JP6560056B2 - H3 equine influenza A virus - Google Patents

Description

State of Government Rights This invention was made in part with grants from the US Government (2001-35204-10184 from the US Agricultural Department). The government has certain rights in this invention.

Background Influenza is a major respiratory disease in mammals, including horses, and causes substantial mortality and economic loss each year. In addition, influenza virus infections can cause serious systemic diseases in poultry, resulting in death. The sorted nature of the influenza virus genome causes fragment reassembly during viral replication in cells infected with two or more influenza viruses. Fragment reassembly can be combined with gene mutations and drifts to produce large numbers of influenza virus branches over time. The new strain shows antigenic changes in its hemagglutinin (HA) and / or neuraminidase (NA) protein, and in particular the gene encoding the HA protein has a high degree of variability. The main current method of influenza prevention is vaccination. Most commonly, whole virus vaccines are used. Because the influenza HA protein is the main target antigen for the host's protective immune response to the virus and is highly variable, the identification and characterization of viral HA antigens associated with influenza virus isolation and recent development Important for vaccine production. Based on epidemics and predictions, vaccines are designed to stimulate protective immune responses against major and predicted influenza virus strains (Park et al., 2004).

  There are three general types of influenza viruses, types A, B and C, identified by the absence of serum cross-reactivity between their internal proteins. Influenza A viruses are further classified into subtypes, HA and NA proteins, depending on the antigenicity and general differences of their glycoproteins. All known HA and NA subtypes (H1-H15 and N1-N9) are isolated from waterfowl and are thought to act as natural reservoirs of influenza. H7N7 and H3N8 type A viruses are the most common causes of equine influenza, and these subtypes are commonly incorporated into equine influenza vaccines.

  Accordingly, there continues to be a need to isolate new influenza virus isolates, such as those for vaccines.

SUMMARY OF THE INVENTION The present invention provides an isolated H3 equine influenza A virus. It is isolated from a foal that dies from fatal pneumonia, and the virus has characteristic substitutions at residues 78 and 159 of HA (position numbering is done with the mature protein lacking the 15 amino acid signal peptide) Have That is, the residue at position 78 of HA is not valine and the residue at position 159 is not asparagine. In one embodiment, an isolated H3 influenza A virus of the invention is a conservative substitution at residue 78, such as a valine to alanine substitution, and a non-conservative substitution at residue 159, such as from aspartic acid. Has serine substitution. In one embodiment, an isolated H3 influenza A virus of the invention has a residue other than methionine at position 29, such as a non-conservative substitution, a residue other than lysine at position 54, such as a non-conservative substitution, position. Residues other than serine at 83, such as non-conservative substitutions, residues other than asparagine at position 92, such as non-conservative substitutions, residues other than leucine at position 222, such as non-conservative substitutions, other than alanine at position 272 Has a residue, eg, a conservative substitution, and / or a residue other than threonine at position 328, eg, a conservative substitution. Conservative amino acid substitution refers to the possibility of substitution between residues having similar side chains. For example, the amino acid group having an aliphatic side chain is glycine, alanine, valine, leucine and isoleucine; the amino acid group having an amide-containing side chain is asparagine and glutamine; and the amino acid group having an aromatic side chain is Phenylalanine, tyrosine and tryptophan: the amino acid groups with basic side chains are lysine, arginine and histidine; the amino acid groups with sulfur-containing side chains are cysteine and methionine. In one embodiment, the conservative amino acid substitution groups are: threonine-valine-leucine-isoleucine-alanine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic acid-aspartic acid; and asparagine-glutamine.

  In one embodiment, the influenza virus of the invention has substantially the same amino acid sequence as one of SEQ ID NOs: 1-8, 17, and / or 18, so long as HA has a characteristic substitution at residues 78 and 159. Contains one or more viral proteins (polypeptides). An amino acid sequence that is substantially identical to a reference sequence has at least 95%, such as 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid of the reference sequence, and a defective sequence, such as substantially Sequences that result in a defective viral protein that has substantially the same activity or can be expressed at substantially the same level as the corresponding full-length, mature viral protein; insert, eg, has substantially the same activity or corresponds A sequence that results in a modified viral protein that can be expressed at substantially the same level as the mature viral protein; and / or substitutions, eg, having substantially the same activity or substantially identical to the reference protein It may contain sequences that give rise to viral proteins that can be expressed at the level. In one embodiment, the one or more residues that are not identical to the reference sequence residue may be conservative or non-conservative substitutions, wherein one or more substitutions have a substitution level (s) with protein expression levels or Does not substantially alter activity and / or does not substantially alter virus levels obtained from cells infected with a virus having the protein. As used herein, “substantially the same expression level or activity” is a detectable protein level that is about 80%, 90% or more, a protein that is about 30%, 50%, 90%, eg, up to 100% or more. Level or measurable activity, including the activity of a full-length mature polypeptide corresponding to one of SEQ ID NOs: 1-8, 17 or 18. In one embodiment, the virus has one or more, for example 2, 5, 10, 15, 20 or more amino acid substitutions, such as full-length, mature residues of a polypeptide having SEQ ID NOs: 1-8, 17 or 18. Of polypeptides having up to 5% of conservative substitutions. The isolated virus of the present invention is used alone or in combination with one or more other isolates, such as other influenza virus isolates, in vaccines to produce virus-specific sera in gene therapy and / or diagnosis. . Therefore, the present invention provides a host infected with the virus of the present invention, and an isolated antibody specific for the virus.

  The invention also provides nucleic acid fragments corresponding to at least one of the viral proteins of the invention, substantially the same level as the corresponding polypeptide encoded by one of SEQ ID NOs: 1-8, 17 or 18. An isolated nucleic acid molecule (polynucleotide) comprising a portion of a nucleic acid fragment for an active viral protein, or a complementary strand of a nucleic acid molecule is provided. In one embodiment, the isolated nucleic acid molecule is at least 95%, such as 96%, identical to a polypeptide having a substantially identical amino acid sequence, such as one of SEQ ID NOs: 1-8, 17, or 18. Encodes a polypeptide having a contiguous amino acid sequence of 97%, 98% or 99%. In one embodiment, the isolated nucleic acid molecule is at least 50%, such as 60%, 70%, identical to a substantially identical nucleotide sequence, such as one of SEQ ID NOs: 9-16 or its complementary strand. At least 95%, for example 96%, 97%, 98% or at least 95% identical to a polypeptide comprising one or more of SEQ ID NOS: 1-8, 17 or 18 It encodes a polypeptide having 99% contiguous nucleic acid sequence.

  The isolated nucleic acid molecules of the present invention can be used in vectors to express influenza proteins, for example to produce recombinant protein vaccines, create antisera as nucleic acid vaccines for use in diagnosis, or for vRNA production, Chimeric genes are prepared using other viral genes, including, for example, other influenza virus genes, and / or recombinant viruses (see, eg, Neumann et al. (1999), incorporated herein by reference). Can be prepared. Thus, the present invention also includes host cells that are in contact with isolated viral polypeptides, recombinant viruses and nucleic acid molecule (s), and / or recombinant viruses of the present invention, and, for example, infected with one or more viruses. An isolated virus-specific antibody obtained from a mammal immunized with an isolated viral polypeptide or polynucleotide encoding a viral polypeptide is provided.

