HK1082422B - Multi plasmid system for the production of influenza virus - Google Patents

Description

Multi-plasmid system for preparing influenza virus

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority and benefit from the following applications: U.S. provisional application No. 60/375,675 filed on 26/4/2002; 60/394,983 filed on 7/9/2002; 60/410,576 filed on 12/9/200; 60/419,802 filed on 18.10.2002; 60/420,708 filed on day 10, 23, 2002; 60/457,699 filed 24/3/2003 (attorney docket number 26-000250 US); and an application entitled "Multiplasmid System for influenza Virus production" filed on 10.4.2003, attorney docket number 26-000260US, the specification of each of which is incorporated herein by reference in its entirety.

Background

Influenza viruses consist of an inner ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope, which is held together by a matrix protein. Both influenza a and influenza B viruses contain 8 segments of negative polarity single stranded RNA. The influenza a virus genome encodes at least 11 polypeptides. Segments 1-3 encode 3 polypeptides, which make up the viral RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB 2. Additional polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza a virus strains also encodes a small protein PB1-F2, produced by another reading frame within the PB1 coding region. Segment 4 encodes the Hemagglutinin (HA) surface glycoprotein, which is involved in cell adhesion and entry of the virus into cells during the infection phase. Segment 5 encodes a nucleocapsid Nucleoprotein (NP) polypeptide, a major structural protein associated with viral RNA. Segment 6 encodes the ceramidase (NA) envelope glycoprotein. Segment 7 encodes two matrix proteins, designated M1 and M2, which are translated from mRNA that is spliced differently. Segment 8 encodes NS1 and NS2(NEP), two non-structural proteins, translated from otherwise spliced mRNA.

The 8 genomic segments of influenza B virus encode 11 proteins. The three largest genes encode components PB1, PB2, and PA of RNA polymerase. Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6 encodes the NA protein and the NB protein. Both the NB and NA proteins are translated from overlapping reading frames of bicistronic mRNA. Segment 7 of influenza B also encodes two proteins: m1 and BM 2. The smallest segment encodes two products: NS1 was translated from full-length RNA and NS2 was translated from a spliced mRNA variant.

Vaccines that produce specific protective immune responses against influenza virus have been prepared for over 50 years. These vaccines include whole virus vaccines, split virus vaccines, surface antigen vaccines and attenuated live vaccines. Suitable formulations of any of these types of vaccines can produce a systemic immune response, while live attenuated vaccines can also stimulate local mucosal immunity in the respiratory tract.

FluMist is a live attenuated vaccine that protects children and adults from influenza (Belshe et al, (1998), "Effect of cold-adapted trivalent intranasal attenuated live influenza vaccine on children" (The efficacy of live attenuated live influenza vaccine in children), N Engl J Med 338: 1405-12; Nichol et al, (1999), "Effect of intranasal attenuated live influenza vaccine in healthy, working adults: random control clinical trials" (effective, affected intracellular influenza in humans), JAMA 282: 137-44). The FLUMIST vaccine strain contains the HA and NA gene segments from the currently circulating wild-type virus strain and six gene segments from the common Master Donor Virus (MDV): PB1, PB2, PA, NP, M, and NS. MDV of influenza A strain of FluMist (MDV-A) was obtained by serial passaging of a wild-type A/Ann Arbor/6/60 strain (A/AA/6/60) in primary chicken kidney tissue culture under conditions of continuously reduced temperature (Maassab (1967) "Adaptation and growth characteristics of influenza virus at 25 ℃" (Adaptation and growth characteristics of influenza virus at 25 degreeC) Nature 213: 612-4). MDV-A replicates efficiently at 25 deg.C (ca, cold acclimation), but its growth is inhibited at 38 deg.C and 39 deg.C (ts, temperature sensitive). In addition, the virus is unable to replicate in the lungs of infected ferrets (att, attenuated). This temperature-sensitive phenotype is believed to be responsible for limiting its replication at sites other than the coldest region in the human respiratory tract, resulting in reduced toxicity. Animal model tests and clinical tests show that this property is quite stable. Unlike this ts phenotype of influenza strains prepared by chemical mutagenesis, the ts properties of MDV-A are not reduced by passage in infected hamsters or by this property of passaged isolates isolated from children (for a recent review, see Murphy & Coelingh (2002) 'principle of preparation of Cold-adapted influenza A and B live attenuated vaccines and uses thereof' (Principles underlying the same and uses of live-attached cold-adapted influenza A and B viruses) Viral Immunol 15: 295-.

The clinical trials of more than 20,000 adults and children with 12 different 6: 2 reassorted strains revealed that these vaccines were attenuated, safe and effective (Belche et al, (1998) "Effect of cold-adapted trivalent intranasal attenuated live influenza Vaccine on children" (The infection of live infected, cold-adapted, three, endogenous influenza Vaccine in children) N Engl J Med 338: 1405-12; Boyce et al, (2000) "Safety and immunogenicity of adjuvanted and unadjuvanted subunit influenza vaccines in healthy humans by intranasal administration" (Safety of infected and unadjuvanted viral infection and unadjuvanted basic influenza Vaccine of inactivated Vaccine of infected and infected animals) 18: 19, 18-26 th follow-up of clinical trials of infected and inactivated influenza Vaccine J-217, 18-26 th experiment of infected and unadjuvanted influenza Vaccine in humans), (1999) "effect of intranasal attenuated live influenza vaccine on healthy, working adults: random control clinical trial "(affected of live, affected refractory inorganic virus vaccine in health, work addts: arnndomized controlled clinical) JAMA 282: 137-44). The rearranged strain (6: 2 rearranged strain) carrying the 6 internal genes of MDV-A and the two HA and NA gene segments of the wild-type virus was able to maintain the ca, ts and att phenotypes at all times (Maassab et al (1982) "evaluation of Cold-recombinant influenza Virus vaccines" with ferrets (Evaluationof a cold-recombinant influenza Virus vaccines in vaccines) J infection Dis 146: 780-.

To date, all commercial influenza vaccines in the united states have been bred in embryonated chicken eggs. Although influenza viruses grow well in eggs, the preparation of vaccines is dependent on the supply of eggs. The supply of eggs must be organized and the virus strains from which the vaccines are prepared must be screened several months before the next flu season, limiting the flexibility of this approach and often resulting in delays or shortages in vaccine preparation and distribution.

Systems for the Production of influenza viruses in cell culture have been developed in recent years (see, furminger, "Vaccine preparation" (Vaccine Production), Nicholson et al (ed.), (Textbook of influenza for cell culture), pp.324-. Generally, these methods employ the use of a selected viral strain to infect a suitable immortalized host cell. The preparation of vaccines according to established tissue culture methods, while not presenting many of the difficulties encountered in the preparation of vaccines from chicken eggs, not all influenza pathogenic strains grow and are prepared well. In addition, many viral strains with desirable properties, such as attenuated virulence, temperature sensitivity and cold adaptation, which are required for the preparation of live attenuated vaccines, cannot be successfully propagated in tissue culture using established methods.

The preparation of influenza virus by recombinant DNA can significantly improve the flexibility and the practicability of preparing influenza vaccine by a tissue culture method. Recently, it has been reported that viruses are prepared using recombinant plasmids having inserted cDNA encoding the viral genome (see Neumann et al, (1999) "influenza A virus from cloned cDNAs is prepared completely using cloned cDNA". Proc Natl Acad Sci USA 96: 9345-9350; Fodo et al, (1999) "influenza A virus from recombinant NDA is recovered" (Rescue of influenza A virus from recombinant DNA), J.Virol 73: 9679-9682; Hoffmann et al, (2000) "DNA transfection system for Generation of influenza A virus using 8 plasmids" (A DNA transfection system for Generation of influenza A virus from) Proc Natl AcSci USA 97: 6108; WO 01/83794). Recombinant viruses and reassortant viruses expressing the immunogenic HA and NA proteins of any selected virus strain can be prepared by these systems. However, unlike influenza a viruses, the use of pure plasmid systems for the preparation of influenza B viruses has not been reported.

In addition, there is no pure plasmid system available for the production of attenuated, temperature sensitive, cold adapted virus strains suitable for the preparation of attenuated live vaccines. The present invention provides an 8 plasmid system for the production of influenza B virus starting from cloned cDNA; and methods for preparing live attenuated influenza a and B viruses suitable for use in vaccine formulations, such as for intranasal administration of live virus vaccine formulations, numerous other advantages will become apparent upon reading the present specification.

Summary of The Invention

The present invention relates to a multi-vector system for the preparation of influenza viruses in cell culture and to a method for the preparation of recombinant influenza viruses and reassortant viruses, including attenuated (att), cold adapted (ca) and/or temperature sensitive (ts) influenza viruses suitable as vaccines, including live attenuated influenza vaccines, such as those suitable for use in intranasal vaccine formulations.

The first aspect of the invention relates to vectors and methods for the production of recombinant influenza B virus in cell culture in the absence of helper virus (i.e. helper virus-free cell culture systems). The methods of the invention comprise introducing a set of vectors, each of which can insert a portion of influenza B virus into a population of host cells capable of supporting viral replication. The influenza virus can be obtained by culturing the host cell under conditions suitable for virus growth. In certain embodiments, the influenza virus is an attenuated virus, a cold-adapted virus, and/or a temperature sensitive virus. For example, in one embodiment, the vector-derived recombinant influenza B virus is an attenuated, cold-adapted, temperature-sensitive virus suitable for preparation into an attenuated live vaccine intranasal formulation for use. In a typical embodiment, the virus is prepared by introducing a population of vectors inserted into all or part of the B/Ann Arbor/1/66 influenza virus genome, i.e., the ca B/AnnAlbor/1/66 virus genome.

For example, in certain embodiments, the influenza B virus is an artificially engineered influenza virus in which one or more amino acid substitutions are inserted to alter the biological properties of the influenza virus strain ca B/Ann ARBOR/1/66. This influenza virus comprises a mutation which leads to position PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴Of one or more amino acids, e.g. PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G). Any mutation (at one or more of these positions), either alone or in combination, which results in increased temperature sensitivity, cold adaptation or attenuation relative to the wild-type virus, is a suitable mutation of the present invention.

In certain embodiments, at least 6 internal genomic segments of one influenza B strain and one or more genomic segments of another influenza strain encoding an immunogenic influenza virus surface antigen are inserted into a set of vectors, which are introduced into a population of host cells. For example, a selected attenuated, cold-adapted and/or temperature-sensitive influenza B virus strain, i.e., the ca, att, ts strain of B/Ann Arbor/1/66, or at least 6 internal genomic segments of an artificially engineered influenza B virus strain containing one or more amino acid changes at the above positions, are introduced into a population of host cells together with one or more segments encoding an immunogenic antigen from another virus strain. Immunogenic surface antigens generally refer to one or both of Hemagglutinin (HA) and/or ceramidase (NA) antigens. In certain embodiments, only one segment encoding an immunogenic surface antigen is introduced, in which case the other 7 complementary segments of the selected virus are also introduced into the host cell.

In certain embodiments, a population of plasmids inserted into a genome segment of an influenza B virus is introduced into a population of host cells. For example, 8 plasmids, each of which contains a different genomic segment, are used to introduce the complete influenza B virus genome into a host cell. In addition, more plasmids with smaller genomic sequences inserted can be used.

In general, the plasmid vectors of the present invention are all bidirectional expression vectors. The bidirectional expression vectors of the present invention generally comprise a first promoter and a second promoter, wherein the first and second promoters are linked to one strand of the same double-stranded cDNA encoding a viral nucleic acid comprising an influenza genomic segment. In addition, the bidirectional expression vector may further comprise a polyadenylation signal and/or a termination sequence. For example, polyadenylation signals and/or termination sequences flank the influenza genome segment between the two promoters. A preferred polyadenylation signal for the present invention is the SV40 polyadenylation signal. A typical plasmid vector of the present invention is pAD3000, shown in FIG. 1.

The vector is introduced into a host cell that is capable of supporting replication of the influenza virus under the control of the vector promoter. Preferred examples of host cells include Vero cells, per.c6 cells, BHK cells, PCK cells, MDCK cells, MDBK cells, 293 cells (e.g., 293T cells), and COS cells. When used in conjunction with the pAD3000 plasmid vectors described herein, Vero cells, 293 and COS cells are preferred. In certain embodiments, co-culture of a mixture of at least two cell lines comprises a population of host cells, such as a mixture of COS and MDCK cells or a mixture of 293T and MDCK cells.

Host cells comprising influenza B virus vectors are grown in culture under conditions suitable for viral replication and assembly. In general, host cells comprising the influenza B virus plasmids of the invention are cultured at 37 ℃ or less, preferably 35 ℃ or less. The cells are generally cultured at a temperature between 32 ℃ and 35 ℃. In certain embodiments, the cells are cultured at a temperature between about 32 ℃ and 34 ℃, such as at about 33 ℃. The recombinant and/or rearranged virus is harvested after culturing for a suitable period of time to allow the virus to replicate to high titers. The harvested virus may also be inactivated.

The invention also relates to a method for producing recombinant influenza viruses in cell culture, which method has a wide range of applicability, said method comprising introducing a set of vectors inserted into the genome of an influenza virus into a population of host cells capable of supporting replication of the influenza virus, culturing the cells at a temperature of less than or equal to 35 ℃, and then harvesting the influenza virus.

In certain embodiments, a population of plasmids inserted into an influenza virus genomic segment is introduced into a population of host cells. In certain particular embodiments, 8 plasmids are used to introduce the complete influenza B virus genome into a host cell, wherein each plasmid comprises a different genomic segment. In general, the plasmid vectors of the present invention are all bidirectional expression vectors. A typical plasmid vector of the present invention is pAD3000, shown in FIG. 1.

In certain embodiments, the influenza virus is an influenza B virus. In other embodiments, the influenza virus is an influenza a virus. In certain particular embodiments, the methods comprise harvesting recombinant and/or reassortant influenza viruses that elicit an immune response when administered to a subject, such as by intranasal administration. In certain embodiments, the virus is inactivated prior to administration, and in other embodiments, a live attenuated virus is administered. Recombinant and reassortant influenza a and influenza B viruses prepared according to the methods of the invention are also a feature of the invention.

In certain embodiments, the virus comprises an attenuated influenza virus, a cold-adapted influenza virus, a temperature-sensitive influenza virus, or an influenza virus having any combination of these several characteristics. In one embodiment, the influenza virus is a B/Ann Arbor/1/66 influenza virus strain, i.e., a cold-adapted, temperature sensitive attenuated strain B/Ann Arbor/1/66. In other embodiments, the influenza virus is a A/Ann Arbor/6/60 influenza virus strain, i.e., a cold-adapted, temperature sensitive attenuated strain A/Ann Arbor/6/60. In another embodiment, the virus is an artificially engineered influenza virus,wherein there is one or more amino acid change(s) affecting the biological properties of the ca A/Ann Arbor/6/60 or ca B/Ann Arbor/1/66 virus, preferably a unique amino acid of ca A/Ann Arbor/6/60 or caB/Ann Arbor/1/66, for the type A influenza virus strain: PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G); for influenza B strains there are: PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V). Likewise, other types of amino acid substitutions at these positions that produce temperature-sensitive, cold-adapted, and/or attenuated properties are also within the scope of the viruses and methods of the present invention.

Alternatively, the reassortant virus may be prepared by introducing a vector containing 6 internal genes of a selected virus strain and a genomic segment encoding the surface antigens (HA and NA) of a selected pathogenic strain, wherein the selected virus strain HAs properties suitable for vaccine preparation. For example, the HA segment is preferably selected from pathogenic strains H1, H3 or B, all of which can be achieved by conventional methods of vaccine preparation. Likewise, the HA segment may be selected from dominant pathogenic strains such as the H2 strain (e.g., H2N2), the H5 strain (e.g., H5N1), or the H7 strain (e.g., H7N 7). In addition, the 7 complementary genomic segments of the first strain may be introduced together with the HA or NA encoding segment. In certain embodiments, the B/Ann Arbor/1/66 or A/Ann Arbor/6/60 influenza virus strain.

In addition, the present invention relates to the preparation of novel influenza viruses having desirable properties, such as temperature sensitivity, attenuation properties and/or cold adaptation, which properties are relevant for the preparation of vaccines, and to a method for preparing influenza vaccines comprising these novel influenza viruses. In certain embodiments, novel influenza a strains are prepared by introducing mutations that result in substitutions of amino acids at one or more specific positions that have been demonstrated to be important for temperature-sensitive phenotypes, such as PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴. For example, at nucleotide position PB1¹¹⁹⁵、PB1¹⁷⁶⁶、PB1²⁰⁰⁵、PB2⁸²¹And NP¹⁴⁶Or other nucleotide mutations that result in amino acid substitutions at specific amino acid positions. Any mutation (at one or more of these positions), either alone or in combination, which results in increased temperature sensitivity, cold adaptation or attenuation relative to the wild-type virus, is a suitable mutation of the present invention. For example, PB1 is preferred³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G) these mutations were introduced into the genome of the wild-type influenza a virus strain, PR8, to prepare a temperature-sensitive mutant suitable for use as an attenuated live vaccine. To increase the stability of the desired phenotype, a set of mutations is typically introduced. After introduction of the selected mutation into the genome, the mutated influenza virus genome replicates under conditions suitable for virus production. For example, a mutated influenza virus genome can replicate in chicken eggs. In addition, the influenza virus genome can also replicate in cell culture. In the latter case, the virus may be further amplified in eggs to increase virus titer. Temperature sensitive, attenuated and/or cold adapted viruses prepared according to the methods of the invention are also a feature of the invention, as are vaccines comprising such viruses. Also, it is contained in PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴Novel recombinant viral nucleic acid exhibiting one or more mutations at positions, i.e. the mutation PB1 selected from³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G); as well as polypeptides having such amino acid substitutions are also a feature of the invention.

Furthermore, the methods described herein are suitable for preparing novel influenza B virus strains that are temperature sensitive, weakly toxic, and/or cold adapted by introducing one or more specific mutations into the influenza B virus genome. For example, one or more ofAt PB2⁶³⁰、PA⁴³¹、PA⁴⁹⁷、NP⁵⁵、NP¹¹⁴、NP⁴¹⁰、NP⁵¹⁰、M1¹⁵⁹Or M1¹⁸³Mutations with amino acid substitutions at positions are introduced into influenza B virus strains to produce temperature-sensitive influenza B virus genomes. Examples of amino acid substitutions include: PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V). As indicated above, vaccines comprising such viruses and nucleic acids and polypeptides incorporating these mutations and amino acid substitutions are a feature of the present invention.

Accordingly, the influenza virus inserted with the mutation of the present invention is a feature of the present invention regardless of the method of preparing the same. That is, the invention encompasses influenza virus strains having the mutations of the invention, i.e., any influenza a virus having an amino acid substitution at one or more of the following positions relative to the wild-type virus strain: PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴Or any influenza B virus in which the amino acid substitution occurs at one or more of the following positions relative to the wild-type virus strain: PB2⁶³⁰；PA⁴³¹；PA⁴⁹⁷；NP⁵⁵；NP¹¹⁴；NP⁴¹⁰；NP⁵¹⁰；M1¹⁵⁹And M1¹⁸³(ii) a However, the strains caA/Ann Arbor/6/60 and B/Ann Arbor/1/66 are not characteristic of the present invention. In certain preferred embodiments, the influenza a virus contains a set of mutations, including: PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G); influenza B virus contains a group of mutations including PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V)。

In one embodiment, a set of plasmid vectors containing an influenza virus genome is introduced into a host cell. For example, an influenza virus genomic segment may be inserted into at least 8 plasmids. In a preferred embodiment, the influenza virus genomic segment is inserted into 8 plasmids. For example, each of these 8 plasmids has a different influenza virus genome segment inserted.

The vector of the present invention may be a bidirectional expression vector. The bi-directional expression vectors of the invention generally comprise a first promoter and a second promoter, wherein the first promoter and the second promoter are operably linked to both strands of the same double-stranded viral nucleic acid comprising an influenza viral genome segment. In addition, the bidirectional expression vector may further comprise a polyadenylation signal and/or a termination sequence. For example, polyadenylation signals and/or termination sequences flank the influenza genome segment between the two promoters. A preferred polyadenylation signal for the present invention is the SV40 polyadenylation signal. A typical plasmid vector of the present invention is pAD3000, shown in FIG. 1.

Any host cell capable of supporting replication of influenza virus under the control of a vector promoter is included within the scope of the present invention. Preferred examples of host cells include Vero cells, per.c6 cells, BHK cells, PCK cells, MDCK cells, MDBK cells, 293 cells (e.g., 293T cells), and COS cells. When used in conjunction with the pAD3000 plasmid vectors described herein, Vero cells, 293 cells and COS cells are preferred. In certain embodiments, co-culture of a mixture of at least two cell lines comprises a population of host cells, such as a mixture of COS and MDCK cells or a mixture of 293T and MDCK cells.

