Reverse genetics vector and associated method of use

Abstract

A modified pHW2000 vector comprising an inactive T7 promoter is provided, as is a method for preparing an influenza virus vaccine using the modified vector.

Description

REVERSE GENETICS VECTOR AND ASSOCIATED METHOD OF USEINTRODUCTION

[0001] This application claims the benefit of priority from U.S. Provisional Application Serial Number 63 / 587,504, filed October 3, 2023, the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (name: SJ0106WO_ST26.xml; size: 17,182 bytes; and date of creation: August 13, 2024) is herein incorporated by reference in its entirety.BACKGROUND

[0003] The influenza A virus reverse genetics (RG) system (Hoffman et al. ( (2000) Proc. Natl. Acad. Sci. USA 97 (11) : 6108-6113; US 6, 951,754 B2; FIG. 1) provides a system for studying point mutations and the identification of phenotypes conferred by a single nucleotide or amino acid substitution. This system uses the bidirectional expression vector, pHW2000, which has a human pol I promoter with a murine terminator, a human cytomegalovirus (CMV) pol II promoter with a bovine growth hormone polyadenylation signal, and a T7 promoter downstream of the CMV promoter. In addition, the pHW2000 vector encodes (3-lactamase (AmpR) as the antibiotic selection marker. The orientation of the pol I and CMV pol II promoters allows the respective synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. To generate RG viruses, cDNAs from all eight influenza gene segments are cloned into pHW2000 using BsmBI or Bsal restriction sites resulting in eight plasmids.All eight plasmids are then transfected into human embryonic kidney (HEK) 293T cells or a coculture of HEK 293T cells and Madin Darby Canine Kidney (MDCK) cells. This dual promoter system expresses the necessary gene products for generating RG viruses.

[0004] After cloning nucleic acid inserts into the pHW2000 vector, and transforming competent bacteria, leaky expression of flu transcripts in bacteria occurs from the T7 and / or CMV pol II promoters. Expression of specific influenza gene segments are deleterious resulting in bacterial pressure. This bacterial pressure can manifest as frameshift or nonsense mutations and nonhomologous recombination between the cDNA insert and bacterial chromosome.

[0005] Modifications to the pHW2000 vector have been previously suggested. For example, US 9,187,732 B2 teaches the generation of vector pKW2000, which was obtained by deleting the CMV promoter in pHW2000. Thus, upon transfection only vRNA is transcribed from pKW2000 derivatives.

[0006] US 2016 / 0030547 Al discloses a similar type of vector as pHW2000, wherein the human Pol I promoter has been replaced with the Porcine Pol I promoter. This vector includes a CMV promoter, a murine polymerase I terminator sequence, a cloning site for genes to be transcribed, the swine polymerase I promoter sequence, and a bovine growth hormone polyadenylation signal.

[0007] Mostafa et al. ((2013) <J. Vir. Meth. 193:603-610) describe the generation of the pMPccdB vector using pHW2000 as a starting point, wherein the pMPccdB vector encodes the ccdB gene whose expression is lethal to most Escherichia coli strains .

[0008] pAD3000 is described by Hoffman et al. ( (2002) PNAS 99 (17) : 11411-11416) as being a variant of pHW2000, whereinthe bovine growth hormone ( BGH) polyadenylation signal is replaced with the SV40 late mRNA polyadenylation signal . Wang et al . ( ( 2007 ) Virol . J. 4 : 102 ) describe the preparation of a variant of pAD3000, wherein the human RNA pol I promoter sequence is replaced with the MDCK canine promoter sequence .SUMMARY

[0009] This invention provides a vector including a multiple cloning site flanked by a human polymerase I promoter and a human cytomegalovirus polymerase I I promoter, wherein the vector further includes an inactive T7 promoter operably linked to the multiple cloning site . In some aspects , the vector is a variant of pHW2000 . In other aspects, the vector further includes an influenza virus transgene in the multiple cloning site . In further aspects , the influenza virus transgene encodes an influenza PB2 , FBI , PA, HA, NP, NA, M or NS protein . A method for producing an influenza virus vaccine using the vector is also provided .BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG . 1 is a schematic representation of a method for the construction of the eight expression plasmids using the conventional pHW2000 vector . Viral RNA is extracted from virus particles . RT-PCR is performed with primers containing segment-specific nucleotides and sequences for the type I l s restriction endonucleases BsmBI (and its isoschizomer Bsp3 l ) or Bsal . The eight viral PCR fragments are digested with BsmBI or Bsal and inserted into pHW2000 (linearized with BsmBI ) . This insertion resulted in eight expression constructs where the viral cDNAs are precisely fused to the pol I promoter and terminator ( the viral terminal sequences AGC . . . ACT are shown for the PB2 segment in the rectangles ) .

[0011] FIG. 2A shows the point mutation in the T7 promoter of the pHW2000 vector resulting in the modified pPW2000 vector .

[0012] FIG. 2B shows the genetic elements controlling dual expression in pHW2000 and pPW2000 vectors.