  The present invention further provides at least one of the following isolated vectors, such as one or more isolated influenza virus vectors, or a composition comprising one or more vectors: wherein the vector binds to a transcription termination sequence. For a PA having substantially the same amino acid sequence as SEQ ID NO: 5, comprising a promoter operably linked to influenza virus PA DNA, the vector substantially has the same amino acid sequence as SEQ ID NO: 3 that binds to a transcription termination sequence The vector contains a promoter operably linked to influenza virus PB1 DNA and the vector operates on influenza virus PB2 DNA with respect to PB2 having substantially the same amino acid sequence as SEQ ID NO: 4 that binds to a transcription termination sequence. The vector contains the promoter that can bind, and the vector is identical to SEQ ID NO: 1 that binds to the transcription termination sequence For a NP having substantially the same amino acid sequence as SEQ ID NO: 6, which comprises a promoter operably linked to influenza virus HA DNA A promoter comprising a promoter operably linked to influenza virus NP DNA, wherein the vector is operably linked to influenza virus NA DNA with respect to NA having substantially the same amino acid sequence as SEQ ID NO: 2 linked to a transcription termination sequence And the vector is operably linked to influenza virus M DNA for M having substantially the same amino acid sequence as SEQ ID NO: 7 (M1) and / or SEQ ID NO: 17 (M2), which binds to a transcription termination sequence The promoter comprises and / or the vector binds to a transcription termination sequence SEQ ID NO: 8 (NS1) and For NS having substantially the same amino acid sequence as SEQ ID NO: 18 (NS2), a promoter operably linked to influenza virus NS DNA is included. Optionally, the two vectors include a promoter that operably binds to an influenza virus that binds to a transcription termination sequence, such as a vector that includes a promoter operably linked to an influenza virus M1 DNA that binds to a transcription termination sequence, and It can be employed in place of a vector comprising a promoter operably linked to influenza virus M2 DNA that binds to a transcription termination sequence. Optionally, the two vectors contain a promoter that operably binds to influenza virus NS DNA that binds to the transcription termination sequence, eg, a promoter that operably binds to influenza virus NS1 DNA that binds to the transcription termination sequence. It can be used in place of a vector and a vector comprising a promoter operably linked to influenza virus NS2 DNA that binds to a transcription termination sequence. Influenza virus vectors are those that contain at least 5 'and 3' non-coding influenza virus sequences.

  The present invention thus provides vectors, such as plasmids encoding influenza virus proteins and / or encoding influenza vRNA, wild type and recombinant vRNA. Therefore, the vector of the present invention may encode influenza virus protein (sense) or vRNA (antisense). A suitable promoter or transcription termination sequence can be employed to express a protein or peptide, such as a viral protein or peptide, a non-viral pathogen protein or peptide, or a therapeutic protein or peptide. In one embodiment, for the expression of vRNA, the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, RNA polymerase III promoter, T3 promoter or T7 promoter. Optionally, the vector includes a transcription termination sequence m such as an RNA polymerase I transcription termination sequence, an RNA polymerase II transcription termination sequence, an RNA polymerase III transcription termination sequence or a ribozyme.

  The composition of the invention may comprise a gene of interest or an open reading frame, eg, a foreign gene encoding an immunogenic peptide or protein useful as a vaccine. Accordingly, another embodiment of the invention comprises a composition of the invention as described above wherein one of the influenza viruses in the vector replaces a foreign gene, or the composition comprises in addition to the influenza virus gene a desired nucleic acid sequence, for example Further included is a vector containing a promoter that binds to a 5 ′ influenza virus sequence that binds to a DNA of interest that binds to a 3 ′ influenza virus sequence that binds to a transcription termination sequence. This nucleic acid sequence optionally produces a recombinant virus when contacted with a host cell that permits influenza virus replication. In one embodiment, the subject DNA is in the antisense orientation. A subject DNA that is a vRNA production vector or a protein production vector encodes an immunogenic epitope, for example, an epitope useful for cancer therapy or vaccine, or a peptide or polypeptide useful for gene therapy. May be.

  Multiple vectors of the present invention may be physically linked, or each vector may be present on an individual plasmid or other linear nucleic acid delivery vehicle.

The present invention also provides a method for preparing influenza virus. The method comprises the step of contacting a cell, such as a poultry or mammalian cell, with an isolated virus of the invention or a number of vectors of the invention, for example sequentially or simultaneously. For example, a composition containing an effective amount of a large number of vectors is employed to produce an infectious influenza virus. The invention also includes isolating the virus from cells infected with the virus or cells contacted with the vector and / or composition. The invention further provides a host cell infected with a virus of the invention, or a host cell contacted with a composition or vector of the invention. In one embodiment, the host cell is infected with an attenuated (eg, cold acclimated) donor virus and a virus of the invention to prepare a cold conditioned reassortant virus useful as a cold conditioned live virus vaccine.

  The invention also induces an immune response in a mammal, eg against one or more pathogens, eg against a virus of the invention and optionally against bacteria, heterologous viruses, or parasites or other antigens. A method for immunizing is provided. The immune response to the composition or vaccine is, for example, an administered viral preparation, polypeptide or administered nucleic acid that can prevent or inhibit infection with the virus or a closely (structurally) related virus. It occurs in the host organism of the polypeptide encoded by the molecule, cells against the viral polypeptide and / or an antibody-mediated immune response. Usually, such responses are directed specifically to the antigen (s) contained in the subject composition or vaccine, such as antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells. Consisting of a subject that produces The method prevents or infects a host organism, such as a mammal, with an effective amount of an influenza virus, such as an attenuated, live virus of the present invention, eg, a mammal, such as a mammal, by the virus or antigenically related virus. Administering in an amount effective to alleviate, optionally in combination with an adjuvant and / or carrier. In one embodiment, the virus is administered intramuscularly, while in another embodiment, the virus is administered intranasally. In some dosing protocols, all doses can be administered intramuscularly or intranasally, and in other protocols a combination of intramuscular and intranasal administration is employed. The vaccine may also be some other isolate of influenza virus, including recombinant influenza virus, other pathogen (s), additional biologics or microbial organisms, such as for forming multivalent vaccines. In one embodiment, intranasal vaccination with inactivated equine influenza virus and mucosal adjuvants such as non-toxic B chain of cholera toxin induces virus-specific IgA and neutralizing antibodies and serum IgG in the nasopharynx To do.

  The equine influenza vaccine can be employed with other antiviral agents such as amantadine, rimantadine and / or neuraminidase, and can be administered separately, eg, before, simultaneously and / or after, in combination with the antiviral agent.

  Furthermore, a diagnostic method using the virus of the present invention, the isolated viral protein encoded thereby, or an antiserum specific for the virus or protein, for detecting a virus-specific antibody or virus-specific protein is provided. To do.

FIG. 1A shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1B shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1C shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1D shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1E shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1F shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1G shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1H shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1I shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1J shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1K shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1L shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1M shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1N shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 10 shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1P shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1Q shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 1R shows the sequence of A / Equine / Wisconsin / 1/03. SEQ ID NOs: 1-8, 17 and 18 show the deduced amino acid sequences of HA, NA, PB1, PB2, PA, NP, M1, NS1, M2 and NS2 of A / Equine / Wisconsin / 1/03, respectively. SEQ ID NOs: 9-16 are A / Equine / Wisconsin / 1/03 HA, NA, PB1, PB2, PA, NP, M1, NS1, M (M1 and M2) and NS (NS1 and NS2) mRNAs, respectively. The sense nucleotide sequence is shown. FIG. 2 shows the sequence alignment of HA-1 of A / Equine / NewYork / 99 (SEQ ID NO: 19) and A / Equine / Wisconsin / 1/03 (SEQ ID NO: 20).

Detailed Description of the Invention
Definitions As used herein, the term “isolated” refers to a nucleic acid molecule, such as a vector or plasmid of the present invention, that is not associated with, or substantially isolated from, in vitro material. Refers to the in vitro preparation and / or separation of peptides or polypeptides (proteins) or viruses. Isolated virus preparations are generally obtained by in vitro culture and propagation and are substantially free of other infectious agents.