One feature of the present invention is that host cells containing the plasmids of the present invention are cultured at 37 ℃ or less, preferably 35 ℃ or less. The cells are generally cultured at a temperature between 32 ℃ and 35 ℃. In certain embodiments, the cells are cultured at a temperature between about 32 ℃ and 34 ℃, such as at about 33 ℃.

Another aspect of the invention relates to a novel method for recovering recombinant or rearranged influenza a or B viruses (i.e. wild type influenza a or influenza and mutants thereof) from cultured Vero cells. A set of vectors containing influenza virus genomes is introduced into a population of host cells by electroporation. The cells are grown under conditions suitable for viral replication, i.e. cold adapted, attenuated, temperature sensitive virus strains, Vero cells are cultured at less than 37 ℃, preferably 35 ℃ or less. The cells are generally cultured at a temperature between 32 ℃ and 35 ℃. In certain embodiments, the cells are cultured at a temperature between about 32 ℃ and 34 ℃, such as at about 33 ℃. Additionally (as for vaccine preparation) Vero cells were grown in serum-free medium without any animal-derived products.

In the methods described above, the virus is obtained by culturing a host cell containing an influenza virus genomic plasmid. In certain embodiments, the harvested virus is a recombinant virus. In other embodiments, the harvested virus is a reassortant virus having a background of multiple parental virus strain genes. In addition, the harvested recombinant virus or the reassortant virus may be further amplified in cultured cells or in eggs.

In addition, the harvested virus may be inactivated. In certain embodiments, the harvested virus constitutes an influenza vaccine. For example, the harvested influenza vaccine can be a reassortant influenza virus (i.e., a 6: 2 or 7: 1 reassortant virus) having HA and/or NA antigens derived from a or B strain of the selected influenza virus. In certain preferred embodiments, the reassortant influenza virus has an attenuated phenotype. In addition, the reassortant virus can be a cold-adapted and/or temperature-sensitive influenza B virus, i.e., an attenuated, cold-adapted and/or temperature-sensitive influenza B virus having one or more amino acid substitutions in table 17. Such influenza viruses are useful as live attenuated vaccines to elicit an immune response against a particular pathogenic influenza virus strain. Influenza viruses, such as attenuated reassortant viruses, made according to the methods of the invention are a feature of the invention.

In another aspect, the invention relates to a method of making a recombinant influenza virus vaccine, saidThe method comprises introducing a population of vectors comprising an influenza virus genome into a population of host cells capable of supporting influenza virus replication, culturing the host cells at a temperature of less than or equal to 35 ℃, and harvesting the influenza virus capable of eliciting an immune response upon administration to a subject. The vaccine of the invention can be influenza A virus strain and also influenza B virus strain. In certain embodiments, the influenza vaccine virus comprises an attenuated influenza virus, a cold-adapted influenza virus, or a temperature-sensitive influenza virus. In certain embodiments, the virus has a combination of these properties. In one embodiment, the influenza virus comprises the A/AnnArbor/6/60 influenza virus strain. In another embodiment, the influenza virus comprises the B/Ann Arbor/1/66 influenza virus strain. In addition, vaccines include engineered influenza A or B viruses that contain at least one substituted amino acid that affects the biological properties of ca A/Ann Arbor/6/60 or ca/B/AnnArbor/1/66, such as the unique amino acids of these strains. For example, vaccines encompassed by the present invention include artificially engineered recombinant and rearranged influenza a viruses that have a mutation at least one position that results in an amino acid substitution at: PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴(ii) a And artificially engineered recombinant and rearranged influenza B viruses having a mutation at least one position resulting in an amino acid substitution at the following positions: PB2⁶³⁰；PA⁴³¹；PA⁴⁹⁷；NP⁵⁵；NP¹¹⁴；NP⁴¹⁰；NP⁵¹⁰；M1¹⁵⁹And M1¹⁸³。

In certain embodiments, the viruses include reassortant influenza viruses (i.e., 6: 2 or 7: 1 reassortant strains) that contain genomic segments derived from multiple influenza virus strains. For example, a preferred reassortant influenza vaccine comprises HA and/or NA surface antigens derived from a selected influenza a or B strain and internal genome segments derived from a viral strain having the desired characteristics associated with vaccine preparation. Usually locally or locally in anticipation of the pathogenic strain (as described above)Influenza virus strains of HA and/or NA coding segment origin are preferred on a worldwide epidemic basis. In some cases, the virus strain containing the internal genomic segment is an attenuated, cold-adapted and/or temperature-sensitive influenza virus strain, such as a/Ann Arbor/6/60, B/Ann Arbor/1/66 or an artificially engineered influenza virus strain having one or more substituted amino acids to have a desired phenotype, such as an influenza a virus comprising at least one mutation resulting in an amino acid substitution at the following positions: PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴(ii) a And influenza B viruses comprising at least one mutation resulting in an amino acid substitution at the following positions: PB2⁶³⁰；PA⁴³¹；PA⁴⁹⁷；NP⁵⁵；NP¹¹⁴；NP⁴¹⁰；NP⁵¹⁰；M1¹⁵⁹And M1¹⁸³. For example, preferred reassortant viruses comprise an artificially engineered influenza a virus having one or more of the following amino acid substitutions: PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G); and an artificially engineered influenza B virus comprising one or more of the following amino acid substitutions: PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V)。

If desired, the influenza virus may be inactivated after harvesting.

Influenza vaccines, including live attenuated vaccines, prepared by the methods of the invention are also a feature of the invention, and in certain preferred embodiments, the influenza vaccine is a reassortant virus vaccine.

Another aspect of the invention relates to a bidirectional expression plasmid vector. The bidirectional expression vector of the present invention contains a first promoter located between a second promoter and a polyadenylation site, the SV40 polyadenylation site. In one embodiment, the first promoter and the second promoter are in opposite orientations and flank at least one cloning site. A typical vector of the present invention is plasmid pAD3000, shown in FIG. 1.

In certain embodiments, at least one segment of the influenza genome is inserted into a cloning site to form a double-stranded nucleic acid. For example, the vectors of the present invention comprise a plasmid in which a first promoter is located between a second promoter and the SV40 polyadenylation site, wherein the first promoter and the second promoter are in opposite orientations and flank at least one influenza genome segment.

Kits comprising one or more expression vectors of the invention are also a feature of the invention. Typically, the kit further comprises one or more cell lines capable of supporting replication of influenza virus, buffers, cell culture media, instructions, packaging materials, and containers. In certain embodiments, the kit comprises a set of expression vectors, each expression vector comprising at least one segment of an influenza virus genome. For example, kits comprising a set of expression vectors, each expression vector containing an internal genomic segment of a selected viral strain having properties relevant to vaccine preparation or use are also a feature of the invention. For example, the selected virus strain may be an attenuated, cold-adapted and/or temperature sensitive virus strain, such as a/Ann Arbor/6/60, B/Ann Arbor/1/66 or other virus strains having desired characteristics, such as an artificially engineered virus strain having one or more amino acid substitutions as described in table 17 herein. In one embodiment, the kit comprises an expression vector comprising a member of a library of nucleic acids encoding mutated HA and/or NA antigens.

Also featured are cell cultures capable of proliferative growth at a temperature of less than or equal to 35 ℃, which cell cultures include at least one cell comprising a set of vectors comprising an influenza virus genome. The composition may also include a cell culture medium. In certain embodiments, a set of vectors includes a bidirectional expression vector, i.e., comprising a first promoter inserted between a second promoter and the SV40 polyadenylation site. For example, the first promoter and the second promoter are oppositely oriented and flank at least one segment of the influenza virus. The cell culture of the invention may be cultured at a temperature of less than or equal to 35 ℃, such as between about 32 ℃ and 35 ℃, typically between about 32 ℃ and 34 ℃, such as about 33 ℃.

The invention also relates to a cell culture system comprising a cell culture as described above capable of proliferative growth comprising at least one cell, wherein the cell comprises a set of vectors comprising an influenza virus genome; and a modulator to maintain growth of the cell culture at less than or equal to 35 ℃. For example, the modulator preferably maintains the growth of the cells at a temperature of between about 32 ℃ and 35 ℃, typically between about 32 ℃ and 34 ℃, such as about 33 ℃.

Another feature of the invention is an artificially engineered recombinant influenza virus or reassortant virus comprising one or more substituted amino acids that affect the temperature sensitive influenza virus, cold adaptation and/or attenuation properties. For example, artificially engineered influenza a viruses that exhibit amino acid substitutions at the following positions: PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴(ii) a And an artificially engineered influenza B virus with amino acid substitutions at the following positions: PB2⁶³⁰；PA⁴³¹；PA⁴⁹⁷；NP⁵⁵；NP¹¹⁴；NP⁴¹⁰；NP⁵¹⁰；M1¹⁵⁹And M1¹⁸³Are preferred embodiments of the present invention. Typical embodiments include influenza a viruses having amino acids substituted for one or more of the following: PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G); and influenza B viruses having one or more of the following amino acid substitutions: PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V). In certain embodiments, the virus comprises a set of mutations, e.g.1, 2, 3, 4, 5,6, 7, 8 or 9 substituted amino acids at the above positions. Correspondingly, in all 5 positions mentioned above, i.e. PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G) an artificially modified influenza a virus with an amino acid substitution; and in the above 8 or all 9 positions, i.e. PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V) an artificially modified influenza B virus having an amino acid substitution is also included in the scope of the present invention. In addition, the virus may also contain one or more other types of substituted amino acids not mentioned above.

In certain embodiments, the artificially engineered influenza virus is a temperature sensitive influenza virus, a cold-adapted and/or an attenuated influenza virus. For example, temperature sensitive influenza viruses of the invention typically exhibit about a 2.0 to 5.0log reduction in growth at 39 ℃ as compared to wild-type influenza viruses₁₀. For example, the growth of a temperature sensitive influenza virus is reduced by at least about 2.0log at 39 ℃ compared to a wild-type influenza virus₁₀About 3.0log₁₀、4.0log₁₀Or 4.5log₁₀. In general, but not necessarily, temperature sensitive influenza viruses retain the most vigorous growth characteristics at 33 ℃. The attenuated influenza viruses of the present invention typically have about a 2.0 to 5.0log reduction in comparison to wild-type influenza viruses as found by ferret attenuation tests₁₀. For example, the attenuated influenza viruses of the present invention exhibit at least about a 2.0log reduction in growth as compared to wild-type influenza virus as demonstrated by ferret attenuation testing₁₀Usually about 3.0log reduction₁₀Preferably about 4.0log reduction₁₀。

Brief Description of Drawings

FIG. 1: description of pAD3000 plasmid

FIG. 2: micrographs of infected cells

FIG. 3: genotyping of rMDV-A and 6: 2H1N1 reassortants obtained by plasmid transfection

FIG. 4: description of the preparation of 8 plasmid System for influenza B Virus

FIG. 5: RT-PCR analysis of the characteristics of the recombinant MDV-B virus; RT-PCR analysis of characteristics of recombinant B/Yamanashi/166/98

FIG. 6: GeneBank-form pAD3000 sequence

FIG. 7: sequence alignment of MDV-B and 8 plasmids

FIG. 8: RT-PCR products obtained by simultaneous amplification of HA and NA segments of influenza B virus

FIG. 9: the bar graph illustrates the relative titers of recombinant and reassortant viruses

FIG. 10: the bar graph illustrates the relative titers of reassortant viruses at permissive and limiting temperatures (temperature sensitive influenza virus)

FIG. 11: the graph illustrates the rearranged virus inserted with specific mutations (knockins) associated with temperature sensitive influenza virus (left panel) and the relative titers at permissive and limiting temperatures (temperature sensitive influenza virus) (right panel).

FIG. 12: the ts mutation was determined using a micro-genomic analysis. HEp-2 cells were transfected with PB1, PB2, PA, NP, and pPlu-CAT, incubated at 33 ℃ or 39 ℃ for 18 hours, and cell extracts were used to analyze CAT reporter gene expression. B. Analysis of CAT mRNA expression Using primer extension assay

FIG. 13: the figures illustrate a three-gene recombinant virus with wild-type residues on the PA, NP and M1 proteins

FIG. 14: table showing growth of single and double gene recombinant viruses

FIG. 15: the table indicates the amino acid residues of the nucleoprotein corresponding to the non-ts phenotype

FIG. 16: schematic of recombinant PR8 mutant strain. Black dots indicate mutations introduced in the PB1 and/or PB2 genes

FIG. 17: the bar graph illustrates the relative titres at 33 ℃ and 39 ℃

FIG. 18: micrographs of plaque morphology of the PR8 mutant at various temperatures. MDCK cells were transfected with virus as indicated and incubated at 33 deg.C, 37 deg.C and 39 deg.C for 3 days, respectively. Plaques were visualized by immunostaining and photographed.

FIG. 19: protein synthesis at permissive and non-permissive temperatures. MDCK cells were transfected with virus as indicated and incubated overnight at 33 ℃ or 39 ℃ respectively. The radiolabeled polypeptides were separated by SDS-PAGE electrophoresis and autoradiography was performed. Indicated in the figure are the viral proteins HA, NP, M1 and NS.

FIG. 20: A. the line graph illustrates the difference between MDA-V and MDV-B replication in Per.C6 cells and MDCK cells. B. The line graphs illustrate the differential replication of MDV-a monogene rearrangement virus in per.c6 cells.

Detailed Description

Many pathogenic influenza viruses have poor growth status in tissue culture, and strains suitable for preparing live attenuated vaccines (i.e., temperature sensitive influenza viruses, cold adapted and/or attenuated influenza viruses) have not been successfully grown in cultured cells to produce commercially useful products. The present invention provides a multi-plasmid system that allows the growth and recovery of influenza strains that are not able to grow under standard cell culture conditions.

In a first aspect, the method of the invention relates to vectors and methods for the preparation of recombinant influenza B viruses in cell culture, entirely from cloned viral DNA. In another aspect, the methods of the invention are based in part on the improvement of tissue culture conditions that can support the growth of strains of virus (influenza a and B strains) having vaccine production-related properties (i.e., attenuated pathogenicity, cold-adapted, temperature-sensitive influenza, etc.) in cells cultured in vitro. Influenza viruses can be prepared by introducing a set of vectors carrying cloned viral genome segments into host cells and culturing at temperatures not exceeding 35 ℃. Recombinant viruses suitable as vaccines can be prepared by standard purification methods when transfected with vectors containing influenza virus genomes. With the vector system and method of the present invention, reassortant viruses can be prepared in tissue culture with high efficiency incorporating the internal gene segments of 6 viral strains with the desired properties and the immunogenic HA and NA segments of selected pathogenic strains. Thus, the systems and methods described herein can be used to rapidly prepare recombinant and reassortant influenza a and B viruses in cell culture, including viruses suitable as vaccines, including live attenuated vaccines, such as vaccines suitable for intranasal use.

Generally, one Master Donor Virus (MDV) strain is selected for each subtype a and B. For live attenuated vaccines, the primary donor virus is generally selected for its desirable properties, such as temperature sensitive influenza virus, cold adaptation and/or attenuation properties associated with vaccine preparation. For example, typical primary donor viruses include such temperature sensitive, attenuated and cold adapted strains of A/Ann Arbor/6/60 and B/Ann Arbor/1/66, respectively. The present invention is described below in the context of mutations that result in these strains to produce the ca, ts and att phenotypes, and provides methods for producing novel influenza strains suitable as donor strains for recombinant and reassortant vaccines.

For example, a selected master donor type A virus (MDV-A) or master donor type B virus (MDV-B) can be prepared starting from a set of cloned viral cDNAs containing the viral genome. In a typical embodiment, the recombinant virus is prepared starting from 8 cloned viral cdnas. The 8 viral cDNAs representing selected MDV-A or MDV-B sequences PB2, PB1, PA, NP, HA, NA, M, and NS are cloned into a bidirectional expression vector such as a plasmid (e.g., pAD3000) so that viral genomic RNA is transcribed from one strand under the control of the RNA polymerase I (pol I) promoter and viral mRNA is synthesized from the other strand under the control of the RNA polymerase II (pol II) promoter. In addition, any gene segment may be modified to include an HA segment (e.g., to remove multi-base cleavage sites).

The plasmids carrying the 8 viral cDNAs are then transfected into suitable host cells, such as Vero cells, co-cultured MDCK/293T or MDCK/COS7 cells, from which infectious recombinant MDV-A or MDV-B viruses can be harvested. Using the plasmids and methods described herein, the present invention can be used to prepare a 6: 2 reassortant influenza virus vaccine by co-transfecting the 6 internal genes (PB1, PB2, PA, NP, M, and NS) of a selected virus (e.g., MDV-A, MDV-B) and HA and NA from different corresponding types of influenza viruses (A or B). For example, the HA segment is preferably selected from pathogenic strains of H1, H3 or B, which are conventional in vaccine preparation. Likewise, the HA segment may also be selected from strains associated with pathogenic strains, such as the H2 strain (H2N2), the H5 strain (H5N1) or the H7 strain (H7N 7). Rearranged virus strains (7: 1 reassortant viruses) containing 7 genomic segments of MDV and the HA or NA genes of the selected strains can also be prepared. In addition, the system can also be used to determine the molecular basis of phenotypic characteristics such as attenuation properties associated with vaccine preparation (att), cold-adapted (ca), and temperature-sensitive (ts) influenza viruses.

Definition of

Unless defined otherwise, all scientific and technical terms are to be understood as having the same meaning as commonly understood in the art to which they pertain. The following terms are specifically defined for the purposes of the present invention.

The terms "nucleic acid", "polynucleotide sequence" and "nucleic acid sequence" refer to a single-or double-stranded deoxyribonucleotide or ribonucleotide polymer, or a chimera or analog thereof. As described herein, the term may also include polymers of natural nucleotide analogs having the basic properties of natural nucleotides, which can hybridize to single-stranded nucleic acids in the same manner as natural nucleotides. Unless otherwise specified, a particular nucleic acid sequence of the invention comprises a complementary sequence in addition to the sequence explicitly indicated.

The term "gene" is a widely used concept and refers to any nucleic acid sequence having a biological function. Thus, a gene includes coding sequences and/or regulatory sequences required for its expression. The term "gene" applies to a particular genomic sequence, as well as to the cDNA or mRNA encoded by the genomic sequence.

Genes also include non-expressed nucleic acid segments, such as recognition sequences that form other proteins. Non-expressed regulatory sequences include "promoters" and "enhancers" to which regulatory proteins, such as transcription factors, bind, resulting in transcription of adjacent or nearby sequences. A "tissue-specific" promoter or enhancer refers to a promoter or enhancer that regulates transcription in a particular type of tissue or cell.

The term "vector" refers to a tool that can be used to propagate and/or transfer nucleic acids between microorganisms, cells, or cell components. Vectors include plasmids, viruses, phages, proviruses, phagemids, transposons, artificial chromosomes and the like, and can replicate autonomously or integrate into the chromosome of the host cell. The vector may also be naked RNA, naked DNA, a polynucleotide composed of DNA and RNA in the same strand, polylysine-bound DNA or RNA, peptide-linked DNA or RNA, liposome-linked DNA, or the like, which cannot replicate autonomously. The most commonly used vector of the present invention is a plasmid.

An "expression vector" is a vector, such as a plasmid, that is capable of promoting expression and replication of the nucleic acid it carries. Generally, the nucleic acid to be expressed is linked to a promoter and/or enhancer, the transcription of which is controlled by the promoter and/or enhancer.

A "bidirectional expression vector" typically has two promoters in opposite directions, with a nucleic acid located between the two promoters, to express either the sense strand (+) or the antisense strand (-) which initiates transcription of RNA in either direction. Alternatively, the bidirectional expression vector may be an ambisense vector, and viral mRNA and viral genomic RNA (as cRNA) are expressed from the same strand.

According to the present invention, the term "free" refers to biological material, such as nucleic acids or proteins, that is free of components with which it is associated or interacts in its natural environment. The free material may also comprise materials not found in the natural environment, such as cells. For example, if the material is present in a natural environment, such as a cell, the material is present in the cell at a different location (e.g., a genome or genetic element) than the location in the natural environment. For example, a native nucleic acid (e.g., a coding sequence, promoter, enhancer, etc.) becomes an episomal nucleic acid if it is introduced in a non-native manner into a genome (i.e., a vector, such as a plasmid or viral vector, or a replicon) at a site that is different from the native site at which the nucleic acid is located. Such nucleic acids are also referred to as "heterologous nucleic acids".

The term "recombinants" refers to materials (e.g., nucleic acids or proteins) that have been artificially engineered or synthesized. Such changes may be made in or removed from their natural environment or state. When the term refers to a virus, such as an influenza virus, the virus is a recombinant virus meaning that the virus is produced by expression of a recombinant nucleic acid.