[0013] FIG. 3 is a schematic representation of cloning an influenza A transgene segment into pPW2000 and pHW2000.

[0014] FIGS. 4A-4F show the measured differences comparing transformation efficiency between pPW2000 and pHW2000. Comparison of transformation efficiency of DH5-ot and HB101 bacteria when transformed with supercoiled vector lacking cDNA insert (FIG. 4A) . Transformation efficiencies from ligation reactions of Penn / 1370 matrix (M) gene (FIG. 4B) or hemagglutinin (HA) gene (FIG. 4C) in pPW2000 or pHW2000 vectors. To ensure that subtle differences in vector quantity were not a contributing factor, increased amounts of vector were included in ligation reactions (FIG. 4D) . Comparison of sequence accuracy between HB101 bacteria transformed with pPW2000 or pHW2000 vectors encoding the Penn / 1370 HA and M gene segments (FIG. 4E) . HA titers from harvested virus rescue supernatant following propagation in 10-day old embryonated chicken eggs (FIG. 4F) . Each black dot denotes a single egg. All reported CFU were normalized according to the bacterial stock titer. Statistical significance was measured using 2- Way ANOVA with Bonferroni correction (FIGS. 4A-4C) , multiple 2-sided unpaired t-tests (FIG. 4D) , and Fischer's Exact test (FIG. 4E) where, *p<0.05 and **P<0.01.

[0015] FIGS. 5A-5B show PGR results of RNA samples, treated with DNase I treated or subjected to mRNA pull-down, amplifying p-lactamase (FIG. 5A) , indicating that the mRNA samples were void of contaminating plasmid DNA. RT-qPCR was used to compare leaky expression in HB101 bacteria harboringeither pHW2000 or pPW2000 vectors ( FIG . 5B ) . Plasmid-free mRNA template was then subj ected to real-Time qPCR to quantify differences in leaky expression of M gene transcripts using the method . p-lactamase was used to normalize mRNA input . Differences in fold-change are relative to M gene expression in the pPW2000 vector . P values were calculated using a two- tailed Mann-Whitney U test . **, P < 0 . 01 .DETAILED DESCRIPTION

[0016] , This disclosure provides a modified pHW2000 vector of the reverse genetics system described in US 6, 951 , 754 B2 , which is incorporated herein by reference in its entirety . The pHW2000 vector described provides a dual promoter for the production of negative strand segmented viruses ( e . g . , influenza A, influenza B , Bunyaviridae) . Because the vector uses a single viral cDNA template for both protein and viral RNA synthesis , this vector reduces the total number of plasmids required for virus production and allows the development of vaccines quickly and cheaply . To decrease expression of viral transgenes in bacteria, the pHW2000 vector has been modified to knockout the T7 promoter . This modified vector, referred to herein as pPW2000, allows for easier cloning of viral transgenes . Advantageously, the pPW2000 vector provides for reduced selective pressure of E. coli to recombine or introduce mutations into the viral transgene inserted into the pPW2000 vector and reduced expression of gene products that are deleterious to virus rescue .

[0017] The invention provides a bidirectional vector and expression system including the same . The terms "vector, " "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e . g . , a foreign gene ortransgene) can be introduced into a host cell , so as to transform the host and promote expression (e . g . , transcription and translation) of the introduced sequence . Plasmids are preferred vectors of the invention . In accordance with the vector of this disclosure , a viral transgene is inserted between an RNA polymerase I (pol I ) promoter and terminator sequences ( inner transcription unit ) . This entire pol I transcription unit is flanked by an RNA polymerase I I (pol II ) promoter and a polyadenylation site (outer transcription unit ) . In the bidirectional vector, the pol I and pol II promoters are on opposite sides of a multiple cloning site for introducing a viral transgene ( e . g . , a cDNA) into the vector, wherein an upstream pol II promoter produces positive-sense capped mRNA and a downstream pol I promoter produces negative-sense uncapped viral RNA (vRNA) . This pol I-pol I I system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters . The pol I promoter and pol I terminator sequence in the bidirectional vector may be referred to as including a "downstream-to-upstream orientation" whereas the pol I I promoter and polyadenylation signal in the bidirectional vector may be referred to as including an "upstream-to- downstream orientation" .

[0018] A promoter , terminator or polyadenylation signal is "upstream" of a transgene if it is proximal to the start of the transgene ( e . g . , the first codon) and distal to the end of the transgene (e . g . , the termination codon ) . A promoter, terminator or polyadenylation signal is "downstream" of a transgene if it is proximal to the end of the transgene and distal to the start of the transgene . Promoters in the vector of the invention , which are functionally associated with atransgene, are oriented to promote transcription of a sense or an antisense strand of the transgene .

[0019] A "promoter" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a sequence . For purposes of defining the present invention , a promoter that is located upstream of a transgene (e . g . , cDNA) is bounded at its 3 ' terminus by a transcription initiation site and extends upstream ( 5 ’ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background . A promoter that is located downstream of a transgene (to express a (- ) RNA) is bounded at its 5 ' terminus by a transcription initiation site and extends downstream ( 3 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background . The bidirectional system of the invention includes both upstream and downstream promoters . Within the promoter sequence will be found a transcription initiation site ( conveniently defined for example, by mapping with nuclease SI ) , as well as protein binding domains ( consensus sequences ) responsible for the binding of RNA polymerase .

