1 to 17, wherein the composition induces a heterotypic immune response against heterologous NDV genotypes.19. The vaccine composition of any one of forms 1 to 18, wherein the inactivated NDV is an inactivated whole virus.20. The vaccine composition of any one of forms 1 to 19, wherein the inactivated influenza is an inactivated whole virus.21. The vaccine composition of any one of forms 1 to 20, wherein the composition induces a heterotypic immune response against heterologous NDV genotypes.22. The vaccine composition of any one of forms 1 to 21, wherein the composition induces a heterotypic immune response against heterologous influenza strains.23. The vaccine composition of any one of forms 1 to 22, wherein the inactivated influenza virus in an avian influenza virus.24. The vaccine composition of any one of forms 1 to 23, wherein the vaccine composition comprises an immunogenic component consisting of (a) inactivated Newcastle Disease Virus (NDV), and (b) inactivated influenza virus.25. The vaccine composition of any one of forms 1 to 24, further comprising at least one pharmaceutically-acceptable excipient, diluent and / or carrier.26. A pharmaceutical composition comprising the vaccine composition of any one of forms 1 to 25.27. A method for inducing or enhancing an immune response against an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26.28. A method for treating or preventing an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26.29. A method of vaccinating a subject against influenza, the method comprising administration of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26.30. 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 of any one of forms 1 to 25 or pharmaceutical composition of form 26.31. 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 forms 1 to 25 or pharmaceutical composition of form 26.32. 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 forms 1 to 25 or pharmaceutical composition of form 26.33. A method for inducing or enhancing an immune response against an NDV infection and an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26.34. A method for treating or preventing an NDV infection and an influenza infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26.35. A method of vaccinating a subject against NDV and influenza, the method comprising administration of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26.36. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for inducing or enhancing an immune response against an influenza infection in a subject.37. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for treating or preventing an influenza infection in a subject.38. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for vaccinating a subject against influenza.39. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for inducing or enhancing an immune response against an NDV infection in a subject.40. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for treating or preventing an NDV infection in a subject.41. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for vaccinating a subject against NDV.42. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for inducing or enhancing an immune response against an NDV infection and an influenza infection in a subject.43. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for treating or preventing an NDV infection and an influenza infection in a subject.44. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 in the manufacture of a medicament for vaccinating a subject against NDV and influenza.45. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for inducing or enhancing an immune response against an influenza infection in a subject.46. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for treating or preventing an influenza infection in a subject, the method comprising administering to the subject.47. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for vaccinating a subject against influenza.48. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for inducing or enhancing an immune response against an NDV infection in a subject.49. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for treating or preventing an NDV infection in a subject.50. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for vaccinating a subject against NDV.51. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for inducing or enhancing an immune response against an NDV infection and an influenza infection in a subject.52. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for treating or preventing an NDV infection and an influenza infection in a subject.53. A therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for vaccinating a subject against NDV and influenza.54. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for inducing or enhancing an immune response against an influenza infection in a subject.55. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for treating or preventing an influenza infection in a subject.56. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for vaccinating a subject against influenza.57. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for inducing or enhancing an immune response against an NDV infection in a subject.58. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 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 forms 1 to 25 or pharmaceutical composition of form 26 for vaccinating a subject against NDV.60. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for inducing or enhancing an immune response against an NDV infection and an influenza infection in a subject.61. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for treating or preventing an NDV infection and an influenza infection in a subject.62. Use of a therapeutically effective amount of the vaccine composition of any one of forms 1 to 25 or pharmaceutical composition of form 26 for vaccinating a subject against NDV and influenza.63. The method, use or composition of any one of forms 27-62, wherein the method, use or composition induces a heterotypic immune response against a NDV genotype different to the photon-irradiated NDV in the subject and / or induces protection against a plurality of NDV genotypes in the subject.64. The method, use or composition of any one of forms 27-62, wherein the method, use or composition induces a heterotypic immune response against an influenza strain different to the photon-irradiated influenza virus and / or induces protection against a plurality of influenza strains in the subject.65. The method, use or composition of any one of forms 27-62, wherein the method, use or composition induces increased protection against influenza, increased survival against influenza, decreased influenza symptoms, increased influenza-specific antibody response, increased influenza-specific neutralising antibody response, and / or an increased neuraminidase neutralising response compared to administering a therapeutically effective amount of influenza virus inactivated by photon-irradiation in the absence of NDV inactivated by photon-irradiation.66. The method, use or composition of any one of forms 27-62, wherein the influenza infection is an avian influenza infection.67. The method, use or composition of any one of forms 27-62, wherein the method, use or composition induces a mucosal immune response.68. The method, use or composition of any one of forms 27-62, wherein the inactivated NDV enhances the immune response against influenza virus.69. The method, use or composition of any one of forms 27-62, wherein the inactivated NDV provides an adjuvant effect for the inactivated influenza virus.67. The method, use or composition of any one of forms 27-62, wherein the administration is intramuscular, subcutaneous, intranasal, ocular or conjunctival administration.68. The method, use or composition of any one of forms 27-62, wherein the administration is intranasal, ocular or conjunctival administration.69. The method, use or composition of any one of forms 27-62, wherein the administration is mucosal administration.70. The method, use or composition of any one of forms 27-62, wherein the subject is an avian subject.71. A method of producing a vaccine composition according to any one of forms 1 to 25, the method comprising inactivating a preparation of NDV by photon-irradiation and inactivating a preparation of influenza virus by photon-irradiation.72. The method of form 71, wherein the inactivating of NDV comprises treatment with a dose of gamma radiation of between 2 and 100 kGy.73. The method of form 71 or 72, wherein the inactivating of influenza virus comprises treatment with a dose of gamma radiation of between 2 and 50 kGy.74. The method of form 71 or 72, wherein the method further comprises purifying the NDV and / or the influenza virus prior to photon irradiation.75. A vaccine composition inactivated Newcastle Disease Virus (NDV) and inactivated influenza virus produced by the method of any one of forms 71-74.[000353] 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. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples andembodiments are presented by way of example only with the scope of the invention intended to be limited only by the claims set forth herein as follows.

