MANUFACTURE THEREOF

Technical Field

The present disclosure relates to the use of reverse genetics for manufacturing or constructing influenza vaccines. More specifically, the present disclosure relates to the production of vaccines and vaccine compositions for vaccination against influenza A, as well as methods for the manufacture of said vaccines and vaccine compositions.

Background

Influenza viruses, more particularly influenza A and B viruses, are the causation of the influenza disease (commonly referred to as influenza or flu) in both human beings and animals. Influenza is an infectious disease having common symptoms including chills, fever, sore throat, coughing, weakness, and general discomfort. Influenza can be transmitted between living organisms (e.g., between human beings or between poultry) in various ways, for example through contact with bodily fluids such as saliva and nasal secretions, as well as with airborne aerosols containing the virus.

Influenza is generally regarded as a serious global health concern. Influenza typically spreads around the world in seasonal epidemics. In addition, influenza pandemics occur at irregular time intervals and result in a significantly increased level of illness and death. Influenza pandemics are historically due to novel viral subtypes or viral strains (e.g., novel strains of influenza A), which can be created by a reassortment of the segmented genome of the influenza viruses (i.e., antigenic shift). For instance, the influenza pandemic in Asia in the 1990s was caused by a novel avian strain of the influenza A virus (also known as the avian influenza A virus).

Classification of the influenza A, B, and C viruses can be done on the basis of antigenic differences in viral nucleocapsid (NP) and matrix (M) proteins. Influenza A viruses contain a ribonucleoprotein (RNP) consisting of eight negative-sense RNA strands that encode at least 10 polypeptides, including polymerase proteins, hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (MP), and non-structural protein (NS). The influenza A viruses can be classified into subtypes on the basis of antigenic differences in hemagglutinin (HA) and neuraminidase (NA). There are presently sixteen different subtypes of the serum hemagglutinin (HA) proteins, designated HAl to HA 16, as well as nine different subtypes of neuraminidase (NA) proteins, designated NA1 to NA9. The HA and NA proteins are displayed on the surface of the influenza A virus and determine the immune response triggered by the influenza A virus. The immune response triggered by a particular influenza A virus is specific for the particular subtype of the influenza A virus. Therefore, the immune response triggered by a particular influenza A virus is determined by the type of HA and NA proteins displayed on the influenza A virus's surface.

The influenza A virus is able to mutate rapidly. Vaccine preparation (e.g., manufacturing of vaccines) can be time consuming, particularly where the emerging viral strain reproduces slowly in culture. Accordingly, waiting for an influenza outbreak before isolating the particular virus strain causing that influenza outbreak and formulating a vaccine against the particular virus strain may not be a viable option. However, vaccine preparation ahead of, or before, a viral outbreak is generally difficult due to the unpredictability of the emergence of any particular strain or subtype of the influenza A virus.

The principle of vaccination or immunization is generally well known. Typically a vaccine that includes specific HA and NA antigens (e.g., viral strains displaying particular HA and NA subtypes) is injected into the body. The injected HA and NA antigens activate the body's immune system, triggering production of antibodies by white blood cells and secretion of such antibodies into the body's circulatory system. The antibodies that are produced and secreted are specific for the HA and NA antigens (e.g., the viral strains displaying particular HA and NA subtypes) that are injected into the body. The availability of such antibodies within the body helps to combat a subsequent infection by an identical or similar virus strain.

Typically, immunity resulting from any particular vaccine is around the order of 70% (i.e., any particular vaccine provides protection against subsequent infection and disease in seven out of ten cases on average). A vaccine does not provide absolute immunity because the viral strain causing a next epidemic (or pandemic) is seldom exactly identical as the viral strain causing the previous epidemic (or pandemic). The more the new viral strain (or variant) differs from the previous one, the less protection the vaccine provides. This is because the body does not simply produce one antibody against each of the recognized HA and NA antigens (e.g. H5 and Nl antigens). A HA antigen (e.g., H5 antigen) may have or present different chemical structures (also known as epitopes) depending on the virus strain from which it is isolated. Each epitope may trigger the production of an antibody specific only for that epitope. In addition, the different epitopes of the same HA antigen (e.g., H5 antigen) may have differing abilities for triggering antibody production within the body during infection of the body by an influenza virus. For example, epitopes displayed by H5 derived from A/tern/South Africa/1961 (H5N3) may be different from epitopes displayed by H5 derived from A/turkey/Canada/26/66 (H5N2). When a particular epitope's ability to trigger antibody production against a specific HA (H) or NA (N) antigen is weak, viral infection will be significantly easier and more damaging. The selection of particular viral strain(s) used for the manufacture of vaccines is important for enhancing the effectiveness of the manufactured vaccine against a subsequent epidemic (or pandemic).

In view of the above complications associated with vaccination, it will be appreciated that enhanced or improved vaccines or vaccine compositions, as well as methods for the manufacture thereof, can be beneficial for global public health.

Summary

In accordance with a first aspect of the present disclosure, there is disclosed a vaccine composition comprising an inactivated influenza virus produced by reverse genetics. The inactivated influenza virus includes a H5 hemagglutinin derived from a Southeast Asian strain of influenza A virus and a neuraminidase subtype derived from an influenza A virus, the neuraminidase subtype being one of Nl, N2, N5, N6, N7, and N8 neuraminidase.

In accordance with a second aspect of the present disclosure, there is disclosed a method for producing a recombinant influenza A virus including extracting RNA from an influenza virus strain of a H5 hemagglutinin subtype and extracting RNA from an influenza virus strain of one of a Nl, N2, N5, N6, N7, and N8 subtype. The method further includes producing cDNA from the extracted RNA, cleaving cDNA from at a predetermined lineage position to reduce pathogenicity of the recombinant influenza A virus, and cloning cleaved cDNA into expression plasmids. In addition, the method includes transfecting 293 cells with the expression plasmids comprising the cleaved cDNA in a 293 cell culture, obtaining supernatant of the 293 cell culture, transfecting MDCK cells with the supernatant of the 293 cell culture in a MDCK cell culture, and collecting supernatant of the MDCK cell culture to thereby obtain the recombinant influenza A virus therefrom.

In accordance with a third aspect of the present disclosure, there is disclosed a method for preventing an influenza A virus outbreak including administering to a living organism a dose of vaccine. The vaccine includes inactivated viruses that include a H5 hemagglutinin derived from an influenza virus of a Southeast Asian origin and a neuraminidase subtype, the neuraminidase subtype being one of Nl, N2, N5, N6, N7, and N8 neuraminidase. The dose of the vaccine is between approximately 0.3 ml and approximately 0.5 ml.

Brief Description of the Drawings

Several embodiments of the present disclosure are described in association with the drawings, in which: FIG. 1 is a flowchart of a process for manufacturing a vaccine including inactivated H5N6 influenza virus in accordance with an embodiment of the present disclosure.

Detailed Description

The more a new or future virus strain differs from virus(es) present in a vaccine that has been administered to a living organism, the less protection that vaccine provides against infection, and disease, caused by said new virus strain. Typically, to provide adequate protection against infection by a future influenza virus (or a future influenza epidemic or pandemic), a vaccine should include an influenza virus having similar hemaglutinin (HA) and neuraminidase (NA) subtypes as said future virus strain. However, the highly mutagenic nature of the genome of influenza viruses (e.g., by antigenic shift) makes it difficult to predict the HA and NA subtypes that will be carried by a new or future virus strain. In addition, each HA subtype (also known as HA antigen or H antigen) and NA subtype (also known as NA antigen or N antigen) may have different chemical structures or sites (also known as epitopes). The epitope of a particular HA subtype (e.g., H5 antigen) differ when derived from different viruses, for example viruses that are isolated from different organisms, and/or in different countries, and/or in different years. Different epitopes of each HA and NA subtype can have differing capabilities for triggering production of antibodies specific for that HA and NA subtype. As used in the context of the present disclosure, an epitope is a portion that is immunodominant for antibody or T cell receptor recognition, or a portion used to generate an antibody against a molecule (e.g. HA molecule) it is carried by.

Vaccine development targeting anticipated future influenza outbreaks (e.g., influenza epidemics or pandemics) should seek to produce vaccines that provide antigenic structures (e.g., HA and NA subtypes) that correspond as comprehensively, and as closely, as possible to antigenic structures carried by new virus strains causing said anticipated future influenza outbreaks. In light of the significant global public health threat that influenza presents, it is important to be able to produce vaccines that are effective against new influenza virus strains.

Embodiments of the present disclosure relate to vaccines, vaccine compositions, and vaccine formulations (hereinafter referred collectively to vaccines), as well as to methods, processes, and techniques for the manufacture, production, or construction of particular vaccines in association with the present disclosure.

Embodiments of the present disclosure rely on the finding that a particular hemagglutinin (HA) subtype (also known as HA antigen or H antigen), for example a H5 antigen, may present different epitopes when derived from different viruses, for example viruses that are isolated from different organisms, and/or in different countries, and/or in different years. Different epitopes of a particular HA subtype (and NA subtype) can have differing capabilities for triggering production of antibodies specific for that particular HA and NA subtype.