[0020] A coding sequence is "under the control of" , " functionally associated with" or "operatively associated with" transcriptional and translational control sequences (e . g . , a pol I or pol II promoter) in a cell when RNA polymerase transcribes the coding sequence into RNA, e . g . , mRNA or vRNA.

[0021] The vector of this disclosure is a variant of the pHW2000 vector described in US 6, 951 , 754 B2 . The pHW2000 vector includes the human pol I promoter with a murine terminator, a human cytomegalovirus (CMV) pol I I promoter with a bovine growth hormone polyadenylation signal , and anactive T7 promoter adjacent to the CMV pol II promoter and operably associated with the multiple cloning site. The variant of the pHW2000 vector, referred to herein as the pPW2000 vector, includes an inactive T7 promoter. As used herein, the "T7 promoter" refers to the nucleotide sequence 5 ' -TAATACGACTCACTATAG-3 ' (SEQ ID NO:1) that is recognized by T7 RNA polymerase. A "knockout of the T7 promoter," "T7 promoter knockout," or "inactive T7 promoter" refers to a nucleotide sequence that is no longer recognized by T7 RNA polymerase. Alternatively stated, T7 RNA polymerase no longer binds to an inactive T7 promoter or vector containing the same. In certain aspects, the T7 promoter knockout has at least one nucleotide substitution or deletion so that T7 RNA polymerase no longer binds. In other aspects, the T7 promoter knockout has two or more nucleotide substitutions or deletions so that T7 RNA polymer no longer binds. In some aspects, the T7 promoter knockout has one or a combination of the substitutions listed in Table 1.TABLE 1See, e.g., Ikeda et al. (1992) Nucl . Acids Res. 20(10) :2517- 2524

[0022] The pol I-pol II system of the vector of this invention facilitates the design and recovery of both recombinant and reassortment viruses and is applicable to the recovery of RNA viruses entirely from cloned cDNA . In some aspects, the vector is used in the production of a recombinant pathogenic virus . The term "pathogenic virus" is used herein to refer to any virus strain that is capable of causing disease ; preferably, the virus is on the current World Health Organization (WHO) , Centers for Disease Control and Prevention (CDC) , or other public health authority list of likely circulating viruses . Such viruses may include hepatitis viruses (e . g . , those of the Picornaviridae, Flaviviridae , and Hepeviridae families,) , as well as members of the Orthomyxovirus family, the Paramyxovirus family, the Filoviridae family, the Bornaviridae family, the Hantaviridae family, the Arenaviridae family, Coronaviridae family, or the Rhabdovirus family . The invention advantageously provides for inserting the genes for the primary antigens from such strains into a plasmid-based system, in which the remaining viral genes have desired culture and / or attenuation characteristics , thus providing for production of quantities of virus of the appropriate antigenic background for vaccine production . For example, the genes of the paramyxovirus, human parainfluenza virus 3 (nucleocapsid gene, phosphoprotein gene, matrix gene, fusion gene, hemagglutininneuraminidase protein gene and large gene) , may conveniently be placed in the plasmids of the invention for production of parainfluenza virus 3 virions .

[0023] Table 2 includes a non-limiting list of RNA viruses which may be produced using the modified vector and system disclosed herein .TABLE 2

[0024] In certain aspects , the vector and system are used to produce an influenza virus . The genome of influenza A virus typically encodes at least 10 dif ferent proteins : PB2 , PB1 , and PA are the three polymerase proteins that associate together making the polymerase complex, HA which is described as hemagglutinin , NP, which is described as an RNA binding nucleoprotein , NA which is described as a neuraminidase or sialidase, M1 / M2 which are described as a matrix protein and an integral membrane protein, respectively, and NS1 / NEP, formerly NS2 , which are nonstructural proteins affecting RNAprocessing and transport . PB1 , PB2 , NP and PA are described as being part of an influenza virus transcriptional polymerase complex . Thus , in some aspects , the modified vector and associated system of this invention may include viral transgenes (e . g . , influenza cDNA) encoding PB2 , PB1 , PA, HA, NP, NA, M or NS that are flanked by the RNA polymeraseI (pol I ) promoter for vRNA synthesis and the RNA polymeraseI I (pol I I ) promoter for viral mRNA synthesis .

[0025] A "viral transgene" is preferably a cloned cDNA corresponding to a genomic RNA molecule from the RNA virus genome . Although cDNA is preferred, any other type of nucleic acid that encodes a viral gene which is to be expressed may be used . For example, PCR-amplif ied products or restriction fragments including viral genes may be used . Furthermore , the transgenes expressed by the vector of the invention may be fused to or tagged with other genes such as purif ication / detection tags ( e . g . , glutathione-S transferase, polyhistidine , green fluorescent protein, myc tags and FLAG tags ) . The present invention also provides embodiments wherein partial gene sequences are used in the vectors herein .