Claims

CLAIMS1. A vaccine composition comprising(a) an inactivated paramyxovirus, and(b) an inactivated influenza virus.

2. The vaccine composition of claim 1, wherein the inactivated paramyxovirus is irradiation- inactivated paramyxovirus.

3. The vaccine composition of claim 1 or claim 2, wherein the inactivated influenza virus is irradiation-inactivated influenza.

4. The vaccine composition of any one of claims 1 to 3, wherein the paramyxovirus is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy.

5. The vaccine composition of any one of claims 1 to 4, wherein the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.

6. The vaccine composition of any one of claims 1 to 5, wherein the composition excludes a supplementary adjuvant.

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

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

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

10. The vaccine composition of any one of claims 1 to 9, wherein the inactivated paramyxovirus is Newcastle Disease Virus (NDV).

11. The vaccine composition of any one of claims 1 to 10, wherein the influenza virus is a type A influenza virus.

12. The vaccine composition of any one of claims 1 to 11, wherein the inactivated paramyxovirus enhances the immune response against influenza virus and / or provides an adjuvant effect for the inactivated influenza virus.

13. A method for inducing or enhancing an immune response against an influenza infection in a subject, the method comprising(i) administering to the subject a therapeutically effective amount of a vaccine composition comprising (a) inactivated paramyxovirus, and (b) inactivated influenza virus; or(ii) co-administering to the subject a therapeutically effective amount of two vaccine compositions, the vaccine compositions comprising a first vaccine composition comprising inactivated paramyxovirus, and a second composition comprising inactivated influenza virus.