Many embodiments of the present disclosure rely on the finding that an epitope of a HA subtype (e.g., H5 antigen) from an influenza A virus isolated locally (i.e., in a same or neighboring country of viral origin/source) may be more effective in triggering production of antibodies against a future or new influenza A virus that also originates locally (i.e., in a same or neighboring country). In other words, many embodiments of the present disclosure relies on the finding that a vaccine including a HA antigen (e.g., a H5 antigen) derived from an influenza virus isolated locally (e.g., from Thailand) may be more effective in preventing infection by a future influenza A virus originating in a same (e.g., Thailand) or neighboring country (e.g., Malaysia, Vietnam, and Cambodia).

Many embodiments of the present disclosure rely on finding that vaccines including inactivated influenza A viruses derived from influenza A viruses of recent influenza A outbreaks (e.g., recent influenza epidemics or pandemics), are more effective than vaccines including inactivated influenza A viruses derived from influenza A viruses of less recent (i.e., older) influenza A outbreaks. Some embodiments of the present disclosure rely on the finding that vaccines including inactivated influenza A viruses derived from influenza A viruses of recent and local influenza A outbreaks are more effective in preventing infection by a future or new influenza A virus.

As used in the context of the present disclosure, a hemagglutinin (HA) (also known as HA antigen or H antigen) or neuraminidase (NA) (also known as NA antigen or N antigen) is said to be "derived from" a particular influenza virus if the HA or NA is encoded by a polynucleotide that is cloned, reverse transcribed, amplified, or otherwise artificially synthesized from the influenza virus in question, or if the HA or NA is obtained from the influenza virus in question through a reassortment process. Many embodiments of the present disclosure relate to vaccines that include an immunologically effective amount of inactivated influenza viruses. The term immunologically effective refers to an amount of the inactivated influenza virus that will induce at least a partial immunity in a treated living organism (e.g., a human being or animal) against a future or subsequent challenge with a virulent strain of influenza virus. In other words, the term immunologically effective amount refers to an amount of inactivated influenza viruses that is capable of preventing or treating infection by an influenza A virus, or preventing, treating, or mitigating clinical effect(s) of infection by the influenza A virus (i.e., influenza disease), as determined by one of in-vitro tests, visual inspection, or other methods or techniques known to a person of ordinary skill in the art.

In most embodiments, the inactivated influenza A viruses include or display a H5 hemaglutinin (HA) (also known as a H5 antigen). Hemagglutinin (HA) is a viral surface glycoprotein comprising approximately 560 amino acids and representing 25% of the total virus protein. HA is primarily responsible for adhesion of virus to, and virus penetration into, host cells. In most embodiments, the inactivated viruses include or carry at least one neuraminidase (NA) subtype (also known as NA antigen or N antigen). The at least one NA subtype is selected from a group including the Nl, N2, N5, N6, N7, and N8 neuraminidases. In many embodiments, the NA subtype of the inactivated viruses is the N6 subtype or N7 subtype. In several embodiments, the inactivated influenza A viruses include or carry at least two different NA subtypes, the at least two NA subtypes being selected from the group including the Nl, N2, N5, N6, N7, and N8 neuraminidases. In selected embodiments, the inactivated influenza A viruses include or carry the N6 and N7 neuraminidases (also known as N6 and N7 antigens). Neuraminidase (NA) is a second membrane glycoprotein of influenza A viruses. NA is typically a 413 amino acid protein encoded by a gene of 1413 nucleotides. NA is primarily responsible for destruction of cellular receptors for viral hemagglutinin by cleaving between the sialic acid molecule and the hemagglutinin to thereby ease liberation of viral progeny from host cells. NA is acknowledged as a significant antigenic determinant that is subject to antigenic variations.

INFLUENZA VIRUSES

The classification and nomenclature of influenza viruses is based on a recommendation made by the WHO. Examples of names of influenza viruses include:

(i) A/chicken/Thailand/73/2004, wherein A refers to the influenza virus type, chicken represents the name of the organism or species from which the influenza virus is isolated, Thailand is the country of isolation of the influenza virus, 73 refers to the isolate number issued by a country, or a laboratory within the country, in which the influenza virus is isolated, and 2004 is the year of isolation of that influenza virus; and

(ii) A/Puerto Rico/8/1934 (Nl), wherein A refers to the influenza virus type, Puerto

Rico is the country of isolation of the influenza virus, 8 is the isolate number issued by the country, or a laboratory within the country, 1934 is the year of isolation of the influenza virus, and Nl refers to the subtype of neuraminidase or N antigen that is carried by the influenza virus.

In most embodiments, the H5 hemagglutinnin is derived from a naturally occurring influenza virus that has or carries the H5 hemagglutinin. In many embodiments, the H5 hemagglutinin is derived from a naturally occurring influenza virus of an Asian origin. In some embodiments, the H5 hemagglutinin is derived from a naturally occurring influenza virus of a Southeast Asian origin. In a number of embodiments, the H5 hemaglutinin is derived from a naturally occurring influenza virus isolated from poultry and being of Southeast Asian origin. In selected embodiments, the H5 hemagglutinin is derived from a naturally occurring influenza virus of a recent influenza A outbreak in poultry in a Southeast Asian country (e.g., Thailand).

In some embodiments, the H5 hemagglutinin is derived from A chicken Thailand/73/2004. In some embodiments, the use of H5 hemaglutinin derived from influenza viruses originating in Thailand facilitates or effectuates an increased ability of the inactivated influenza viruses, and hence the vaccine, to trigger production of antibodies against new or future (i.e., emerging) influenza viruses displaying H5 hemaglutinin. More specifically, the use of H5 hemaglutinin derived from influenza viruses originating in Thailand facilitates or effectuates an increased ability of the inactivated influenza viruses, and hence the vaccine, to trigger production of antibodies against a new or future (i.e., emerging) influenza viruses originating from an Asian, more particularly Southeast Asian, country (e.g., Thailand).

In specific embodiments, the H5 hemagglutinin is derived from (i.e., encoded by) a polynucleotide sequence as shown below: Nucleotide sequence for the H5 hemagglutinin