The term "rearranged" when referring to a virus indicates that the virus comprises genetic and/or polypeptide elements derived from multiple parental viral strains or sources. For example, a 7: 1 reassortant virus comprises 7 genomic segments (or gene segments) from a first parent virus and a complementary viral genomic segment from a second parent virus, such as a genomic segment encoding hemagglutinin or ceramidase. The 6: 2 reassortant virus comprises 6 genomic segments, most commonly 6 internal genes, from the first parental virus; and two complementary fragments of the other parent virus, hemagglutinin and ceramidase.

The term "introduced" when used with respect to a heterologous or episomal nucleic acid refers to the incorporation of the nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid is inserted into the genome of the cell (e.g., chromosomal, episomal, plastid, or mitochondrial DNA) and transformed to form an autonomous replicon or transiently expressed (e.g., transfected mRAN). The term includes methods such as "infection", "transfection", "transformation" and "transduction". In the present invention, various methods can be used for introducing a nucleic acid into a prokaryotic cell, such as electroporation, calcium phosphate precipitation, lipid-mediated transfection (lipofection), and the like.

The term "host cell" refers to a cell that contains a heterologous nucleic acid, such as a vector, and that supports replication and/or expression of the nucleic acid, and that can also produce one or more encoded nucleic acids, including polypeptides and/or viruses. The host cell may be a prokaryotic cell such as E.coli, or a eukaryotic cell such as yeast, insect cell, amphibian cell, avian cell or mammalian cell, including human cells. Typical host cells for use in the present invention include Vero cells (Vero cells), per.c6 cells (human embryonic retinal cells), BHK cells (baby hamster kidney cells), primary chicken kidney cells (PCK), Madin-Darby dog kidney (MDCK) cells, Madin-Darby bovine kidney (MDBK) cells, 293 cells (e.g., 293T cells), and COS cells (e.g., COS1, COS7 cells). The term host cell encompasses a mixture of cells, such as a mixed culture of different types of cells or cell lines.

The terms "temperature sensitive", "cold-adapted" and "attenuated" are well known in the art. For example, the term "temperature sensitive" ("ts") for a type a influenza strain indicates a 100-fold or greater reduction in the titer of the virus at 39 ℃ relative to 33 ℃, and for a type B influenza strain indicates a 100-fold or greater reduction in the titer of the virus at 37 ℃ relative to 33 ℃. For example, the term "cold adaptation" ("ca") refers to a virus that grows at 25 ℃ within 100-fold of its growth at 33 ℃. For example, the term "attenuated" ("att") means that the virus replicates in the upper respiratory tract of ferrets but is not detectable in lung tissue, nor does it cause cold-like illness in animals. It is also understood that viruses having an intermediate phenotype, i.e., a virus titer that decreases less than 100-fold at 39 ℃ (for strain a) or 37 ℃ (for strain B), a growth that is more than 100-fold (e.g., within 200-fold, 500-fold, 1000-fold, 10000-fold) greater at 25 ℃ than it grows at 33 ℃), and/or a growth that is weaker in the lungs of ferrets than it grows in the upper respiratory tract (i.e., a reduction in toxic moieties) and/or that causes cold-like illness in animals, are also useful viruses encompassed by the present invention having one or more amino acid substitutions as described herein. Growth refers to an increase in viral load by measuring titer, plaque size or morphology, particle density, or other methods known to those skilled in the art.

The term "artificially engineered" as used herein means that the virus, viral nucleic acid or virus-encoded product, e.g., polypeptide, vaccine, contains at least one mutation introduced by recombinant means, e.g., directed mutagenesis, PCR mutation, and the like. The term "artificially engineered" when used with respect to a virus (or viral component or product) containing one or more nucleotide mutations and/or amino acid substitutions means that the viral genome or genomic segment encoding the virus (or viral component or product) is not derived from a natural source, such as a pre-existing laboratory virus strain, either naturally occurring or prepared by non-recombinant means (e.g. progressive passage at 25 ℃), i.e. a wild-type or cold-adapted influenza a/Ann Arbor/6/60 or B/Ann Arbor/1/66S virus strain.

Influenza virus

The genome of influenza virus consists of 8 segments of linear (-) ribonucleic acid (RNA) strands, encoding immunogenic Hemagglutinin (HA) and ceramidase (NA) proteins, respectively; and 6 internal core polypeptides: nuclear shell Nucleoprotein (NP), matrix protein (M), nonstructural protein (NS), and 3RNA polymerase (PA, PB1, PB2) proteins. During replication, genomic viral RNA is transcribed in the nucleus of the host cell into (+) strand messenger RNA and (-) strand genomic cRNA. The 8 genomic segments are packaged into a ribonucleoprotein complex that contains, in addition to RNA, NP and polymerase complexes (PB1, PB2 and PA).

In the present invention, each of the respective viral genomic RNAs in the 8 segments is inserted into a recombinant vector for manipulation and production of influenza virus. Various vectors, including viral vectors, plasmids, cosmids, phages and artificial chromosomes can be used in the present invention. For ease of manipulation, viral genome segments are typically inserted into plasmid vectors which contain one or more origins of replication capable of functioning in bacterial and eukaryotic cells, and which may also contain selectable marker genes to facilitate selection of cells transfected with the plasmid sequences. A typical vector is pAD3000 shown in figure 1.

The most commonly used plasmid vectors of the present invention are bi-directional expression vectors that allow transcription of the inserted viral genome segment to be initiated in either direction, resulting in the synthesis of (+) and (-) strand RNA. To increase the efficiency of bidirectional transcription, each viral genome segment is inserted into a vector containing at least two independent promoters such that copies of viral genomic RNA are transcribed from one strand by a first RNA polymerase promoter (i.e., Pol I) and viral mRNA is synthesized by a second RNA polymerase promoter (i.e., Pol II). Accordingly, the two promoters are arranged in opposite orientations, flanking at least one cloning site (i.e., a restriction enzyme recognition sequence), preferably a unique cloning site, for insertion of the viral genomic RNA fragment. Alternatively, an "ambisense" vector may be used in which the (+) strand mRNA and (-) viral RNA (as cRNA) are expressed from the same strand.

Expression vector

The segment of the influenza virus genome that is desired to be expressed is linked to an appropriate transcriptional control sequence (promoter) that controls the synthesis of mRNA. Various promoters are suitable for use in the transcriptional regulation of influenza genomic segments in expression vectors. In certain embodiments, the vector is plasmid pAD3000, and the promoter used is the Cytomegalovirus (CMV) DNA-dependent RNA polymerase II (PolII) promoter. Other promoters may be substituted to induce transcription of the RNA under specific conditions, or in specific tissues or cells, if desired, for example, for the purpose of regulating conditional expression. Many viral promoters or mammalian, e.g., human, promoters may be used, and may be isolated for a particular use. For example, promoters derived from the genomes of animal and human viruses include promoters derived from adenovirus (e.g., adenovirus 2), papilloma virus, hepatitis b virus, polyoma virus and simian virus 40(SV40), as well as various retroviral promoters. Mammalian promoters include actin promoters, immunoglobulin promoters, heat shock promoters, and the like. Alternatively, a bacteriophage promoter may be used in combination with a homologous RNA polymerase, such as the T7 promoter.

Transcription can also be enhanced by the addition of enhancer sequences. Enhancers are generally short, about 10-500bp, cis-acting DNA elements that act in concert with a promoter to increase transcription. Many enhancer sequences have been isolated from mammalian genes (hemoglobin, elastase, albumin, alpha-fetoprotein, and insulin) and eukaryotic viruses. Enhancers can be ligated into the vector at a position 5 ' or 3 ' to the heterologous coding sequence, but are typically inserted 5 ' to the promoter. In general, the chosen promoter, as well as additional transcription enhancing sequences, optimize the expression of heterologous DNA in the host Cell into which it is introduced (Scharf et al, (1994) "Heat stress promoters and transcription factors" (Heat stress promoters and transcription factors) Results sheet Cell Differ 20: 125-62; Kriegler et al, (1990) "Assembly of enhancers, promoters and splice sites to control the expression of transferred genes" (Assembly of promoters, and splice sites) Methods in Enzymol 185: 512-27). In addition, the amplicon may also contain a ribosome binding site or Internal Ribosome Entry Site (IRES) to facilitate initiation of translation.

The vectors of the invention also preferably contain sequences required for transcription termination and mRNA stabilization, such as polyadenylation sites or terminator sequences. Such sequences are typically placed in the 5 'untranslated region, and sometimes in the 3' untranslated region, of eukaryotic or viral DNA or cDNA. In one embodiment, the one comprising plasmid pAD3000, the polyadenylation signal is provided by the SV40 polyadenylation sequence.

In addition, as described above, the expression vector may also contain one or more selectable marker genes to provide phenotypic traits desirable for selection of transformed cells, and in addition to the genes listed above, markers such as dihydrofolate reductase or neomycin resistance are suitable for selection of eukaryotic cell cultures.

Vectors containing the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform host cells which permit expression of the protein. Although the vectors of the present invention are replicable in bacteria, in most cases these vectors need to be introduced into mammalian cells, such as Vero cells, BHK cells, MDCK cells, 293 cells, COS cells, to obtain expression.

Other expression elements

In the case of large numbers, the genomic segment encoding the influenza virus protein includes its expression, including any additional sequences required for translation into a functional viral protein, in other cases minigenes or other artificial constructs encoding viral proteins such as HA or NA proteins may also be used. In such cases, it is often desirable to include specific initiation signals to facilitate efficient translation of the heterologous coding sequence. These signals include the ATG initiation codon and its adjacent sequences. To ensure translation of the entire insert, the initiation codon should be inserted into the correct reading frame for the viral protein. The foreign transcription element and the initiation codon can be of various origins, either natural or synthetic. Expression efficiency can be enhanced by adding enhancers appropriate for the cell system.

If desired, polynucleotide sequences encoding additional expression elements, such as signal sequences, secretion or localization sequences, and the like, can be inserted into the vector, usually in frame with the polynucleotide sequence of interest, to target the expressed polypeptide to the desired cell compartment, membrane or organelle, or secreted into the cell culture medium. Such sequences are well known to those skilled in the art and include secretory leader peptides, organelle targeting sequences (e.g., nuclear localization sequences, endoplasmic reticulum retention signals, mitochondrial transport defects), membrane localization/anchoring sequences (e.g., stop transport sequences, GPI anchoring sequences), and the like.

Influenza virus vaccine

Influenza vaccines have previously been prepared by empirically predicting the virus strain to be circulating and then selecting the virus strain to prepare in embryonated eggs. Recently, reassortant strains have been prepared which contain selected hemagglutinin and ceramidase antigens and are the dominant strains of attenuated, temperature-sensitive influenza viruses that have been approved. After multiple subcultures in eggs, influenza virus is harvested, and formaldehyde and/or beta-propiolactone can be selected for inactivation. However, there are several significant disadvantages to preparing influenza vaccines in this manner. The contaminants remaining from the eggs are highly antigenic and are produced by high heat, often with significant side effects after use. More importantly, designing the virus strain to be prepared must be done several months before the next epidemic season comes in order to have enough time to prepare and inactivate the influenza vaccine. Attempts to prepare recombinant and reassortant vaccines in cell culture have also been hampered by the problem that neither virus strains approved for vaccine production can grow under standard cell culture conditions.

The present invention provides vector systems and methods for the production of recombinant and reassortant viruses in cell culture, which enable the rapid production of vaccines based on one or more selected antigenic virus strains. In particular, the conditions and viral strains provided by the present invention allow for efficient virus production from a multiple plasmid system in cell culture. In addition, the virus may be further amplified in eggs if desired.

For example, growth of influenza B virus main strain B/AnnArbor/1/66 using standard cell culture conditions such as at 37 ℃ is not possible. In the method of the invention, each plasmid of the multi-plasmid system contains a segment of the influenza genome, and this system is introduced into suitable cells, and the cells are then cultured at a temperature of less than or equal to 35 ℃. In general, the cell culture may be cultured at a temperature of between about 32 ℃ and 35 ℃, preferably between about 32 ℃ and 34 ℃, such as about 33 ℃.

Usually, the cell culture is cultured in a system such as a cell incubator, controlled humidity and CO₂The temperature being maintained within a constant range by means of a temperature-regulating device, e.g. a thermostat, to ensure that the temperature does not exceedPassing through 35 ℃.

The reassortant virus can be readily obtained by introducing a set of vectors comprising a genomic segment of the main influenza virus and a complementary segment derived from the strain of interest (i.e. the antigenic strain of interest). In general, the selection of the main virus strain is based on whether it has properties relevant to vaccine use. For example, for vaccine preparation, i.e., preparation of an attenuated live vaccine, the primary donor virus is selected to have an attenuated phenotype, a cold-adapted and/or a temperature-sensitive influenza virus. Thus, influenza A virus strain ca A/Ann Arbor/6/60; influenza B strain ca B/AnNarbor/1/66; or other influenza virus strains having the desired phenotypic properties, i.e., attenuated, cold-adapted and/or temperature-sensitive, such as the artificially engineered influenza a virus strains described in example 4; or one or more substituted amino acids listed in table 17, are all preferably the main donor virus strain.

In one embodiment, a plasmid containing 6 internal genes of the mainstream influenza virus strain (i.e., PB1, PB2, PA, NP, M1, NS1, and NS2) and antigenic virus strains, i.e., virus strain-derived hemagglutinin and neuraminidase fragments predicted to be likely to cause significant local or global influenza, is transfected into a suitable host cell. The reassortant virus is replicated in cell culture at a temperature suitable for efficient growth, i.e., less than or equal to 35 ℃, such as between about 32 ℃ and 35 ℃, for example between about 32 ℃ and 34 ℃, or about 33 ℃, and then harvested. Alternatively, the harvested virus may be inactivated with a denaturant such as formaldehyde or beta-propiolactone.

Attenuated, temperature sensitive and cold adapted influenza virus vaccines

In one aspect, the invention is based on identifying mutations that result in a preferred master donor virus strain producing the ts phenotype. To determine the functional importance of single nucleotide changes to the genome of the MDV strain, temperature sensitive influenza viruses were analyzed for reassortant viruses of highly related strain origin within the a/AA/6/60 virus line. The isogenic nature of the two parental viruses allows the effect of single nucleotide changes on the ts phenotype to be evaluated. Accordingly, the genetic basis of the ts phenotype of MDV-A at the nucleotide level was mapped to specific amino acids within PB1, PB2, and NP.

Previous researchers have attempted to map the genetic basis of the ts phenotype of ca A/AA/6/60 using coinfection/reassortment techniques to produce monogenic and polygenic reassortant viruses between A/AA/6/60 and unrelated wild-type strains. These studies indicate that PB2 and PB1 are both related to the ts phenotype (Kendal et al, (1978) "Biochemical properties of recombinant viruses at suboptimal temperatures-evidence indicates that ts lesions are present in RNA segments 1 and 3, that RNA1 encodes a viral proto-transcriptase" (Biochemical characteristics of viral viruses: evidence that ts are present in the region of viral fragments 1 and 3, and that RNA1 codes for the viral transcription enzyme, p.734-743, B.W.J.Mahy and R.D.Barry (eds.),Negative Strand Virusesacademycpress; kendal et al, (1977) "comparative study of wild-type and cold mutant (temperature sensitive influenza virus) influenza viruses: family of matrix proteins (M) and nonstructural proteins (NS) of recombinant cold-adapted virus H3N2 (combinatorial of wild-type and cold-reactive) infiluenzavities: genetic of the matrix (M) and the non-structural (NS) proteins induced coded H3N2viruses),J Gen Virol37: 145-159; kendal et al, (1979) "comparative study of wild-type and cold mutant (temperature sensitive influenza virus) influenza viruses: the temperature sensitive influenza virus of viral replication and the temperature sensitive influenza virus of viral proto-transcriptase activity are independent of each other during recombination of the mutant A/Annar/6/60 with the wild-type H3N2strain "(comprehensive students of wild-type and cold-mutant) inflectiona viruses: independent growth of temporal-sensitivity of viral replication from temporal-sensitivity of viral replication and synthesis of mutant A/Ann Arbor/6/60with wild-type H3N2strains),J Gen Virol,44: 443-; snyder et al, (1988) "4 viral gene pairs live influenza A/AnnArbor/6/60 (H2)N2) Cold-adapted reassortant viral vaccine to attenuation activity is independent "(Four viral genes indendedly controlled to attenuation of live influenza A/Annrbor/6/60 (H2N2) cold-adapted recombinant viruses vaccines),J Virol62: 488-95). The results of these studies are, however, confounded by the interplay effects caused by the mixed gene segments from the two different influenza a strains. Interaction may be attenuated by alteration between A/AA/6/60 and the wild-type gene segment, rather than other strains expressing the ts phenotype with A/AA/6/60 gene background. The interplay effects also show that the interpretation of the interrelationship between M genome segments and att phenotypes can be confounded (Subbarao et al, (1992) "the attenuated phenotype of A/Korea/82(H3N3) reassorted by influenza A/Ann Arbor/6/60Cold-adapted virus (H2N2) M genes is due to the gene interplay effect" (the attenuation phenotypical by M gene of the influenza A/Ann Arbor/6/60cold-adapted virus (H2N2) on the A/Korea/82(H3N2) refractory virus from the fusion effect),Virus Res，25：37-50)。

in the present invention, the resulting amino acid is PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴Mutations that are substituted at positions have been identified as functionally significant for the temperature-sensitive phenotype of the MDV-A strain virus. As will be appreciated by those skilled in the art, at PB1¹¹⁹⁵、PB1¹⁷⁶⁶、PB1²⁰⁰⁵、PB2⁸²¹And NP¹⁴⁶Nucleotide mutations at positions which may lead to PB1, respectively³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴Amino acid substitutions at positions occur. Thus, any nucleotide change that results in an amino acid substitution at these positions is a feature of the present invention. Typical mutations include the single mutation PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G), preferably a combination mutation, which results in a temperature sensitive phenotype of the virus. Again, these mutations were lost upon reverting to the wild-type ts phenotypeThese mutations can be introduced into viruses with a wild-type genetic background to confer the ts phenotype. Consistent with the stability of these phenotypes during virus passaging, a single change does not return the viral temperature-sensitive influenza virus to the level of wild-type virus. And these changes are coordinated together to fully express the ts phenotype. This discovery allows us to engineer other temperature sensitive influenza a strains into master donor viruses suitable for the preparation of live attenuated influenza vaccines.

Similarly, each amino acid change on the master donor virus B strain was associated with the ts phenotype listed in table 17. Thus, the methods described herein are suitable for the preparation of novel influenza B virus strains having a temperature-sensitive phenotype, which may also have an attenuated phenotype and/or a cold-adapted phenotype by introducing one or more specific mutations within the genome of the influenza B virus strain. For example, a temperature sensitive influenza B virus is prepared by introducing into the genome of an influenza B strain one or more mutations that result in amino acid substitutions at the following positions: PB2⁶³⁰；PA⁴³¹；PA⁴⁹⁷；NP⁵⁵；NP¹¹⁴；NP⁴¹⁰；NP⁵¹⁰；M1¹⁵⁹And M1¹⁸³. Typical amino acid substitutions include: PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V)。

Regardless of the method of preparation, influenza viruses containing the mutations of the invention are a feature of the invention. That is to say that the invention includes influenza virus strains comprising the mutations of the invention, i.e. any influenza a virus in which an amino acid substitution occurs at one or more of the following positions relative to the wild type: PB1³⁹¹、PB1⁵⁸¹、PB1⁶⁶¹、PB2²⁶⁵And NP³⁴Or any influenza B virus in which an amino acid substitution occurs at one or more of the following positions relative to wild type: PB2⁶³⁰；PA⁴³¹；PA⁴⁹⁷；NP⁵⁵；NP¹¹⁴；NP⁴¹⁰；NP⁵¹⁰；M1¹⁵⁹And M1¹⁸³However, the strains ca A/AnnArbor/6/60 and B/Ann Arbor/1/66 are not characteristic of the present invention. In certain preferred embodiments, the influenza a virus contains a set (i.e., 2, 3, 4, 5 or more) of the following mutations: PB1³⁹¹(K391E)、PB1⁵⁸¹(E581G)、PB1⁶⁶¹(A661T)、PB2²⁶⁵(N265S) and NP³⁴(D34G); influenza B viruses contain the following set of mutations: PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V). For example, in addition to providing a virus with a phenotype associated with vaccine preparation, a virus containing a set of mutations, such as 1, 2, 3, 4, or 5 selected mutations, can also be used to study the effect of other mutations on the virus phenotype. In certain embodiments, the influenza virus contains at least one additional non-wild-type nucleotide (which may result in additional amino acid changes) that may also confer or further contribute to the desired phenotype of the virus.

Cell culture

Propagation of the virus is generally in the medium in which the host cells are cultured. Host cells suitable for influenza virus replication include Vero cells, per.c6 cells, BHK cells, MDCK cells, and COS cells, such as 293T cells, COS7 cells. Co-cultures of the two cell lines described above, such as MDCK cells and 293T or COS cells, are commonly co-cultured in a 1: 1 ratio to increase the efficiency of replication. The cells are typically cultured in standard commercial media, such as Dulbecco's modified Eagle's Medium supplemented with serum (e.g., 10% fetal bovine serum), or serum-free Medium, with controlled humidity and CO₂To maintain a suitable neutral buffered pH (i.e., a pH between 7.0 and 7.2). In addition, antibiotics can be added to the medium to prevent bacterial growth, such as penicillin, streptomycin, etc., and nutrients such as L-glutamine, sodium pyruvate, non-essential amino acids, and to optimize cell growthAdditives in the form of e.g. trypsin, beta-mercaptoethanol, etc.