  As used herein, “substantially purified” means that the species of interest is the main species, for example, on a molar basis, more abundant than any other individual species in the composition, preferably It means at least about 80% of the species present, and optionally 90% or more of the species present in the composition, such as 95%, 98%, 99% or more.

  As used herein, “substantially absent” means below the level of detection for a particular infectious agent using standard detection methods for that agent.

  A “recombinant virus” is a virus that has been manipulated in vitro, eg, using recombinant DNA technology, to introduce mutations into the viral genome.

  As used herein, the term “recombinant nucleic acid” or “recombinant DNA sequence or portion” is derived or isolated from a nucleic acid, eg, a source, and then the sequence is not native or the sequence is native. A DNA that can be chemically altered in vitro to correspond to a native sequence that is not arranged to be located in the genome. An example of DNA from one origin is a DNA sequence that is identified as a useful fragment and then chemically synthesized in essentially pure form. Examples of such DNA “isolated” from a source can be further manipulated, eg, by chemical means, eg, restriction endonucleases, so that it can be amplified for use in the present invention by genetic engineering methods. Useful DNA sequences that are cut or removed from the source by use.

Structure and growth of influenza A virus
Influenza A has a genome of 8 single-stranded negative sense viral RNAs (vRNAs) that encode at least 10 proteins. The influenza virus life cycle begins with hemagglutinin (HA) binding to sialic acid-containing receptors on the surface of the host cell, followed by receptor-mediated endocytosis. The low pH of the terminal endosomes causes structural changes in HA, resulting in the N-terminus of the HA2 subunit (so-called fusion peptide). The fusion peptide gives rise to viral and endosomal membranes, and the matrix protein (M1) and RNP complex are released into the cytoplasm. RNP (s) consist of a nucleoprotein (NP) that envelopes vRNA and a viral polymerase complex formed by PA, PB1 and PB2 proteins. RNPs are transported to the nucleus where transcription and replication occur. The RNA polymerase complex catalyzes three reactions: synthesis of mRNA with a 5 'cap and 3' polyA structure, synthesis of full-length complementary RNA (cRNA), and synthesis of genomic vRNA using cRNA as a template. . The newly synthesized vRNA (s), NP and polymerase protein are associated with RNP (s), removed from the nucleus and transported to the plasma membrane, where offspring virus particles germinate. Neuraminidase (NA) protein plays a decisive role at the end of infection by removing sialic acid from sialyl oligosaccharides, thereby releasing new associated virions from the cell surface and suppressing self-aggregation of virus particles. Viral association involves protein-protein and protein-vRNA interactions, but the nature of these interactions is largely unknown.

Any cell, such as any poultry or mammalian cell, such as a human, dog, cow, horse, cat, pig, sheep, mink, such as a MvLu1 cell, or a non-human primate cell, including a mutant cell, Supports efficient replication and can be used to isolate and / or propagate influenza viruses. The isolated virus can be used to prepare a reassortant virus, such as an attenuated virus. In one embodiment, the host cell for vaccine production is a cell found in poultry eggs. In another embodiment, the host cell for vaccine production is a continuous mammalian or poultry cell line or cell type. It is preferred to establish a complete characterization of the cells used to include a suitable test of the purity of the final product. The data that can be used to characterize the cells are: (a) information about its origin, derivation and history; (b) information about its growth and morphological characteristics; (c) test results of accidental drugs; (d A) prominent features that one cell can clearly distinguish from other cell lines, such as biochemical, immunological and cytogenetic patterns; and (e) tumor formation test results. Preferably, the level of progression or population doubling of the host cells used is probably low.

  Virus produced by the host cell is highly purified prior to vaccine or gene therapy formulation. In general, purification methods result in extensive removal of cellular DNA, other cellular components and accidental agents. A method of reducing or denaturing DNA on a large scale can also be used.

Detection of Equine Influenza Virus Diseases that cause equine influenza viruses are generally H7N7 (equal to 1) and H3N8 (equal to 2) subtype influenza A viruses. These are generally different from the subtypes that cause infection in humans (H1N1, H2N2 and H3N2). Equine influenza is infected by inhalation or contact with secretions (eg, physiological fluids) containing live viruses. The virus infects epithelial cells in the upper and lower airways and causes pili loss over a wide area of the airways within 4-6 days. As a result, the mucociliary clearance mechanism is impaired and the rate of tracheal clearance decreases for up to 32 days before infection. Bronchitis and broncholitis develop and interstitial pneumonia occurs, which is accompanied by congestion, edema, leukocyte infiltration. In general, H3N8 virus causes considerably more severe disease than H7N7 virus; H3N8 subtype viruses are more pulmonary and are also associated with myocarditis.

  Clinical signs in animals that have not previously experienced influenza are easily discernable. Influenza is characterized by sudden onset with an incubation period of 1-3 days. The first sign is an increase in body temperature (up to 41 ° C), usually biphasic. Causes severe dry cough that releases large amounts of virus into the atmosphere. This dry cough is often accompanied by a nasal discharge that becomes mucinous due to secondary bacterial infection. The other most commonly observed clinical signs are myalgia, anorexia and hypertrophy of the submandibular lymph nodes. Leg and scrotal edema are also rarely seen. The severity of the disease varies with dose, virus strain and horse immune status.

  Although coughing may last for a long time, healthy, immunocompetent adult horses usually recover from simple influenza within 10 days. If a secondary bacterial infection occurs, the recovery period will be longer. However, relatively high mortality rates have been recorded in foals, poor animals and donkeys. If the maternal antibody is not present during the exposure period, the foal develops viral pneumonia that leads to death. Adult death is usually the result of a secondary bacterial infection that causes pleurisy, purulent pneumonia, or rarely hemorrhagic purpura. Equine influenza sequelae include chronic pharyngitis, chronic bronchiolitis, myocarditis and alveolar emphysema, causing vomiting, secondary sinus and throat sac infections.

  The clinical signs of partial immunity in animals as a result of vaccination or previous infection are more difficult to recognize due to little or no cough or fever. Infections with no experience have always had a prevalence of infection, but horses vaccinated 18 days before recognizable clinical signs have the potential to become infected.

  The development of infectious respiratory disease occurs by various drugs such as equine herpesvirus, rhinovirus, adenovirus and arteritis virus, Streptococcus equi or Streptococcus zooepidemicus It may be. A putative diagnosis of influenza based on clinical signs should be confirmed by virus isolation or detection, or serum tests. Experimental confirmation of a clinical diagnosis is a viral antigen, viral nucleic acid or virally infected cell, either by typical isolation of the virus from nasopharyngeal swabs or serology to demonstrate seroconversion, or within respiratory secretions Can be done by rapid diagnostic tests to detect the presence of. In rapid diagnostic tests, despite the simplicity and ease of use, little or no information about the genetic or antigenic nature of the infecting virus strain is available, and the virus cannot be isolated.

  Viral detection or isolation nasopharyngeal swabs need to be collected as quickly as possible. Experimental load test results suggest that the highest virus titer is obtained 24 to 48 hours after the onset of fever 2-3 days after infection, and the virus incubation period is usually within 4 or 5 days . A nasal swab sample is obtained by passing the swab as far as possible through the abdominal tract into the horse's nasopharynx to absorb respiratory secretions. Swabs need to be quickly transferred to containers using virus delivery media and placed on ice to maintain virus survival. Viruses are probably not viable when harvesting dry swabs, and the chance of contamination is increased when using bacterial transport media. Nasal swab samples can be inoculated into the allantoic (or amnion) cavity of 9-11 instar larval eggs. After incubation at 33-35 ° C for 3 days, urine is collected and tested for hemagglutination activity. Alternatively, the virus can be isolated using cell cultures. Influenza infection can also be diagnosed by comparing the serum test results of an acute serum sample taken as soon as possible after the onset of clinical signs with the serum test results of a convalescent serum sample taken 2-4 weeks later .