[0026] Viral transgene sequences are introduced into the vector via a multiple cloning site . As is conventional in the art , a "multiple cloning site" is a nucleic sequence of the vector that includes a variety of singular restriction enzyme recognition sequences so that a suitable insertion point for a transgene is available .

[0027] In other aspects, the vector will incorporate gene segments for antigens required to produce a protective immunological response . A "protective immunological response" includes a humoral (antibody) or cellular component , or both, effective to eliminate infectious virions and infected cellsin an immunized (vaccinated) subj ect . Thus , a protective immune response can prevent or resolve an RNA virus , e . g . , influenza virus , infection . Preferably, the antigens are "surface antigens, " i . e . , expressed on the surface of the virion or the surface of infected cells . More preferably, the surface antigens are glycoproteins . For influenza , the primary glycoprotein antigens are hemagglutinin (HA or H) and neuraminidase (NA or N) .

[0028] As used herein, the term "immunogenic" means that an antigen is capable of eliciting a humoral or cellular immune response , and preferably both . An immunogenic composition is a composition that elicits a humoral or cellular immune response , or both, when administered to an animal . A molecule is "antigenic" when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin ( antibody) or T cell antigen receptor . An antigenic polypeptide contains an epitope of at least about five, and preferably at least about 10, amino acids . An antigenic portion of a polypeptide, also called herein the epitope, can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conj ugating the antigenic portion to a carrier polypeptide for immunization . A molecule that is antigenic need not be itself immunogenic, i . e . , capable of eliciting an immune response without a carrier .

[0029] In some aspects, the vector and system of this invention is used in the design and recovery of reassortment viruses . A "reassortment" virus is a virus in which gene segments encoding antigenic proteins from a pathogenic virus are combined with gene segments encoding viral polymerase complex or other similar genes (e . g . , non-glycoprotein genes ,including M genes and NS genes ) from viruses adapted for growth in culture (or attenuated viruses ) . The reassortment virus thus carries the desired antigenic characteristics in a background that permits efficient production in a host cell . Such a reassortment virus is a desirable "virus seed" for production of virions to produce vaccine . In addition to incorporating genes for viral antigens in a genetic background of a virus strain adapted to grow well in culture , the invention permits creating cross-species reassortments , e . g . , an influenza B antigen in an influenza A background .

[0030] The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of recombinant RNA virions , preferably negative strand segmented RNA virions, by the cell . Exemplary host cells include , but are not limited to, Madin-Darby Canine Kidney (MDCK) cells, VERO cells , CV1 cells, COS-1 and COS-7 cells, and BHK-1 cells , for example and not by way of limitation . In some aspects , a transient co-culture is preferred for producing virions . Co-culturing permits ef ficient transfection of a receptive cell , such as a 293T cell, with subsequent infection of a permissive cell for viral growth, such as an MDCK cell . The term "cell culture" or "co-culture" as used herein refers to an acceptable method for propagating virus for vaccine production, e . g . , embryonated hens ' eggs , and cell culture .

[0031] As indicated herein, the present vector and associated system provides an efficient and economic strategy for production of vaccines for treating or preventing RNA viral infections, preferably, negative strand RNA virus infections . Accordingly, this invention provides a method for using the pPW2000 vector to prepare a vaccine , in particular an influenza virus vaccine against seasonal influenza strains ,pandemic strains , and highly pathogenic avian influenza strains . The vector of the invention eliminates the need for selection and provides close control of reassortment viruses . For the production of an inactivated influenza vaccine six plasmids containing the non-glycoprotein segments (e . g . , PB1 , PB2 , PA, NP, M and NS) from a high yield strain can be cotransfected with two expression plasmids encoding HA and NA from the candidate vaccine virus . Since no helper virus is required, the generated virus is an influenza virus with the desired gene constellation . An analogous approach may be used to produce any variety of inactivated, reassortment RNA virus for use in a vaccine . Expression vectors harboring viral gene segments for a target virus ( e . g . , nonglycoprotein segments ) may be co-transfected with other expression vectors encoding proteins corresponding to a given infectious viral subtype ( e . g . , a viral subtype which is currently circulating in the population ) . Virus produced in accordance with the invention can be used in traditional or new approaches to vaccination, particularly in the development of live , attenuated vaccines .

[0032] Three types of inactivated influenza vaccines are conventionally available : whole virus, split-product , and surface antigen vaccines . Because the present vector permits the rapid development of a desired reassortment virus with acceptable growth characteristics in culture, it advantageously positions a vaccine manufacturer to generate a sufficient quantity of vaccine to meet public health needs and ensure standardi zation, which is an important requirement currently mitigated by the need to produce clinical quantities of vaccine, usually an 8 to 9 month period .