Methods for culturing mammalian cells have been reported and are well known to those skilled in the art. The general cultivation procedure is described in the following documents: freshney (1983) culture of animal cells: basic technical methods "(Culture of Animal Cells: Manual of Basic Technique), Alan R.Liss, New York; paul (1975) Cell and Tissue Culture (Cell and Tissue Culture), fifth edition, Livingston, Edinburgh; adams (1980) Laboratory Techniques in Biochemistry and Molecular Biology-cell culture for Biochemists (Laboratory Techniques in Biochemistry and Molecular Biology-cell culture for Biochemistry), Work and Burdon (eds.), Elsevier, Amsterdam. Additional details regarding specific tissue culture methods for the preparation of influenza viruses in vitro are described in Merten et al, (1996) "preparation of influenza viruses in cell culture for vaccine preparation" (Production of influenza viruses in cell culture), Cohen and Shafferman (eds.), "New Strategies for the Design and preparation of Vaccines" (Novel strains in designs and Production of Vaccines), all of which are incorporated herein by reference in their entirety. In addition, modifications to these methods to adapt them to the present invention are also readily achievable by routine experimentation.

The cells used for preparing influenza virus can be cultured in serum-containing or serum-free medium. In some cases, such as in the preparation of purified viruses, it is desirable that the host cells are grown in serum-free media. The cells can be cultured on a small scale, such as in a petri dish or flask containing less than 25ml of culture medium, in a large flask, such as in a spinner flask, with shaking, or in microcarrier microspheres (e.g., DEAE-Dextran microcarrier microspheres, such as Dormacell, Pfeifer)&Langen; superbead, Flow Laboratories; styrene copolymer-tris-methylamine microspheres, such as Hillex, Solohill, Ann Arbor) were surface cultured using culture flasks or reactors. The microcarrier microsphere is a tiny sphere (with a diameter in the range of 100-200 microns) that is adherent to cells in a unit volume of cell cultureProvides a larger surface area. For example, 1 liter of medium may contain more than 2 million microcarrier microspheres, providing a growth surface in excess of 8000mm². For commercial use in virus production, such as vaccine production, cells are typically cultured in a bioreactor or fermentor. Bioreactors with volumes as small as below 1 liter and as large as above 100 liters, such as Cyto3 bioreactor (Osmonics, Minnetonka, MN); NBS bioreactor (New Brunswick Scientific, Edison, n.j.); laboratory and commercial scale bioreactors from b.braun Biotech international (b.braun Biotech, Melsungen, Germany).

In the present invention, regardless of the size of the culture volume, it is important that the culture temperature be maintained at or below 35 ℃ to ensure efficient harvesting of the recombinant virus and/or the reassortant virus using the multi-plasmid system described herein. For example, cells are cultured at a temperature of between about 32 ℃ and 35 ℃, typically between about 32 ℃ and 34 ℃, and most typically about 33 ℃.

Typically, a regulator such as a thermostat or other device capable of detecting and maintaining the temperature of the cell culture system is used to ensure that the temperature does not exceed 35 ℃ during virus replication.

Introduction of vectors into host cells

Vectors carrying influenza virus genome segments are introduced (e.g., transfected) into host cells to introduce heterologous nucleic acids into eukaryotic cells according to methods well known in the art, including calcium phosphate co-precipitation, electroporation, microinjection, liposome-mediated transfection, and transfection using polyamine transfection reagents. For example, vectors such as plasmids can be introduced into host cells, such as COS cells, 293T cells or a mixture of COS or 293T cells and MDCK cells, using the polyamine transfection reagent TransIT-LT1(Mirus) according to the manufacturer's instructions. About 1. mu.g of the vector required about 2. mu.l TransIT-LT1 to be diluted into 160. mu.l of medium, preferably serum-free medium, in a total volume of 200. mu.l, for introduction into the host cell population. DNA: the transfection reagent mixture was incubated at room temperature for 45 minutes and then 800. mu.l of medium was added. The transfection mixture was added to the host cells, which were then cultured as described above. As calculated herein, in the preparation of recombinant or reassortant viruses in cell culture, vectors containing 8 genomic segments (PB1, PB2, PA, NP, M, NS, HA and NA) were mixed with approximately 20. mu.l TransIT-LT1 and transfected into host cells. Alternatively, serum-containing media can be replaced with serum-free media, such as Opti-MEM I, prior to transfection, and then incubated for 4-6 hours.

Alternatively, a vector containing an influenza virus genome segment may be introduced into a host cell by electroporation. For example, a plasmid vector containing influenza a virus or influenza B virus is preferably introduced into Vero cells by electroporation according to the following procedure: harvest 5X 10⁶Vero cells grown in Modified Eagle Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) were resuspended in 0.4ml OptiMEM, and then the cells were added to the electroporation cuvette. Mu.g of DNA was mixed to 25. mu.l, then added to the cell-containing cup and gently mixed by tapping. Electroporation was carried out according to the manufacturer's instructions (BioRad Gene pulser II with Capacitance Extender Plus connected) at 300 volts, 950 microfarads for 28-33 seconds. The cells were remixed by gentle tapping, and 0.7ml of 10% FBS-containing MEM was directly added to the cuvette about 1 to 2 minutes after electroporation. The cells were then transferred to 2 wells of a standard 6-well tissue culture plate containing 2ml of MEM + 10% FBS or serum-free OPTI-MEM. The cuvette was washed to recover the remaining cells and the wash suspension was split into two wells. The final volume was about 3.5. The cells are then incubated under conditions suitable for virus growth, i.e., the cold adapted strain is incubated at about 33 ℃.

The virus is typically recovered from the culture medium in which the infected (or transfected) cells are grown. The roughly isolated medium is generally clarified before concentrating the influenza virus. Common methods include filtration, ultrafiltration, barium sulfate adsorption and elution, and centrifugation. For example, the medium from the crude separation in infected cultures is first clarified by centrifugation at 1000-2000 Xg for a time sufficient to remove cell debris and other large particulate matter, e.g., for 10 to 30 minutes. In addition, the culture medium can also be filtered with a 0.8 μm cellulose acetate filter to remove intact cells and other large particulate matter. Alternatively, the clarified culture supernatant may be recentrifuged to pellet the influenza virus, e.g., 15,000 Xg for about 3-5 hours. After resuspending the viral pellet in a suitable buffer such as STE (0.01M Tris-HCl; 0.15M NACI; 0.0001M EDTA) or Phosphate Buffered Saline (PBS) pH7.4, the virus is concentrated by density gradient centrifugation using sucrose (60% -12%) or potassium tartrate (50% -10%). Continuous or stepwise centrifugation may be used, e.g. a sucrose gradient between 12% and 60% with 4 12% steps. The gradient centrifugation is performed at a sufficient speed and time to concentrate the virus into a visible band for recovery of the virus. In addition, for the largest scale commercial applications, density gradient centrifugation is not used and a continuous mode ribbon centrifuge rotor is used. Additional details described in the following literature are sufficient to instruct those skilled in the art to prepare influenza viruses in tissue culture: furminger, "vaccine preparation" (vaccine preparation), Nicholson et al (eds.), (Textbook of influenza viruses) p.324-; merten et al, (1996) "preparation of influenza viruses in cell culture for vaccine preparation" (Production of influenza viruses in cell cultures for vaccine delivery), Cohen and Shafferman (eds.), (noveltheres in Design and Production of Vaccines), p.141-; and U.S. patent No. 5,690,937. If desired, the recovered virus can also be stored at-80 ℃ using sucrose-phosphate-glutamate (SPG) as a stabilizer.

Methods and compositions for use of prophylactic vaccines

Recombinant and reassortant viruses of the invention may be mixed in suitable carriers or vehicles for prophylactic use to stimulate an immune response specific for one or more influenza virus strains. The carrier or excipient is typically a pharmaceutically acceptable carrier or excipient, such as sterile water, saline solution, buffered saline solution, dextrose solution, glycerol aqueous solution, ethanol, allantoic fluid from uninfected eggs (i.e., normal allantoic fluid "NAF"), or a mixture of solutions. Such solutions are prepared according to methods known in the art to ensure sterility, proper pH, isotonicity, and stability. The carrier or excipient is generally selected to minimize allergic or other adverse reactions and to suit the particular route of administration, e.g., subcutaneous, intramuscular, intranasal, etc.

The influenza viruses of the present invention are generally administered in a sufficient amount to stimulate an immune response specific for one or more influenza virus strains. The use of influenza viruses is preferred to elicit a protective immune response. Dosages and methods for eliciting a protective immune response against one or more influenza strains are well known to those skilled in the art. For example, inactivated influenza virus is administered at a dose of about 1 to 1000HID₅₀(human infectious dose), i.e. about 10 in each dose⁵-10⁸pfu (plaque forming unit). In addition, about 10-50 μ g, such as about 15 μ g, of HA is administered without adjuvant, or with adjuvant added in smaller doses. Generally, the dosage within this range can be adjusted depending on age, physical condition, body weight, sex, diet, time of administration, and other clinical factors. The prophylactic vaccine formulation can be administered using a needle and syringe, or a needle-free injection device, subcutaneously or intramuscularly. Alternatively, the vaccine formulation may be administered intranasally using drops, large particle aerosols (greater than 10 microns), or sprayed into the upper respiratory tract by spraying. Although the above routes of administration may elicit protective systemic immune responses, intranasal administration has the additional advantage that mucosal immunity may be elicited at the site of entry of the influenza virus. For intranasal administration, live attenuated virus vaccines are generally preferred, i.e., attenuated, cold-adapted and/or temperature sensitive influenza virus recombinant or reassortant influenza viruses. While it is preferred to stimulate a protective immune response with a single dose, additional doses administered by the same or different routes of administration may also achieve the desired protective effect.

In addition, immune responses can also be elicited by targeted interaction of dendritic cells with influenza viruses in vitro or in vivo. For example, contacting a proliferating dendritic cell with a virus in a sufficient dose and for a sufficient time can cause the dendritic cell to capture influenza virus antigen. The cells are then transferred to the immunized subject by standard vein graft methods.

In addition, the formulation of the protective influenza virus or subunit thereof may further comprise one or more adjuvants to enhance the immune response against influenza virus antigens. Suitable adjuvants include: saponins, mineral gums such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille calmette-guerin (BCG), corynebacterium parvum and synthetic adjuvants QS-21 and MF 59.

If desired, the prophylactic influenza virus vaccine can also be used with one or more immunostimulatory molecules. Immunostimulatory molecules include various cytokines, lymphokines, and chemokines, which have immunostimulatory, immunopotentiating, and pro-inflammatory activity, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM) -Colony Stimulating Factor (CSF)); and other immunostimulatory molecules such as macrophage inflammatory factor, Flt3 ligand, B7.1, B7.2, and the like. The immunostimulatory molecule may be in the same formulation as the influenza virus or may be administered separately from the influenza virus. The protein or expression vector encoding the protein may be used to produce an immunostimulatory effect.

In another embodiment, the vectors of the invention containing the influenza viral genome segment can be used to introduce a heterologous nucleic acid into a host bacterium or host cell, such as a mammalian cell, including cells of human origin, in combination with a suitable pharmaceutical carrier or excipient as described above. The heterologous nucleic acid is typically inserted into a non-essential region of the M gene of a gene or gene segment, such as segment 7. The heterologous polynucleotide sequence encodes a polypeptide or peptide, or an RNA, such as an antisense RNA or ribozyme. The heterologous nucleic acid is then introduced into the host or host cell by preparing a recombinant virus carrying the heterologous nucleic acid, which is used as described above.

Alternatively, the heterologous nucleic acid-containing vector of the present invention can be introduced into influenza virus-infected cells by means of co-transfection and expressed in host cells. The cells may then be returned to the recipient, typically to the site of origin of the cells. In certain applications, the cells may be transplanted to a tissue, organ or system site of interest (as described above) by established cell transfer or transplantation procedures. For example, stem cells of a hematopoietic cell line, such as bone marrow, cord blood, or peripheral blood-derived hematopoietic stem cells, can be transferred to a recipient by standard transfer or infusion techniques.

Alternatively, a virus containing a heterologous nucleic acid may be transferred into a cell in the recipient. Such methods generally involve injection of carrier particles into a target cell population (blood cells, skin cells, liver cells, nerve (including brain) cells, kidney cells, uterine cells, muscle cells, intestinal cells, neck cells, vaginal cells, prostate cells, etc., as well as tumor cells of various cell, tissue, organ origins). Viral particles can be administered systemically by intravenous injection, or they can be directly delivered to the site of interest by a variety of methods, such as injection (using a needle or syringe), needleless vaccine transfer, topical administration, or by pushing into a tissue, organ, or skin site. For example, viral vector particles can be delivered by inhalation, orally, intravenously, subcutaneously, subdermally, intradermally, intramuscularly, intraperitoneally, intratracheally, vaginally, or rectally, or by placing the viral particles in the body's lacuna or other sites during surgery.

The above methods may be used for the therapeutic and/or prophylactic treatment of a disease or disorder by introducing a vector of the invention comprising a heterologous polynucleotide encoding a therapeutic or prophylactic polypeptide (or peptide) or RNA (into an antisense RNA or ribozyme) into a target cell population in vitro or in vivo. Polynucleotides encoding a polypeptide (or peptide) or RNA of interest are typically linked to appropriate regulatory sequences as described above in the sections "expression vector" and "other expression elements". In addition, one vector or virus may be inserted with multiple heterologous coding sequences. For example, in addition to encoding a therapeutically or prophylactically active polypeptide or RNA, the vector may also contain other therapeutically or prophylactically active polypeptides, such as antigens, co-stimulatory molecules, cytokines, antibiotics, and/or the like, and/or screening markers, and the like.

The methods and vectors of the invention are useful for preventing or treating a variety of diseases, including genetic diseases and acquired diseases, such as diseases caused by viruses, bacteria, etc., in the form of vaccines.

Reagent kit

To facilitate the use of the vectors and vector systems of the present invention, any vector, such as plasmids containing influenza virus consensus sequences, plasmids containing mutated influenza virus polypeptides, influenza virus polypeptide library plasmids, and the like, and other components used to package and infect experimental or therapeutic influenza viruses, such as buffers, cells, culture media, can be packaged in the form of a kit. Generally, in addition to the components described above, the kit will contain other materials such as instructions for using the methods of the invention, packaging materials, and containers.

Manipulation of viral nucleic acids and proteins

Within the scope of the present invention, influenza nucleic acids and/or proteins may be manipulated according to known molecular biological techniques. Many such methods, including amplification, cloning, mutation, transformation, etc., are described in detail in the following references: ausubel et al, Current Molecular Biology manipulation techniques (supplement 2000), John Wiley & Sons, New York ("Ausubel"); sambrook et al, Molecular Cloning-A Laboratory Manual (second edition), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook"); and Berger and Kimmel molecular cloning guide, methods in enzymology, Vol 152, Academic Press, Inc., San Diego, Calif. (Berger).

In addition to the above-mentioned documents, other methods of operation of in vitro amplification techniques which can be used to amplify the cDNA probes of the invention, such as Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Q-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA) can be found in the following documents: mullis et al, (1987) U.S. Pat. No. 4,683, 202; the PCR technology-instructions for Methods and applications (PCR Protocols A guides to Methods and applications) (eds., Innis et al), Academic Press Inc. san Diego, CA (1990) ("Innis"); arnheim and Levinson (1990) C & EN 36; the Journal Of NIH Research (1991) 3: 81; kwoh et al, (1989) Proc Natl Acad Sci USA86, 1173; guatelli et al, (1990) Proc NatlAcad Sci USA 87: 1874; lomell et al, (1989) J Clin Chem 35: 1826; landegren et al, (1988) Science 241: 1077; van Brunt (1990) Biotechnology 8: 291; wu and Wallace (1989) Gene 4: 560; barringer et al, (1990) Gene 89: 117 and soknanan and Malek, (1995) Biotechnology 13: 563. other methods that can be used to clone the nucleic acids of the invention are described in Wallace et al, U.S. Pat. No. 5,426,039. Improved methods for amplifying large fragments of nucleic acids by PCR are described in review by Cheng et al (1994) Nature 369: 684 and references therein.

Certain polynucleotides, such as oligonucleotides, of the invention can be synthesized using a variety of solid phase techniques, including mononucleotide-and/or trinucleotide phosphoramidite coupling chemistry. For example, nucleic acid sequences can be synthesized by adding activated monomer or trisomy sequences to an extended polynucleotide chain, see Caruthers, M.H., et al, (1992) Meth Enzymol 211: 3.

in addition to synthesizing the desired sequences, basic nucleic acid sequences can be ordered from various merchants, such as Midland verified Reagent Company (R) ((R))mcrcoligos.com)，The Great American Gene Company(www.genco.com)，ExpressGen，Inc.(www.expressgen.com)，Operon Technologies，Inc.(www.operon.com) And the like.

In addition, substitution of a given amino acid on a viral polypeptide can be achieved by site-specific mutagenesis. For example, viral polypeptides containing substituted amino acids functionally related to phenotypic characteristics, such as an attenuated phenotype, cold-adapted, temperature-sensitive influenza virus, can be prepared by introducing specific mutations into the viral nucleic acid segment encoding the polypeptide. Methods for site-specific mutagenesis are well known in the art, see the description of Ausubel, Sambrook and Berger, supra. A number of site-specific mutagenesis kits are commercially available, such as the Chameleon site-directed mutagenesis kit (Stratagene, La Jolla), which can be manipulated according to the manufacturer's instructions to introduce one or more of the amino acid substitutions described in Table 6 or Table 17 into a genomic segment encoding an influenza A or B polypeptide.

Examples

Example 1: construction of pAD3000

Plasmid pHW2000(Hoffmann et al, (2000)' DNA transfection System for the preparation of influenza A Virus from 8 plasmids ` (A DNA transfection system for the generation of influenza A virus from plasmids) Proc Natl Acad Sci USA 97: 6108-.

The SV 40-derived sequences were amplified by Taq MasterMix (Qiagen) using the following oligonucleotides in the 5 'to 3' direction:

polyA.1：AACAATTGAGATCTCGGTCACCTCAGACATGATAAGATACATTGATGAGT(SEQ ID NO：1)

polyA.2：TATAACTGCAGACTAGTGATATCCTTGTTTATTGCAGCTTATAATGGTTA(SEQ ID NO：2)

plasmid pSV2His was used as template. A175 bp product consistent with the prediction was amplified using Topo TA cloning vector (Invitrogen) according to the manufacturer's instructions and then cloned into pcDNA3.1. The desired 138bp fragment containing the SV40 polyadenylation signal was excised from the resulting plasmid using EcoRV and BstEII, separated by agarose gel electrophoresis, and cloned into the unique PvuII and BstEII sites of pHW2000 using conventional methods (see Ausubel, Berger, Sambrook). The resulting plasmid pAD3000 (FIG. 1) was sequenced and found to contain the SV40 polyadenylation site and to be correctly oriented. Nucleotide 295-423 on pAD3000 relative to nucleotide 2466-2594 on SV40 strain 777(AF 332562).

Example 2: 8 plasmid system for preparing MDV-A

Influenza A vaccines for intranasal administration are usually prepared using cold adapted mutant A/AA/6/60 of influenza A virus as the main donor virus. This virus strain is a typical Master Donor Virus (MDV) in the present invention. For simplicity, this A/AA/6/60 mutant was named MDV-A herein. MDV-A viral RNA was extracted using RNeasy Mini kit (Qiagen) and 8 corresponding cDNA fragments were amplified by RT-PCR using the primers listed in Table 1.

TABLE 1 primer sequences for cloning of MDV-A8 segments

The remaining 6 genes were amplified with primers containing the BsmB I restriction enzyme recognition site, except that the influenza virus genome segment encoding HA and PB2 was amplified with primers containing the Aar I restriction enzyme recognition site. Both AarI and BsmB I cDNA fragments can be cloned between two BsmB I sites in the pAD3000 vector.

Sequence analysis showed that all cloned cDNA fragments contained mutations associated with the MDV-A consensus sequence, which were probably introduced during cloning. The mutations found in each gene segment are summarized in table 2.