  The hemagglutination inhibition (HI) test measures the ability of influenza-specific antibodies present in serum samples to inhibit the aggregation of erythrocytes by the virus. Serum is heat inactivated and pretreated to reduce non-specific reactions and serially diluted prior to incubation with standard virus doses in U-bottom microtiter plates. After addition of a red blood cell suspension and further incubation, agglutination is tested. Viral infectious antibodies increased 4 fold, indicating infection. Although all viral antigens may be used for H7N7 viruses, antigens broken with Tween 80-ether are usually required to improve the test sensitivity of H3N8 viruses. In repeatedly vaccinated horses, infection cannot stimulate a 4-fold increase in HI titer.

  The one-way radiation hemolysis (SRH) test is less strain specific but more reproducible and less error prone than the HI test, and is a more linear and linear test, so it is more sensitive and heavily vaccinated horses A small increase in antibody induced by infection can be detected. The SRH test is based on the ability of influenza-specific antibodies to lyse virus-coated erythrocytes in the presence of complement. Test serum is added to perforated wells in agarose containing coated erythrocytes and complement and the agarose is allowed to diffuse for 20 hours. The clear area of haemolysis around the well is comparable to the level of influenza antibody present in the serum sample.

  When horses are vaccinated on the infected surface, it is probably not possible to use the HI and SRH tests to determine whether the increase in antibody levels is due to vaccination or infection.

Influenza vaccine The vaccine of the present invention comprises the isolated influenza virus of the present invention, and optionally other isolated influenza viruses, West Nile virus, equine herpes virus, equine arteritis virus, equine infectious anemia lentivirus, rabies virus One or more immunogenic proteins or glycoproteins of eastern and / or western and / or Venezuelan equine encephalitis virus, one or more isolated influenza viruses or one or more other pathogens, eg one or more bacteria-derived immunogenicity One or more comprising an isolated nucleic acid encoding one or more viral proteins (eg DNA vaccines) comprising one or more immunogenic proteins of a protein, a non-influenza virus, yeast or fungus, or an isolated influenza virus of the invention Includes other isolated viruses. In one embodiment, the influenza virus of the invention may be a vaccine vector for influenza virus or other pathogens.

  Complete virion vaccines can be concentrated by ultrafiltration and then purified by zonal centrifugation or chromatography. For example, it is inactivated before or after purification using formalin or β-propiolactone.

  Subunit vaccines contain purified glycoproteins. Such a vaccine can be prepared as follows: purified using, for example, ultracentrifugation, using a virus suspension fragmented by treatment with a detergent. Thus, this subunit vaccine mainly contains HA protein and NA. The surfactants used are cationic surfactants such as hexadecyltrimethylammonium bromide (Bachmeyer, 1975), anionic surfactants such as ammonium deoxycholate (Laver and Webster, 1976); or for example under the name TRITONX100 A commercially available nonionic surfactant may be used. Hemagglutinin can be isolated after treating the virion with a protease such as bromelin and purified by the method described by Grand and Skehel (1972).

  Split vaccines contain virions that can be processed into drugs that dissolve lipids. A split vaccine can be prepared as follows: an aqueous suspension of purified virus obtained as described above, which may or may not be inactivated, is associated with a surfactant. The mixture is treated with stirring in a fat-soluble solvent such as ethyl ether or chloroform. Dissolution of the viral envelope lipid results in fragmentation of the viral particles. A split vaccine, which is mainly composed of hemagglutinin and neuraminidase along with its original lipid environment to be removed, and an aqueous layer containing the nucleus or its degradation products is recovered. Thereafter, the residual infectious particles are inactivated if they still cannot be inactivated.

Inactivated vaccines Inactivated influenza virus vaccines are provided by inactivating replicating viruses using known methods, not limited to formalin or β-propiolactone treatment. Inactivated vaccine species that can be used in the present invention can include whole virus (WV) vaccines or subvirion (SV) (split) vaccines. WV vaccines contain intact inactivated viruses, while SV vaccines contain purified viruses that have been chemically inactivated by residual viruses after breaking with a detergent that dissolves the lipid-containing viral envelope.

  In addition, vaccines that can be used include isolated HA and NA surface proteins referred to as surface antigens or subunit vaccines.

Live Attenuated Virus Vaccine A live attenuated influenza virus vaccine can be used to prevent or treat influenza virus infection. Attenuation can be achieved in a single step by transport of the attenuated gene from the attenuated donor virus to a replicating isolate or reassortant virus according to known methods (see, for example, Murphy, 1993). Since resistance to influenza A virus is mainly mediated by the development of an immune response to HA and / or NA glycoproteins, the genes encoding these surface antigens can be obtained from reassortant viruses or clinical isolates. Must be done. The attenuated gene is derived from the attenuated parent. In this method, the attenuated gene preferably does not encode HA and NA glycoproteins.

Viruses (donor influenza viruses) that can attenuate influenza viruses with good reproducibility can be used. For example, cold (ca) donor viruses can be used to produce attenuated vaccines. A live attenuated reassortant virus vaccine can be made by combining ca donor virus with virulent replicating virus. The reassortant progeny are then selected at 25 ° C. (restricting virulent virus replication) in the presence of a suitable antiserum. This inhibits viral replication resulting in surface antigens of attenuated ca donor virus. Useful reassortants are: (a) infectious, (b) attenuated serologically non-adult and immunologically treated adults, (c) immunogenicity, and (D) Genetically stable. The immunogenicity of ca reassortant is comparable to its replication level. Thus, acquisition of the ca donor virus 6 transfer gene by the novel wild type virus reproducibly attenuates these viruses for use in adult and non-adult mammal vaccination.

Other attenuated mutants can be introduced into influenza viruses by site-directed mutagenesis to rescue infectious viruses carrying these mutant genes. Attenuating mutants can be introduced into non-coding and coding regions of the genome. Such attenuated mutants can be introduced into genes other than HA or NA, such as the PB2 polymerase gene (Subbarao et al., 1993). Thus, new donor viruses can also produce attenuated mutations introduced by site-directed mutations, and such new donor viruses are live attenuated in a manner similar to that described above for the ca donor virus. It can be used to produce a reassortant vaccine candidate. Similarly, other known suitable attenuated donor strains, and a reassortant with influenza virus, it is possible to obtain a suitable attenuated vaccine for use in the vaccination of mammals (Enami et al., 1990; Muster et al., 1991; Subbarao et al., 1993).

  Preferably, such attenuated viruses maintain genes from viruses that encode antigenic determinants that are substantially similar to the antigenic determinants of the original clinical isolate. This is because the purpose of the attenuated vaccine is to provide substantially the same antigenicity as the original clinical isolate of the virus, while minimizing the chance that the vaccine will cause severe disease in the vaccinated mammal. This is because it lacks pathogenicity to the extent that it can be minimized.

  Thus, the virus can be formulated and administered attenuated or inactivated according to methods known as vaccines that induce immune responses in animals such as mammals. Methods for determining whether such attenuated or inactivated vaccines maintain an antigenicity similar to that of clinical isolates or hyperproliferative strains derived therefrom are well known in the art. is there. Such known methods include the use of antisera or antibodies to remove viruses that present the antigenic determinants of the donor virus; chemical selection (eg, amantadine or rimantidine); HA or NA activity and inhibition; and antigenic determinants Nucleic acid screening (eg, probe hybridization or PCR) to confirm that the donor gene encoding (eg, the HA or NA gene) is not present in the attenuated virus. See, for example, Robertson et al., 1988; Kilbourne, 1969; Aymard-Henry et al., 1985; Robertoson et al., 1992.