[0033] As used herein, a "vaccine" is a composition that can elicit protective immunity when administered to a subj ect . The term "vaccine" refers to a composition containing virus,inactivated virus, attenuated virus, split virus, or viral protein, i.e. , a surface antigen, that can be used to elicit protective immunity in a recipient. It should be noted that to be effective, a vaccine can elicit immunity in a portion of the population, as some individuals may fail to mount a robust or protective immune response, or, in some cases, any immune response. This inability may stem from the individual's genetic background or because of an immunodeficiency condition (either acquired or congenital) or immunosuppression (e.g., treatment with immunosuppressive drugs to prevent organ rejection or suppress an autoimmune condition) . Efficacy can be established in animal models.

[0034] A "protective dose" of a vaccine is an amount, alone or in conjunction with an adjuvant, effective to elicit a protective immune response in a recipient subject. Protection can also depend on the route of administration, e.g. , intramuscular (preferred for an inactivated vaccine) or intranasal (preferred for an attenuated vaccine) .

[0035] The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specif ically enhances the immune response. Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Coryn eba ct er i um parvum, An example of a preferred synthetic adjuvant is QS-21. Alternatively, or in addition, immunostimulatory proteins can be provided as anadjuvant or to increase the immune response to a vaccine. Preferably, the adjuvant is pharmaceutically acceptable.

[0036] The phrase "pharmaceutically acceptable" refers to molecular-entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans . The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Sterile water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin .

[0037] Vaccination effectiveness may be enhanced by coadministration of an immunostimulatory molecule such as an immunostimulatory, immunopotentiating, or pro-inflammatory cytokine, lymphokine, or chemokine with the vaccine, particularly with a vector vaccine. For example, cytokines or cytokine genes such as interleukin IL-1, IL-2, IL-3, IL'S, IL-12, IL-13, granulocyte-macrophage (GM) -colony stimulating factor (CSF) and other colony stimulating factors, macrophage inflammatory factor, Flt3 ligand, as well as some key costimulatory molecules or their genes (e.g. , B7.1, B7.2) can be used. These immunostimulatory molecules can be delivered systemically or locally as proteins or byexpression of a vector that codes for expression of the molecule .

[0038] Mucosal vaccine strategies are particularly effective for many pathogenic viruses since infection often occurs via the mucosa. Thus, mucosal vaccination strategies for inactivated and attenuated virus vaccines are contemplated. While the mucosa can be targeted by local delivery of a vaccine, various strategies have been employed to deliver immunogenic proteins to the mucosa. In some aspects, the vaccine can be administered in an admixture with, or as a conjugate or chimeric fusion protein with, cholera toxin, such as cholera toxin B or a cholera toxin A / B chimera. In another aspect, an admixture with heat labile enterotoxin (LT) can be prepared for mucosal vaccination.

[0039] Other mucosal immunization strategies include encapsulating the virus in microcapsules (See, e.g., US 5,075,109, US 5,820,883, US 5,853,763) and using an immunopotentiating membranous carrier (WO 98 / 0558) . Immunogenicity of orally administered immunogens can be enhanced by using red blood cells (RBC) or RBC ghosts (US 5, 643,577) , or by using blue tongue antigen (US 5,690,938) .Example 1 : Materials and Methods

[0040] Mammalian Cell Culture. Monolayer cell cultures of human embryonic kidney (HEK) 293T cells were grown in opti- minimum essential media (MEM) supplemented with 5% fetal bovine serum grown at standard conditions of 37 °C with 5% CO2.

[0041] Bacterial Culture. Competent strains of E. coli HB101 and DH5-CX were obtained from commercial vendors (Table 3) . Bacterial stock titers were determined by making 10-fold serial dilutions and plating on Luria-Bertani (LB) agarovernight at 37 °C and determining the colony forming units (CPU) at 16-hours post spreading to calculate bacterial stock titer .TABLE 3

[0042] Plasmid Isolation. Plasmid DNA was isolated from 2 mL of transformed bacteria in logarithmic growth phase using the QIAprep® spin miniprep kit. Plasmid stocks were made by lysing bacteria and isolating plasmid DNA from 120 mL of transformed bacteria grown in the presence of ampicillin (100 pg / mL) . Both Mini preps and Maxi preps were performed according to the manufacturer's recommendation.

[0043] Site-Directed Mutagenesis . Approximately 50 ng of pHW2000 without insert was subjected to mutagenesis using the T7 KO forward primer 5' -CTTATCGAAATTAATACGGCTCACTATAGGGAGACC-3' (SEQ ID NO: 10) and the T7 KO reverse primer 5' -GGTCTCGCTATAGTGAGCCGTATTAATTTCGATAAG-3' (SEQ ID NO: 11) using the Phusion high fidelity DNA polymerase (NEB) . Two pL of ligation reaction was then used to transform competent bacterial strains DH5-a or HB101 cells in the presence of ampicillin. The resulting mutant pHW2000 vector, referred to herein as pPW2000 is set forth in SEQ ID NO: 12.