TABLE 2 mutations introduced into the MDV-A clone of pAD3000

Gene segment	Mutation site (nt)	Amino acid changes
			PB2	A954(G/C/T)，G1066A，T1580C，T1821C	The gene is silenced, and the gene is not expressed,gly to Ser, Val to Ala, Gene silencing
PB1	C1117T	Arg to terminator
			PA	G742A，A1163G，A1615G，T1748C，C2229del	Gly to Ser, Asp to Gly, Arg to Gly, Met to Thr, noncoding sequence
HA	A902C，C1493T	Asn to His, Cys to Arg
			NP	C113A，T1008C	Thr to Asn, Gene silencing
NA	C1422T	Pro to Leu
			M	A191G	Thr to Ala
NS	C38T	Gene silencing

All mutations were corrected back to the MDV-A consensus sequence using the QuikChange site-directed mutagenesis kit (Stratagene) and the synthetic oligonucleotide primers listed in Table 3.

TABLE 3 primers used to correct mutations in MDV-A clones

	HJ67	PB2A954G	5/P/gcaagctgtggaaatatgcaaggc(SEQ ID NO：19)
					HJ68	PB2A954G.as	gccttgcatatttccacagcttgc(SEQ ID NO：20)
	HJ69	PB2G1066A	5/P/gaagtgcttacgggcaatcttcaaac(SEQ ID NO：21)
				PB2	HJ70	PB2G1066A.as	gtttgaagattgcccgtaagcacttc(SEQ ID NO：22)
	HJ71	PB2T1580A	5/P/cctgaggaggtcagtgaaacac(SEQ ID NO：23)
					HJ72	PB2T1580A.as	gtgtttcactgacctcctcagg(SEQ ID NO：24)
	HJ73	PB21821C	5/P/gtttgttaggactctattccaac(SEQ ID NO：25)
					HJ74	PB21821C.as	gttggaatagagtcctaacaaac(SEQ ID NO：26)
PB1	HJ75	PB1C1117T	gacagtaagctccgaacacaaatac(SEQ ID NO：27)
					HJ76	PB11117T.as	gtatttgtgttcggagcttcatgc(SEQ ID NO：28)
	HJ77	PA-G742A	5/P/cgaaccgaacggctacattgaggg(SEQ ID NO：29)
					HJ78	PA-G742A.as	ccctcaatgtagccgttcggttcg(SEQ ID NO：30)
	HJ79	PA-A1163G	5/P/cagagaaggtagatttgacgactg(SEQ ID NO：31)
					HJ80	PA-A1163G.as	cagtcgtcaaagtctaccttctctg(SEQ ID NO：32)
PA	HJ81	PA-A1615G	5/P/cactgacccaagacttgagccac(SEQ ID NO：33)
					HJ82	PA-A1615G.as	gtggctcaagtcttgggtcagtg(SEQ ID NO：34)
	HJ83	PA-T1748C	5/P/caaagattaaaatgaaatggggaatg(SEQ ID NO：35)
					HJ84	PA-T1748C.as	cattccccatttcattttaatctttg(SEQ ID NO：36)
	HJ85	PA-C2229	5/P/gtaccttgtttctactaataacccgg(SEQ ID NO：37)
					HJ86	PA-C2230.as	ccgggttattagtagaaacaaggtac(SEQ ID NO：38)
	HJ87	HA-A902C	5/P/ggaacacttgagaactgtgagacc(SEQ ID NO：39)
				HA	HJ88	HA-A902C.as	ggtctcacagttctcaagtgttcc(SEQ ID NO：40)
	HJ89	HA-C1493T	5/P/gaattttatcacaaatgtgatgatgaatg(SEQ ID NO：41)
					HJ90	HA-C1493T.as	cattcatcatcacatttgtgataaaattc(SEQ ID NO：42)
	HJ91	NP-C113A	5/P/gccagaatgcaactgaaatcagagc(SEQ ID NO：43)
				NP	HJ92	NP-C113A.as	gctctgatttcagtttcattctggc(SEQ ID NO：44)
	HJ93	NP-T1008C	5/P/ccgaatgagaatccagcacacaag(SEQ ID NO：45)
					HJ94	NP-T1008C.as	cttgtgtgctggattctcattcgg(SEQ ID NO：46)
	HJ95	NA-C1422T	catcaatttcatgcctatataagctttc(SEQ ID NO：47)
				NS	HJ96	NA-C1422T.as	gaaagcttatataggcatgaaattgatg(SEQ ID NO：48)
	HJ97	NS-C38T	cataatggatcctaacactgtgtcaagc(SEQ ID NO：49)
					HJ98	NS-C38T.as	gcttgacacagtgttaggatccattatg(SEQ ID NO：50)
PA	HJ99	PA6C375T	ggagaatagattcatcgagattggag(SEQ ID NO：51)
					HJ100	PA6C375T.as	ctccaatctcgatgaatctattctcc(SEQ ID NO：52)

Example 3: preparation of infectious recombinant MDV-A and reassortant influenza viruses

Madin-Darby dog kidney (MDCK) cells and human COS7 cells were cultured in Modified Eagle Medium (MEM) containing 10% Fetal Bovine Serum (FBS). Human embryonic kidney cells (293T) were cultured in Opti-MEM I (Life technologies) containing 5% FBS. MDCK was co-cultured with either COS7 or 293T cells in a 1: 1 ratio in 6-well plates and used for transfection when the cells were grown to about 80% confluence. 293T and COS7 cells have high transfection efficiency, but do not allow influenza virus replication. Co-culture with MDCK cells ensures efficient replication of the recombinant virus. Serum-containing medium was replaced with serum-free medium (Opti-MEM I) before transfection, followed by incubation for 4-6 hours. Transfection of plasmid DNA 1. mu.g of each of 8 kinds of plasmid DNA (PB1, PB2, PA, NP, M, NS, HA and NA) was mixed with TransIT-LT1 using TransIT-LT1(Mirus), and then diluted into 160. mu.l of Opti-MEM I medium in a total volume of 200. mu.l. DNA: the transfection reagent mixture was incubated at room temperature for 45 minutes and 800. mu.l of Opti-MEM I medium was added. The transfection mixture was then added to co-cultured MDCK/293T or MDCK/COS7 cells. Transfected cells were incubated at 35 ℃ or 33 ℃ for 6 to 24 hours, e.g., overnight, and then the transfection mixture was replaced by adding 1ml of Opti-MEM I per well. After 24 hours of incubation at 35 ℃ or 33 ℃, 1ml of Opti-MEM I containing 1. mu.g/ml TPCK-trypsin was added to each well and incubated for 12 hours. The harvested virus is then amplified in confluent MDCK cells or directly in embryonated chicken eggs. MDCK cells were transfected with 0.2ml of transfection mix in 12-well plates for 1 hour at room temperature, and then the transfection mix was removed and replaced with 2ml of Opti-MEMI containing 1. mu.g/ml TPCK-trypsin. Cells were incubated at 35 ℃ or 33 ℃ for 3-4 days. The amplified virus is added with SPG stabilizer and stored at-80 ℃, or plaque purification and amplification are carried out in MDCK cells or eggs containing embryos.

Functional expression of MDA-A Polymer proteins

The functional activity of the 4 MDV-a polymerase proteins PB2, PB1, PA and NP was analyzed by testing their ability to replicate the influenza virus minigenome encoding the EGFP reporter gene. A set of 8 plasmid recovery systems for recovery of influenza A viruses, and selection for control of influenza viruses (elevation plasmid recovery system for influenza A viruses; Options for the control of influenza A viruses) a set of vectors containing 8 expression plasmids (see Table 4) (Hoffmann et al, (2001) containing cDNA for the A/PR/8/34 strain (H1N1) and an influenza minigenome carrying a reporter gene encoding enhanced green fluorescent protein (EGFP, pHW 72-EGFP).

MDV-A PB1, PB2, PA and NP, or PB1, PA and NP (PB2 as negative control) were transfected into co-cultured MDCK/293T cells together with a plasmid expressing the influenza A virus EGFP minigenome (pHW72-EGFP) (Hoffmann et al, (2000) ' Ambisense ' method for preparation of influenza A virus by synthesis of vRNA and mRNA ' from one template for Viogl RNA and RNA synthesis from one template for Virology15 (267-310). Transfected cells were observed under phase contrast or fluorescence microscopy 48 hours after transfection. Additionally, flow cytometry can be used to detect EGFP expression.

As shown in FIG. 2, green fluorescence was observed in cells transfected with PB2, PB1, PA and NP of MDV-A, indicating that the EGFP minigenome is expressed and no fluorescence was observed in cells transfected with only 3 polymer proteins. This indicates that the MDV-A polymerase protein within pAD3000 is functional.

In other assays, a minigenome containing the Chloramphenicol Acetyltransferase (CAT) gene, designated pFlu-CAT, was used to detect polymerase activity. Expression of CAT at the protein level (e.g. by ELISA) or RNA level was used in this assay as an indicator of minigenome replication.

Analysis of MDV-A plasmid by Single Gene rearrangement assay

The results of the rearrangement experiments performed by cotransfection of a single gene segment of MDV-A with the other 7 complementary segments of the control strain A/PR/8/34 showed that the 8MDV-A genomic segments cloned into pAD3000 were functional. Cotransfection of all 8 plasmids containing a single genomic segment with the complement of the control strain produced infectious reassorted viruses that caused cytopathological effects in infected MDCK cells, suggesting that all 8 plasmids encode functional MDV-A proteins.

TABLE 4.7+1 reassortant viruses recovered by plasmid

Viral gene segments	PB2	PB1	PA	NP
					1	PMDV-A-PB2	pHW191-PB2	pHW191-PB2	pHW191-PB2
2	PHW192-PB1	pMDV-A-PB1	pHW192-PB1	pHW192-PB1
					3	P14W193-PA	pHW193-PA	pMDV-A-PA	pHW193-PA
4	PHW195-N	pHW195-N	pHW195-N	pMDV-A-NP
					5	PHW197-M	pHW197-M	pHW197-M	pHW197-M
6	PHW198-NS	pHW198-NS	pHW198-NS	pHW198-NS
					7	PHW194-HA	pHW194-HA	pHW194-HA	pHW194-HA
8	PHW-196-NA	pHW-196-NA	pHW-196-NA	pHW-196-NA
					CPE	(+)	(+)	(+)	(+)
Viral gene segments	M	NS	HA	NA
					1	PHW191-PB2	pHW191-PB2	pHW191-PB2	pHW191-PB2
2	PHW192-PB1	pHW192-PB1	pHW192-PB1	pHW192-PB1
					3	PHW193-PA	pHW193-PA	pHW193-PA	pHW193-PA
4	PHW195-NP	pHW195-NP	pHW195-NP	pHW195-NP
					5	PMDV-A-M	pHW197-M	pHW197-M	pHW197-M
6	PHW198-NS	pMDV-A-NS	pHW198-NS	pHW198-NS
					7	PHW194-HA	pHW194-HA	pMDV-A-HA	pHW194-HA
8	PHW-196-NA	pHW-196-NA	pHW-196-NA	pMDV-A-NA
					CPE	(+)	(+)	(+)	(+)

To further determine the packaging limitations of influenza a viruses, the NS segment is divided into two gene segments: one encoding the NS1 genomic segment and the other encoding the NS2 genomic segment. 9 plasmids containing 9 genome segments of influenza A virus were transfected into the MDCK/COS cells described above, and the harvested virus was amplified with embryonated chicken eggs prior to titer determination using MDCK cells. As a result, the plaque produced by the 9-plasmid system was found to be smaller than that produced by the 8-plasmid system described above. RT-PCR analysis showed that only the NS2 segment was present within the virus particle, whereas the NS1 gene was not packaged.

Harvesting of MDV-A and 6: 2 reassortant viruses

Culture supernatants were collected for transfection of new MDCK cells 3 days after transfection with 8MDV-A plasmids (recombinant viruses) or plasmids containing 6 MDV-A internal genes and HA and NA genes from A/PR/8/34 (6: 2 reassorted viruses) according to the procedure described above, and the transfected cells were incubated in the presence of 1. mu.g/ml TPCK-trypsin for 3 days at 33 ℃. The cytoplasmic effect of the recombinant virus was observed using a microscope. Expression of viral hemagglutinin was analyzed by a standard Hemagglutination Assay (HA). HA assay was performed by mixing 50 μ l of serial double-diluted culture supernatant with 50 μ l of 1% chicken red blood cells in a 96-well plate. The HA titers detected for the transfected 8MDV-A plasmid-derived amplified virus and the 6: 2 reassortant virus ranged from about 1: 254 to 1: 1024. Transfection reactions with the 8A/PR/8/34 plasmid as a gift from doctor E.Hoffman served as a positive control. The infectious influenza viruses prepared by these 3 transfection reactions are listed in table 5.

TABLE 5 plasmids for recovery of A/PR/8/34, MDV-A and 6: 2 reassortant viruses

Viral gene segments	A/PR/8/34(H1N1)	rMDV-A(H2N2)	6: 2 reassortant viruses
				1	pHW191-PB2(AD731)	pMDV-A-PB2#2(AD760)	pMDV-A-PB2#2(AD760)
2	pHW1982-PB1(AD732)	pMDV-A-PB1(AD754)	pMDV-A-PB1(AD754)
				3	pHW193-PA(AD733)	pMDV-A-PA(AD755)	pMDV-A-PA(AD755)
4	pHW195-NP(AD735)	pMDV-A-NP#1(AD757)	pMDV-A-NP#1(AD757)
				5	pHW197-M(AD737)	pMDV-A-M(AD752)	pMDV-A-M(AD752)
6	pHW198-NS(AD738)	pMDV-A-NS(AD750)	pMDV-A-NS(AD750)
				7	pHW194-HA(AD734)	pMDV-A-HA(AD756)	pHW194-HA(AD734)
8	pHW-196-NA(AD735)	pMDV-A-NA#4(AD759)	pHW196-NA(AD736)
				CPE	+	+	+

The genotype of the recovered virus was determined by RT-PCR. Viral RNA was isolated from infected cell culture supernatant using RNeasy mini kit (Qiagen) and 8 influenza virus segments were amplified by RT-PCR using primers specific for each MDV-a gene segment and H1 and N1 specific primers. As shown in FIG. 3, the rMDV-A contains PB2, PB1, NP, PA, M and NS which are MDV-A specific, and HA and NA which are H2 and N2 subtype specific. The 6: 2 reassortant virus contained 6 internal genes from MDV-A and HA and NA from A/PR/8/34(H1N 1). This indicates that the virus prepared with the transfected plasmid has the correct genotype.

The titer of the recovered virus was analyzed by a plaque formation assay using MDCK cells, and whether plaques were formed by influenza virus was determined by immunostaining with chicken serum against MDV-A. MDCK cells were infected with 100 μ l of 10-fold serial dilutions of virus when grown to 100% confluence in 12-well plates with gentle shaking for 1 hour at room temperature. The inoculum was removed and the cells were covered with 1 XL 15 containing 0.8% agarose and 1. mu.g/ml TPCK-trypsin. The plates were incubated at 35 ℃ or 33 ℃ for 3 days, then fixed with 100% methanol, blocked with 5% milk in PBS, incubated with 1: 2000 diluted chicken anti-MDV-A antiserum for 1 hour, and then incubated with HRP-linked rabbit anti-chicken IgG for another 1 hour. Plaques were visible upon addition of HRP substrate solution (DAKO). All recovered viruses were positive for immunostaining.

Example 4: mapping the Gene basis of the ca, ts, att phenotypes of MDV-A

MDV-a influenza vaccine strains have several phenotypes associated with vaccine preparation, namely attenuated live vaccines: cold adaptation (ca), temperature sensitive influenza virus (ts), and attenuation properties (att). Sequence comparison of the MDV-A strain with the non-ts virulent wild-type A/AA/6/60 strain revealed a 17nt difference between the two strains (Table 6). Several changes in the MDV-A sequence were found to be unique to this strain by comparison with all influenza A viruses in the GeneBank database, suggesting that one or more of these substituted amino acids are associated with the att, ca and ts phenotypes. At PB2⁸²¹The single amino acid change at this position is the only nucleotide position that has been previously reported to determine the MDV-A ts phenotype (Subbarao et al, (1995)' introduction of a Temperature-Sensitive Influenza Missense mutation in the PB2Gene of Influenza A Transfectant virus Can affect the Increase in Temperature-Sensitive Influenza virus and Attenuation activity and make the design of an engineered live Influenza A virus vaccine more rational "(Addition of Temperature-Sensitive Influenza virus mutation in the PB2Gene of infection of Influenza A Transfectant live vaccine and mutation of MDV-A phenotyped Permits the Rational Design ofa Genetically Engineered Live Influenza A Virus Vaccine)J.Virol.69：5969-5977)。

To pinpoint the minimal substitution involved in the MDV-A phenotype, the different nucleotides on MDV-A from wild-type A/AA/6/60 were individually changed to nucleotides of wild-type A/AA/6/60 (i.e., "inversions"). Each inverted gene segment is then introduced into the host cell along with other segments of MDV-A to recover the monogene rearrangement virus. Alternatively, the inverted gene segments and corresponding MDV-A fragments may also be transfected with other wild-type virus strains, such as A/PR/8/34, to assess the contribution of each gene segment to the virus phenotype. Non-ts reassortant viruses were prepared by site-specific mutagenesis using the recombinant MDV-A plasmid system described above to further alter the 6 internal genes. A total of 15 nucleotide substitutions were mutably introduced into 6 MDV-A plasmids, representing the recombinant wild-type A/AA/6/60 genome listed in Table 6 (rWt, Flu 064). Madin-Darby dog kidney (MDCK) cells and COS-7 cells were cultured and transfected as described above. The recovered virus was passaged once in MDCK cells and then amplified in the allantoic cavity of embryonated chicken eggs. Transfection and virus amplification in MDCK and hen eggs was performed at 33 ℃ which minimizes temperature selection pressure for both ca and wt viruses. The genotype of the virus is determined by sequencing a cDNA fragment amplified from the viral RNA.

TABLE 6 sequence comparison of "wild-type" A/AA/6/60 and MDV-A

RNA segment	Base (amino acid) position	E10SE2	MDV-A	rWT(Flu044)
					PB2	141	A	G	A
	821(265)	A(Asn)	G(Ser)	A
						1182	A	T	T
	1212	C	T	T
						1933	T	C	T
PB1	123	A	G	G
						1195(391)	A(Lys)	G(Glu)	A
	1395(457)	G(Glu)	T(Asp)	G
						1766(581)	A(Glu)	G(Gly)	A
	2005(661)	G(Ala)	A(Thr)	A
						2019	C	T	C
PA	20	T	C	T
						1861(613)	A(Lys)	G(Glu)	G
	2167/8(715)	TT(Leu)	CC(Pro)	TT
					NP	146(34)	A(Asp)	G(Gly)	G
	1550	‘5A’	‘6A’	‘6A’
					M	969(M2-86)	G(Ala)	T(Ser)	G
NS	483(NS1-153)	G(Ala)	A(Thr)	G

The bold numbers represent the difference between rMDV-A and rWt.

The bolded word (15) represents the change between rmdv-a and rwt.

Phenotypic characteristics are determined by methods well known in the art, as described in Parkin, U.S. Pat. No. 6,322,967, entitled "Recombinant Tryptophan mutant of influenza Virus," which is hereby incorporated by reference in its entirety. Briefly, temperature-sensitive influenza viruses of recombinant viruses were identified by plaque formation assays on MDCK cells at 33 ℃, 38 ℃ and 39 ℃. MDCK cells in 6-well plates were infected with 400 μ l of 10-fold serial dilutions of virus and adsorbed for 60 min at room temperature. The inoculum was removed and the cells were covered with 1 XL 15/MEM containing 0.8% agarose and 1. mu.g/ml TPCK-trypsin. CO at 33 ℃ of infected cells₂Incubation in an incubator, or in a circulating water bath containing 5% CO₂The sealed water vessel of (1) was incubated at 38. + -. 0.1 ℃ or 39. + -. 0.1 ℃ (Parkin et al (1996) Temperature sensitive variants of influenza A virus produced by reverse genetics and clusterized charged to alanine genetics. Vir. Res.46: 31-44). After 3 days of incubation, monolayersCells were immunostained with chicken polyclonal antibody against MDV and the number of plaque formations was counted. The number of plaques obtained at each stabilization was compared to evaluate the ts phenotype of each virus, with a minimum of 3 replicates per experiment. The shutdown temperature was defined as the lowest temperature, and its titer was reduced by 100-fold or more compared to 33 ℃.

Infectious viruses obtained from co-cultured COS-7/MDCK cells infected with 8 plasmids (pMDV-PB2, pMDV-PB1, pMDV-PA, pMDV-NP, pMDV-HA, pMDV-NA, pMDV-M and pMDV-NS) were amplified in embryonated chicken eggs, and these viruses were shown to have the same ts phenotype as the biologically prepared non-recombinant MDV-A (Table 7). Neither MDV-A nor rMDV-A formed significant plaques at 39 deg.C, although they formed significant plaques at 33 deg.C.