Pharmaceutical compositions of the present invention suitable for vaccination, eg nasal, parenteral or oral administration, comprise one or more influenza virus isolates, eg one or more attenuated or inactivated influenza viruses, subunits thereof The isolated protein (s) and / or an isolated nucleic acid encoding the one or more proteins. Optionally further comprises a sterile aqueous or non-aqueous solution, suspension or emulsion. The composition can further comprise adjuvants or excipients known in the art. See, for example, Berkow et al., 1987; Avery's Drug Treatment , 1987; Osol, 1980. The compositions of the invention are generally provided in the form of individual doses (unit doses).

Conventional vaccines generally contain about 0.1-200 μg, eg, 30-100 μg, of HA from each strain that enters the composition. The vaccine that forms the main component of the vaccine composition of the invention may comprise a single influenza virus, or a combination of influenza viruses, for example at least 2 or 3 influenza viruses including one or more reassortants .

  Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and / or emulsions, and may include adjuvants or excipients known in the art. Examples of water-insoluble solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may include liposomal solutions that generally include liquid dosage forms. Suitable dosage forms for suspended liposomes include inert diluents commonly used in the art, such as emulsions, suspensions, solutions, syrups and elixirs containing purified water. In addition to the inert diluent, such compositions may also contain adjuvants, humidifiers, emulsifiers and suspending agents, or sweeteners, flavoring or flavoring agents. See, for example, Berkow et al., 1992; Avery's, 1987; and Osol, 1980.

  When the composition of the present invention is used for administration to an individual, it may further comprise a salt, buffer, adjuvant or other substrate desirable to improve the efficacy of the composition. For vaccines, adjuvants and substrates that can increase the specific immune response can be used. Usually, the adjuvant or composition is mixed before being presented to the immune system or presented separately, but presented at the same site of the organism to be immunized. An example of a material suitable for use in a vaccine composition is provided in Osol (1980).

  Vaccine heterogeneity can be provided by mixing replicating influenza viruses for at least two influenza virus strains, such as 2-20 strains or any range or value thereof.

  The pharmaceutical composition according to the invention comprises at least one chemotherapeutic compound, for example for gene therapy an immunosuppressant, anti-inflammatory or immunopotentiator, for vaccines gamma-globulin, amantadine, guanidine, hydroxybenzimidazole, α -Interferon, β-interferon, γ-interferon, α-tumor necrosis factor, thiosemicarbazone, methisazone, rifampin, ribavirin, pyrimidine analog, purine analog, phoscarnet, phosphonoacetate, acyclovir, dideoxynucleosidase, protease inhibitor Or a chemotherapeutic agent including but not limited to ganciclovir.

  The composition may include variations except small amounts of formaldehyde that do not contain endotoxin, and preservatives that have been found to be safe and undesired to the organism to which the composition is administered.

Administration of a pharmaceutical purpose composition (or antisera it induces) may be for either “prevention” or “treatment” purposes. When provided prophylactically, a composition of the invention that is a vaccine is provided before any symptoms or clinical signs of pathogenic infection become apparent. Prophylactic administration of the composition serves to prevent or attenuate any subsequent infection. When provided prophylactically, the gene therapy composition of the invention is provided before any symptoms or clinical signs of disease become apparent. Prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with the disease.

  Where provided therapeutically, attenuated or inactivated viral vaccines are provided for the detection of symptoms or clinical signs of a specific infection. The therapeutic administration of the compound (s) serves to attenuate any specific infection. See, for example, Berlow et al., 1992; and Avery, 1987. When provided therapeutically, gene therapy compositions are provided for the detection of disease symptoms or clinical signs. The therapeutic administration of the compound (s) serves to attenuate the symptoms or clinical signs of the disease.

  Thus, the attenuated or inactivated vaccine composition of the present invention can be provided before the start of infection or after the start of a specific infection (to prevent or attenuate the expected infection). Similarly, for gene therapy, the composition can be provided before any symptom or clinical sign of the disorder or disease becomes apparent, or after one or more symptoms are detected.

  A composition is said to be “pharmacologically acceptable” if its administration is tolerated by a recipient mammal. An agent is said to be administered in a “therapeutically effective amount” if the amount administered is physiologically significant. When a detectable change occurs in the physiology of the recipient patient, e.g., when at least one primary or secondary humoral immune response or cellular immune response is enhanced against at least one strain of infectious influenza virus The composition of the present invention is physiologically significant.

  The “protection” provided need never be absolute. That is, if there is a statistically significant improvement over the control group or mammal group, the influenza infection need not be completely prevented or extinct. Protection may be limited to relief of the onset of symptoms of influenza virus infection or the severity or rapidity of clinical signs.

Pharmaceutical Administration The compositions of the present invention provide resistance to one or more pathogens, eg, one or more influenza virus strains, either by passive immunity or active immunity. In active immunity, an inactivated or attenuated live vaccine composition is administered prophylactically to a host (eg, a mammal) and the host immune response to administration protects against infection and / or disease. For passive immunization, induced antisera can be collected and administered to a recipient suspected of causing infection by at least one influenza virus strain. The gene therapy composition of the present invention provides a prophylactic or therapeutic level of the desired gene product by active immunity.

  In one embodiment, the vaccine is administered for a time and in an amount sufficient to cause the generation of an immune response that serves to protect both females and fetuses or neonates (through passive uptake of antibodies through the placenta or in breast milk). Under conditions, provided to female mammals (before pregnancy or childbirth).

  Accordingly, the present invention includes methods for preventing or reducing a disorder or disease, eg, infection by at least one pathogen strain. As used herein, where administration results in either total or partial alleviation (ie suppression) of clinical signs or symptoms of disease, or the individual's total or partial immunity to the disease, Vaccines are said to prevent or reduce disease. As used herein, where administration results in either total or partial alleviation (ie suppression) of clinical signs or symptoms of disease, or the individual's total or partial immunity to the disease, Gene therapy compositions are said to prevent or reduce disease.

  At least one influenza virus isolate of the invention comprises a virus that is activated or attenuated, one or more isolated viral proteins thereof, one or more isolated nucleic acids encoding the one or more viral proteins. It can be administered by any method that achieves the intended purpose, including molecules, or combinations thereof.

  For example, administration of such compositions can be by various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be accomplished by bolus injection or prolonged gradual infusion.

  A typical dosage regimen for preventing, suppressing or treating an influenza virus-related pathogen involves the administration of an effective amount of the vaccine composition described herein. The composition is administered as a single treatment or repeated as incremental or additional doses for a period of up to 1 week to about 24 months, or in any range or number as described herein.

  According to the present invention, an “effective amount” of the composition is an amount sufficient to achieve the desired effect. It will be appreciated that the effective amount will depend on the recipient species, age, sex, health status and weight, type of co-treatment, frequency of treatment, if any, and nature of desired effect. The following effective dose ranges are not intended to limit the invention, but are intended to indicate dose ranges.

The dose of live attenuated or killed virus vaccine for animals such as adult mammals can be about 10 ² to 10 ¹⁵ , such as 10 ³ to 10 ¹² plaque forming units (PFU) / kg, or any range or number thereof. Inactivated vaccine doses can range from about 0.1 to 1000, eg, 30 to 100 μg of HA protein. However, the dosage should be a safe and effective amount determined by conventional methods with the vaccine present as a starting point.