14. The method of claim 13, wherein the paramyxovirus is inactivated by irradiation.

15. The method of claim 13 or claim 14, wherein the influenza is inactivated by irradiation.

16. The method of any one of claims 13 to 15, wherein the paramyxovirus is inactivated by a dose of gamma radiation of between 2 kGy and 100 kGy.

17. The method of any one of claims 13 to 16, wherein the influenza virus is inactivated by a dose of gamma radiation of between 2 kGy and 50 kGy.

18. The method of any one of claims 13 to 17, wherein the paramyxovirus comprises irradiation-inactivated, protease treated paramyxovirus.

19. The method of claim 18, wherein the protease treatment occurred following the irradiation inactivation of the paramyxovirus.

20. The method of claim 18 or claim 19, wherein the protease is trypsin.

21. The method of any one of claims 13 to 20, wherein the inactivated paramyxovirus is a NDV.

22. The method of any one of claims 13 to 21, wherein the inactivated influenza is a type A influenza virus.

23. The method of any one of claims 13 to 22, wherein the administering is selected from the group consisting of mucosal administration, intranasal administration and intramuscular administration.

24. The method of any one of claims 13 to 23, wherein the method induces or enhances a protective immune response that treats, prevents or reduces the severity of an influenza infection in the subject.

25. The method of any one of claims 13 to 24, wherein the method induces a cross- protective immunity against an influenza strain different to the inactivated influenza virus and / or induces protection against a plurality of influenza strains in the subject.

26. The method of any one of claims 13 to 25, wherein the method induces increased protection against influenza, increased survival against influenza, decreased influenza symptoms, an increased influenza-specific antibody response, increased influenzaspecific neutralising antibody response, and / or an increased neuraminidase neutralising response compared to administering a therapeutically effective amount of an inactivated influenza virus in the absence of the inactivated paramyxovirus.

27. The method of any one of claims 13 to 26, wherein the inactivated paramyxovirus enhances the immune response against influenza virus and / or provides an adjuvant effect for the inactivated influenza virus.

28. The method of any one of claims 13 to 27, wherein the method further comprises inducing an immune response against a paramyxovirus infection in the subject or treating, preventing or reducing the severity of a paramyxovirus infection in the subject.

29. The method of any one of claims 13 to 28, wherein the method induces a paramyxovirusspecific antibody response or a paramyxovirus-specific neutralising antibody response.

30. The method of any one of claims 13 to 29, wherein the subject is an avian subject.

31. The method of any one of claims 13 to 29, wherein the subject is human.

32. A method of producing a vaccine composition, the vaccine composition comprising (a) an irradiation-inactivated paramyxovirus and (b) irradiation-inactivated influenza virus vaccine composition, the method comprising:(i) obtaining a preparation of the paramyxovirus, and optionally clarifying and / or washing the paramyxovirus;(ii) irradiating the paramyxovirus preparation to obtain an irradiation-inactivated paramyxovirus preparation; and optionally protease-treating the irradiation-inactivated paramyxovirus preparation;(iii) obtaining a preparation of influenza virus, and optionally clarifying and / or washing the influenza virus;(iv) irradiating the influenza preparation to obtain an irradiation-inactivated influenza preparation; and(v) optionally, combining the irradiation-inactivated paramyxovirus preparation obtained from step (ii) with the irradiation-inactivated influenza preparation obtained from step (iv).

33. The method of claim 32, wherein the irradiation-inactivated paramyxovirus is irradiated with a dose of gamma radiation of between 2 kGy and 100 kGy.

34. The method of claim 32 or 33, wherein the irradiation-inactivated influenza is irradiated with a dose of gamma radiation of between 2 kGy and 50 kGy.

35. The method of any one of claims 32 to 34, wherein the protease is trypsin.

36. The method of any one of claims 32 to 35, wherein the paramyxovirus is NDV.

37. The method of any one of claims 32 to 36, wherein the influenza virus is a type A influenza virus.

38. A vaccine composition produced by the method of any one of claims 32 to 37.

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