[0044] Cloning and Virus Rescue. The cDNA from all eight genomic segments from A / Chicken / Pennsylvania / 1370 / 1983 (H5N2) (Penn / 1370) and A / Puerto Rico / 8 / 1934 (H1N1) (PR / 8) were cloned into the influenza A virus RG expression vectorspHW2000 and pPW2000 . RG viruses were generated as 7+1 viruses where HA, NA and internals (excluding M) originated from PR / 8 and the M was derived from Penn / 1370 . All of the PR / 8 genomic segments were cloned into pHW2000 while the M from Penn / 1370 was cloned into both pHW2000 and pPW20000 as previously described (Hof fman et al . (2000 ) Proc . Natl . Acad. Sci . USA 97 ( 11 ) : 6108- 6113 ) . Briefly, plasmids encoding cDNA of all eight viral RNA segments were transfected into human embryonic kidney (HEK) 293T cells using the transfection reaction sold under the tradename LIPOFECTAMINE® 3000 reagent (Thermo Fisher) . At 48-hours post-transfection, cell supernatant was harvested and 200 pL of cell supernatant was inj ected into 10-day old embryonated chicken eggs to propagate virus and incubated at 35 °C . Virus rescue was considered successful with a positive hemagglutinin agglutination titer >2 following egg propagation .

[0045] Restriction Digest and Liga tion . Approximately 100 ng of pHW2000 or pPW2000 and the M and HA genes from Penn / 1370 that were previously cloned into pHW2000 were digested out with the restriction enzymes Sall and Apal ( FIG . 3 ) . Because the HA insert and vector closely migrate on an agarose gel , this reaction was subj ected to a triple digest with Sall , Apal , and Ncol in order to further digest the vector and facilitate extraction of the HA insert from the agarose gel . Restriction digest fragments were recovered using the GFX™ PCR DNA and Gel Band Purification Kit (Cytiva) and eluted in 50 pL of nuclease free water . Ligation reactions were carried out in a 20 pL total volume, which included 1 pL of digested vector, 4 pL of HA insert or 2 μL of M insert , and NEB Ligase according to manufacturer' s recommendation .

[0046] Transformation . Approximately 30 pL of competent bacteria were mixed with 2 pL of ligation reaction . Bacteriawere heat-shocked for 90 seconds, allowed to undergo one round of replication, spread on LB agar supplemented with 100 pg / mL of ampicillin (Amp+) , and grown overnight at 37 ° C . Transformation efficiency was determined by transforming bacteria with pHW2000 or pPW2000 lacking cDNA insert and determining the number of CFU / pg of DNA . Transformation efficiency ( % ) of ligation reactions was determined by dividing the number of transformed CFUs by the total number of CFUs in the bacterial stock .

[0047] Sanger Sequencing. Plasmids were sequenced by the Sanger method by the Hartwell Center at St . Jude Children' s Research hospital by capillary electrophoresis using an ABI prism capillary sequencer (Applied Biosystems ) . Genes were assembled with DNASTAR Lasergene version 17 . 0 . 2 and aligned against its homologous sequence previously determined by Illumina paired short reads sequencing .

[0048] Conventional PCR . Phusion high fidelity DNA polymerase (NEB) was used to amplify p-lactamase using the same primers and conditions used in RT-qPCR . However, the reverse transcription step was omitted . Amplicons were visualized by running DNA on a 2% agarose gel stained with intercalating agent GelRed® .

[0049] Real Time PCR. Total RNA was subj ected to DNase I digestion ( Invitrogen) and mRNA pull down (Qiagen RNeasy pure mRNA bead kit ) . Primers and probes used for reverse transcription quantitative polymerase chain reaction (RT- qPCR) were synthesized by integrated DNA technologies ( IDT) . PCR was done using Superscript™ II I Platinum™ One-Step qRT- PCR kit with ROX as a passive reference in the 7500 system . p-Lactamase was used as an endogenous control . PCR ef ficiency was determined usingPrimer and probesequences for p-lactamase were homologous to vector sequence . Data were analyzed using the 2-AACtmethod .

[0050] Hemaggl utinin Agglutina tion (HA) Assay. This assay was performed as previously described ( Killian (2014 ) Methods Mol . Biol . 1161 : 3- 9 ) . Briefly, 100 pL of virus is added to a 96-well plate . Two-fold dilutions in phosphate-buf fered saline ( PBS ) were performed by adding 50 pL of sample to 50 pL of PBS . Then 50 pL of 0 . 5% chicken red blood cells were added to each well and incubated at room temperature for 30 minutes . HA titer was determined as the highest dilution with agglutination present .

[0051] RNA Extraction . Total RNA was extracted by adding 750 pL of Trizol™ reagent to 250 pL of bacteria in exponential growth phase harboring either pHW2000 or pPW2000 . One-hundred and fi fty pL of chloroform was added to the Trizol™ reagent for RNA isolation by phase separation . RNA was recovered from the aqueous phase and then precipitated with isopropanol and then washed two times with 75% ethanol . RNA pellets were air dried and then resuspended in 50 pL of RNase-free water . RNA quantity and quality was determined by Nanodrop® UV-Vis spectrophotometry .