1 CTGTCAAATG GAGAAAATAG TGCTTCTTTT TGCAATAGTC AGTCTTGTTA AAAGTGATCA

61 GATTTGCATT GGTTACCATG CAAACAACTC GACAGAGCAG GTTGACACAA TAATGGAAAA

121 GAACGTTACT GTTACACATG CCCAAGACAT ACTGGAAAAG ACACACAACG GGAAGCTCTG

181 CGATCTAGAT GGAGTGAAGC CTCTAATTTT GAGAGATTGT AGTGTAGCTG GATGGCTCCT

241 CGGAAACCCA ATGTGTGACG AATTCATCAA TGTGCCGGAA TGGTCTTACA TAGTGGAGAA

301 GGCCAATCCA GTCAATGACC TCTGTTACCC AGGGGATTTC AATGACTATG AAGAATTGAA

361 ACACCTATTG AGCAGAATAA ACCATTTTGA GAAAAT CAG ATCATCCCCA AAAGTTCTTG

421 GTCCAGTCAT GAAGCCTCAT TAGGGG GAG CTCAGCATGT CCATACCAGG GAAAGTCCTC

481 CTTTTTCAGA AATGTGG AT GGCTTATCAA AAAGAACAGT ACATACCCAA CAATAAAGAG

541 GAGCTACAAT AATACCAACC AAGAAGATCT TTTGGTACTG TGGGGGATTC ACCATCCTAA

601 TGATGCGGCA GAGCAGACAA AGCTCTATCA AAACCCAACC ACCTATATTT CCGTTGGGAC

661 ATCAACACTA AACCAGAGAT TGGTACCAAG AATAGCTACT AGATCCAAAG TAAACGGGCA

721 AAGTGGAAGG ATGGAGTTCT TCTGGACAAT TTTAAAACCG AATGATGCAA TCAACTTCGA

781 GAGTAATGGA AATTTCATTG CTCCAGAATA TGCATACAAA ATTGTCAAGA AAGGGGACTC

841 AACAA TATG AAAAGTGAAT TGGGATATGG TAACTGCAAC ACCAAGTGTC AAACTCCAAT

901 GGGGGCGATA AACTCTAGTA TGCCATTCCA CAATATACAC CCTCTCACCA TCGGGGAATG

961 CCCCAAATAT GTGAAATCAA ACAGATTAGT TCTTGCGACT GGGCTCAGAA ATAGCCCTCA

1021 AAGAGAGACT CGAGGACTAT TTGGAGCTAT AGCAGGTTTT ATAGAGGGAG GATGGCAGGG

1081 AATGGTAGAT GGTTGGTATG GGTACCACCA TAGCAATGAG CAGGGGAGTG GGTACGCTGC

1141 AGACAAAGAA TCCACTCAAA AGGCAATAGA TGGAGTCACC AATAAGGTCA ACTCGATCAT

1201 TGACAAAATG AACACTCAGT TTGAGGCCGT TGGAAGGGAA TTTAACAACT TAGAAAGGAG

1261 AATAGAGAAT TTAAACAAGA AGATGGAAGA CGGGTTCCTA GATGTCTGGA CTTATAATGC

1321 TGAACTTCTG GTTCTCATGG AAAATGAGAG AACTCTAGAC TTTCATGACT CAAATGTCAA

1381 GAACCTTTAC GACAAGGTCC GACTACAGCT TAGGGATAAT GCAAAGGAGC TGGGTAACGG

1441 TTGTTTCGAG TTCTATCATA AATGTGATAA TGAATGTATG GAAAGTGTAA GAAACGGAAC

1501 GTATGACTAC CCGCAGTATT CAGAAGAAGC AAGACTAAAA AGAAAGGAAA TAAGTGGAGT

1561 AAAATTGGAA TCAATAGGAA TTTACCAAAT ACTGTCAATT TATTCTACAG TGGCGAGTTC

1621 CCTAGCACTG GCAATCATGG TAGCTGGTCT ATCCTTATGG ATGTGCTCCA ATGGGTCGTT

1681 ACAATGCAGA ATTTGCATTT AAATTTGTGA GTTCAGAA

As previously mentioned, the inactivated influenza viruses of vaccines according to most embodiments of the present disclosure include a N A hemagglutinin selected from a group that includes the Nl, N2, N5, N6, N7, and N8 neuraminidases. In some embodiments, the inactivated influenza viruses include or carry either the N6 or N7 neuraminidase.

Many embodiments of the present disclosure are based on the finding that the use of the N6 or N7 neuraminidase facilitates or effectuates an enhanced (e.g., increased) production of antibodies when the vaccine is administered into living organisms (e.g., human being or animals). In other words, many embodiments are based on finding that use of the N6 or N7 neuraminidase facilitates or effectuates enhanced prevention or treatment against infection by a future or new influenza A virus strain. In addition, some embodiments of the present disclosure are based on the finding that use of N6 or N7 neuraminidase facilitates or effectuates higher rate of replication of influenza viruses (before inactivation to form the inactivated influenza viruses) in chicken egg cells, thereby contributing to an increased yield of manufactured vaccine.

In selected embodiments, the inactivated influenza viruses include or carry at least two of the Nl , N2, N5, N6, N7, and N8 neuraminidases. In most embodiments, the Nl , N2, N5, N6, N7, and N8 neuraminidases are each derived from a group of influenza viruses derived from birds (i.e., organisms of the class Aves). In selected embodiments, the Nl neuraminidase is derived from A/Puerto Rico/8/1934, the N2 neuraminidase is derived from A/Singapore/1/57, the N5 neuraminidase is derived from A/Duck/ Alberta/60/1976, the N6 neuraminidase is derived from A/gull Maryland/704/77, the N7 neuraminidase is derived from A/chicken Germany/N/49, and the N8 neuraminidase is derived from A/duck Australia/341/83.

In some embodiments, each of the Nl, N2, N5, N6, N7, and N8 neuraminidases, more particularly the N6 and N7 neuraminidases, can be derived from an influenza virus of a more recent influenza A outbreak. In selected embodiments, each of the Nl, N2, N5, N6, N7, and N8 neuraminidases, more particularly the N6 and N7 neuraminidases, can be derived from an alternative influenza virus as desired, for example based on a country in which the vaccine is to be used, administered, or given, or a species of living organism that the vaccine is to be administered to.

In most embodiments of the present disclosure, the inactivated influenza viruses, and accordingly the vaccine, are constructed or manufactured using reverse genetics methods, processes, techniques, and/or methodologies.

REVERSE GENETICS METHODS, METHODOLOGIES, AND PROCESSES

In most embodiments of the present disclosure, the inactivated viruses are manufactured or constructed by applying reverse genetics methods, methodologies, and/or processes. In many embodiments, the inactivated influenza virus is constructed by combining a HA gene from a first high pathogenicity influenza virus (e.g., avian influenza virus) and a NA gene from a second lower, or low, pathogenicity influenza virus into a backbone gene sequence from a third low pathogenicity influenza virus, the backbone gene sequence including remaining influenza A viral genes, for example genes encoding for viral nucelocapsid (NP) and matrix (M) proteins. The third low pathogenicity virus may be similar to the second low pathogenicity influenza virus. In many embodiments, the backbone gene sequence is derived from the H1N1 avian influenza virus designated A/Puerto Rico/8/34.

In many embodiments of the present disclosure, the vaccine includes inactivated influenza A viruses constructed by combining a HA gene (or HA gene sequence) corresponding to the H5 hemagglutinin with a NA gene (or NA gene sequence) corresponding to one of the Nl, N2, N5, N6, N7, and N8 neuraminidases. In some embodiments, the vaccine includes at least one inactivated influenza A virus constructed by combining gene sequences encoding for the H5 hemagglutinin and the N6 neuraminidase, and at least one inactivated influenza A virus constructed by combining gene sequences encoding for the H5 hemagglutinin and the N7 neuraminidase.

In several embodiments, the inactivated influenza A viruses are constructed by combining a H5 gene from A/chicken/Thialand/73/2004, a N6 gene from A/gull/Maryland/704/77, and a backbone gene sequence from A/Puerto Rico/8/1934. In numerous embodiments, the inactivated influenza A viruses are constructed by combining a H5 gene from A/chicken/Thialand/73/2004, a N7 gene from A/chicken/Germany/N/49, and a backbone gene sequence from A/Puerto Rico/8/1934.

Recently developed reverse genetics systems have allowed the manipulation of the influenza viral genome (Palese et al., Proc. Natl. Acad. Sci. U.S.A. 93:1354 (1996); Neumann and Kawaoka, Adv. Virus Res. 53:265 (1999); Neumann et al., Proc. Natl. Acad. Sci. U.S.A. 96:9345 (1999); Fodor et al., J. Virol. 73:9679 (1999)). For example, it has been demonstrated that the plasmid driven expression of influenza mRNAs from a pol I promoter and all mRNAs from a pol II promoter results in the formation of infectious influenza A viruses (Hoffmann et al., Proc. Natl. Acad. Sci. USA 2000, 97:6108), which is incorporated by reference in its entirety for its description of a minimal plasmid reverse genetics system, and its description of genetic engineering methods. Reverse genetics methods allow for specific production of influenza virus types with specific alterations to the polypeptide amino acid sequence. Techniques to isolate and modify specific nucleic acids and proteins are known to a person of ordinary skill in the art. In addition, embodiments of the present disclosure may employ conventional molecular biology, microbiology, and recombinant DNA techniques known to a person of ordinary skill in the art.

As a non-limiting example, a cDNA copy of each of a H5 hemagglutinin gene and a N6 neuraminidase gene can be obtained (e.g., through reverse transcription) from isolated RNA of particular influenza A viruses and cloned into expression vectors or expression plasmids. The expression plasmids, referred to hereinafter as recombinant expression plasmids, express the H5 hemagglutinin and N6 neuraminidase cDNA along with other influenza virus genes (e.g., backbone gene sequence which can be obtained in a similar manner) from the same or a different influenza A virus to produce recombinant influenza A viruses containing the H5 hemagglutinin and N6 neuraminidase of the particular influenza A viruses used.

The recombinant expression plasmids are transfected into cell cultures, for example 293 cell cultures and MDCK cell cultures, to support the production of the recombinant influenza A viruses. In most embodiments, the recombinant expression plasmids include a first recombinant expression plasmid that expresses H5 hemagglutinin, a second recombinant expression plasmid expressing one of the Nl, N2, N5, N6, N7, and N8 neuraminidases, and one or more recombinant expression plasmids that express PB1, PB2, PA, NP, Ml, M2, NS1, and NS2 proteins. In certain embodiments, the PB1, PB2, PA, NP, Ml, M2, NS1, and NS2 proteins are each expressed from a separate recombinant expression plasmid. Alternatively, more than one of the PB 1 , PB2, PA, NP, Ml , M2, NS 1 , and NS2 proteins are expressed from a single recombinant expression plasmid.

Transfection of the recombinant expression plasmids into cell cultures can be performed using Lipofectamine. Transfection techniques and methodologies have been discussed in the above- cited references, as well as in Plesechka S. et al., "A plasmid based reverse genetics system for influenzae A virus", J. Virol. 1996; 70(6): 4188-192, as well as Sambrook J. and Russell DW, Molecular Cloning, 3 ^rd Edition, New York.Could Harbor Laboratory Press; 2001. In numerous embodiments of the present disclosure, the recombinant influenza viruses are amplified or multiplied using egg-based techniques in which the supernatant obtained from the cell cultures, for example the 293 cell culture or the MDCK cell culture, are injected into an egg cell (e.g., a chicken egg cell). Recombinant influenza viruses are then recovered from harvested allantoic fluid of the egg cells.