TABLE 7 replication of MDV/Wt reassortant viruses at various temperatures

Viruses with wild-type genes	33℃	38℃	33℃/38℃	39℃	33℃/39℃
						MDV	8.91	6.10	2.82	<4.0	>4.91
rMDV-A	8.72	6.19	2.53	<4.0	>4.72
						Wt(E1OSE2)	8.86	8.87	-0.01	8.87	-0.01
rWT(Flu064)	9.02	9.07	-0.05	8.96	0.06
						Wt-PB2	8.46	7.87	0.59	5.80*	2.66
Wt-PB1	8.92	8.74	0.18	7.86*	1.06
						Wt-NP	8.40	7.24	1.15	<4.0	>4.40
Wt-PA	8.57	6.10	2.48	<4.0	>4.57
						Wt-M	8.80	6.68	2.12	<4.0	>4.80
Wt-NS	8.72	6.10	2.62	<4.0	>4.72
						Wt-PB1/PB2	8.94	8.89	0.05	8.10*	0.85
Wt-PB1/PB2/NP	8.52	8.38	0.14	8.41	0.1

Indicates a reduction in plaques compared to rWt

Underlined at 10^-4No plaques were detected at double dilution

To systematically detail the genetic basis of the MDV-A ts phenotype, the sequences of several closely related non-ts, non-att wild-type A/AA/6/60 strains were compared to the sequences of ca A/AA/6/60, including the highly related isolate wt A/AA/6/60E10SE2, with a 17-48nt difference. There was a total difference of 19nt between E10SE2 and MDV-A (Table 6). E10SE2 was non-ts (table 7) and non-att in ferrets. To prepare recombinant non-ts viruses, 15-19 mutations were introduced in the MDV-a plasmid by site-specific mutagenesis, representing 10 amino acid changes. The 4 nucleotide positions PB2-1182, 1212, PB1-123 and NP-1550, which differ between MDV-A and E10SE2, do not change in the MDV-A Sequence, since these nucleotides are also present in other non-ts isolates of A/AA/6/60, and therefore the nucleotides at these positions may not have an effect on ts expression (Herlocher et al, (1996) "Sequence comparison of A/AA/6/60 influenza Virus: mutations affecting the attenuation phenotype" (Sequence comprisons of A/AA/6/60 influenza Virus: mutations in the attenuation phenotype.: Virus while mass restriction; Virus Research 42: 11-25). Recombinant virus encoding 15 nucleotide changes (rWt, Flu064) was obtained from co-cultured COS-7/MDCK cells transfected with 8 plasmids, pWt-PB2, pWT-PB1, pWt-PA, pWt-NP, pWt-M, pWt-NS, pMDV-HA and pMDV-NA. Sequence analysis indicated that rWt contained a pre-designed genetic alteration, was non-ts phenotypic at 39 ℃ and was consistent with the biologically produced wild-type A/AA/6/60. These results demonstrate that the ts phenotype is due to the change in these 15nt nucleotides.

Contribution of 6 internal Gene segments to the ts phenotype of the Virus

The effect of each wild-type gene segment on the MDV-A ts phenotype was evaluated by generating recombinant single-gene rearrangement viruses (Table 7). The introduction of wild type PB2 into rMDV-A resulted in a non-temperature sensitive phenotype of the virus only at 38 ℃ but retained a temperature sensitive phenotype at 39 ℃. By plaque formation assay in MDCK cells, it was found that virus titers were reduced by 0.6log at 38 ℃ and 39 ℃ respectively₁₀And 2.7log₁₀(compare 33 ℃). The reassortant virus containing the wild type PB1 gene segment is a non-temperature sensitive influenza virus which can form plaques at both 38 ℃ and 39 ℃. However, the size of plaques formed by this recombinant virus was affected by the increased temperature and was significantly reduced at 39 ℃ compared to rWt. The introduction of the wild-type NP gene into rMDV-A also resulted in a non-temperature-sensitive phenotype of the virus at 38 ℃, but this virus containing the wild-type NP gene segment was unable to form plaques at 39 ℃ compared to the wild-type PB2 recombinant virus. Introduction of the wild-type PA, M or NS gene segment alone into rMDV-A did not alter its temperature-sensitive phenotype, indicating that these 3 gene segments had little effect on maintaining this phenotype.

Since expression of wild-type PB1, PB2 or NP alone on an MDV-A background did not produce significant plaques, which were not as large as non-wild-type rWt, these gene segments were introduced into MDV-A in various combinations. The combined introduction of wild type PB1 and wild type PB2 resulted in a non-temperature sensitive phenotype of the virus at 38 ℃ and 39 ℃ (Table 7). Although the size of plaques was larger than that of any of the single gene reassortant viruses, it was significantly smaller than that of rWt. Thus, the wild-type PB2, PB1 or NP gene segments alone only partially reversed the temperature sensitive phenotype, whereas the 3 wild-type gene segments together reversed the temperature sensitive phenotype completely to the same non-temperature sensitive phenotype as rWt.

To determine whether these 3 genomic segments could confer rWt a temperature sensitive phenotype of MDV-A, 6 internal genes from MDV-A were introduced separately or in combination into rWt. Introduction of PB1, PB2, or NP alone into rWt resulted in a reduction in viral titer at 38 ℃ and more at 39 ℃, but none of these monogene reassortant viruses were as high temperature limited as rMDV-a (fig. 10). The MDV-A derived PA, M and NS gene segments were unable to affect the rWt non-temperature sensitive phenotype. Consistent with the previous results, the introduction of both MDV-A PB1 and PB2 genes into the rWt background greatly increased the temperature sensitive phenotype of the virus at 38 ℃, but the complete reversal of the temperature sensitive phenotype of the virus required the addition of the NP gene. Thus, the MDV-A derived PB1, PB2, and NP gene segments are equally important in conferring a fully temperature sensitive phenotype.

Mapping gene locus determining MDV-A temperature-sensitive phenotype

rWt, PB1, PB2 and NP and rMDV-A, PB1, PB2 and NP all list specific differences to determine which alterations play a significant role in the temperature sensitive phenotype. The difference between the NP gene of rMDV-A and the NP gene of rWt was only at position 146 (G34D, Table 6). The difference between PB2gene of rMDV-a and PB2gene of rWt was at 3 sites, but only the change of nt821 resulted in an amino acid change (N265S, table 6), thus inferring that this site represents a temperature sensitive site on the PB2gene segment. The difference between the PB1 gene of rMDV-A and the PB1 gene of rWt was at 6 positions, of which there were 4 changes in the encoded amino acids (Table 6). Each wild-type amino acid residue was individually substituted onto the PB1 gene segment of rMDV-A to evaluate its effect on the ts phenotype. 1395G (Glu-457) and 2005G (Ala) do not affect the temperature sensitive phenotype of DV-A. 1195A (Lys-391) and 1766A (Glu-581) resulted in a slight decrease in the stable sensitive phenotype at 38 ℃ but no effect at 39 ℃ (Table 8). These data suggest that 1195A and 1766A may be temperature sensitive sites on the PB1 gene segment. However, 1195A and 1766A combined did not produce a temperature-sensitive phenotype consistent with wild-type PB1 (table 6). The addition of 2005G to PB1-1195A/1766A instead of 1395A further reduced the temperature sensitive phenotype of the virus at 39 ℃ indicating that 2005A also had an effect on the expression of the temperature sensitive phenotype of the MDV-A PB1 fragment.

Table 8: mapping of residues determining ts phenotype in PB1

Viruses with wild-type sequences 33 deg.C, 38 deg.C, 39 deg.C, 33 deg.C, 39 deg.C

log₁₀PFU/mL

rMDV-A 8.67 6.00 2.67 <4.0 >4.67

rWt 9.04 9.01 0.03 9.03 0.01

PB1-1195A 8.06 6.68 1.38 <4.0 >4.06

PB1-1395G 8.72 5.88 2.85 <4.0 >4.72

PB1-1766A 8.07 6.70 1.37 <4.0 >4.07

PB1-2005G 8.76 6.31 2.45 <4.0 >4.76

PB1-1195A1766A 8.65 7.60 1.05 5.98* 2.68

PB1-1195A1395G1766A 8.84 8.13 0.71 6.38* 2.46

PB1-1195A1766A2005G 8.79 8.12 0.66 7.14* 1.64

PB1/PB2/NP 8.26 8.63 0.12 8.59 0.16

PB2/NP 8.81 8.21 0.59 7.56* 1.25

PB1-1195A/PB2/NP 8.86 8.81 0.05 7.60* 1.26

PB1-1766A/PB2/NP 9.33 8.84 0.50 8.71* 0.62

PB1-1766A2005G/PB2/NP8.30 8.22 0.08 8.11* 0.18

PB1-1766A1395G/PB2/NP8.88 8.85 0.03 8.39* 0.49

PB1-1195A1766A/PB2/NP8.45 8.48 0.06 8.10 0.35

Indicates a reduction in plaques compared to rWt

Underlined at 10^-4No plaques were detected at double dilution

Single site mutated PB1 was introduced into rMDV-A together with wild type PB2 and NP. Wild type PB2/NP and rMDV-A reassortant viruses are non-temperature sensitive influenza viruses at 38 ℃ and have a 1.25log reduction in titer at 39 ℃₁₀But the formation of plaques was significantly reduced compared to rWt. Addition of PB1-1195A or 1766A did not significantly alter the phenotype of the wild-type PB2/NP reassortant virus. Only the combination of PB1-1195A and 1766A together with wild-type PB2 and NP led to a virus with the same non-temperature sensitive phenotype as wild-type PB1/PB2/NP and rMDV-A reassortants (Table 8). The introduction of PB1-1395G or 2005G into wild type PB1-1766/PB2/NP also failed to convert the virus to the rWt non-temperature sensitive phenotype. Thus, these data indicate that the 4 amino acids distributed in the PB1, PB2, and NP genes completely reversed the stable sensitive phenotype of MDV-A.

Host cell restriction of MDV-A and reassortant viruses

In addition to having a temperature sensitive influenza virus and an attenuated phenotype, MDV-a viruses and reassortant viruses having one or more segments of MDV-a origin are host cell restricted because the virus grows at a lower level in per.c6 cells than in MDCK cells. C6 cells showed significantly reduced levels of growth of MDV-a and reassortant viruses with MDV-a derived PB1 and PB2 segments in per cells compared to MDCK cells, as shown in fig. 20A and 20B.

Modification of temperature sensitive attenuated viral strains

To determine whether the PB1, PB2, and 5 amino acids of the NP gene segment of MDV-A were able to repair the temperature-sensitive and attenuated phenotypes of MDV-A, PB1-391E, 581G661T, PB2-265S, NP-34G was introduced into a wild-type virus strain (A/PR/8/34; "PR 8") and the virus titer was found to decrease by 1.9log at 38 ℃₁₀Decrease of 4.6log at 39 ℃₁₀The results were very similar to those of rMDV-A (FIG. 11).

Sequence comparison of the PB1, PB2 and NP genes of ca A/AA/6/60(MDV-A) and A/PR/8/34 revealed that the 4 substituted amino acids in the PB1 and PB2 genes of MDV-A were unique. NP34 was conserved between MDV-A and PR8, therefore, 3 temperature sensitive sites PB1 on PB1 gene of MDV-A were mutated site-specifically³⁹¹(K391E)、PB1⁵⁸¹(E581G) and PB1⁶⁶¹(A661T) was introduced into A/PR/8/34 and PB2 was introduced²⁶⁵(N265S) was introduced into PB2 of A/PR/8/34. Mutations introduced on PB1 and PB2 were confirmed by sequence analysis. The primer pairs used for the mutation reactions are listed in table 9. These viruses are schematically indicated in FIG. 16. .

TABLE 9 primers for introducing ts mutation into PR8PB1 gene and PB2gene

HJ240 PR8-PB1A1195G 5’GAAAGAAGATTGAAGAAATCCGACCGCTC(SEQ ID NO：79)

HJ241 PR8-PB1A1195G.as 5’GAGCGGTCGGATTTCTTCAATCTTCTTTC(SEQ ID NO：80)

HJ242 PR8-PB1A1766G 5’GAAATAAAGAAACTGTGGGGGCAAACCCGTTCC(SEQ ID NO：81)

HJ243 PR8-PB1A1766G.as 5’GGAACGGGTTTGCCCCCACAGTTTCTTTATTTC(SEQ ID NO：82)

HJ244 PR8-PB1G2005A 5’GTATGATGCTGTTACAACAACACACTCC(SEQ ID NO：83)

HJ245 PR8-PB1G2005.as 5’GGAGTGTGTTGTTGTAACAGCATCATAC(SEQ ID NO：84)

HJ246 PR8-PB2A821G 5’ATTTGCTGCTAGGAGCATAGTGAGAAGAGC(SEQ ID NO：85)

HJ247 PR8-PB2A821G.as 5’GCTCTTCTCACTATGCTCCTAGCAGCAAT(SEQ ID NO：86)

To examine whether temperature sensitive mutations introduced into PB1 and PB2 of PR8 could lead to a temperature sensitive phenotype in vitro, a minigenome analysis was performed. The influenza minigenome reporter, known as pFlu-CAT, contains an antisense CAT gene under the control of the pol I promoter. The expression of CAT protein is dependent on the expression of influenza PB1, PB2, PA and NP proteins.

Briefly, HEp-2 cells were transfected with 1. mu. gPB1, PB2, PA, NP, and pflur-CAT minigenomes via lipofectamine 2000(Invitrogen), respectively. After incubation overnight (about 18 hours) at 33 ℃ or 39 ℃, cell extracts were analyzed for CAT protein expression using a CAT ELISA kit (Roche Bioscience). The expression of CAT mRNA was detected by elongation analysis. 48 hours after transfection, total cellular RNA was extracted using TRIZOlreagent (Invitrogen), 1/3RNA was mixed with excess DNA primer (5 '-ATGTTCTTTACGATGCGATTGGG) and T4 Polynucleotide kinase in 6. mu.l water, where the primers were used at the 5' end using-³²P]-an ATP marker. After denaturation at 95 ℃ for 3 minutes, 50U of reverse transcriptase (Invitrogen) was added to the reaction buffer containing 0.5mM dNTP for primer extension at 42 ℃ for 1 hour. The transcripts were analyzed electrophoretically on 6% polyacrylamide gels containing 8M urea in TBE, followed by autoradiography.

As shown in FIGS. 12A and 12B, PB1 carrying 3 substituted amino acids (PR8-3s)³⁹¹(K391E)、PB1⁵⁸¹(E581G) and PB1⁶⁶¹(A661T) PB1 gene showed a decrease in activity at 33 ℃ compared to PR8 control. The decrease in CAT protein expression at 39 ℃ was more pronounced in this mutant (FIG. 12A), indicating that the PB1 gene with 3 introduced MDV-A temperature sensitive sites replicates as a temperature sensitive influenza virus in an in vitro assay. PB2²⁶⁵(N265S) introduced PR8 with little effect on its activity at permissive (33 ℃) and non-permissive (39 ℃) temperatures. The combination of PB1-3s and PB2-1s can reduce the activity of protein significantly (PR8-4s) and is even stronger than temperature-sensitive influenza virus of MDV-A. As expected, in cells transfected with the MDV-A derived PB1, PB2, PA and NP genes,its activity at 39 ℃ is much lower (15%) than wild type A/AA/6/60(wt A/AA).

PR8 mutant viruses were prepared and recovered as described above. Briefly, co-cultured COS7 and MDCK cells were transfected with 8 plasmids encoding PR 8-derived PR8HA, NA, PB1, PB2, PA, NP, M and NS genes. For the preparation of a virus containing 4 temperature sensitive sites (PR8-4s), 3 altered PB1-3s at positions nt1195(K391E), nt1766(E581G) and nt2005(A661T) of PB1 and one altered PB1-1s at position 821(N265S) of PB2 were used. In addition, PR8(PR8-3s) with 3 mutations on PB1 or PR (PR8-1s) with 1 mutation on PB2 were also recovered, respectively. These viruses are schematically indicated in FIG. 16. All 4 recombinant mutant PR8 viruses grew at very high titers in eggs, reaching 9.0log₁₀pfu/ml or higher as shown in Table 10.

To examine the synthesis of viral proteins in infected cells, MDCK cells were infected with a virus with an m.o.i. of 5 and 7 hours after transfection³⁵S-Trans was labeled for 1 hour. The labeled cell lysates were electrophoresed on a 1.5% polyacrylamide gel containing SDS and autoradiographed. Protein synthesis was analyzed by western blot assay. Virus infected cells were harvested 8 hours post transfection and electrophoresed in a 4-15% gradient gel. Labeled with anti-M1 antibody or chicken polyclonal antibody against MDV-a, and then incubated with HRP-labeled secondary antibody. The antibody-bound protein bands were detected with a chemiluminescent detection system (Invitrogen) and then exposed to X-ray film.

As shown in FIG. 19, all cells had similar protein synthesis levels at 33 ℃ but the protein synthesis levels in PR8-1 s-infected cells were slightly reduced at 39 ℃ while the protein synthesis levels in PR8-3s and PR8-4 s-infected cells were significantly reduced. Western blot experiments also indicated that the order of decreasing levels of protein synthesis was PR8-4s > PR8-3s > PR8-1 s. Thus, the reduction in the level of replication of a temperature-sensitive mutant may be the result of a reduction in its replication under nonpermissive stability.

Temperature-sensitive influenza viruses of the PR8 mutant virus were assayed by plaque-forming assay using MDCK cells at 33 deg.C, 37 deg.C, 38 deg.C and 39 deg.C, respectively. The harvested lentinus edodes is amplified in eggs containing embryos and then introduced into cells according to the method. After the virus-infected cells were cultured at the set temperature for 3 days, the monolayer cells were immunostained with chicken polyclonal antibody against MDV, and the number of plaque formation was counted. The number of plaques obtained at each temperature was compared to assess the temperature sensitive phenotype of each virus. The shutdown temperature was defined as the lowest temperature, and its titer was reduced by 100-fold or more compared to 33 ℃.

As shown in FIGS. 10 and 17, all mutants replicated efficiently at 33 ℃ although the virus titer was slightly reduced. At 38 ℃, titers of all mutants were significantly reduced. At 39 ℃, the titer of viruses (PR8-3s and PR8-4s) with 3 temperature sensitive sites on the PB1 gene is reduced by more than 4.0log₁₀. PR8-1s is also a temperature sensitive influenza virus at 39 ℃. The temperature sensitive phenotype of PR8-4s is very similar to that of MDV-A, with a 4.6log reduction in titer at 39 ℃ compared to 33 ℃₁₀. Although all 3 PR8 mutants did not show more than 2.0log reduction in titer at 37 deg.C₁₀However, its plaque morphology is significantly different from that at 33 ℃. As shown in FIG. 18, plaque size of each mutant strain at 33 ℃ was reduced only a little compared to PR 8. The plaques formed by PR8-3s at 37 ℃ are obviously reduced, and the plaques formed by PR8-4s are smaller. Plaques formed at 37 ℃ in PR8-1s did not appear to shrink significantly. At 39 ℃, PR8-3s and PR8-4s can only form plaques of a few needlepoint sizes. The plaque size formed by PR8-1s was approximately 30% of that of wild-type PR 8.

TABLE 10 temperature sensitive influenza viruses with PR8 having an introduced ts site

Viral titer (log)₁₀pfu/ml)

The virus was at 33 deg.C, 37 deg.C, 38 deg.C, 39 deg.C

MDV-A 8.6 7.0 6.4 4*

Wt A/AA 8.7 8.7 8.9 8.3

PR8 9.6 9.5 9.5 9

PB8-1s 9.4 8.9 7.7 7.4

PB8-3s 9.2 8.8 7.8 5.2

PB8-4s 9.5 7.8 7.1 4.4

No virus detection at 10,000 fold dilution was considered to be a virus titer of 4.0

The attenuating activity of the mutated PR8 virus was determined in ferrets. Briefly, 9-10 week old male ferrets were used to evaluate viral replication in the respiratory tract of animal hosts. Feeding ferrets separately, inoculating 8.5log in nose₁₀pfu virus. Animals were anesthetized with ketamine hydrochloride 3 days after infection and their lungs and turbinates (NTs) were removed. Virus titers were determined in eggs containing day 10 embryos after serial dilution of lung tissue homogenates. Viral titer in lung (log)₁₀pfu/ml) was calculated by the Karber method. Virus replication in NT was analyzed by plaque formation assay and expressed as log₁₀pfu/ml。

The level of virus replication in the lungs and nasal turbinates was examined by EID50 or plaque formation assays (table 11). 3 days after infection, PR8 replicated to 5.9log in lung tissue₁₀Level of pfu/ml. However, the replication level of PR8-1s in the lung of ferret was reduced by 3.0log₁₀pfu/ml, whereas PR8-3s replicated very little. No replication of PR8-4s was detected in both groups infected with PR8-4 s. The lower limit detectable by EID50 test was 1.5log₁₀Therefore, the titer of PR8-4s was assigned as log₁₀EID 50. As a control, MDV-A failed to replicate in the lungs of ferrets, and the titer of wild-type A/AA/6/60 was 4.4log₁₀. Replication of the virus in the turbinate (NT) was analyzed by plaque formation assays in MDCK cells. The intranasal replication titer of PR8 reached 6.6log₁₀pfu/g. There was only a slight decrease in the titers of PR8-ls and PR8-3 s. 2.2log reduction in the titer of PR8-4s (A)₁₀And the titer of PR8-4s (B) was reduced by 4.3log₁₀The virus had an alteration in the PB1 gene (E390G). The significant reduction in the level of replication of PR8-4s (b) was consistent with its temperature sensitive influenza virus at 37 ℃. 8.5log is used herein₁₀Infectious dose replacement of pfu 7.0log₁₀And the latter is commonly used in the evaluation of the attenuated phenotype of MDV-a derived influenza virus vaccines. This result indicates that PR8 containing 4 MDV-a derived temperature sensitive sites has reduced replication activity in the lower respiratory tract of ferrets.