[0052] mRNA Isola tion . Total RNA isolated via the Trizol® / chloroform method was subj ected to the RNeasy pure mRNA bead kit (Qiagen) . Briefly, 5 pg of total RNA was suspended in 250 pL of nuclease-free water and mixed with 275 pL of polyA-oligo-dT beads . RNA was incubated for 2 minutes at 70 °C to break RNA secondary structures . RNA and polyA- oligo-dT mixture were then incubated for 10 minutes at room temperature to allow hybridi zation of the mRNA poly A tail and oligo-dT beads . The mRNA-oligo-dT was then centrifuged and resuspend in washing buffer and added to a spin column that did not allow oligo-dT beads to pass through . mRNA-oligo-dT was washed, eluted by adding 20 pL of elution buffer pre-warmed to 65 °C to denature the mRNA-oligo-dT hybrid, and centrifuged to collect isolated mRNA. This was done two times to increase mRNA yield in a total elution volume of approximately 40 pL . Three pL of purified mRNA was then subj ected to RT-qPCR to quantify M gene transcripts .

[0053] DMA Extraction . Total DNA was extracted from 12 mL of transformed bacteria in logarithmic growth phase using the phenol : chloroform: isoamyl ( PCI ) alcohol ( 25 : 24 : 1 v / v) method . Briefly, bacteria were centrifuged at 5000 x g for 7 minutes . Bacterial broth was decanted and the bacterial pellet was resuspended in 250 pL sterile PBS supplemented with MgC12 and RNase A and then incubated at room temperature for 30 minutes . Fifty pL of 10% SDS was added, mixed, and incubated at 55 ° C for 1 hour with gentle mixing every 20 minutes . DNA was extracted by adding 750 pL of UltraPure™ PCI that was saturated with Tris-HCl ( Invitrogen ) and subj ected to centrifugation at 12 , 000 x g for 15 minutes . DNA was collected from the aqueous phase and equal amounts of PCI was added and centrifuged using previous conditions . This was done two additional times and then DNA was then precipitated with 100% ethanol and 3 M sodium acetate . The DNA pellet was washed two times with 75% ethanol . DNA quantity and purity was assessed using a Nanodrop™ UV-Vis spectrophotometer . This DNA served as a positive control when evaluating DNA contamination in RNA samples .

[0054] Virus Propagation . Viruses were propagated in 10-day old embryonated chicken eggs . RG viruses were made on the backbone of PR / 8 and propagated in chicken eggs for 72 hours and then chilled overnight at 4 °C . After chilling, approximately 10 mL of virus was harvested from each egg and virus was subj ected to HA assay for titer determination .Viruses with the highest HA titer were pooled and aliquoted for storage at -80 °C .Example 2 : Results

[0055] During cloning of transgenes into the pHW2000 vector , the T7 promoter likely facilitates Influenza A virus gene expression in bacteria . Some of these gene products can be toxic to the bacteria resulting in pressure to induce mutations in the cloned transgene . Mutations occur most often when making a plasmid stock . To overcome this , the pHW2000 expression vector was modified by knocking out the T7 promoter using site-directed mutagenesis ( FIG . 2A) . The resulting vector is referred to herein as pPW2000 . With the exception of the mutation of the T7 promoter, pPW2000 does not include any changes in the regions flanking the cDNA insert ( FIG . 2B ) . To determine whether pPW2000 had less nucleotide mutations or insert ions / deletions , the cDNA from HA and M from Penn / 1370 were generated by PCR amplification, then inserted into the multiple cloning site of pHW2000 or pPW2000 , and the sequences were confirmed by Sanger sequencing . The impact of the loss of T7 promoter function on cloning efficiency, virus rescue , and leaky transcription was then determined .

[0056] Two different bacterial strains, DH5-a and HB101, were used in this analysis . The latter is a more robust bacterial strain for cloning toxic gene segments . Three different experiments were conducted to determine if loss of T7 promoter function impacted cloning efficiency . The first experiment compared transformation efficiency of undigested, supercoiled plasmid (pHW2000 and pPW2000 ) lacking cDNA insert . The second experiment used ligation reactions containing digested vector and M gene cDNA, which is considered a nontoxic gene andrelatively easy to clone . Lastly, ligation reactions were carried out with an HA ( Penn / 1370 ) gene segment that is difficult to clone and tends to recombine with the bacterial chromosome . No significant difference in transformation efficiency was found between pHW2000 and pPW2000 in DH5-a or HB101 cells when transformed with undigested, supercoiled plasmid ( FIG . 4A) . Transformation efficiencies between pHW2000 and pPW2000 encoding the Penn / 1370 M segment were significantly (p<0 . 05 ) different when transformed into DH5-a competent bacteria ( FIG . 4B) . However, no significant differences were detected in the HB101 strain . Significant differences in transformation efficiencies between pHW2000 and pPW2000 from ligation reactions were observed when the HA cDNA was encoded ( FIG . 4C) . The largest dif ference in transformation efficiency was found when comparing DH5-ot cells transformed with the pHW2000 encoding HA to HB101 bacteria transformed with pPW2000 encoding the HA (p<0 . 05 ) . In comparison , no significant difference in transformation efficiency was observed between DH5-ot cells transformed with HA-containing pPW2000 compared to HB101 bacteria transformed with HA-containing pHW2000. This indicated that loss of T7 promoter function can partially compensate for using a less efficient bacterial strain .