In many embodiments, the recovered recombinant influenza viruses are then inactivated to obtain inactivated influenza viruses. Inactivation of influenza viruses can be performed using methods, processes, techniques, or procedures known to a person of ordinary skill in the art. For instance, influenza viruses may be inactivated by chemical inactivation using chemical inactivating agents such as binary ethyleneimine, beta-propiololactone, formalin, gluteraldehyde, sodium dodecyl sulfate, or a like chemical substance or mixture. Alternatively, influenza viruses may be inactivated using heat or psoralen in the presence of ultraviolet light.

As previously described, most embodiments of the present disclosure relate to vaccines including inactivated viruses that include or carry the H5 hemagglutinin and a NA subtype selected from the group including the Nl, N2, N5, N6, N7, and N8 neuraminidases. In some embodiments, the vaccine includes inactivated viruses that include or carry the H5 hemagglutinin and either the N6 neuraminidase or the N7 neuraminidase. The vaccine can be administered to living organisms (e.g., human beings and animals) for facilitating or effectuating production of antibodies specific for the inactivated influenza A viruses carried by the vaccine, preventing or treating infection by future or new influenza A viruses, and/or protecting the living organisms from climcal signs or symptoms associated with infection by influenza viruses.

VACCINE DOSES, VACCINE FORMULATIONS, AND VACCINE ADMINISTRATION Most embodiments of the present disclosure provide vaccines that include an immunologically effective amount or quantity of the inactivated influenza virus. As previously mentioned, the term immunologically effective refers to an amount of the inactivated influenza virus that will induce at least a partial immunity in a treated living organism (e.g., human being or animal) against a future or subsequent challenge with a virulent strain of influenza virus. In other words, the term immunologically effective amount refers to an amount of the inactivated influenza viruses that is capable of preventing or treating infection by an influenza A virus, or preventing, treating, or mitigating clinical effect(s) of infection by the influenza A virus (i.e., influenza disease), as determined by one of in-vitro tests, visual inspection, or other methods or techniques known to a person of ordinary skill in the art. In addition, for purposes of the present disclosure, a dose of vaccine (e.g., vaccine composition and vaccine formulation) relates to a particular amount or quantity of the vaccine that is administered at a particular point in time. Alternatively, a dose of vaccine can also be a quantity of vaccine that is gradually administered to the living organism using an extended release formulation and/or apparatus.

In most embodiments of the present disclosure, a dose is between approximately 0.1 OmL to 2.5mL, for example 0.25mL, 0.50mL, 0.75mL, l.OmL, 1.5mL, 2.0mL, and 2.5 mL. In some embodiments, two or more doses (i.e., multiple doses) of the vaccine are administered to the living organism at different time points. For example, a second and possibly subsequent doses of the vaccine may be administered 2, 3, 4, 5, 10, 15, 20, 30, 40, 60, 90, or more days after administration of a first dose of the vaccine.

As stated above, an immunologically effective amount of the inactivated influenza virus is an amount of the inactivated influenza virus that will induce at least a partial immunity in a treated living organism against subsequent challenge with a virulent strain of influenza virus. Complete or partial immunity can be assessed by observing, either qualitatively or quantitatively, the clinical symptoms of influenza virus infection in a vaccinated (or treated) living organism as compared to an unvaccinated (or untreated) living organism after being challenged with a virulent strain of influenza virus. Where the clinical symptoms of influenza virus infection in a vaccinated living organism after challenge are reduced, lessened, or eliminated as compared to the clinical symptoms observed in an unvaccinated living organism after a similar or identical challenge, the amount or quantity of the vaccine that was administered to the vaccinated living organism is regarded as an immunologically effective amount. In some embodiments of the present disclosure, the immunologically effective amount of the vaccine corresponds to between approximately 10 ^" and approximately 10 ^" EID50 /0.5ul (dose) of vaccine. In several embodiments, the immunologically effective amount of the vaccine corresponds to approximately 10 ^"7 EID50 for a dose of vaccine of between approximately between 0.3ul and 0.5ul. In selected embodiments, the immunologically effective amount of the vaccine corresponds to approximately 10 ^"7 EID50 /0.5ul (dose) of vaccine.

In some embodiments of the present disclosure, immunologically effective amounts are expressed in terms of hemagglutinin units (or HA units). Additionally or alternatively, immunologically effective amounts can be expressed in terms of neuraminidase units (or NA units). In many embodiments, the immunologically effective amounts can be varied depending on characteristics of the living organism (e.g., age, size, living environment, and species of the living organism) the vaccine is to be administered.

Non-limiting representative amounts of the inactivated influenza virus that can be considered immunologically effective amounts include between approximately 1 HA and approximately 1000 HA (e.g. between approximately 1 H5 and approximately 1000 H5 hemagglutinin) units per dose of the vaccine. In some embodiments, immunologically effective amounts include approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 950 H5 units per dose of the vaccine. In several embodiments, immunologically effective amounts include approximately 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and 950 N2, N5, and/or N6 units per dose of the vaccine.

Although specific doses, and immunologically effective amounts of the vaccine, are disclosed herein, a person of ordinary skill in the art will understand that alternative doses and/or immunologically effective amounts of the vaccine are also included in the present disclosure. For example, the dose and/or immunologically effective amount of the vaccine can be altered depending on species, size, age, and/or physiological condition of the living organism being administered the vaccine. In several embodiments of the present disclosure, the vaccine can further include one or more of pharmaceutically acceptable carriers, adjuvants, lipopolysaccharides, sapponins, excipients, stabilizers, additives, preservatives, and other chemicals, compounds, or mixtures known in the art. In some embodiments, PBS solution is used for dilution in order to obtain a desired vaccine concentration or dosage. In selected embodiments, Montanide ISA 70 is also used to dilute or adjust the vaccine to a final desired concentration.

Examples of pharmaceutically acceptable carriers include water, saline, or phosphate buffers. Examples of suitable adjuvants include carbopol, dimethyl dioctadecyl ammonium bromide (DDA), aluminum hydroxide, aluminum phosphate, and other aluminum or metal salts. Examples of lipopolysaccharides include bacterial lipopolysaccharides or bacterial lipopolysaccharide derivatives, for example monophosphoryl lipid A and 3-O-Deacylated monophosphoryl lipid A (3D-MPL). Saponins are steroid or triterpene glycosides that are widely found in plant and marine animal kingdoms. Most saponins are widely accepted for their ability to form colloidal solutions in water, for precipitating cholesterol, and for creating pore like structures in membranes of cells to thereby cause the cell membrane to burse. Saponins are commonly used as adjuvants in vaccines for systemic administration. For example, the haemolytic saponins QS21 and QS17 (HPLC purified fractions of Quil A) have been used as systemic adjuvants.

Examples of excipients include surfactants (e.g., sorbitan mono-oleate esters, ethylene oxide/propylene oxide block copolymers, and nonionic surfactants such as Triton X-45, Triton X-100, Triton X-114, and Triton N-101), wetting agents, and other known vaccine formulation aids. There are many known stabilizers, additives, and preservatives known in the art, for example formalin, and carbohydrates such as sorbitol, mannitol, starch, sucrose, dextrin, and glucose. In most embodiments of the present disclosure, the vaccine is administered to living organisms (e.g., human beings and animals). Administration of the vaccine may be performed by any one of numerous methods of administration as desired, for example intranasal delivery and intradermal delivery by use of one of conventional syringes, jet injection devices, and ballistic powder/particle delivery devices.

ISOLATED SERUM SAMPLES

Serological assays are widely used in the determination of influenza diagnosis. In many embodiments of the present disclosure, serological assays (e.g., using isolated serum samples), for example HI assays, show that particular vaccines provided by embodiments of the present disclosure are capable of triggering the production of antibodies in living organisms to which the vaccine is administered.