  The dosage of immune response HA at each dose of replicating viral vaccine includes a suitable amount, such as 30-100 μg or any range or number thereof, or an amount recommended by a government agency or understood by a specialized agency. Has been standardized. However, the amount of NA in which this glycoprotein is stable during purification and storage is also standardized.

Equine Influenza Virus Compositions and Doses Equine influenza vaccines generally include representative strains of H7N7 and H3N8 subtypes, either as inactivated whole virus or subunits thereof. It provides protection against influenza and / or potentially important cell-mediated immune responses against other viral proteins by inducing antibodies to surface glycoproteins, particularly HA, which is essential for virus binding and cell transfer . Vaccination is useful for preventing influenza, but protection is short-lived (using conventional inactivated virus vaccines) because the frequency of vaccination varies according to how the horse comes into contact with the virus. 3-4 months) (see Table 1). The general method of primary course is vaccination with a single dose, followed by vaccination with a second dose 3-6 weeks later. Vaccine manufacturers recommend booster vaccinations every 6-12 months. Alternatively, the first is a 1-2 ml dose, such as an intramuscular (IM) injection, the second is a 1-2 ml dose, such as an IM injection, and optionally the third is at a different injection site after 3-4 weeks. Horses are administered by a 1-2 ml dose, eg IM or intranasal (IN). Each 1-2 ml dose of vaccine may contain about 1-500 billion virus particles, preferably 100 billion virion particles. For example, horses that come into contact with a large number of horses in board stables, training centers, racetracks, shows and other such events are frequently vaccinated every two to three months. The 3-dose primary system was found to induce higher and sustained immunity than the recommended 2-dose system regardless of age.

  It is preferred to vaccinate young horses, especially racehorses and other race horses, with conventional vaccines, at intervals of 4-6 months for several years after the primary course of vaccination. Inclusion of additional booster vaccinations of the second to third vaccinations recommended by vaccine manufacturers is beneficial for young horses. Annual booster vaccination may be sufficient for older horses that have been vaccinated regularly since foal, such as show jumpers and mares. Vaccinations for those presently occurring are sometimes performed but are probably not effective unless there is at least a 7-10 day interval before the newly vaccinated horse is exposed to the infection. Although equine influenza episodes are not as seasonal as humans, they are often associated with sails or races where horses from different fields gather and mix. It is therefore advantageous to perform additional booster vaccinations in time prior to such an event.

  The mare pedigree should be vaccinated late in pregnancy, but prior to 2 weeks before the foal is born, to ensure adequate supply of colostrum antibodies to the foal. The foal should be vaccinated at 3-6 months of age, boosted in April-July, boosted again in May-August, and if the foal is at high risk of exposure Should be repeated every 3 months.

  Influenza vaccines may be mixed with tetanus or herpes virus antigens, and other pathogens such as equine pathogens. The immune response caused by tetanus toxin is much more sustained than the response induced by influenza antigens. Therefore, in a powerful influenza vaccination program, only vaccines containing influenza are preferred.

  Antibody levels measured by the SRH test required for horse protection are identified from vaccination and challenge tests and data in the field. Because vaccine-induced antibody responses to HA in horses are extremely short lived, adjuvants such as aluminum hydroxide or carbomers are usually included to increase the amplification and duration of immune responses to whole virus vaccines. Subunit equine influenza vaccines containing immunostimulatory complexes (ISCOMs) are also immunogenic.

  Empirically, the amount of antigen in an inactive vaccine has been expressed in terms of HA chick cell aggregation (CCA) units, and efficacy has been expressed in terms of HI antibody responses induced by guinea pigs and horses, Neither gives reproducible results. The single radical dispersion (SRD) test is an improved in vitro potency test that measures the concentration of immunologically active HA (expressed in milligrams of HA) and is used in process tests prior to the addition of adjuvant.

  The invention is further illustrated by the following non-limiting examples.

  A 36-year-old Morgan / Friesian foal was referred to a large animal hospital at the University of Wisconsin for evaluation of abnormal mental effects (mental state) that were found immediately after birth. Although no birth was observed, the foal was found to be separated from the mare by a fence at the age of several hours. When first seen, the foal was able to walk and breastfeed, but in the next 36 hours it lost its sense of direction, was not clearly visible and wandered without purpose. SNAP immunoglobulin G (IgG) test at 24 hours of age (Idexx Labratories, Weatbrook, ME) showed IgG concentrations> 800 mg / dL, at which time CBC was normal. Prior to referral, foals were treated twice with 1 g / kg IV of dimethyl sulfoxide diluted in 5% dextrose.

  At the time of the examination, the foal wandered for no purpose, hit the object and seemed dull and invisible, but the pupil responded to the light. When placed near the mare, the foal was breastfed successfully. There was no problem in the physical examination. CBC and serum biochemical tests were routine, including a 937 mg / dL serum IgG concentration measured by radioimmunodiffusion.

  Initial treatment of presumed oxygenemia, ischemic brain injury included 1 hour, 250 mL loading dose of 20% magnesium sulfate, followed by constant rate infusion of 42 mL / hour and thiamine hydrochloride 2.2 mg / kg IV q 24 hours. Antibacterial therapy consists of amikacin 20 mg / kg IV q 24 hours and procaine penicillin G 22,000 U / kg IM q 12 hours. Omeprazole 1 mg / kg PO q 24 hours was also administered to foals to prevent the development of gastric ulcers.

  The mental state of the foal remained calm for the next 24 hours, and mannitol 1 g / kg IV q 24 hours and dexamethasone sodium phosphate 0.1 mg / kg IV q 24 hours after admission on the 2nd and 3rd days. Additional treatment was not related to improvement. On day 3, the foal received normal anesthesia for computed tomography of the skull and proximal spinal cord, but was normal. Cerebrospinal fluid samples were obtained from the lumbosacral space, but cytological evaluation was normal and had normal protein concentrations.

  On the fourth day after admission, the foal tilted its head to the right, but remained calm on the fifth day after admission. On the fifth day, magnesium sulfate therapy was discontinued, but the rest of the treatment plan remained unchanged. On the sixth day, the foal had two short general seizures. This was controlled with midazolam 0.05 mg / kg IV. During the seizure, the foal was fine, feverish, and lactated.

On the 7th day after hospitalization, the foal has a fever (40 ° C), drains mucus purulent nasal discharge, and when auscultated, an accidental ticking and seeing sound diffuses. Had developed. During the previous 7 days, fever and cough were observed in several other mares and foals in the newborn care unit. Antibacterial therapy was changed to ticarcillin / clavulanic acid 50 mg / kg IV q 8 hours with gentamicin 6.6 mg / kg IV q 24 hours and foals were treated with polyionic liquid. However, the foal was still breastfeeding. In 8-10 days, the foal's nerve condition continued to improve, the head ceased to tilt and returned to normal mental state, but tachypnea, dyspnea and accidental lung sounds worsened. Chest radiography at this point was poor and showed a bronchial interstitial pattern. Aminophylline 0.5 mg / kg IV q 12 hours with slow infusion and nasal inhalation of oxygen began on days 9 and 10 of hospitalization. Continuous arterial blood gas analysis identified hypoxemia (PaO ₂ , 52 mmHg), hypercapnia (PaCO ₂ , 68.4 mmHg) and hypoxia saturation (76%) at the end of day 10. The foal was then placed in a mechanical ventilator. Ventilator support and systemic parenteral nutrition were continued for 48 hours, when arterial blood gas levels were standardized with 100% oxygen. Antimicrobial therapy continued as before. In a 13-day stress test excluding ventilator support, the foal developed severe dyspnea and cyanosis and was euthanized at the owner's request. The aerobic culture of transtracheal extracts obtained on day 13 grew ticarcillin / clavulanic acid and gentamicin resistant Klebsiella pneumoniae and E. coli.