[0057] To ensure these results were not from unequal amounts of vector input, which could alter CFU formation and erroneously affect the results , increasing amounts of vector were used in ligation reactions ( FIG . 4 D) . However , the large difference was unexpected when transforming the M gene , which is considered a nontoxic gene . In all three strains, increased CFUs were observed when transformed with the pPW2000 vector . Even when three times the amount of pHW2000 was added to the ligation reactions , increased CFUs were consistently observedfollowing transformation with pPW2000. Additionally, no CPUs were observed when vector was ligated without insert .

[0058] It was subsequently determined whether a functional T7 promoter would significantly af fect the sequence accuracy of cloned gene segments . These experiments included the use of pHW2000 and pPW2000 that encode cDNA of the M or HA segments . Twelve M segments and 29 HA segments were then sequenced . A comparison of cloned M gene sequences (n = 12 ) indicated that 100% of the sequences were correct , regardless of the vector used . However, comparison of HA sequences (n = 29) revealed disparities in sequence accuracy when HA was encoded by pHW2000 ( 25 / 29 ) compared to pPW2000 ( 28 / 29 ) . Although , this difference in sequence accuracy was found to be statistically insignificant (p=0. 1762 ) in HB101 bacteria ( FIG . 4E) . When the number of sequences that had mutations disrupting their open reading frame (ORF) were compared, pHW2000 had one recombination event between the cDNA insert and the bacterial chromosome .

[0059] It was subsequently ensured that the knockout of the T7 promoter did not have a deleterious effect on virus rescue . Plasmid stocks of pHW2000 and pPW2000 that encoded the Penn / 1370 M gene segment were used . The other seven genes originated from PR / 8 and were cloned into pHW2000 . The expression plasmids were co-transfected into 293T cells and at 48 hours post-transfection, cell supernatant was harvested and inj ected into embryonated chicken eggs . Virus was harvested in the form of allantoic fluid and the viral HA titer was determined ( FIG . 4 F) . It was found that loss of T7 promoter function had no impact on virus rescue efficacy .

[0060] To determine whether knocking out the T7 promoter reduced influenza gene expression in the bacterial cell, real-time quantitative PCR (RT-qPCR) targeting the Penn / 1370M gene and (3-lactamase was used. Total RNA was extracted from bacteria, and mRNA was purified using an mRNA-pull down. To ensure plasmid contamination was not interfering with the results, a DNase I digestion and a mRNA-pulldown were both carried out (FIG. 5A) . The purified mRNA was then subjected to conventional PCR using only high-fidelity DNA polymerase, which would only allow amplification of contaminating plasmid. HB101 bacteria transformed with pPW2000 were used as a positive control. The mRNA pull-down samples were subsequently used for transcript expression analysis by real time qPCR (FIG. 5B) . A dose-dependent response was observed in the real-time PCR assay, which was utilized as a standard curve. The PCR efficiency for p-lactamase and Ml were 99.136% (r2= 0.998) and 95.052 (r2= 0.994) , respectively. The amount of template added to the PCR reactions were normalized to [3- lactamase expression, which was also encoded on the RG plasmids pHW2000 and pPW2000. It was found that pHW2000 had increased expression of Ml transcripts compared to pPW2000 (p<0.01) . Collectively, these results indicated that the influenza A virus RG vector, pHW2000, was improved by rendering the T7 promoter nonfunctional. The improved vector pPW2000 had significantly reduced expression of cloned gene segments compared to pHW2000. Loss of T7 promoter function increased cloning and transformation efficiencies, which has pertinent applications for generating future influenza vaccines .

Claims

CLAIMS1 . A vector comprising a multiple cloning site flanked by a human polymerase I promoter and a human cytomegalovirus polymerase I I promoter, the vector further comprising an inactive T7 promoter operably linked to the multiple cloning site .2 . The vector of claim 1 , wherein the vector is a variant of pHW2000 .3 . The vector of claim 1 , wherein the vector comprises the nucleotide sequence of SEQ ID NO : 12 .4 . The vector of claim 1 , further comprising an influenza virus transgene in the multiple cloning site .5 . The vector of claim 4 , wherein influenza virus transgene encodes an influenza PB2 , PB1 , PA, HA, NP, NA, M or NS protein .

6. A method for producing an influenza virus vaccine comprising introducing into a host cell a vector of claim 1 comprising a transgene encoding influenza PB2 protein, a vector of claim 1 comprising a transgene encoding influenza PB1 protein, a vector of claim 1 comprising a transgene encoding influenza PA protein, a vector of claim 1 comprising a transgene encoding influenza HA protein, a vector of claim 1 comprising a transgene encoding influenza NP protein, a vector of claim 1 comprising a transgene encoding influenza NA protein, a vector of claim 1 comprising a transgeneencoding influenza M protein, and a vector of claim 1 comprising a transgene encoding influenza NS protein, and isolating influenza virus produced by the host cells .

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