A hemagglutination inhibition (HI) assay is the standard method for serologic detection of influenza virus infection in living organisms. The principle or basis of the HI assay is that antibodies to a particular influenza virus will prevent attachment of the influenza virus to red blood cells. Therefore hemagglutination is inhibited when antibodies to that influenza virus are present. Where antibodies to the influenza virus are present, hemagglutination will only occur when the antibodies are sufficiently diluted (i.e., when the serum including the antibodies is sufficiently diluted). The highest dilution of serum that prevents hemagglutination is referred to as the HI titer of the serum. The higher the HI titer of the serum, the more antibodies to the influenza virus are present in serum, and correspondingly, the more effective a particular vaccine is. In many embodiments, the isolated serum samples (i.e., isolated serum samples extracted from living organisms that were administered at least one dose of the vaccine) exhibit a HI titer of at least 4 when tested using an influenza A virus including or carrying the H5 hemagglutinin. A HI titer of at least 4 represents a positive result in HI assays. Accordingly, obtaining HI titers of at least 4 indicates that the vaccine is capable of inducing antibody production against influenza A viruses that include or carry the H5 hemagglutinin. In some embodiments, the isolated samples exhibit a HI titer of 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, or more when tested against an influenza A virus including or displaying a H5 hemagglutinin. In some embodiments, a neuraminidase inhibition (NI) assay is used for differentiating between vaccinated living organisms (e.g., chickens) and living organisms (e.g., chickens) that are infected or challenged with an influenza A virus (e.g., influenza A virus of a H5N1 subtype). The NI assay typically enables identification and classification of influenza (e.g., influenza A) viruses based on differing NA subtypes being carried by the influenza A viruses. In several embodiments, the NI assay was used to distinguish between living organisms (e.g., chickens) administered the vaccine and chickens that have been naturally or artificially infected with an influenza A virus. The DIVA (i.e., differentiation of infected from vaccinated animals) technique, as described in published PCT WO 03/086453 and incorporated by reference in its entirety herein, is used in association with the present disclosure for differentiating between vaccinated animals (e.g., chickens) and infected animals (e.g., chickens). As described above, embodiments of the present disclosure provide vaccines and vaccine compositions for preventing or treating influenza A virus infection. Particular vaccines of the present disclosure include inactivated influenza viruses (e.g., inactivated recombinant influenza viruses) that include or carry the H5 hemagglutinin derived from A/chicken/Thailand/704/2004. In addition, the inactivated influenza viruses (e.g., inactivated recombinant influenza viruses) further include or carry at least one NA subtype. In many embodiments, the at least one NA subtype carried by the inactivated influenza viruses is the N6 neuraminidase or the N7 neuraminidase.

Representative examples of the particular vaccines, inactivated influenza viruses, and methods for manufacture of particular vaccines in association with the present disclosure are provided in the following disclosure. Although particular examples are described below, a person of ordinary skill in the art will understand that modifications, variations, and adaptations may be made to the examples within the scope of the present disclosure. EXAMPLE ONE

An Exemplary Vaccine or Vaccine Composition

A vaccine or vaccine composition that includes inactivated influenza viruses is provided by the present disclosure, wherein the inactivated viruses include the H5 hemagglutinin (HA) subtype (also known as H5 antigen). In addition, the inactivated viruses include a neuraminidase (NA) subtype, the neuraminidase subtype being selected from a group including Nl, N2, N5, N6, N7, and N8 neuraminidases.

The H5 hemagglutinin is derived from an influenza A virus of Southeast Asian origin. Derivation of the H5 hemagglutinin from an influenza A virus of Southeast Asian origin facilitates or effectuates an enhanced (e.g., increased) production of antibodies against future Southeast Asian influenza outbreaks (e.g., influenza epidemics or pandemics) caused by influenza A viruses of the H5 hemagglutinin subtype. Derivation of the H5 hemagglutinin from an influenza A virus of Southeast Asian origin facilitates or effectuates an increased likelihood of the inactivated influenza viruses of the vaccine having similar epitopes to that of future or new influenza virus strains of Southeast Asian origin.

EXAMPLE TWO

Another Exemplary Vaccine or Vaccine Composition

A vaccine or vaccine composition that includes inactivated influenza viruses is provided by the present disclosure, wherein the inactivated viruses include the H5 hemagglutinin (HA) subtype (also known as H5 antigen). In addition, the inactivated viruses include either the N6 neuraminidase (i.e., N6 antigen) or the N7 neuraminidase (i.e., N7 antigen). The H5 hemagglutinin is derived from A/chicken/Thailand/704/2004. Derivation of the H5 hemagglutinin from A/chicken/Thailand/704/2004 facilitates or effectuates an enhanced (e.g., increased) production of antibodies against future Southeast Asian influenza outbreaks (e.g., influenza epidemics or pandemics) caused by influenza A viruses of the H5 hemagglutinin subtype. Derivation of the H5 hemagglutinin from A/chicken/Thailand/704/2004 facilitates or effectuates an increased likelihood of the inactivated influenza viruses of the vaccine having similar epitopes to that of future or new influenza virus strains of Southeast Asian origin. EXAMPLE THREE

Another Exemplary Vaccine or Vaccine Composition

A vaccine or vaccine composition that includes inactivated influenza viruses is provided by the present disclosure, wherein the inactivated viruses include the H5 hemagglutinin (HA) subtype (also known as H5 antigen). In addition, the inactivated viruses include either the N6 neuraminidase (i.e., N6 antigen) or the N7 neuraminidase (i.e., N7 antigen).

The H5 hemagglutinin is derived from A/chicken/Thailand/704/2004. Derivation of the H5 hemagglutinin from A/chicken/Thailand/704/2004 facilitates or effectuates an enhanced (e.g., increased) production of antibodies against future Southeast Asian influenza outbreaks (e.g., influenza epidemics or pandemics) caused by influenza A viruses of the H5 hemagglutinin subtype. Derivation of the H5 hemagglutinin from A/chicken/Thailand/704/2004 facilitates or effectuates an increased likelihood of the inactivated influenza viruses of the vaccine having similar epitopes to that of future or new influenza virus strains of Southeast Asian origin.

The N6 neuraminidase is derived from A/gull/Maryland/704/77 and the N7 neuraminidase is derived from A/chicken/Germany/N/49. In some embodiments, influenza A viruses including the H5 hemagglutinin that is derived from A/chicken/Thailand/703/2004, and either the N6 neuraminidase or N7 neuraminidase, facilitates or effectuates an increased production of antibodies against a future or new influenza epidemic (or pandemic). In some embodiments, influenza A viruses including the H5 hemagglutinin that is derived from A/chicken Thailand/703/2004, and either the N6 neuraminidase or N7 neuraminidase, facilitates or effectuates unexpected and significant reduction in tracheal and cloacal shedding in vaccinated living organisms (e.g., chickens).

EXAMPLE FOUR

Manufacture or generation of vaccine or vaccine composition including inactivated H5N6 Influenza virus

Representative processes for manufacturing, generating, or constructing a vaccine, more particularly vaccine including inactivated H5N6 influenza virus, utilizes reverse genetics methodology or techniques, for example the reverse genetics methodology described in Hoffman et al., Vaccine 20:3165-3170 (2002). FIG. 1 shows a flowchart of a representative process 100 for manufacturing a particular vaccine in association with the present disclosure, more particularly a vaccine including inactivated H5N6 influenza viruses.

In a first process portion 110, portions of RNA are isolated or extracted from influenza A viruses, more specifically from A/chicken/Thailand/73/2004, A/Puerto Rico/8/1934, and A/gull/Maryland/704/77. The isolation of RNA can be performed using techniques known to a person of ordinary skill in the art. For example, RNA can be isolated using the RNAeasy kit (Qiagen).

In a second process portion 115, cDNA is produced from the extracted RNA using appropriate primers and an enzyme known as reverse transcriptase (which is a DNA polymerase enzyme). In a third process portion 120, the cDNA is amplified using segment specific primers, for example using the technique described in Hoffmann et al., Arch. Virol. 146:2275-2289, 2001.

In a fourth process portion 125, enzymatic cleavage of the cDNA is performed. The enzymatic cleavage is performed at a specific lineage site to attenuate or to reduce the pathogenicity of produced or manufactured influenza A viruses of the vaccine. The lineage site used for the influenza A virus of A/chicken/Thailand/73/2004 is 1055-1067, which corresponds to an amino acid sequence of RKKR. It will be appreciated by a person of ordinary skill in the art that cDNA obtained from RNA of other influenza viruses may be cleaved at different cleavage sites as desired, for example to reduce the pathogenicity of correspondingly produced influenza viruses.

In a fifth process portion 130, the cDNA is cloned into expression plasmids, for example pHW2000, forming recombinant plasmids. Cloning of cDNA into the expression plasmid is performed using techniques known to a person of ordinary skill in the art. In a sixth process portion 135, the recombinant plasmids are placed into cultures of 293 cells. Transfection was carried out using techniques or methods known to a person of ordinary skill in the art. In a seventh process portion 140, the media from the cultures of 293 cells is harvested. In an eighth process portion 145, the media harvested from the cultures of 293 cells are then cultured with MDCK cells. Transfection can be carried out using techniques or methods known to a person of ordinary skill in the art.

The sixth to eighth process portions (i.e., 135 to 145) represents a deviation from standard laboratory protocol for using for manufacture of recombinant viruses, which typically places the recombinant plasmids in a co-culture including both 293 cells and MDCK cells. The sequential use of 293 cells followed by MDCK cells facilitates at least one of enhanced cell transfection and generation of recombinant viruses as compared to simultaneous use of both the 293 cells and MDCK cells. This enhanced cell transfection and generation of recombinant viruses associated with the sequential performance of the sixth to eighth process portions can be considered unexpected by a person of ordinary skill in the art. In a ninth process portion 150, supernatant of the MDCK cell culture is retrieved. The supernatant includes recombinant viruses of the H5N6 strain, which are hybrids of the influenza viruses used for their production. More specifically, the recombinant viruses include H5 hemagglutinin derived from A/chicken/Thailand/73/2004, N6 neuraminidase derived from A/gull/Maryland/704/77, and other influenza viral proteins such as PB1, PB2, PA, NP, M, and NS derived from A/Puerto Rico/8/1934.