  Complete and histopathological anatomy and real-time quantitative polymerase chain reaction (PCR) assessment for the presence of equine herpesvirus (EHV) -1 and EHV-4 in nasal discharge samples; exposure to equine viral arteritis virus Serum tests to determine; and Directigen Flu A test (Bectin Dickinson and Co., Franklin, NJ) and virus isolation from nasal juice samples to test for the presence of influenza virus. Nasal juice samples were collected with Dacron Swab and placed in 2 ml of virus transport medium containing phosphate buffered saline, 0.5% bovine serum albumin, and penicillin G, streptomycin, nystatin and gentamicin. Nasal swab samples were collected on the 8th day of hospitalization. Follow-up assessment of influenza virus is based on the presence of influenza nucleoprotein (NP) expression, quick frozen, immunohistochemistry for formalin-fixed lung, viscera and central nervous system tissue, virus isolation from frozen lung tissue and Virus sequence analysis was included. Macroscopic postmortem examination identified severe diffuse interstitial pneumonia and subdural hematoma on the ventral caudal surface of the brain surrounding the pituitary gland. There was no evidence of sepsis or pathogens in other organs. Histopathological examination of the lungs identified necrotizing bronchitis and bronchiolitis, diffuse squamous metaplasia, and multifocal interstitial pneumonia. Mild mononuclear infiltration covered the lower respiratory tract, possibly covering areas of alveolar destruction associated with congestion and exudation. Evaluation of brain tissue showed white matter vacuole formation in the diffuse cerebrum, especially the cerebellum, with mild dilation of the ventricular system. Multiple fractions were cresyl violet stained for the presence of myelin. Compared to age-matched tissue from age-matched stained controls, it was small except showing brain and spinal cord myelin. Additional histopathological abnormalities in the central nervous system clearly lacked a molecular layer in the cerebellum. Serum tests for equine virus arteritis and real-time PCR tests for EHV-1 and EHV-4 DNA were negative.

  The presence of influenza virus in the nasal discharge was first confirmed by a positive Directigen test. Previous tests document the sensitivity and specificity of this test when applied to equine nasal discharge samples (Morely et al., 1995 and Chambers et al., 1994). Nasal swab transport medium samples were also inoculated into allantoic cavities of chick larval eggs and Madin-Darby canine kidney (MDCK) growing in 24-well cell culture plates. Cytopathogenic effects consistent with influenza virus growth were observed in inoculated MDCK cells, and agents that caused hemagglutination of chicken erythrocytes were isolated from inoculated eggs (Palmar et al., 1975). Positive presence of influenza virus in MDCK cell culture on the same plate, anti-NP monoclonal antibody (Mab) 68D2 (kindly received from Dr. Yoshihiro Kawaoka from the University of Wisconsin-Madison veterinary school) Swine influenza virus inoculation) and negative (mock inoculation) control cells were confirmed by immunocytochemical staining of inoculated cells (Landolt et al., 2003). Identification of the virus as an H3-subtype equine influenza virus was performed by reverse transcription-PCR amplification of the hemagglutination (HA) gene from an isolate using primers described in Olsen et al. (1997), followed by Confirmed by a cycle sequence of the full-length protein encoding the region and a pairwise comparison to the viral sequences available at GenBank (DNASTAR software, version 4.0, Win32, Bestfit, Madison, WI). The virus used the maximum syntax bootstrap analysis of the HA sequence (PAUP software, version 4.0B6; David Swofford, Smithsonian Institution, Washington, DC), comparing the reference virus strain with the first discovery search of 1,000 bootstrap replicas. Phylogenetic analysis revealed that it was derived from the East American H3 equine influenza virus. Similar analysis of the nucleotide sequence part of the nonstructural protein gene (sequenced 544 nucleotides) and the NP gene (sequenced 885 nucleotides) confirms the identity of the virus as an East American equine influenza virus did. Here, the virus was identified as A / Equine / Wisconsin / 1-03. FIG. 1 provides the sequence of the coding region of each gene of the virus.

  The presence of influenza virus was also evaluated in foal lungs and other tissues. Specifically, immunohistochemistry with Mab 68D2 showed influenza virus NP expression in a diffusible, widely dispersed region (mainly around the airways) in frozen and formalin-fixed lung tissue. NP expression was not seen in other visceral or central nervous systems. In addition, influenza virus was isolated from MDCK cells from frozen lung tissue samples (and confirmed by immunocytochemistry and HA gene sequencing).

  Neonatal acute dyspnea syndrome (ARADS) is associated with bacterial sepsis (Wilkins, 2003; Hoffman et al., 1993), perinatal EHV-1 (Frymus et al., 1986; Gilkerson et al., 1999) and EHV-4 (Gilkerson et al.). , 1999), and extremely low-grade respiratory tract disease reported as a result of equine viral arteritis infection (Del Piero et al., 1997) may occasionally occur in adenovirus and EHV-2 infections, especially in immunocompromised patients. Have been reported (Webb et al., 1981; Murray et al., 1996). Bronchial interstitial pneumonia and ARDS are respiratory diseases with higher mortality in older foals with various possible causes including bacterial and viral infections (Lakritz, 1993). It occurs in either newborns experiencing septic shock or older foals with diffuse bronchial interstitial pneumonia, and ARDS is characterized by acute onset, acute progression and severe tachypnea. Increased respiratory effort, exacerbation of cyanosis, and hypercapnia with ARDS are often poorly responsive to aggressive treatment (Wilkins, 2003; Lakritz, 1993). It has a variety of possible causes and generally has a mortality rate of over 30% despite powerful treatment with antibacterials, oxygen, anti-inflammatory agents, bronchodilators and thermoregulatory controls Enter the category of respiratory diseases. Equine influenza is a well-known cause of upper respiratory tract disease throughout the world (Wilkins, 2003; Van Maanen et al., 2002; Wilson 1993), but has been published in the literature on the manifestation of this disease in newborn horses. There is little information. One report describes bronchial interstitial pneumonia in a 7-day-old foal from which type A equine influenza was isolated (Britton et al., 2002); the foal described herein It's similar to.

  The foal detailed in this study was one of a variety of hospitalized horses that had fever, mucus purulent nasal discharge and coughing for 2 or 3 weeks. Clinical signs of other diseased horses, including neonates at high risk, are generally limited to the upper respiratory tract, except for mild systemic symptoms of fever and anorexia. The cause of severe lung failure in this foal is unknown. Treatment included doxamethasone, a powerful immunosuppressant, and general anesthesia for diagnostics, all of which made pneumonia more likely to develop in foals. The impact of foal neurological disease on the development and progression of respiratory disease is also unclear. The histological discovery of diffuse airborne formation, central myelin depletion and the absence of molecular layers in the cerebellum are not compatible with any particular clinical or histopathological diagnosis. The foal had impaired central control of breathing. This is because the brain regions (bridges and medulla oblongata) that are involved in the control of respiration show diffuse arsenal formation and reduced myelin staining. Any resulting ventilation disturbance may have been the final event that resulted in normalization of ventilation function by days after admission. However, inborn abnormal mental states, artillery formation, decreased myelin formation in the central nervous system, and cerebellar abnormalities have compromised the ability of the foal to respond to worsening respiratory function. It is a sign. The localized bleeding observed in the ventral tail of the brain was mild and probably the result of injury in one of the seizures experienced by the foal.