TABLE 11 replication of PR8 mutant in ferrets

Virus	Ferret	Dosage (log)10pfu)	Viral titer in lung (log)10EID50/g±SE)	Viral titer in turbinate (log)10/g±SE)
					PR8	4	8.5	5.9±0.3	6.6±0.1
PR8-1s	4	8.5	3.8±0.4	5.9±0.2
					PR8-3s	4	8.5	1.7±0.1	5.8±0.3
PR8-4s(A)	4	8.5	1.5±0.0a	4.6±0.2
					PR8-4s(B)b	4	8.5	1.5±0.0	2.3±0.3
MDV-A	4	8.5	1.5±0.0	4.6±0.1
					Wt A/AA	4	8.5	4.4±0.1	5.4±0.1

a) No virus detection considered a 1.5log titer₁₀EID50/g

b) The virus also contained additional alterations PB1-1193(E390G)

In temperature sensitive and attenuated experiments, the PR8 mutant virus has a temperature sensitive phenotype and an attenuated phenotype very similar to MDV-A. These data indicate that the unique substituted amino acids of plasma MDV-a introduced into different influenza strains can confer the desired temperature sensitive influenza virus and attenuated phenotype on the virus to facilitate the preparation of attenuated live vaccines. In addition, temperature sensitive attenuated PR-8 viruses grow at high titers and are therefore suitable as the main donor virus for the preparation of live attenuated or inactivated influenza vaccines. These results indicate 5 MDV-A mutations: PB1-391E, PB1-581G, PB1-661T, PB2-265S and NP-34G can confer a temperature sensitive phenotype and an attenuated phenotype on any influenza strain. Likewise, novel temperature-sensitive attenuated B strains suitable for vaccine preparation can also be prepared by introducing mutations of MDV-B into influenza B strains. In addition to the preparation of live attenuated vaccines, the introduction of these mutations into donor virus strains can also be used to prepare safe inactivated vaccines.

Example 5: 8 plasmid system for preparing MDV-B

Cold-adapted influenza mutant B/Ann Arbor/1/66(CA/Master Ann Arbor/1/66P1Aviron10/2/97) is a typical Master donor strain of influenza B virus (MDV-B), and viral RNA was extracted from 100. mu.l of embryonated egg allantoic fluid infected with the virus using RNeasy kit (Qiagen, Valencia, Calif.) and the extracted RNA was dissolved in 40. mu.l of water. Genomic segments were amplified by RT-PCR using a One-Step RT-PCR kit (One Step RT-PCR kit) (Qiagen, Valencia, Calif.) according to the instructions, using 1. mu.l of extracted RNA per reaction. The RT reaction was carried out at 50 ℃ for 50 minutes and then at 94 ℃ for 15 minutes. The PCR reaction was performed in 25 cycles, each cycle consisting of 94 ℃ for 1 minute, 54 ℃ for 1 minute, and 72 ℃ for 3 minutes. Two fragments were obtained as a result of amplifying the P gene using segment-specific primers containing BsmB I site (Table 12).

TABLE 12 RT-PCR primers for amplification of 8 viral RNAs of influenza virus ca B/Ann Arbor/1/66

Sequences complementary to influenza virus sequences are indicated in bold. The 5' -end has a recognition sequence for the restriction enzymes BsmBI (Bm) or BsaI (Ba).

Cloning of plasmids

The PCR fragment was isolated, digested with BsmBI (NP with BsaI), and inserted into the BsmBI site of pAD3000 (a derivative of pHW2000, transcribable antisense vRNA and sense mRNA) described above. From 2 to 4 clones of each plasmid were sequenced and the RT-PCR fragments were compared to the consensus sequence of MDV-B based on direct sequencing. Plasmids with nucleotide changes resulting in amino acid changes inconsistent with consensus sequences were repaired by cloning of the plasmids or using a Quikchange kit (Stratagene, La Jolla, Calif.). The resulting B/Ann Arbor/1/66 plasmids were designated pAB121-PB1, pAB122-PB2, pAB123-PA, pAB124-HA, pAB125NP, pAB126-NA, pAB127-M, and pAB 128-NS. Using this bi-directional transcription system, all viral RNA and proteins can be produced intracellularly, resulting in infectious influenza B virus (FIG. 4).

Notably, pAB121-PB1 and pAB124-HA contained 2 silent nucleotide substitutions and pAB128-NS contained 1 silent nucleotide substitution compared to consensus sequence (Table 13). These nucleotide changes do not produce amino acid changes and are not involved in affecting virus growth and recovery. These silent substitutions are retained to facilitate genotyping of recombinant viruses.

TABLE 13 plasmid set representing 8 fragments of B/Ann Arbor/1/66(MDV-B)

To construct plasmids with nucleotide substitutions in the PA, NP and M1 genes, the nucleotides were mutated using the Quikchange kit (Stratagene, La Jolla, Calif.) using plasmids pAB123-PA, pAB125-NP and pAB127-M as templates. In addition, two fragments were amplified by PCR method using primers containing the desired mutation, then digested with BsmBI, and then inserted into pAD3000-BsmBI by 3-fragment ligation. The resulting plasmid was sequenced to ensure that the cDNA did not contain unwanted mutations.

The sequence of the template DNA was determined using the Rhodamine or dRhodamine staining-terminator cycle sequencing Ready kit (Perkin-Elmer Applied Biosystems, Inc, Foster City, Calif.) containing AmpliTaq DNA polymerase FS. The samples were separated by electrophoresis and analyzed on a PE/ABI 373-type, 373-type Stretch-type or 377-type DNA sequencer.

In another experiment, viral RNA from the influenza strain B/Yamanshi/166/98 was amplified and cloned into pAD3000 in a manner similar to that described above, except that the reaction was carried out in 25 cycles, each cycle consisting of 94 ℃ for 30 seconds, 54 ℃ for 30 seconds, and 72 ℃ for 3 minutes. The same primers were used to amplify segments of the B/Yamanashi/166/98 strain, except that the primers for amplifying the NP and NA segments were as follows: MDV-B5' BsmBI-NP: TATTCGTCTCAGGGAGCAGAAGCACAGCATTTTCTTGTG (SEQ ID NO: 75); MDV-B3' BsmBI-NP: ATATCGTCTCGTATTAGTAGAAACAACAGCATTTTTTAC (SEQ ID NO: 76); Bm-NAb-1: TATTCGTCTC AGGGAGCAGAAGCAGAGCA (SEQ ID NO: 77); Bm-NAb-1557R: ATATCGTCTCGTATTAGTAGTAACAAGAGCATTTT (SEQ ID NO: 78). The B/Yamanashi/166/98 plasmids were designated pAB251-PB1, pAB252-PB2, pAB253-PA, pAB254-HA, pAB255-NP, pAB256-NA, pAB257-M, and pAB 258-NS. The 3 silent nucleotide differences identified in PA favour genotyping of recombinant and rearranged B/Yamanashi/166/98 virus.

Example 6: preparation of infectious recombinant influenza B and reassortant influenza B viruses

To overcome the difficulty of influenza virus growth in helper virus-free cell culture systems, the present invention provides novel vectors and methods for the preparation of recombinant and reassortant B-strain influenza viruses. The vector system for recovering influenza B virus was developed based on the method for preparing influenza A virus (Hoffmann et al, (2000)' DNA transfection System for preparing influenza A virus starting from 8 plasmids ` (A DNA transfection system for generating of influenza A virus from plasmids) ` Proc Natl Acad Sci USA 97: 6108-6113 ` `Hoffmann et al `&Webster (2000) "Single-directional RNA polymerase I-polymerase II transcription System for the preparation of influenza A viruses starting from 8 plasmids" (unidirectional RNA polymerase I-polymerase II transcription system for the generation of inflenza A viral from one plasmid) J Gen Virol 81: 2843-7). 293T or COS-7 cells (primate cells with high transfection efficiency and pol I activity) were co-cultured with MDCK (allowing influenza virus replication), 293T cells were cultured in OptiMEM I-AB medium containing 5% FBS, and COS-7 cells were cultured in DMEM I-AB medium containing 10% FBS. MDCK cells were cultured with 1 × MEM supplemented with 10% FBS, antibiotics, and antimycotic reagents. Cells were washed once with 5ml PBS or FBS-free medium before transfection with viral genomic vector. 10ml Trypsin-EDTA added to 75cm²On confluent cells in flasks (MDCK cells incubated for 20-45 min, 293T cells for 1 min). The cells were centrifuged and then resuspended in 10ml of OptiMEM I-AB. Then 1ml of each cell suspension was diluted into 18ml of OptiMEM I-AB and mixed well. Cells were added to 6-well plates, 3ml per well. After 6-24 hours, 1. mu.g of each plasmid was added to a 1.5ml Eppendorf tube containing OptiMEM I-AB (200. mu.l of x. mu.l plasmid + x. mu.l OptiMEM I-AB + x. mu.l TransIT-LT 1); mu.g of plasmid DNA was treated with 21TransIT-LT 1. The mixture was incubated at room temperature for 45 minutes. Then 800. mu.l of OptiMEM I-AB was added. Removing the medium and transfectingThe mixture was added to the cells (t ═ 0) and incubated at 33 ℃ for 6-15 hours. The transfection mixture was then slowly removed from the cells, 1ml of OptiMEM I-AB was added, and the cells were incubated at 33 ℃ for 24 hours. 48 hours after transfection, 1ml of OptiMEM I-AB containing 1. mu.g/ml TPCK-trypsin was added to the cells. 96 hours after transfection, 1ml of OptiMEM I-AB containing 1. mu.g/ml TPCK-trypsin was added to the cells.

1ml of cell culture supernatant was aspirated between 4 and 7 days post-transfection and monitored by HA assay or plaque formation assay. Briefly, 1ml of the supernatant was added to a centrifuge tube and centrifuged at 5000rpm for 5 minutes. Mu.l of the supernatant was transferred to a new tube, serially diluted and added to MDCK cells at 500. mu.l per well (e.g., in 12-well plates). The supernatant was removed after 1 hour of incubation with the cells and replaced with infection medium (1 XMEM) containing 1. mu.g/ml TPCK-trypsin. HA analysis or plaque formation assays were then performed. For example, for plaque formation assays, supernatants were titered on MDCK cells, which were covered with 0.8% agarose for 3 days at 33 ℃. For infected eggs, supernatants from infected cells were harvested at 6 or 7 days post-transfection, and 100. mu.l of the supernatant diluted with Opti-MEM I was injected into eggs containing 11-day embryos and cultured at 33 ℃. TCID on MDCK cells 3 days after inoculation₅₀The assay was performed to determine the titer of the virus.

To prepare MDV-B, co-cultured 293T-MDCK or COS-7-MDCK cells were infected with 1. mu.g of plasmid. When tested 5-7 days post-infection, co-cultured MDCK cells exhibited cytopathic effects (CPE), indicating that infectious MDV-B virus was produced from the cloned cDNA. No CPE was observed in cells transfected with 7 plasmids (table 14). To determine the efficiency of the DNA transfection system for virus production, cell supernatants 7 days after transfection were titered using MDCK cells and virus titers were determined by plaque formation assays. The titer of the co-cultured 293T-MDCK supernatant was 5.0X 10⁶The titer of the supernatant of pfu/ml, COS7-MDCK cells was 7.6X 10⁶pfu/ml。

TABLE 14 preparation of infectious influenza B viruses using 8 plasmids

Transient co-cultured 293T-MDCK (1, 2) or COS7-MDCK cells (3, 4) were transfected with 7 or 8 plasmids. Cytopathic effect (CPE) on co-cultured MDCK cells was monitored 7 days post-transfection. Supernatants from transfected cells were dropped onto MDCK cells 7 days after transfection. The pfu/ml data represent the average of multiple (e.g., 3 or 4) transfection experiments.

Similar results were obtained with the transfection assay using the B/Yamanashi/166/98 plasmid vector. These results indicate that the transfection system is capable of amplifying influenza B virus starting from 8 plasmids.

Genotyping of recombinant influenza B viruses

After serial passages in MDCK cells, the supernatants of infected cells were amplified by RT-PCR to determine whether the virus was produced. All 8 segments were amplified using segment-specific primers RT-PCR (Table 12). As shown in FIG. 5A, PCR products were amplified for all segments. Direct sequencing of PCR products of PB1, HA and NS revealed that the 4 nucleotides analyzed were identical to those seen on plasmids pAB121-PB1, pAB124-HA and pAB 128-NS. These results confirm that the resulting virus was prepared from the designed plasmid, excluding any possible laboratory contamination of the parental virus (except for negative controls) 9 fig. 5B).

Similarly, the virus was recovered after transfection with the B/Yamanashi/166/98 plasmid vector to amplify the region containing PA-segment nucleotides 1280-1290. Sequencing revealed that the recovered virus was identical to the plasmid-derived recombinant virus B/Yamanashi/166/98 (FIGS. 5C and 5D).

Phenotyping of rMDV-B

MDV-B virus has two characteristic phenotypes: temperature sensitive influenza virus (ts) and cold adaptation (ca). A difference of 2 logs (or more) in virus growth at 37 ℃ compared to 33 ℃ is defined as having a temperature-sensitive phenotype, and a difference of less than 2 logs in virus growth level at 25 ℃ compared to 33 ℃ is defined as having a cold-adapted phenotype. Primary Chicken Kidney (PCK) cells were transfected with parental MDV-B virus or plasmid-derived virus to determine virus growth at 3 temperatures.

Plaque formation assays were performed with MDCK cells (ECACC) pooled in 6-well plates. The virus dilutions were incubated at 33 ℃ for 30-60 min. Cells were covered with 0.8% agarose. Infected cells were incubated at 33 ℃ or 37 ℃.3 days after infection, cells were stained with 0.1% crystal violet solution and the number of plaque formations counted.

TCID by virus samples at 25 ℃, 33 ℃ and 37 ℃₅₀Titrations were performed to analyze the temperature sensitive-cold adapted phenotype. TCID₅₀The titer was determined by detecting the cytopathic effect (CPE) of the influenza virus in primary chicken kidney monolayers in 96-well plates at different temperatures (25 ℃, 33 ℃, 37 ℃). The results of this assay are independent of plaque morphology, which varies with temperature and strain, and depend only on the stability of influenza virus replication and the resulting CPE effect. Primary Chicken Kidney (PCK) cells were suspended in MEM (Earl's) medium containing 5% FCS to prepare a cell suspension by trypsinizing the primary tissue. The PCK cells were seeded into a 96-well culture plate and cultured for 48 hours to prepare a confluency ratio>90% of the cells in the monolayer. After 48 hours, the PCK monolayer cells were washed with serum-free MEM medium containing 5Mm L-glutamine, antibiotics, non-essential amino acids (referred to as Phenotypic Analysis Medium (PAM)) for 1 hour. The 10-fold serial dilutions of the virus samples were then added to PAM-containing 96-well plates. Each diluted virus sample was infected with the diluted virus in 6 duplicate wells. Uninfected cells as controls were also plated in 6 duplicate wells per sample. The titer of the virus was determined by taking 2-4 wells per sample. Phenotypic control viruses with predetermined titers at 25 ℃, 33 ℃ and 37 ℃ were included in each assay. To determine the temperature sensitive phenotype of the virus samples, plates were incubated at 5% CO₂The culture was carried out in an incubator at 33 ℃ and 37 ℃ for 6 days, respectively. To determine cold-adapted phenotypic characteristics, plates were incubated at 25 ℃ for 10 days. Virus titers were calculated by the Karber method, log₁₀TCID₅₀Mean titer (n-4)/ml + standard deviation. In the figureThe standard deviation of the virus titers appearing in 1-3 is between 0.1 and 0.3. The difference in virus titre at 33 ℃ and 37 ℃ was used to determine the temperature sensitive phenotype and the difference in virus titre at 25 ℃ and 33 ℃ was used to determine the cold adapted phenotype.

Plasmid-derived recombinant MDV-B (recMDV-B) viruses express two characteristic phenotypes in cell culture as expected-cold-adapted and temperature-sensitive influenza viruses. The virus is able to replicate efficiently at 25 ℃, that is to say the difference in the titre of the virus at 25 ℃ and at 33 ℃ is less than or equal to 2log when analysed using PCK cells₁₀I.e., the virus is considered to have a cold-adapted phenotype. Both parental MDV-B and recMDV-B have cold-adapted phenotypes; the difference in titre at 25 ℃ and 33 ℃ was 0.3 and 0.4log, respectively₁₀(Table 15). The temperature sensitive phenotype is also determined by measuring the titer in PCK cells at different temperatures; however, for the temperature sensitive phenotype, titers at 37 ℃ should be 2log greater than those at 33 ℃₁₀Or more. The difference in titre at 33 ℃ and 37 ℃ for parental MDV-B and recMDV-B was 3.4 and 3.7log, respectively₁₀(Table 15). Thus, plasmid-derived recombinant MDV-B expresses a cold-adapted phenotype and a temperature-sensitive phenotype.

TABLE 15 phenotypic analysis of MDV-B and rMDV-B prepared with plasmids

Primary chicken kidney cells were infected with the parental MDV-B virus and a plasmid-derived recombinant virus (recMDV-B). Virus titers were determined at 3 different temperatures.

Example 7: preparation of reassortant Virus B/Yamanashi/166/98

The HA and NA segments, which can represent several different strains of the major influenza B virus lineage, were amplified and then cloned into pAD3000 as described above. The primer combinations were optimized for simultaneous RT-PCR amplification of HA and NA segments. Influenza B virus HA and NA genes were discovered by comparing viral RNA terminal sequences representing non-coding regions of segment 4(HA) and segment 6(NB/NA)Between 20 nucleotides at the 5 'end and 15 nucleotides at the 3' end. The primer pairs used for RT-PCR were (the underlined part is specific for influenza B virus): Bm-NAb-1: TAT TCGTCT CAG GGA GCA GAA GCA GAG CA(SEQ ID NO：79)；Bm-NAb-1557R：ATA TCG TCT CGTATT AGT AGT AAC AAG AGC ATT TT(SEQ ID NO: 80), which was used to simultaneously amplify the HA and NA genes of various influenza B virus strains (FIG. 8). HA and NA PCR segments of B/Victoria/504/2000, B/Hawaii/10/2001, and B/Hong Kong/330/2001 were isolated, digested with BsmBI, and inserted into pAD 3000. The results show that the primers can be used for effectively amplifying plasmids containing influenza B HA and NA genes of several different wild-type viruses, and the several wild-type viruses represent the main pedigrees of the influenza B viruses. RT-PCR products were used for sequencing and/or cloning into expression plasmids.

To demonstrate that B/Yamanashi/166/98 (a B/Yamagata/16/88-like virus) can be used to efficiently express various influenza B lineage-derived antigens, a reassortant virus containing PB1, PB2, PA, NP, M, NS of B/Yamanashi/166/98 and HA and NA representing Victoria and Yamagata lineages (6+2 reassortant virus) was prepared. Transient co-cultured COS7-MDCK cells were transfected with 6 plasmids representing B/Yamanashi/166/98 and plasmids containing HA and NA segment cDNAs from two strains of B/Ving Kong/330/2001 and B/Hawaii/10/2001 of the B/Victoria/2/87 lineage and one strain of B/Victoria/504/2000 of the B/Yamagata/16/88 lineage, as described above. Supernatants were titrated on fresh MDCK cells 6 to 7 days post transfection. All 6+2 reassortant viruses had titers ranging from 4 to 9X 10⁶pfu/ml (Table 16). These data indicate that the 6 internal genes of B/Yamanashi/166/98 and the HA and NA genes of both influenza B strains are effective in forming infectious viruses.

The supernatant of COS7-MDCK cells was titrated 6 or 7 days after transfection and the virus titer was determined by plaque formation assay using MDCK cells.