In a tenth process portion 155, the supernatant is injected into the allantoic cavity of embryonated chicken egg cells. Recombinant influenza viruses are recovered from the harvested allantoic fluid. The tenth process portion enables an increase in the quantity of the recombinant influenza viruses (i.e., increases yield of the recombinant influenza virus). It is found in the present disclosure that the use of N6 neuraminidase, or use of A gull/Maryland/704/77, facilitates or effectuates an unexpectedly high production, or yield, of recombinant influenza viruses from the chicken egg cells. In an eleventh process portion 160, the recombinant influenza viruses are inactivated using formalin to produce inactivated influenza viruses. It will be appreciated by a person of ordinary skill in the art that other methods for inactivation of produced recombinant influenza viruses may alternatively be used. Inactivation of the recombinant influenza viruses extracted from the harvested allantoic fluid from chicken egg cells is important to reduce pathogenicity of the influenza viruses of the manufactured vaccine.

EXAMPLE FIVE

Testing for effectiveness of particular vaccines provided by the present disclosure

Experiments were conducted to evaluate the effectiveness of particular vaccines provided by the present disclosure. More specifically, experiments to determine potency, safety, stability, and sterility were conducted. With specific regard to potency, experiments associated with HI assays, tracheal swabs, and cloacal swabs were conducted.

Experiments With Regard to Potency Experiment (A): Test of vaccine including inactivated influenza A viruses that include H5 derived from A/chicken/Thailand/ ^' / '04/2004 and H6 derived from A/gull/Maryland/704/77

20 specific pathogenic free (SPF) chickens were used in experiment (A). The 20 SPF chickens were divided into two groups in which:

(i) Group 1 : Negative Controls. Consists of 10 chickens administered (e.g., injected) with 0.5ml of (PBS + Adjuvant)

(ii) Group 2: Consists of 10 chickens that were administered with 0.5ml of the vaccine Blood samples were extracted or obtained from the chickens at 21 -day and 42-day time periods. HI assays were performed for each blood sample (i.e., HI titer of each blood sample was determined) at the 21 -day and 42-day time periods.

After 42 days from administration of either (PBS + adjuvant) or vaccine, the chickens were challenged with high pathogenicity viruses of the H5N1 subtype (hereinafter referred to as challenge virus). Approximately lOul of 10 ^{4 5} EID ₅₀ of the challenge viruses were introduced into the chickens intranasally. At days 3, 5, 7, 10, and 20 after the challenge viruses were introduced into the chickens, blood samples were extracted from the chickens and the HI titer of each blood sample was determined.

Extraction of blood samples and determination of HI titers were performed using methods known to a person of ordinary skill in the art.

In addition, tracheal and cloacal swab tests were performed on the chickens at days 3, 5, 7, 10, and 20 after the challenge viruses were introduced into the chickens. Tracheal and cloacal swab tests were performed using methods or techniques known to a person of ordinary skill in the art.

Results for Experiment (A)

As shown in Table 1, HI titers of 0 were obtained for each of the negative controls (i.e., chickens in Group 1). This indicates that no antibodies were produced in the negative controls. The HI titers of vaccinated chickens were on average 6.22 at day 21 and 8.55 at day 42. This indicates that increased quantities of antibodies were present in the vaccinated chickens. Generally, HI titers of more than 4 correspond to increased quantities of antibodies within the body. At days 3, 5, 7, 10, and 20 after introduction of the challenge virus, HI titers could not be determined for the negative controls because all the negative controls died within 48 hours from introduction of the challenge viruses.

However, there were no fatalities (i.e., a 100% survival rate) with the vaccinated chickens at each of days 3, 5, 7, 10, and 20 after the introduction of the challenge virus. The 100% survival rate of vaccinated chickens after the introduction of challenge viruses was unexpected and represents an extraordinarily good immmunological protection provided by particular vaccines in association with the present disclosure.

At days 3, 5, 7, 10, and 20 after introduction of the challenge virus, average HI titers of the vaccinated chickens were 9, 9, 8.77. 8.66, and 9.55 respectively. This indicates increased quantities of antibodies within the vaccinated chickens of group 2. All the vaccinated chickens of group 2 survived past 20 days after introduction with the challenge viruses.

Results from the tracheal and cloacal swab tests are shown in Table 2. Tracheal and cloacal swab tests could not be performed with the negative controls as all the negative controls die within 48 hours. Results from the tracheal and cloacal swab tests show no tracheal shedding and no cloacal shedding in each of the vaccinated chickens at each of days 3, 5, 7, 10, and 20 after introduction of the challenge virus. The total absence of tracheal and cloacal shedding in each of the vaccinated chickens was an unexpected result indicating a surprising level of efficacy for vaccines provided by the present disclosure.

Table 1: HI titers of negative controls and vaccinated chickens at specific time periods

HI titers after introduction of challenge

HI titer (Log) virus (i.e., influenza A virus of H5N1 subtype)

Group After

Before vaccination Day Day

Day 3 Day 5 Day 7

vaccination 10 20

21 day 42 day

Group 1 Chickens died within 48 hours of

0 0 0

(Negative Controls) introduction of challenge virus

Group 2

(Vaccinated 0 6.22 8.55 9 9 8.77 8.66 9.55 chickens)

Table 2: Presence of tracheal shedding and cloacal shedding in negative controls and vaccinated chickens at specific time periods

Conclusion for Experiment (A)

Results show a 100% survival rate of vaccinated chickens after the introduction of challenge viruses. This unexpected result represents an extraordinarily good immmunological protection provided by particular vaccines in association with the present disclosure. The 100% survival rate of vaccinated chickens after the introduction of challenge viruses also suggests that particular vaccines in association with the present disclosure have an enhanced safety profile.

Results indicate that chickens that are vaccinated with a particular vaccine provided by the present disclosure, more specifically the vaccine including inactivated influenza virus with H5 hemagglutinin derived from A/chicken Thailand 704/2004 and N6 neuraminidase derived from A/gull/Maryland/704/77, have increased protection against future infection by an influenza A virus of the H5N1 subtype. Results indicate that chickens that are vaccinated with the vaccine including inactivated influenza virus with H5 hemagglutinin derived from A/chicken/Thailand/704/2004 and N6 neuraminidase derived from A/gull Maryland/704/77, possess or have higher quantities of antibodies specific for H5 hemagglutinin. Accordingly, results indicate that particular vaccines provided by the present disclosure, more specifically the vaccine including inactivated influenza virus with H5 hemagglutinin derived from A/chicken/Thailand/704/2004 and N6 neuraminidase derived from A/gull/Maryland/704/77, are effective or potent for triggering production of antibodies specific for the H5 hemagglutinin, and hence effective for preventing future infection of vaccinated chickens by an influenza A virus of the H5N1 subtype.

Results also reveal unexpected absence of tracheal shedding and cloacal shedding of the inactivated influenza A virus of the vaccine. This indicates an absence of shedding or expulsion of the inactivated influenza A virus (more specifically progeny of the virus) from tracheal and cloacal sites of the body.

Experiment (B): Test of vaccine including inactivated influenza A viruses that include H5 derived from A/chicken/Thailand/704/2004 and H7 derived from A/chicken/Germany/N/49

20 specific pathogenic free (SPF) chickens were used in experiment (B). The 20 SPF chickens were divided into two groups in which:

(iii) Group 1: Negative Controls. Consists of 10 chickens administered (e.g., injected) with 0.5ml of (PBS + Adjuvant)

(iv) Group 2: Consists of 10 chickens that were administered with 0.5ml of the vaccine

Blood samples were extracted or obtained from the chickens at 21 -day and 42-day time periods. HI assays were performed for each blood sample (i.e., HI titer of each blood sample was determined) at the 21 -day and 42-day time periods.

After 42 days from administration of either (PBS + adjuvant) or vaccine, the chickens were challenged with high pathogenicity viruses of the H5N1 subtype (hereinafter referred to as challenge virus). Approximately lOul of 10 ^{4 5} EID5o of the challenge viruses were introduced into the chickens intranasally. At days 3, 5, 7, 10, and 20 after the challenge viruses were introduced into the chickens, blood samples were extracted from the chickens and the HI titer of each blood sample was determined.

Extraction of blood samples and determination of HI titers were performed using methods known to a person of ordinary skill in the art. In addition, tracheal and cloacal swab tests were performed on the chickens at days 3, 5, 7, 10, and 20 after the challenge viruses were introduced into the chickens. Tracheal and cloacal swab tests were performed using methods or techniques known to a person of ordinary skill in the art.