  Mares have been vaccinated against the flu in the last two years with killed products in half a year and boosted in late pregnancy. Considering the fact that foals are sufficiently passively transmitted, these antibodies clearly did not protect foals well. Furthermore, a phylogenetic analysis of an isolate obtained from a foal characterized it as an H3N8 subtype, and the commercial product used to vaccinate late pregnancy mares was the same subtype of influenza It contained a virus strain. This suggests that passive transmission cannot be guaranteed to protect against natural infections under certain circumstances. This lack of vaccine efficacy describes the failure of commercially available H7N7 and H3N8 equine influenza virus vaccines to protect adults against clinical respiratory disease resulting from natural infection with certain H3N8 virus strains Consistent with recent research by Mumford et al. (2003). Recovery via trachea of two bacterial species that are resistant to antimicrobial regimens in place at the time of death confuses the conclusion that influenza was the only cause of death. However, postmortem testing does not identify any macroscopic or histopathological evidence of pneumonia, and synergies between influenza viruses and several bacterial pathogens cause pneumonia with increased mortality (McCullers et al. 2003; Simonsen, 1999). In addition, six days after the virus was first recovered from the nasopharyngeal swab, isolation of infectious virus from lung tissue obtained from cadaver, and immunohistochemical demonstration of viral antigens from the lung tissue were: This provides evidence of a strong pathogen contribution arising from influenza virus with respiratory failure in foals.

  To compare the growth characteristics of poultry, horse, human and porcine viruses in primary canine airway epithelial cells, and to study the effect of species on their growth characteristics, cultured cells were A / Equime / Wisconsin / 1 Virus containing / 03 was used to infect at MOI 3 and incubated for up to 10 hours. Other viruses included 6 type A human and swine influenza virus isolates (A / Phillipines / 08/98, A / Panama / 2002/99, A / Costa Rica / 07/99; A / Swine / North Carolina / 44173/00, A / Swine / Minnesota / 593/99, A / Swine / Ontario / 00130/97) and two equine influenza viruses (A / Equine / Kentucky / 81 and A / Equine / Kentucky / 91). At the end of the experiment, cells were fixed with formalin for immunocytochemistry and flow cytometry analysis.

The six human and swine influenza virus isolates described above infect virtually all (80-90%) canine airway epithelial cells and have high titers (10 ^5.3 to 10 ⁷ TCID _{50 in the} cells). / ml). A / Equine / Kentucky / 81 and A / Equine / Kentucky / 91 are highly restricted in their infectivity, most (10 / ^1.7 TCID ₅₀ / ml for A / Equine / Kentucky / 81) or (A / Equine No virus growth could be observed (for / Kentucky / 91). In contrast, A / Equime / Wisconsin / 1/03 infects a higher percentage (about 30%) of primary canine airway epithelial cells, and within those cells, substantially higher titers (about 10 ^4.8 TCID ₅₀ / ml). This result demonstrated that all influenza A viruses tested can infect primary airway epithelial cells in dogs. However, virus infectivity and replication were strongly strain-dependent.

  Dubovi et al. (2004) reported a recurrent onset of severe respiratory illness characterized by cough and fever in a Florida Kennel Greyhound dog. Most infected dogs recovered, but some died of interstitial pneumonia. Lung tissue from 5 dogs that died of interstitial pneumonia syndrome was transformed into African green monkey kidney epithelial cells (Vero), Madin-Darby canine kidney epithelial cells (MDCK), primary canine kidney epithelial cells, primary canine lung epithelial cells, Virus isolation studies were performed on primary canine testicular epithelial cells, canine tumor fibroblasts (A-72) and human colorectal adenocarcinoma epithelial cells (HRT-18) (Dubovi et al., 2004). The cytopathology of MDCK cells focused on the first passage of a lung homogenate from one dog, and the loss of cytopathology on the subsequent passage of cells cultured without trypsin was associated with hemagglutination activity in the culture supernatant. In combination with the presence of influenza virus (Dubovi et al., 2004). This virus was first identified as an influenza virus by PCR using primers specific for the matrix gene. Canine influenza virus was designated as A / Canine / Florida / 43/04 strain. Based on the virus isolation from the lung, the presence of viral antigens in lung tissue by immunohistochemistry, and the seroconversion data of Dubovi et al. (2004) show that the isolated influenza virus is in a competitive greyhound during Jacksonville 2004. Concludes that it is most likely a causal agent responsible for lethal hemorrhagic pneumonia, and this is the first report of equine influenza virus associated with canine respiratory disease (Dubovi et al. (2004) The HA protein of canine isolates differs from the A / Equine / Wisconsin / 1/03 strain by 6 amino acids.

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  All publications, patents and patent applications are hereby incorporated by reference. In the foregoing specification, the invention has been described in connection with certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but the invention allows for additional embodiments and is described herein. It will be apparent to those skilled in the art that certain details may vary considerably without departing from the basic principles of the invention.

Claims (12)

Containing HA gene segment encoding a polypeptide de having SEQ ID NO: 1, comprises an isolated H3 influenza virus effective amount of a pharmaceutical composition for immunizing a mammal against influenza, wherein the composition, A pharmaceutical composition, which is administered to the mammal.   The pharmaceutical composition according to claim 1, wherein the mammal is a dog or a horse. Isolation H3 influenza virus containing HA gene segment encoding a polypeptide de having SEQ ID NO: 1, in an amount effective to induce a prophylactic or therapeutic response to influenza infection, vaccines.   The pharmaceutical composition according to claim 1, wherein the isolated influenza virus is an attenuated virus, a reassortant virus, or modified by chemical, physical or molecular means. 3. The vaccine according to 3.   The pharmaceutical composition according to claim 1 or the vaccine according to claim 3, further comprising an adjuvant or a pharmaceutically acceptable carrier. Isolation H3 influenza virus containing HA gene segment encoding a polypeptide de having SEQ ID NO: 1. At least one of the following:
An NA gene segment encoding a polypeptide having SEQ ID NO: 2 or having at least 95% amino acid sequence identity with SEQ ID NO: 2,
A PB1 gene segment encoding a polypeptide having SEQ ID NO: 3 or having at least 95% amino acid sequence identity with SEQ ID NO: 3,
A PB2 gene segment encoding a polypeptide having SEQ ID NO: 4 or having at least 95% amino acid sequence identity with SEQ ID NO: 4,
A PA gene segment encoding a polypeptide having SEQ ID NO: 5 or having at least 95% amino acid sequence identity with SEQ ID NO: 5,
An NP gene segment encoding a polypeptide having SEQ ID NO: 6 or having at least 95% amino acid sequence identity with SEQ ID NO: 6,
An M gene segment encoding an M1 polypeptide having SEQ ID NO: 7 or having at least 95% amino acid sequence identity with SEQ ID NO: 7,
An M gene segment encoding an M2 polypeptide having SEQ ID NO: 17 or having at least 95% amino acid sequence identity with SEQ ID NO: 17,
Has an NS gene segment encoding an NS1 polypeptide having SEQ ID NO: 8 or having at least 95% amino acid sequence identity with SEQ ID NO: 8, and / or SEQ ID NO: 18, or at least with SEQ ID NO: 18 7. The isolated influenza virus of claim 6 , comprising an NS gene segment encoding an NS2 polypeptide having 95% amino acid sequence identity. Isolated HA polypeptides de shown in SEQ ID NO: 1, or an immunogenic composition comprising a HA-1 part component shown in SEQ ID NO 2 0 of the polypeptide. Use of the isolated H3 influenza virus according to claim 6 in the manufacture of a medicament.   6. A vaccine or pharmaceutical composition according to claim 5, wherein the carrier is suitable for intranasal or intramuscular administration.   The pharmaceutical composition according to claim 1 or the vaccine according to claim 3, further comprising an antigen of a pathogen other than the isolated influenza virus. 4. The vaccine of claim 3, further comprising another isolated influenza virus.

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