Table 16: plasmids for the preparation of B/Yamanashi/166/98 and 6+2 reassortant viruses

Wild type B/Yamanashi/166/98 gave relatively high titers when replicated in eggs. Tests were used to determine if this property is an inherent phenotype of the 6 "internal" genes of the virus. To assess this property, the yield of wild-type B/Victoria/504/2000, which replicates only moderately in eggs, was compared to the yield of 6+2 reassortant viruses expressing B/Victoria/504/2000HA and NA. All of these viruses, except wild-type and recombinant B/Yamanashi/166/98, were inoculated into eggs containing 3-or 4-day embryos at 100 or 1000 pfu. Allantoic fluid was harvested from eggs 3 days after infection and TCID was determined using MDCK cells₅₀The titer. As a result, the 6+2 reassortant virus was found to be present in the allantoic fluid in an amount similar to that of the wild-type or recombinant B/Yamanashi/166/98 virus strain (FIG. 9). The difference in titers between B/Victoria/504/2000 and 6+2 recombinant viruses was approximately 1.6log₁₀TCID50(0.7-2.5log₁₀TCID₅₀Ml, 95% CI). Significant differences between B/Victoria/504/2000 and 6+2 recombinant viruses were demonstrated by 3 different experiments (P)<0.001). These results demonstrate that HA and NA normally expressed by viral strains that are difficult to replicate in eggs allow B/Yamanashi/166/98 to grow in eggs.

Example 8: molecular basis for the Cold-adapted phenotype of ca B/Ann Arbor//1/66

MDV-B virus (ca B/Ann Arbor/1/66) is attenuated in humans, has an attenuated phenotype in ferrets, and has a cold-adapted phenotype and a temperature-sensitive phenotype in cell culture. The deduced amino acid sequence of the MDV-B internal gene was compared to the sequence of the Los Alamos influenza virus database (website: flu. lan. gov) using the BLAST search algorithm. 8 amino acids unique to MDV-B that were not present in any other strain were identified (Table 17). The genomic segments encoding PB1, BM2, NS1 and NS2 do not have unique substitution residues. There were 2 PA and M1 proteins and 4 unique substituted amino acids for the NP protein (table 17). A substituted amino acid was found at position PB2630 (another strain B/Harbin/7/94(AF170572) also had an arginine residue at position 630).

These results indicate that gene segments PB2, PA, NP and M1 may be involved in the development of the MDV-B attenuated phenotype. In a manner analogous to the MDV-A described above, the 8-plasmid system is used for the preparation of recombinant and recombinant taxonomic viruses (monogenic and/or bigenic reassortant viruses, i.e.7: 1 or 6: 2 reassortant viruses) by cotransfection of the relevant plasmids into cultured cells in a manner analogous to the MDV-A described above, independently of helper viruses. For example, 6 internal genes from B/Lee/40 were transfected with MDV-B derived HA and NA fragments to prepare 6+2 reassortant viruses.

Table 17. unique substituted amino acids of B/Ann Arbor/1/66

The deduced amino acid sequences of 8 ca B/Ann Arbor proteins were used in a BLAST search. The amino acids that are not identical between MDV-B and the alignment sequence are listed in the table. Underlined nucleotides in the codons represent the positions that were replaced.

To determine whether these 8 unique amino acid differences had an effect on the phenotypic characteristics of MDV-B, a recombinant virus was constructed in which all 8 nucleotide positions encode amino acids that are complementary to the background of the wild-type influenza virus gene. A set of plasmids was constructed in which 8 residues in the PA, NP and M1 genes were altered by site-specific mutagenesis to encode wild-type amino acids (as shown in Table 17). The recombinant virus with all 8 nucleotide changes was designated rec53-MDV-B and was obtained by co-transfection of a COS7-MDCK cell co-culture with the constructed plasmid, the cells were cultured at 33 ℃ to ensure that high titers of virus were obtained in the supernatant 7 days after transfection. The supernatants of the transfected cells were titrated in MDCK cells and PCK cells using a plaque formation assay and their titers were determined at 33 ℃ and 37 ℃.

As shown in FIG. 13, recMDV-B expressed a temperature sensitive phenotype in both MDCK cells and PCK cells in two different independent experiments. The three-gene rearrangement virus rec53-MDV-B contains all 8 amino acid changes that determine a non-temperature-sensitive phenotype, and the difference between the titers at 33 ℃ and 37 ℃ in PCK cells is only 0.7log₁₀. This titer is less than 2log required to define a temperature sensitive phenotype₁₀Is also compared to the 3log observed for recMDV-B₁₀The difference in (a) is much smaller. These results indicate that 8 amino acid changes within PA, NP and M1 are sufficient to generate a non-temperature sensitive influenza virus wild-type-like virus carrying homologous and heterologous glycoproteins.

The contribution of each gene segment to the temperature sensitive phenotype was subsequently investigated. Plasmid-derived recombinant viruses having the PA, NP or M gene segments in wild-type amino acid complement were prepared using DNA co-transfection techniques. Growth of all monogene recombinant viruses was restricted at 37 ℃ in MDCK and PCK cells (fig. 14), indicating that changes in individual gene segments cannot reverse the temperature-sensitive phenotype. In addition, recombinant viruses carrying the NP and M or PA and M gene segments also retain a temperature sensitive phenotype. Whereas the difference in titre between the recombinant viruses containing the PA and NP gene segments at 37 ℃ and 33 ℃ is lower than or equal to 2.0log₁₀Similar to rec 53-MDV-B. These results indicate that the NP and PA genes are the major genes affecting the temperature sensitive phenotype.

To determine whether 4 amino acids in the NP protein and 2 amino acids in the PA protein both affected the non-temperature sensitive phenotype, tri-and bi-gene recombinant viruses with mutations in the NP and PA genes were prepared (FIG. 15). Two amino acid substitutions, A114 → V114 and H410 → P410, produce a non-temperature sensitive phenotype. Viruses with single amino acid substitutions H410 → P410 in the nucleoprotein have a non-temperature sensitive phenotype in MDCK cells and PCK cells. On the other hand, viruses with single amino acid substitution A55 → T55 have a temperature sensitive phenotype. These results indicate that P410 on NP is involved in efficient virus growth at 37 ℃. These results indicate that 4 residues out of 6 amino acids of PA and NP contribute to the non-temperature sensitive phenotype.

Based on previous experimental results, the temperature sensitive phenotype and the attenuated phenotype are highly correlated. Previous studies demonstrated that no virus could be detected in lung tissue of ferrets infected with ca B/Ann Arbor/1/66, whereas non-attenuated influenza B virus could be detected in lung tissue following intranasal infection. To determine whether identical mutations are the basis for the temperature-sensitive and attenuated phenotypes, the following experiments were performed.

The recombinant virus obtained after transfection was passaged in embryonated chicken eggs to prepare virus stocks. 9 weeks old ferrets were inoculated intranasally with 0.5ml virus per nostril at titers of 5.5, 6.0, or 7.0log₁₀pfu/ml. Ferrets were sacrificed 3 days after infection and non-tissue and turbinate were examined as described above.

Ferrets (4 animals per group) were infected intranasally with recMDV-B or rec 53-MDV-B. Nasal concha and lung tissues were taken 3 days after infection with virus and tested for the presence of virus. At 7.0log of infection₁₀No virus was detected in lung tissue of ferrets from pfu rec MDV-B. Infection 7.0log₁₀3 of 4 animals of pfu rec53-MDV-B detected virus in lung tissue (the reason why one animal in this group could not be detected was unclear). Low dose of infection (5.5 log)₁₀pfu) rec 53-MDV-B2 of 4 ferrets isolated virus in lung tissue. Thus, the 8 unique amino acid changes on the PA, NP and M1 proteins were sufficient to switch the attenuated phenotype to a non-attenuated phenotype.

Since the results of the cell culture experiments showed a major effect on the temperature sensitive phenotype, ferrets were infected with 6log pfu of rec53-MDV-B (PA, NP, M), rec62-MDV-B (PA), NP rec71-MDV-B (NP) in the second experiment. 2 of 4 animals infected with rec53-MDV-B detected virus in the lungs. Ferrets infected with either the monogene or the bigene reassortant virus did not detect the virus in lung tissue. Thus, in addition to the amino acids on the PA and NP proteins, the M1 protein is also important for the attenuated phenotype. Viruses with wild-type PA and NP were unable to replicate in ferret lungs, suggesting that a set of mutations involved in the attenuated phenotype also had an effect on the temperature-sensitive phenotype.

Thus, the temperature sensitive phenotype of B/Ann Arbor/1/66 is determined by up to3 genes. The substitution of 8 amino acids in the PA, NP and M1 proteins to wild-type residues confers replication capacity to the recombinant virus at 37 ℃. Likewise, the 6+2 recombinant virus containing the 6 internal genes of MDV-B and the HA and NA segments of B/Hong Kong/330/01 HAs a temperature sensitive phenotype, whereas the three-gene recombinant virus is a non-temperature sensitive influenza virus.

Substitution of the above residues, as described above for influenza A strains, e.g. PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵¹⁰(A510T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V) confers a temperature sensitive phenotype and an attenuated phenotype on the virus. Accordingly, the artificially modified influenza B virus strain with one or more substituted amino acids has temperature sensitive phenotype and attenuated phenotype, and is suitable for being used as main donor virus for preparing attenuated live influenza virus vaccine.

Example 9: preparation of influenza viruses from 8 plasmid System by electroporation transduction of Vero cells

It has been previously reported that recombinant influenza A virus can be recovered from Vero cells (Fodor et al, (1999) "recovery of influenza A virus from recombinant DNA using recombinant DNA" J.Virol.73: 9679-82; Hoffmann et al, (2002) "8 plasmid System for Rapid preparation of influenza Vaccine" (Eight-plasmid System for Rapid Generation of influenza Vaccine) Vaccine 20: 3165-. The reported methods require liposome reagents and only one laboratory strain of influenza a virus (a/WSN/33 and a/PR/8/34) with high replication capacity has been reported, and thus its application in the preparation of live attenuated viruses suitable for vaccine preparation is limited. The present invention provides a novel method for recovering recombinant influenza viruses from Vero cells using electroporation. These methods are suitable for the preparation of influenza a and B strains, and can be used to recover cold-adapted, temperature sensitive attenuated viruses from Vero cells grown in serum-free medium to prepare live attenuated vaccines suitable for intranasal use, i.e. intranasal vaccine formulations. In addition to being able to be used with different virus strains, electroporation techniques do not require the addition of other reagents than the medium required for cell growth, thus reducing the risk of contamination. The method is particularly effective in the preparation of recombinant and reassortant viruses using serum-free cultured Vero cells, such as Vero cells which are pathogen-free and suitable for vaccine preparation. This feature makes electroporation a suitable commercial method for introducing DNA into a cellular substrate.

Electroporation techniques are compared with various other methods of introducing DNA into Vero cells, including transfection using liposomal reagents, calcium phosphate precipitation, and microinjection of cells. Although recovery of influenza a virus using liposomal reagents has sometimes been successful, only electroporation techniques can recover influenza B and influenza a virus from Vero cells.

Vero cells reaching 90-100% confluence were dispersed 1 day before electroporation in 9X 10 flasks per T225⁶The cells were inoculated at a density in MEM (MEM, 10% FBS) supplemented with penicillin/streptomycin, L-glutamine, non-essential amino acids, and 10% FBS. On the next day, the cells were trypsinized and resuspended in a T225 flask containing 50ml of Phosphate Buffered Saline (PBS). The cells were then centrifuged and resuspended in a T225 flask containing 0.5ml of OptiMEM I. In addition, ordered OptiMEM media containing components of non-human or animal origin may also be used. Cells were diluted 1: 40, counted on a hemocytometer and cell density determined, 5X 10⁶Cells were added to a 0.4cm electroporation cuvette with a final volume of 0.4ml OptiMEM I. 20. mu.g of DNA, equimolar mixtures of 8 plasmids containing the MDV-A or MDV-B genome, having a volume of not more than 25. mu.l, are then added to the cell-containing cups. Electroporation was performed on a BioRad Gene Pulser II with Capacitance Extender plus connected (BioRad, Hercules, Calif.) by gentle mixing with tapping at 300 volts for a duration of 28-33 seconds at 950 microfarads.

The cells were remixed by gentle tapping and 0.7ml of 10% FBS-containing MEM was added to the cuvette with a 1ml pipette about 1-2 minutes after electroporation. Cells were gently mixed by pipetting up and down several times, and then transferred to 2 wells of a standard 6-well tissue culture plate, containing 2ml MEM + 10% FBS. The cuvette was washed with 1ml of MEM + 10% FBS and the wash suspension was divided into two wells with a final volume of approximately 3.5ml per well.

In another experiment, Vero cells capable of growing in serum-free medium, OptiPro (SFM) (Invitrogen, Carlsbad, Calif.) were electroporated as described above, except that the process was performed in OptiMEM I, and the cells were diluted with OptiPro (SFM), which is the medium in which the cells were cultured for virus recovery.

The electroporated cells are cultured under conditions suitable for replication and recovery of the introduced virus as a cold-adapted master donor virus strain, e.g., 33 ℃. On the next day (i.e., about 19 hours after electroporation), the medium was removed and the cells were washed with OptiMEM or OptiPro (SFM) at 3ml per well. Then 1ml of penicillin/streptomycin-containing OptiMEM I or OptiPro (SFM) was added to each well and the supernatant was collected by replacing the medium every day. The supernatant was stored in SPG at-80 ℃. Virus production typically peaks at 2 to3 days after electroporation.

Table 18: results of recovery of MDV strains with 8 plasmids in different types of cells by different transfection methods

Example 10: influenza virus vector system for gene transfer

The vector of the present invention can also be used as a gene transfer system for gene therapy. In this application, it is desirable to produce recombinant influenza viruses that express foreign proteins, such as recombinant influenza virus type a or recombinant influenza virus type B. For example, since 7 segments of influenza B virus are not linked to each other, it is easy to insert a heterologous nucleic acid sequence. The mRNA contains two cistrons with two open reading frames encoding the M1 and BM2 proteins. The open reading frame of BM2 or M1 may be replaced by a heterologous sequence of interest, such as a gene encoding Enhanced Green Fluorescent Protein (EGFP). The cDNAs encoding the M1-EGFP and BM2 development reading frames were cloned into two different plasmids using the plasmid vector system of the present invention. The open reading frame is flanked by non-coding regions of segment 7, which contain signals required for replication and transcription. In addition, the following two plasmids can be constructed: one containing the ORF of M1 and the other containing EGFP-BM 2. The 9 plasmids are cotransfected to prepare the B type recombinant influenza virus containing a heterologous gene sequence. Also, EGFP may be expressed from the NS1 segment of influenza a virus.

Typical "green" influenza B viruses can be used in standardized viral assays, such as microneutralization assays. The combination of plasmid technology and simple detection of protein expression (fluorescence from EGFP can be observed under a microscope, as shown in fig. 2) allows to optimize the expression of the protein.

While certain details of the invention have been described above for purposes of clarity of understanding, it will be apparent to those skilled in the art from a reading of this specification that various changes in form and detail can be made without departing from the true scope of the invention. For example, all of the techniques and apparatus described above may be applied in various combinations. All publications, patents, patent applications, or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document were individually indicated to be incorporated by reference.

Claims (32)

1. A helper-free method of producing infectious influenza B virus in cell culture, the method comprising:

i) introducing 8 plasmid vectors containing a nucleic acid sequence which transcribes the influenza B virus genome segments PB2, PB1, PA, NP, HA, NA, M and NS into a population of host cells, each plasmid comprising a different genome segment, the population of host cells being capable of supporting replication of influenza B virus;

ii) culturing the population of host cells at a temperature less than or equal to 35 ℃; and

iii) recovering the influenza B virus.

2. The method of claim 1, wherein the influenza B virus comprises at least one of attenuated influenza virus, cold-adapted influenza virus and temperature-sensitive influenza virus.

3. The method of claim 1, wherein the influenza B virus has one or more of the following phenotypic characteristics, attenuation properties, temperature sensitivity and cold adaptation.

4. The method of claim 1, wherein the influenza B virus is suitable for use in the form of an intranasal vaccine formulation.

5. The method of claim 1, wherein the influenza B virus genomic segment is the influenza B/Ann Arbor/1/66 genomic segment or an artificially modified influenza B virus genomic segment comprising at least one substituted amino acid selected from the group consisting of PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵⁰⁹(A509T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V)。

6. The method of claim 5, wherein the artificially engineered influenza B virus comprises a set of substituted amino acids.

7. The method of claim 6, wherein the set of substituted amino acids comprises 2, 3, 4, 5,6, 7, 8, or 9 substituted amino acids.

8. The method of claim 1, wherein the PB2, PB1, PA, NP, M, and NS genomic segments are from a first influenza B virus strain; and at least one genomic segment of HA or NA surface antigen is from a second influenza B strain.

9. The method of claim 1, wherein said PB2, PB1, PA, NP, M, and NS genomic segments are from a first influenza B virus strain that is attenuated, cold-adapted, and/or temperature sensitive; and wherein at least one genomic segment encoding an HA or NA surface antigen is from a second influenza B virus strain.

10. The method of claim 1, wherein said PB2, PB1, PA, NP, M, and NS genomic segments are from a first influenza B virus strain that is attenuated, cold-adapted, and temperature sensitive; wherein at least one genomic segment encoding an HA or NA surface antigen is from a second influenza B virus strain.

11. The method of claim 1, wherein the PB2, PB1, PA, NP, M, and NS genomic segments are from influenza strain B/Ann Arbor/1/66 or an artificially engineered influenza virus containing at least one substituted amino acid selected from PB2⁶³⁰(S630R)、PA⁴³¹(V431M)、PA⁴⁹⁷(Y497H)、NP⁵⁵(T55A)、NP¹¹⁴(V114A)、NP⁴¹⁰(P410H)、NP⁵⁰⁹(A509T)、M1¹⁵⁹(H159Q) and M1¹⁸³(M183V); wherein at least one genomic segment encoding an influenza HA or NA surface antigen is from a different influenza B strain.

12. The method of claim 11 wherein the two genomic segments encoding HA and NA antigens are from influenza strains other than B/Ann Arbor/1/66.

13. The method of claim 1, wherein the population of host cells comprises one or more of Vero cells, Per.C6 cells, MDCK cells, 293T cells, or COS cells.

14. The method of claim 13, wherein the cell population comprises a mixture of at least two of MDCK cells, 293T cells and COS cells.

15. The method of claim 1, comprising culturing the population of host cells at a temperature between 32 ℃ and 35 ℃.

16. The method of claim 1, comprising culturing the population of host cells at a temperature between 32 ℃ and 34 ℃.

17. The method of claim 1, comprising culturing the population of host cells at 33 ℃.

18. The method of claim 1, further comprising inactivating influenza virus.

19. A method of producing an infectious influenza B virus in cell culture, the method comprising:

i) introducing by electroporation into a population of Vero cells 8 plasmid vectors containing nucleic acids which transcribe influenza B virus genomic segments PB2, PB1, PA, NP, HA, NA, M and NS, each plasmid comprising a different genomic segment;

ii) culturing the Vero cell population at a temperature of less than or equal to 35 ℃; and

iii) recovering the influenza B virus population.

20. The method of claim 19, wherein said influenza B virus has one or more of the phenotypic characteristics of attenuation, temperature sensitivity and cold adaptation.

21. The method of claim 19, wherein the influenza B virus comprises an attenuated, cold-adapted, temperature sensitive influenza virus.

22. The method of claim 19, wherein the influenza B virus is suitable for use in the form of an intranasal vaccine formulation.

23. The method of claim 19 comprising culturing Vero cells in serum-free medium.

24. A helper-free method of making a recombinant influenza B virus vaccine, the method comprising:

i) introducing 8 plasmid vectors containing nucleic acids that transcribe influenza B virus genome segments PB2, PB1, PA, NP, HA, NA, M and NS into a population of host cells, each plasmid comprising a different genome segment, the population of host cells capable of supporting replication of influenza B virus;

ii) culturing the host cell at a temperature of less than or equal to 35 ℃; and

iii) recovering influenza B virus capable of eliciting an immune response upon administration to the subject.

25. The method of claim 24, wherein said influenza B virus has at least one of the phenotypic characteristics of temperature sensitivity, cold adaptation and attenuation.

26. The method of claim 24, wherein the influenza B virus vaccine comprises a live attenuated influenza B virus vaccine.

27. The method of claim 24, further comprising inactivating influenza B virus.

28. A composition, comprising:

a cell culture comprising proliferative growth of at least one cell cultured at a temperature of less than or equal to 35 ℃, said at least one cell comprising 8 plasmid vectors comprising nucleic acids that transcribe to produce influenza B virus genomic segments PB2, PB1, PA, NP, HA, NA, M and NS, each plasmid comprising nucleic acid encoding a different genomic segment.

29. The composition of claim 28, further comprising a cell culture medium.

30. The composition of claim 28, wherein the vector comprises a bidirectional expression vector.

31. The composition of claim 28, wherein said bidirectional expression vector carries a plasmid comprising a first promoter located between a second promoter and a polyadenylation site of SV 40; wherein the first and second promoters are in opposite directions and flank at least one influenza segment.

32. The method of claim 1, 19, or 24, wherein the Log of recovered influenza B virus at 33 ℃_lOThe TCID50/ml titer was at least 7.0.