Results for Experiment (B)

As shown in Table 3, HI titers of 0 were obtained for each of the negative controls (i.e., chickens in Group 1). This indicates that no antibodies were produced in the negative controls. The HI titers of vaccinated chickens were on average 3.87 at day 21 and 9.42 at day 42. This indicates that increased quantities of antibodies were present in the vaccinated chickens. Generally, HI titers of more than 4 correspond to increased quantities of antibodies within the body.

At days 3, 5, 7, 10, and 20 after introduction of the challenge virus, HI titers could not be determined for the negative controls because all the negative controls died within 48 hours from introduction of the challenge viruses.

However, there were no fatalities (i.e., a 100% survival rate) with the vaccinated chickens at each of days 3, 5, 7, 10, and 20 after the introduction of the challenge virus. The 100% survival rate of vaccinated chickens after the introduction of challenge viruses was unexpected and represents an extraordinarily good immmunological protection provided by particular vaccines in association with the present disclosure.

At days 3, 5, 7, 10, and 20 after introduction of the challenge virus, average HI titers of the vaccinated chickens were 8.85, 8.85, 8.57, 8.57, and 9.71 respectively. This indicates increased quantities of antibodies within the vaccinated chickens of group 2.

Results from the tracheal and cloacal swab tests are shown in Table 4. Tracheal and cloacal swab tests could not be performed with the negative controls as all the negative controls die within 48 hours. Results from the tracheal and cloacal swab tests show no tracheal shedding and no cloacal shedding in each of the vaccinated chickens at each of days 3, 5, 7, 10, and 20 after introduction of the challenge virus. The total absence of tracheal and cloacal shedding in each of the vaccinated chickens was unexpected.

Table 3: HI titers of negative controls and vaccinated chickens at specific time periods

Table 4: Presence of tracheal shedding and cloacal shedding in negative controls and vaccinated chickens at specific time periods

Conclusion for Experiment (B)

Results show a 100% survival rate of vaccinated chickens after the introduction of challenge viruses. This unexpected result represents an extraordinarily good immmunological protection provided by particular vaccines in association with the present disclosure. The 100% survival rate of vaccinated chickens after the introduction of challenge viruses also suggests that particular vaccines in association with the present disclosure have an enhanced safety profile.

Results indicate that chickens that are vaccinated with a particular vaccine provided by the present disclosure, more specifically the vaccine including inactivated influenza virus with H5 hemagglutinin derived from A/chicken/Thailand/704/2004 and N7 neuraminidase derived from A chicken/Germany/N/49, have increased protection against future infection by an influenza A virus of the H5N1 subtype. Results indicate that chickens that are vaccinated with the vaccine including inactivated influenza virus with H5 hemagglutinin derived from A chicken Thailand/704/2004 and N7 neuraminidase derived from A/chicken/Germany/N/49, possess or have higher quantities of antibodies specific for H5 hemagglutinin.

Accordingly, results indicate that particular vaccines provided by the present disclosure, more specifically the vaccine including inactivated influenza virus with H5 hemagglutinin derived from A/chicken/Thailand/704/2004 and N7 neuraminidase derived from A/chicken/Germany/N/49, are effective or potent for triggering production of antibodies specific for the H5 hemagglutinin, and hence effective for preventing future infection of vaccinated chickens by an influenza A virus of the H5N1 subtype. Results also reveal unexpected absence of tracheal shedding and cloacal shedding of the inactivated influenza A virus of the vaccine. This indicates an absence of shedding or expulsion of the inactivated influenza A virus (more specifically progeny of the virus) from tracheal and cloacal sites of the body. Experiments With Regard to Safety

Experiments or tests were conducted to evaluate the safety of particular vaccine provided by the present disclosure, more specifically each of the vaccines used in experiments (A) and (B) as described above (hereinafter referred to as H5N6 vaccine, and H5N7 vaccine, respectively). 20 specific pathogenic free (SPF) chickens of 3 weeks of age were used for the experiments with regard to safety for the H5N6 vaccine. The 20 SPF chickens were divided into two groups in which:

(i) Group 1: Negative Controls. Consists of 10 chickens administered (e.g., injected) with 0.5ml of (PBS + Adjuvant)

(ii) Group 2: Consists of 10 chickens that were administered with 1.0ml of the H5N6 vaccine

Similarly, 20 specific pathogenic free (SPF) chickens of 3 weeks of age were used for the experiments with regard to safety for the H5N7 vaccine. The 20 SPF chickens were divided into two groups in which:

(i) Group 1: Negative Controls. Consists of 10 chickens administered (e.g., injected) with 0.5ml of (PBS + Adjuvant)

(ii) Group 2: Consists of 10 chickens that were administered with 1.0ml of the H5N7 vaccine

After 2 weeks from administration of either (PBS + adjuvant) or vaccine, the chickens were examined for clinical signs of disease, local lesion(s), or death. Results for experiments with regard to safety

As shown in Tables 5 and 6, all negative controls (i.e., chickens not administered either the H5N6 vaccine or the H5N7 vaccine) showed or displayed no clinical signs, local lesion(s), or death. In addition, chickens that were administered with either the H5N6 vaccine or H5N7 vaccine also showed or displayed no clinical signs, local lesion(s), or death.

Table 5: Safety test with regard to H5N6 vaccine

Conclusion for experiments with regard to safety

Results indicate that particular vaccines provided by the present disclosure, more specifically the H5N6 vaccine and the H5N7 vaccine do not cause clinical signs of disease, local lesion(s), or death in chickens that have been administered with said vaccines. Accordingly, results indicate that the H5N6 vaccine and the H5N7 vaccine do not adversely effect the health or well-being of the chickens that are administered with said vaccines.

Experiments With Regard to Stability

Each of the H5N6 vaccine and the H5N7 vaccine are kept at 4°C (in a liquid form) for a predetermined time period.

At predetermined time periods, more specifically at the 7 ^th month, 10 ^th month, 11 ^th month, and th

12 ^l " month, each of the H5N6 vaccine and the H5N7 vaccine were tested for effectiveness or potency using the HI assay as described above. At each of the 7 ^th month, 10 ^th month, 11 ^th month, and 12 month, each of the H5N6 vaccine and the H5N7 vaccine was injected into 10 SPF chickens, and the HI titers of each of the SPF chickens were thereafter determined.

Results for experiments with regard to stability

As shown in Table 7, with reference to the H5N6 vaccine, the HI titers of chickens infected with the H5N6 vaccine at the 7 ^th month, 10 ^th month, 11 ^th month, and 12 ^th month were 7.80, 9.20, 7.33, and 6.25 respectively. Generally, HI titers of more than 4 correspond to increased quantities of antibodies within the body. Accordingly, results shown that the H5N6 vaccine is still capable of inducing increased production of antibodies specific for the H5 hemagglutinin even after 12-months from manufacture or formulation.

As shown in Table 8, with reference to the H5N7 vaccine, the HI titers of chickens infected with the H5N6 vaccine at the 7 ^th month, 10 ^th month, 11 ^th month, and 12 ^th month were 6.50, 8.83, 8.80, and 6.60 respectively. Generally, HI titers of more than 4 correspond to increased quantities of antibodies within the body. Accordingly, results shown that the H5N7 vaccine is still capable of inducing increased production of antibodies specific for the H5 hemagglutinin even after 12-months from manufacture or formulation.

Table 7: HI titers of chickens infected with H5N6 vaccine at specific time periods

Table 8: HI titers of chickens infected with H5N7 vaccine at specific time periods

HI titer (Log) at specific time periods after vaccination

Vaccine

subtype Before

7 month 10 month 11 month 12 month vaccination

H5N7 0 6.50 8.83 8.80 6.60 Conclusion for experiments with regard to stability

Results indicate that particular vaccines provided by the present disclosure, more specifically the H5N6 vaccine and the H5N7 vaccine, are still capable of inducing increased production of antibodies specific for the H5 hemagglutinin at each of 7, 10, 11, and 12 months after manufacture or formulation of said vaccines. In other words, results indicate that particular vaccines provided by the present disclosure, more specifically the H5N6 vaccine and the H5N7 vaccine, retain their ability to prevent future infection by influenza A viruses of a H5 hemagglutinin subtype at each of 7, 10, 11, and 12 months after manufacture or formulation of said vaccines.

Experiments With Regard to Sterility

Sterility tests for particular vaccines provided by the present disclosure, more specifically each of the H5N6 vaccine and the H5N7 vaccine, were performed using methods known to a person of ordinary skill in the art. Sterility tests involve checking for presence of aerobic bacteria, anaerobic bacteria, salmonella species, and fungi within the vaccines.

Results for experiments with regard to sterility

As shown in table 9, results indicate that no, or no detectable amounts of, aerobic bacteria, anaerobic bacteria, salmonella species, and fungi was present in each of the H5N6 vaccine and the H5N7 vaccine.

Table 9: Absence/Presence of anaerobic bacteria, salmonella species, and fungi in H5N6 vaccine and H5N7 vaccine

Conclusion for experiments with regard to sterility

Results indicate that no, or no detectable amounts of, aerobic bacteria, anaerobic bacteria, salmonella species, and fungi was present in each of the H5N6 vaccine and the H5N7 vaccine. Accordingly, each of the H5N6 vaccine and the H5N7 vaccine is sterile and hence safe for administration into chickens.

While the experiments of example five were performed in chickens, a person of ordinary skill in the art will appreciate that the results of the experiments can be similarly observed with other living organisms (e.g., human beings and other poultry or animals).

EXAMPLE SIX

Test for differentiating between vaccinated chickens and non-vaccinated infected chickens Experiments were conducted to evaluate the ability to differentiate between chickens that were vaccinated with particular vaccines of the present disclosure (hereinafter referred to as vaccinated chickens) and non-vaccinated chickens that have been infected with influenza A viruses of the H5N1 subtype. More specifically, neuraminidase inhibition (NAI) technique or assay was used to differentiate between vaccinated chickens and non-vaccinated infected chickens. The NAI assay is based on the ability to differentiate between different NA subtypes (e.g., between the Nl neuraminidase and the N6 or N7 neuraminidase).

In-vivo experiments were conduced to evaluate the ability to differentiate between chickens that were immunized by a particular vaccine provided by the present disclosure, more specifically the H5N7 vaccine, and chickens that are infected by the influenza A viruses of the H5N1 subtype.

Two groups of chickens were used for the experiments:

(i) Group 1: Negative controls. Consists of ten 2-week old chickens administered (e.g., injected) with 1.0ml of (PBS + Adjuvant)

(ii) Group 2: Consists often 2-week old chickens that were administered with vaccine including inactivated influenza virus including H5 hemagglutinin derived from A/chicken/Thailand/704/2004 and N7 neuraminidase derived from A/chicken/Germany/N/49 (i.e., H5N7 vaccine). The vaccine was administered in two doses of 0.5ml each, the second dose administered 3 weeks after administration of the first dose. Three weeks after administration of the second dose, a first portion of chickens (i.e., 5 chickens) of each of group 1 and group 2 were challenged with influenza A virus of H5N1 subtype via an intranasal injection. The other five chickens (hereinafter referred to as a second portion of chickens) of each of group 1 and group 2 were challenged with influenza A of H5N7 subtype.

Serum samples were obtained from the chickens at predetermined time periods and used to determine NA activity using the NAI assay. NAI assays were performed using protocol adapted from section G of the WHO Animal Influenza Manual. More specifically, the protocol from section G of the WHO Animal Influenza Manual was adapted to decrease, in proportion, the volume of each reagent used in the NAI assay.

NA activity can be determined qualitatively (e.g., by visual examination or observation) or quantitatively (e.g., by retrieving an optical density value from a spectrophotometer calibrated at 549nm). For experiments of example 6, the NAI assays were conducted qualitatively, through visual observation of serum samples obtained or extracted from the chickens at predetermined time intervals.

Results

As shown in Table 10, serum samples obtained from chickens to which the challenge viruses (i.e., influenza A virus of the H5N1 subtype) are not yet introduced are pink in color.

After introduction of the H5N1 virus, the serum samples obtained from the first portion of chickens of each of group 1 and group 2 from such chickens are yellow in color upon performing the NAI assay. This indicates that antibodies specific for Nl neuraminidase are not present in the serum samples of the first portion of chickens from each of group 1 and group 2. This result also shows that the H5N7 vaccine is not capable of inducing production of antibodies specific forNl neuraminidase.

After introduction of the H5N7 virus, the serum samples obtained from the second portion of chickens of group 1 are yellow in color upon performance of the NAI assay, indicating that no antibodies specific for the N7 neuraminidase is present in serum samples obtained from the second portion of chickens of group 1.

However, serum samples obtained from the second portion of chickens of group 2 are pink in color upon performance of the NAI assay, indicating that antibodies specific for the N7 neuraminidase is present in serum samples obtained from the second portion of chickens of group 2. This suggests that the administration of the H5N7 vaccine to chickens induces production of antibodies specific for the N7 neuraminidase. Accordingly, one is able to distinguish between chickens that have been administered with the H5N7 vaccine and chickens infected with the influenza A virus of the H5N1 subtype based on the specificity of antibodies, more specifically NA specificity of antibodies, present in the chickens upon performance of an NAI assay. Table 10: Qualitative results (i.e., color observed) of NAI Assays

Conclusion

When a living organism is naturally infected with H5N1 virus (e.g., during an influenza outbreak or epidemic), there is typically a stimulation or trigger of an increased production of antibodies by the living organism. This increase in production of antibodies caused by naturally infected living organisms during an influenza A outbreak needs to be differentiated from the increase in production of antibodies specifically due to vaccination of the living organism (i.e., administration of vaccine into the living organism). Particular vaccines provided by the present disclosure facilitate differentiation between vaccinated chickens and infected chickens. More specifically, chickens administered with the vaccine including inactivated influenza A viruses of H5 hemagglutinin derived from A chicken/Thailand/703/2004 and N7 neuraminidase derived from A/chicken/Germany N/49 can be differentiated from non-vaccinated chickens infected with an influenza A virus of the H5N1 subtype.

Accordingly, experiments of example six indicate that the administration of particular vaccines provided by the present disclosure, for instance the H5N7 vaccine, in chickens can be verified in the case of an influenza A virus outbreak of a similar HA subtype but different NA subtype. In other words, in the occurrence of an influenza A virus (H5N1) outbreak, chickens which have been administered the H5N7 vaccine can be differentiated from chickens that have not been administered the H5N7 vaccine. As described above, embodiments of the present disclosure provide vaccines or vaccine compositions including inactivated influenza A viruses that include or carry the H5 hemagglutinin, and at least one neuraminidase subtype, which is selected from a group including or consisting of the Nl, N2, N5, N6, N7, and N8 neuraminidases. In many embodiments, the H5 hemagglutinin is derived from A/chicken Thailand73/2004. Derivation of the H5 hemagglutinin from A/chicken/Thailand73/2004 helps to increase vaccine effectiveness against infection by future influenza virus strains originating from Southeast Asian countries (e.g., Thailand, Vietnam, and Malaysia). This is because of the present finding that the H5 hemagglutinin derived from A/chicken/Thailand73/2004 is more likely to have similar epitopes as compared to a future or new local influenza A virus strain (e.g., a future influenza virus originating from Thailand or a neighboring country to Thailand).

Vaccines accordingly to many embodiments of the present disclosure include inactivated influenza A viruses that include or carry either the N6 neuraminidase or the N7 neuraminidase. In some embodiments, the N6 neuraminidase is derived from A/gull/Maryland/704/77 and the N7 neuraminidase is derived from A/chicken/Germany/N/49. In many embodiments, the use of either the N6 neuraminidase or the N7 neuraminidase facilitates or effectuates an increased yield (i.e., increased production) of the influenza A virus (i.e., the influenza A virus including or carrying the H5 hemagglutinin derived from A/chicken/Thailand/703/2004 and either the N6 neuraminidase or the N7 neuraminidase) being replicated in chicken egg cells. The increase in yield was unexpectedly high. The increase in yield facilitates a more rapid manufacture of vaccines that include inactivated influenza A carrying the H5 hemagglutinin derived from A/chicken/Thailand/703/2004 and either the N6 neuraminidase or the N7 neuraminidase. In most embodiments of the present disclosure, the vaccine provides protection against infection by a future influenza virus strain that includes H5 hemagglutinin. The vaccine triggers production of antibodies for the H5 hemagglutinin or H5 antigen within the body of the living organism that has been administered the vaccine. The increased level of antibodies within the body (as detected using HI titers) protects the living organism from infection by a future influenza virus strain that includes H5 hemagglutinin. Inactivated influenza A viruses of particular vaccines provided by the present disclosure show an unexpected absence of tracheal shedding and cloacal shedding in living organisms that are administered the vaccine.

As mentioned above, vaccines provided by many embodiments of the present disclosure include inactivated influenza viruses including the H5 hemagglutinin as well as one of the N6 neuraminidase and the N7 neuraminidase. The use of the one of the N6 neuraminidase and the N7 neuraminidase facilitates differentiation between vaccinated living organisms (e.g., vaccinated chickens) and non-vaccinated living organisms (e.g., non-vaccinated chickens) that have been infected by an influenza virus carrying the H5 hemagglutinin but a different neuraminidase subtype from the N6 or N7 neuraminidase (e.g., Nl neuraminidase).

Although embodiments and particular examples of the present disclosure have been described above, a person of ordinary skill in the art will understand that various modifications, alterations, and variations may be made to the vaccines, vaccine compositions, vaccine formulations, as well as methods of manufacture thereof without departing from the scope and spirit of the present disclosure. In addition, while advantages associated with particular vaccines, and methods of manufacture thereof, have been described in the disclosure, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.